piperidines and capsazepine

The endocannabinoid system modulates a wide variety of pain conditions. Systemically administered AM404, an endocannabinoid reuptake inhibitor, exerts antinociceptive effects via activation of the endocannabinoid system. However, the mechanism and site of AM404 action are not fully understood. Here, we explored the effect of AM404 on neuropathic pain at the site of the spinal cord.. Male Sprague-Dawley rats were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effects of intrathecal administration of AM404 on mechanical and cold hyperalgesia were examined using the electronic von Frey test and cold plate test, respectively. Motor coordination was assessed using the rotarod test. To understand the mechanisms underlying the action of AM404, we tested the effects of pretreatment with the cannabinoid type 1 (CB. AM404 attenuated mechanical and cold hyperalgesia with minimal effects on motor coordination. AM251 significantly inhibited the antihyperalgesic action of AM404, whereas capsazepine showed a potentiating effect.. These results indicate that AM404 exerts antihyperalgesic effects primarily via CB

Topics: Animals; Arachidonic Acids; Capsaicin; Constriction; Disease Models, Animal; Endocannabinoids; Hyperalgesia; Indoles; Male; Neuralgia; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rotarod Performance Test; Spinal Cord; TRPV Cation Channels

Medications that improve pain threshold can be useful in the pharmacotherapy of Parkinson's disease (PD). Pain is a prevalent PD's non-motor symptom with a higher prevalence of analgesic drugs prescription for patients. However, specific therapy for PD-related pain are not available. Since the endocannabinoid system is expressed extensively in different levels of pain pathway, drugs designed to target this system have promising therapeutic potential in the modulation of pain. Thus, we examined the effects of the 6-hydroxydopamine- induced PD on nociceptive responses of mice and the influence of cannabidiol (CBD) on 6-hydroxydopamine-induced nociception. Further, we investigated the pathway involved in the analgesic effect of the CBD through the co-administration with a fatty acid amide hydrolase (FAAH) inhibitor, increasing the endogenous anandamide levels, and possible targets from anandamide, i.e., the cannabinoid receptors subtype 1 and 2 (CB1 and CB2) and the transient receptor potential vanilloid type 1 (TRPV1). We report that 6-hydroxydopamine- induced parkinsonism decreases the thermal and mechanical nociceptive threshold, whereas CBD (acute and chronic treatment) reduces this hyperalgesia and allodynia evoked by 6-hydroxydopamine. Moreover, ineffective doses of either FAAH inhibitor or TRPV1 receptor antagonist potentialized the CBD-evoked antinociception while an inverse agonist of the CB1 and CB2 receptor prevented the antinociceptive effect of the CBD. Altogether, these results indicate that CBD can be a useful drug to prevent the parkinsonism-induced nociceptive threshold reduction. They also suggest that CB1 and TRPV1 receptors are important for CBD-induced analgesia and that CBD could produce these analgesic effects increasing endogenous anandamide levels.

Topics: Amidohydrolases; Analgesics; Animals; Benzamides; Brain; Cannabidiol; Capsaicin; Carbamates; Celecoxib; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Morphine; Nociception; Oxidopamine; Pain; Parkinson Disease; Piperidines; Pyrazoles; Thienamycins

Clinical studies indicate that cannabidiol (CBD), the primary nonaddictive component of cannabis that interacts with the serotonin (5-HT)1A receptor, may possess analgesic and anxiolytic effects. However, its effects on 5-HT neuronal activity, as well as its impact on models of neuropathic pain are unknown. First, using in vivo single-unit extracellular recordings in rats, we demonstrated that acute intravenous (i.v.) increasing doses of CBD (0.1-1.0 mg/kg) decreased the firing rate of 5-HT neurons in the dorsal raphe nucleus, which was prevented by administration of the 5-HT1A antagonist WAY 100635 (0.3 mg/kg, i.v.) and the TRPV1 antagonist capsazepine (1 mg/kg, i.v.) but not by the CB1 receptor antagonist AM 251 (1 mg/kg, i.v.). Repeated treatment with CBD (5 mg/kg/day, subcutaneously [s.c.], for 7 days) increased 5-HT firing through desensitization of 5-HT1A receptors. Rats subjected to the spared nerve injury model for 24 days showed decreased 5-HT firing activity, mechanical allodynia, and increased anxiety-like behavior in the elevated plus maze test, open-field test, and novelty-suppressed feeding test. Seven days of treatment with CBD reduced mechanical allodynia, decreased anxiety-like behavior, and normalized 5-HT activity. Antiallodynic effects of CBD were fully prevented by capsazepine (10 mg/kg/day, s.c., for 7 days) and partially prevented by WAY 100635 (2 mg/kg/day, s.c., for 7 days), whereas the anxiolytic effect was blocked only by WAY. Overall, repeated treatment with low-dose CBD induces analgesia predominantly through TRPV1 activation, reduces anxiety through 5-HT1A receptor activation, and rescues impaired 5-HT neurotransmission under neuropathic pain conditions.

Topics: Action Potentials; Animals; Anxiety; Cannabidiol; Capsaicin; Disease Models, Animal; Exploratory Behavior; Feeding Behavior; Ganglia, Spinal; Hyperalgesia; Lysergic Acid Diethylamide; Male; Maze Learning; Neuralgia; Piperazines; Piperidines; Pyrazoles; Pyridines; Rats; Rats, Wistar; Serotonin; Serotonin Antagonists; Swimming

A recent hypothesis suggesting that the pharmacological target TRPV1 (transient receptor potential vanilloid subfamily, member 1) may function as a tumour suppressor, which potentially impacts the development of TRPV1 antagonist therapeutics for a range of conditions. However, little is known about the long-term physiologic effects of TRPV1 blockade in the skin. In vitro and in vivo studies suggested that the potent TRPV1 competitive antagonist AMG-9810 promoted proliferation in N/TERT1 cells (telomerase-immortalised primary human keratinocytes 1) and tumour development in mouse skin that was mediated through EGFR/Akt/mTOR signalling. We attempted to reproduce the reported in vitro and in vivo findings to further explore this hypothesis to understand the underlying mechanism and the risk associated with TRPV1 antagonism in the skin. In vitro proliferation studies using multiple methods and topical application with AMG-9810 and structurally similar TRPV1 antagonists such as SB-705498 and PAC-14028 were performed. Although we confirmed expression of TRPV1 in primary human epidermal keratinocytes (HEKn) and spontaneously immortalised human keratinocytes (HaCaT), we were unable to demonstrate cell proliferation in either cell type or any clear evidence of increased expression of proteins in the EGFR/Akt/mTOR signalling pathway with these molecules. We were also unable to demonstrate skin tumour promotion or underlying molecular mechanisms involved in the EGFR/Akt/mTOR signalling pathway in a single-dose and two-stage carcinogenesis mouse study treated with TRPV1 antagonists. In conclusion, our data suggest that inhibiting the pharmacological function of TRPV1 in skin by specific antagonists has not been considered to be indicative of skin tumour development.

Topics: Acrylamides; Animals; Anthracenes; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Cell Line; Cell Proliferation; Cell Survival; Cocarcinogenesis; Female; Humans; Keratinocytes; Mice; Mice, Hairless; Piperidines; Primary Cell Culture; Pyridines; Pyrrolidines; Risk; Skin Neoplasms; TRPV Cation Channels; Urea

Despite evidence from psychiatry and psychology clinics pointing to altered cognition and decision making following the consumption of cannabis, the effects of cannabis derivatives are still under dispute and the mechanisms of cannabinoid effects on cognition are not known. In this study, we used effort-based and delay-based decision tasks and showed that ACEA, a potent cannabinoid agonist induced apathetic and impulsive patterns of choice in rats in a dose-dependent manner when locally injected into the anterior cingulate cortex (ACC) and orbitofrontal cortex (OFC), respectively. Pre-treatment with AM251, a selective cannabinoid type 1 (CB1) receptor antagonist, reversed ACEA-induced impulsive and apathetic patterns of choice in doses higher than a minimally effective dose. Unlike CB1 receptor antagonist, pretreatment with capsazepine, a transient receptor potential vanilloid type 1 (TRPV1) channel antagonist, was effective only at an intermediary dose. Furthermore, capsazepine per se induced impulsivity and apathy at a high dose suggesting a basal tonic activation of TRPV1 channels that exist in the ACC and OFC to support cost-benefit decision making and to help avoid apathetic and impulsive patterns of decision making. Taken together, unlike previous reports supporting opposing roles for the CB1 receptors and TRPV1 channels in anxiety and panic behavior, our findings demonstrate a different sort of interaction between endocannabinoid and endovanilloid systems and suggest that both systems contribute to the cognitive disrupting effects of cannabinoids. Given prevalent occurrence of apathy and particularly impulsivity in psychiatric disorders, these results have significant implications for pharmacotherapy research targeting these receptors.

Topics: Animals; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cerebral Cortex; Choice Behavior; Dose-Response Relationship, Drug; Impulsive Behavior; Male; Maze Learning; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

Acetaminophen is one of the most commonly used analgesic and antipyretic drugs. It has been reported that acetaminophen has anticonvulsant effects in several animal models of seizure. An active metabolite of acetaminophen, AM404, inhibits the uptake of the endocannabinoid anandamide. However, the mechanism of the anticonvulsant effect of acetaminophen is unknown.. This study was performed to examine whether or not acetaminophen can protect against pentylenetetrazol-induced kindling in mice and to investigate the precise mechanisms of the anticonvulsant effect of acetaminophen using the fully kindled mouse models.. Repeated administration of acetaminophen significantly delayed the progression of seizure severity induced by pentylenetetrazol. Additionally, acetaminophen showed a dose-dependent anticonvulsant activity against fully pentylenetetrazol-kindled seizures. AM404 also exhibited a dose-dependent anticonvulsant activity in fully kindled animals. The anticonvulsant activity of acetaminophen was antagonized by capsazepine and AMG9810, two transient receptor potential vanilloid-1 (TRPV1) antagonists. However, the transient receptor potential ankyrin 1 (TRPA1) antagonist HC030031 and CB1 receptor antagonist AM251 had no effect.. These findings suggest that acetaminophen has an anticonvulsant effect in pentylenetetrazol-kindled mouse models and TRPV1 mediates the anticonvulsant action.

Topics: Acetaminophen; Acetanilides; Acrylamides; Animals; Anticonvulsants; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Disease Models, Animal; Dose-Response Relationship, Drug; Kindling, Neurologic; Male; Mice; Mice, Inbred ICR; Pentylenetetrazole; Piperidines; Purines; Pyrazoles; Seizures; Time Factors; TRPV Cation Channels

Rimonabant is well recognized as a cannabinoid CB₁ receptor antagonist/inverse agonist. Rimonabant not only antagonizes the effects induced by exogenous cannabinoids and endocannabinoids at CB₁ receptors, it also exerts several pharmacological and behavioral effects independent of CB₁ receptor inactivation. For example, rimonabant can function as a low-potency mixed agonist/antagonist of the transient receptor potential vanilloid receptor 1 (TRPV1). Hence, it is important to explain the underlying mechanisms of the diverse physiological effects induced by rimonabant with caution. Interestingly, CB₁ receptor has recently been suggested to play a role in olfactory functions. Olfaction not only is involved in food intake, visual perception and social interaction, but also is proposed as a putative marker for schizophrenia and autism. Therefore, the present study aimed to investigate whether CB₁ receptor and TRPV1 played a role in olfactory functions. We first used the genetic disruption approach to examine the role of CB₁ receptor in olfactory functions and found that CB₁ knockout mice exhibited olfactory discrimination deficit. However, it is important to point out that these CB₁ knockout mice, despite their normal locomotivity, displayed deficiencies in the olfactory foraging and novel object exploration tasks. These results imply that general exploratory behaviors toward odorant and odorless objects are compromised in CB₁ knockout mice. We next turned to the pharmacological approach to examine the role of CB₁ receptor and TRPV1 in olfactory functions. We found that the short-term administration of rimonabant, injected systemically or directly into the olfactory bulb (OB), impaired olfactory discrimination that was rescued by the TRPV1 antagonist capsazepine (CPZ), via the same route of rimonabant, in wild-type mice. These results suggest that TRPV1 in the OB is involved in rimonabant-induced olfactory discrimination deficit. However, the rimonabant and/or CPZ treatments neither affected locomotivity nor general exploratory behaviors in wild-type mice. Finally, the acute systemic administration of rimonabant, unlike the short-term administration regimen, did not affect olfactory discrimination. Taken together, this study not only is the first one, to the best of our knowledge, suggests that the olfactory TRPV1 plays a role in olfactory functions, but also provides a possible mechanism for the olfactory discrimination deficit induced by rimonabant.

Topics: Animals; Capsaicin; Male; Mice; Mice, Inbred C57BL; Motor Activity; Olfactory Bulb; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Smell; TRPV Cation Channels

The role of cannabinoids in thrombosis remains controversial. In view of the primary importance of tissue factor (TF) in blood coagulation and its involvement in the pathology of several cardiovascular, inflammatory and neoplastic diseases, a regulation of this initial procoagulant signal seems to be of particular interest. Using human umbilical vein endothelial cells (HUVEC) the present study investigated the impact of the synthetic cannabinoid WIN 55,212-2 on interleukin (IL)-1β-induced TF expression and activity. WIN 55,212-2 caused a time- and concentration-dependent suppression of IL-1β-induced TF protein accompanied by decreases in TF mRNA and activity. Inhibition of TF protein expression by WIN 55,212-2 was mimicked by its cannabinoid receptor-inactive enantiomer WIN 55,212-3 but not by structurally unrelated phyto-, endo- and synthetic cannabinoids. In addition, the inhibitory effect of WIN 55,212-2 was not reversed by antagonists to cannabinoid receptors (CB1, CB2) or transient receptor potential vanilloid 1. Mechanistic approaches revealed WIN 55,212-2 to suppress IL-1β-induced TF expression via inhibition of ceramide formation and via decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases. Further inhibitor experiments demonstrated neutral sphingomyelinase (nSMase) to confer ceramide generation upon IL-1β treatment with the parallel IL-1β-mediated activation of MAPKs occurring via an nSMase-independent pathway. Finally, a receptor-independent inhibition of IL-1β-induced TF protein by WIN 55,212-2 was confirmed in human blood monocytes. Collectively, this data provide a hitherto unknown receptor-independent anticoagulatory action of the cannabinoid WIN 55,212-2.

Topics: Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Capsaicin; Cells, Cultured; Ceramides; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Monocytes; Morpholines; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Partial Thromboplastin Time; Phosphorylation; Piperidines; Pyrazoles; RNA, Messenger; Signal Transduction; Sphingomyelin Phosphodiesterase; Thromboplastin; TRPV Cation Channels

Cutaneous current-induced vasodilation (CIV) in response to galvanic current application is an integrative model of neurovascular interaction that relies on capsaicin-sensitive fiber activation. The upstream and downstream mechanisms related to the activation of the capsaicin-sensitive fibers involved in CIV are not elucidated. In particular, the activation of cutaneous transient receptor potential vanilloid type-1 (TRPV1) channels and/or acid-sensing ion channels (ASIC) (activators mechanisms) and the release of calcitonin gene-related peptide (CGRP) and substance P (SP) (effector mechanisms) have been tested. To assess cathodal CIV, we measured cutaneous blood flow using laser Doppler flowmetry for 20min following cathodal current application (240s, 100μA) on the skin of the thigh in anesthetized healthy rats for 20min. CIV was studied in rats treated with capsazepine and amiloride to inhibit TRPV1 and ASIC channels, respectively; CGRP8-37 and SR140333 to antagonize CGRP and neurokinin-1 (NK1) receptors, respectively; compared to their respective controls. Cathodal CIV was attenuated by capsazepine (12±2% vs 54±6%, P<0.001), amiloride (19±8% vs 61±6%, P<0.01), CGRP8-37 (15±6% vs 61±6%, P<0.001) and SR140333 (9±5% vs 54±6%, P<0.001) without changing local acidification. This is the first integrative study performed in healthy rats showing that cutaneous vasodilation in response to cathodal stimulation is initiated by activation of cutaneous TRPV1 and ASIC channels likely through local acidification. The involvement of CGRP and NK1 receptors suggests that cathodal CIV is the result of CGRP and SP released through activated capsaicin-sensitive fibers. Therefore cathodal CIV could be a valuable method to assess sensory neurovascular function in the skin, which would be particularly relevant to evaluate the presence of small nerve fiber disorders and the effectiveness of treatments.

Topics: Acid Sensing Ion Channels; Amiloride; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Electrodes; Electrophysiology; Hydrogen-Ion Concentration; Laser-Doppler Flowmetry; Male; Microcirculation; Peptide Fragments; Piperidines; Quinuclidines; Rats; Rats, Wistar; Receptors, Neurokinin-1; Sensory Receptor Cells; Skin; Substance P; TRPV Cation Channels; Vasodilation; Vasodilator Agents

Studies have suggested that the endocannabinoid system is implicated in the pathophysiology of schizophrenia. We have recently reported that Spontaneously Hypertensive Rats (SHRs) present a deficit in social interaction that is ameliorated by atypical antipsychotics. In addition, SHRs display hyperlocomotion - reverted by atypical and typical antipsychotics. These results suggest that this strain could be useful to study negative symptoms (modeled by a decrease in social interaction) and positive symptoms (modeled by hyperlocomotion) of schizophrenia and the effects of potential drugs with an antipsychotic profile. The aim of this study was to investigate the effects of WIN55-212,2 (CB1/CB2 agonist), ACEA (CB1 agonist), rimonabant (CB1 inverse agonist), AM404 (anandamide uptake/metabolism inhibitor), capsaicin (agonist TRPV1) and capsazepine (antagonist TRPV1) on the social interaction and locomotion of control animals (Wistar rats) and SHRs. The treatment with rimonabant was not able to alter either the social interaction or the locomotion presented by Wistar rats (WR) and SHR at any dose tested. The treatment with WIN55-212,2 decreased locomotion (1mg/kg) and social interaction (0.1 and 0.3mg/kg) of WR, while the dose of 1mg/kg increased social interaction of SHR. The treatment with ACEA increased (0.3mg/kg) and decreased (1mg/kg) locomotion of both strain. The administration of AM404 increased social interaction and decreased locomotion of SHR (5mg/kg), and decreased social interaction and increased locomotion in WR (1mg/kg). The treatment with capsaicin (2.5mg/kg) increased social interaction of both strain and decreased locomotion of SHR (2.5mg/kg) and WR (0.5mg/kg and 2.5mg/kg). In addition, capsazepine (5mg/kg) decreased locomotion of both strains and increased (5mg/kg) and decreased (10mg/kg) social interaction of WR. Our results indicate that the schizophrenia-like behaviors displayed by SHR are differently altered by cannabinoid and vanilloid drugs when compared to control animals and suggest the endocannabinoid and the vanilloid systems as a potential target for the treatment of schizophrenia.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Capsaicin; Disease Models, Animal; Dose-Response Relationship, Drug; Interpersonal Relations; Male; Morpholines; Motor Activity; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Inbred SHR; Rats, Wistar; Rimonabant; Schizophrenia; Schizophrenic Psychology; TRPV Cation Channels

Action potentials trigger synaptic terminals to synchronously release vesicles, but some vesicles release spontaneously. G-protein-coupled receptors (GPCRs) can modulate both of these processes. At cranial primary afferent terminals, the GPCR cannabinoid 1 (CB1) is often coexpressed with transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel present on most afferents. Here we tested whether CB1 activation modulates synchronous, action potential-evoked (eEPSCs) and/or spontaneous (sEPSCs) EPSCs at solitary tract nucleus neurons. In rat horizontal brainstem slices, activation of solitary tract (ST) primary afferents generated ST-eEPSCs that were rapidly and reversibly inhibited from most afferents by activation of CB1 with arachidonyl-2'-chloroethylamide (ACEA) or WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate]. The CB1 antagonist/inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] blocked these responses. Despite profound depression of ST-eEPSCs during CB1 activation, sEPSCs in these same neurons were unaltered. Changes in temperature changed sEPSC frequency only from TRPV1(+) afferents (i.e., thermal sEPSC responses only occurred in TRPV1(+) afferents). CB1 activation failed to alter these thermal sEPSC responses. However, the endogenous arachidonate metabolite N-arachidonyldopamine (NADA) promiscuously activated both CB1 and TRPV1 receptors. NADA inhibited ST-eEPSCs while simultaneously increasing sEPSC frequency, and thermally triggered sEPSC increases in neurons with TRPV1(+) afferents. We found no evidence for CB1/TRPV1 interactions suggesting independent regulation of two separate vesicle pools. Together, these data demonstrate that action potential-evoked synchronous glutamate release is modulated separately from TRPV1-mediated glutamate release despite coexistence in the same central terminations. This two-pool arrangement allows independent and opposite modulation of glutamate release by single lipid metabolites.

Topics: Action Potentials; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Brain Stem; Capsaicin; Dopamine; Excitatory Postsynaptic Potentials; Glutamic Acid; Hot Temperature; In Vitro Techniques; Male; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission; TRPV Cation Channels

The midbrain dorsal periaqueductal gray (dPAG) has an important role in orchestrating anxiety- and panic-related responses. Given the cellular and behavioral evidence suggesting opposite functions for cannabinoid type 1 receptor (CB₁) and transient receptor potential vanilloid type-1 channel (TRPV1), we hypothesized that they could differentially influence panic-like reactions induced by electrical stimulation of the dPAG. Drugs were injected locally and the expression of CB₁ and TRPV1 in this structure was assessed by immunofluorescence and confocal microscopy. The CB₁-selective agonist, ACEA (0.01, 0.05 and 0.5 pmol) increased the threshold for the induction of panic-like responses solely at the intermediary dose, an effect prevented by the CB₁-selective antagonist, AM251 (75 pmol). Panicolytic-like effects of ACEA at the higher dose were unmasked by pre-treatment with the TRPV1 antagonist capsazepine (0.1 nmol). Similarly to ACEA, capsazepine (1 and 10 nmol) raised the threshold for triggering panic-like reactions, an effect mimicked by another TRPV1 antagonist, SB366791 (1 nmol). Remarkably, the effects of both capsazepine and SB366791 were prevented by AM251 (75 pmol). These pharmacological data suggest that a common endogenous agonist may have opposite functions at a given synapse. Supporting this view, we observed that several neurons in the dPAG co-expressed CB₁ and TRPV1. Thus, the present work provides evidence that an endogenous substance, possibly anandamide, may exert both panicolytic and panicogenic effects via its actions at CB₁ receptors and TRPV1 channels, respectively. This tripartite set-point system might be exploited for the pharmacotherapy of panic attacks and anxiety-related disorders.

Topics: Anilides; Animals; Arachidonic Acids; Capsaicin; Cinnamates; Drug Interactions; Electric Stimulation; Male; Microinjections; Panic; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 μM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK₁) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2β-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)₂ and EP₄ receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK₁ receptors, and production of COX products capable of activating relaxant EP₂/EP₄ receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E₂ (PGE₂) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE₂ and the β-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE₂, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE₂.

Topics: Animals; Biphenyl Compounds; Bronchoalveolar Lavage Fluid; Capsaicin; Carbachol; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Indomethacin; Inflammation; Influenza A virus; Isoproterenol; Leukocytes; Male; Mice; Mice, Inbred BALB C; Models, Biological; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Orthomyxoviridae Infections; Piperidines; Prostaglandin Antagonists; Respiratory Mucosa; Specific Pathogen-Free Organisms; Substance P; Trachea; TRPV Cation Channels; Xanthones

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (K(Ca)) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (N(G)-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CB(x)), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB(1), CB(2) and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CB(x) receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CB(x) receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve K(Ca) or the NO pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amidohydrolases; Animals; Anisoles; Apamin; Arachidonic Acids; Benzamides; Benzyl Compounds; Cannabinoid Receptor Antagonists; Capsaicin; Carbamates; Charybdotoxin; Cyclohexanes; Endocannabinoids; Endothelium, Vascular; Imidazoles; Indoles; Indomethacin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Chloride; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Epoprostenol; Tetraethylammonium; TRPV Cation Channels; Vasodilation

While endocannabinoid modulation of both GABAergic and glutamatergic synaptic transmission and plasticity has been extensively investigated, our understanding of the role of endocannabinoids in protecting neurons from harmful insults remains limited. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous ligand and a full agonist for cannabinoid receptors, exhibits anti-inflammatory and neuroprotective effects via a CB1 receptor (CB1R)-mediated mechanism. However, it is still not clear whether 2-AG is also able to protect neurons from β-amyloid (Aβ)-induced neurodegeneration. Here, we demonstrate that exogenous application of 2-AG significantly protected hippocampal neurons in culture against Aβ-induced neurodegeneration and apoptosis. This neuroprotective effect was blocked by SR141716 (SR-1), a selective CB1R antagonist, but not by SR144528 (SR-2), a selective CB2R antagonist, or capsazepine (CAP), a selective transient receptor potential cation channels, subfamily V, member 1 (TRPV1) receptor antagonist. To determine whether endogenous 2-AG is capable of protecting neurons from Aβ insults, hippocampal neurons in culture were treated with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), the enzyme hydrolyzing 2-AG. MAGL inhibition that elevates endogenous levels of 2-AG also significantly reduced Aβ-induced neurodegeneration and apoptosis. The 2-AG-produced neuroprotective effects appear to be mediated via CB1R-dependent suppression of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) phosphorylation and cyclooxygenase-2 (COX-2) expression. Our results suggest that elevation of endogenous 2-AG by inhibiting its hydrolysis has potential as a novel efficacious therapeutic approach for preventing, ameliorating or treating Alzheimer's disease.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Camphanes; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Culture Techniques; Drug Interactions; Endocannabinoids; Glycerides; Hippocampus; Monoacylglycerol Lipases; Nerve Degeneration; Peptide Fragments; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Signal Transduction

Hypothermia occurs in the most severe cases of systemic inflammation, but the mechanisms involved are poorly understood. This study evaluated whether the hypothermic response to bacterial lipopolysaccharide (LPS) is modulated by the endocannabinoid anandamide(AEA) and its receptors: cannabinoid-1 (CB1), cannabinoid-2 (CB2) and transient receptor potential vanilloid-1 (TRPV1). In rats exposed to an ambient temperature of 22◦C, a moderate dose of LPS (25 - 100 μg kg−1 I.V.) induced a fall in body temperature with a nadir at ∼100 minpostinjection. This response was not affected by desensitization of intra-abdominal TRPV1 receptors with resiniferatoxin (20 μg kg - 1 I.P.), by systemic TRPV1 antagonism with capsazepine(40mg kg−1 I.P.), or by systemic CB2 receptor antagonism with SR144528 (1.4 mg kg−1 I.P.).However, CB1 receptor antagonism by rimonabant (4.6mg kg−1 I.P.) or SLV319 (15mg kg−1 I.P.)blocked LPS hypothermia. The effect of rimonabant was further studied. Rimonabant blocked LPS hypothermia when administered I.C.V. at a dose (4.6 μg) that was too low to produce systemic effects. The blockade of LPS hypothermia by I.C.V. rimonabant was associated with suppression of the circulating level of tumour necrosis factor-α. In contrast to rimonabant,the I.C.V. administration of AEA (50 μg) enhanced LPS hypothermia. Importantly, I.C.V. AEAdid not evoke hypothermia in rats not treated with LPS, thus indicating that AEA modulates LPS-activated pathways in the brain rather than thermo effector pathways. In conclusion, the present study reveals a novel, critical role of brain CB1 receptors in LPS hypothermia. Brain CB1 receptors may constitute a new therapeutic target in systemic inflammation and sepsis.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Body Temperature Regulation; Brain; Camphanes; Capsaicin; Disease Models, Animal; Diterpenes; Endocannabinoids; Female; Hypothermia; Injections, Intraperitoneal; Injections, Intravenous; Injections, Intraventricular; Lipopolysaccharides; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Long-Evans; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Signal Transduction; Sulfonamides; Time Factors; TRPV Cation Channels

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at -70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 μM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.

Topics: Alkaloids; Animals; Benzodioxoles; Capsaicin; Excitatory Amino Acid Agents; Glutamates; Neurons; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Rats; Substantia Gelatinosa; Synaptic Transmission; TRPV Cation Channels

The contributions of brain cannabinoid (CB) receptors, typically CB1 (CB type 1) receptors, to the behavioral effects of nicotine (NC) have been reported to involve brain transient receptor potential vanilloid 1 (TRPV1) receptors, and the activation of candidate endogenous TRPV1 ligands is expected to be therapeutically effective. In the present study, the effects of TRPV1 ligands with or without affinity for CB1 receptors were examined on NC-induced depression-like behavioral alterations in a mouse model in order to elucidate the "antidepressant-like" contributions of TRPV1 receptors against the NC-induced "depression" observed in various types of tobacco abuse.. Repeated subcutaneous NC treatments (NC group: 0.3 mg/kg, 4 days), like repeated immobilization stress (IM) (IM group: 10 min, 4 days), caused depression-like behavioral alterations in both the forced swimming (reduced swimming behaviors) and the tail suspension (increased immobility times) tests, at the 2 h time point after the last treatment. In both NC and IM groups, the TRPV1 agonists capsaicin (CP) and olvanil (OL) administered intraperitoneally provided significant antidepressant-like attenuation against these behavioral alterations, whereas the TRPV1 antagonist capsazepine (CZ) did not attenuate any depression-like behaviors. Furthermore, the endogenous TRPV1-agonistic CB1 agonists anandamide (AEA) and N-arachidonyldopamine (NADA) did not have any antidepressant-like effects. Nevertheless, a synthetic "hybrid" agonist of CB1 and TRPV1 receptors, arvanil (AR), caused significant antidepressant-like effects. The antidepressant-like effects of CP and OL were antagonized by the TRPV1 antagonist CZ. However, the antidepressant-like effects of AR were not antagonized by either CZ or the CB1 antagonist AM 251 (AM).. The antidepressant-like effects of TRPV1 agonists shown in the present study suggest a characteristic involvement of TRPV1 receptors in NC-induced depression-like behaviors, similar to those caused by IM. The strong antidepressant-like effects of the potent TRPV1 plus CB1 agonist AR, which has been reported to cause part of its TRPV1-mimetic and cannabimimetic effects presumably via non-TRPV1 or non-CB1 mechanisms support a contribution from other sites of action which may play a therapeutically important role in the treatment of NC abuse.

Topics: Animals; Antidepressive Agents; Arachidonic Acids; Capsaicin; Depression; Dopamine; Endocannabinoids; Hindlimb Suspension; Ligands; Male; Mice; Mice, Inbred ICR; Nicotine; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Stress, Psychological; Swimming; TRPV Cation Channels

Chemosensory signaling by the tongue is a primary determinant of ingestive behavior and is mediated by specific interactions between tastant molecules and G protein-coupled and ion channel receptors. The functional relationship between tastant and receptor should be amenable to pharmacologic methods and manipulation. We have performed a pharmacologic characterization of the taste-directed licking of mice presented with solutions of capsaicin and other transient receptor potential vanilloid-1 (TRPV1) agonists using a brief access taste aversion assay. Dose-response functions for lick-rate suppression were established for capsaicin (EC(50) = 0.5 microM), piperine (EC(50) = 2 muM), and resiniferatoxin (EC(50) = 0.02 microM). Little or no effect on lick rate was observed in response to the full TRPV1 agonist olvanil. Capsaicin lick rates of wild-type and transient receptor potential melastatin-5 (TRPM5) knockout mice were equivalent, indicating that TRPM5, a critical component of aversive signaling for many bitter tastants, did not contribute to the capsaicin taste response. The selective TRPV1 antagonists N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (10 microM) and (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (AMG9810) (10 microM) effectively blocked capsaicin- and piperine-mediated lick suppression. However, (E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)-N-phenylprop-2-enamide (SB 366791) and capsazepine, also TRPV1 antagonists, were without effect at test concentrations of up to 30 and 100 microM, respectively. Our results demonstrate that TRPV1-mediated oral aversiveness presents a pharmacologic profile differing from what has been reported previously for TRPV1 pain signaling and, furthermore, that aversive tastes can be evaluated and controlled pharmacologically.

Topics: Acrylamides; Administration, Oral; Alkaloids; Anilides; Animals; Avoidance Learning; Benzodioxoles; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Cinnamates; Diterpenes; Dose-Response Relationship, Drug; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Polyunsaturated Alkamides; Pyrazines; Pyridines; Taste; TRPM Cation Channels; TRPV Cation Channels

The release of arginine vasopressin (AVP) from the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) is crucial for body fluid homeostasis. The MNC activity is modulated by synaptic inputs and humoral factors. A recent study demonstrated that an N-terminal splice variant of the transient receptor potential vanilloid type 1 (TRPV1) is essential for osmosensory transduction in the SON. In the present study, we examined the effects of mannitol and angiotensin II on miniature EPSCs (mEPSCs) in the supraoptic MNCs using whole-cell patch-clamp recording in in vitro slice preparation. Mannitol (60 mm) and angiotensin II (0.1 microm) increased the frequency of mEPSCs without affecting the amplitude. These effects were attenuated by pre-exposure to a nonspecific TRPV channel blocker, ruthenium red (10 microm) and enhanced by pre-exposure to cannabinoid type1 receptor antagonist, AM251 (2 microm). Mannitol-induced potentiation of mEPSCs was not attenuated by angiotensin II receptor antagonist, losartan (10 microm), indicating independent pathways of mannitol and angiotensin II to the TRPV channels. The potentiation of mEPSCs by mannitol was not mimicked by a TRPV1 agonist, capsaicin, and also not attenuated by TRPV1 blockers, capsazepine (10 microm). PKC was involved in angiotensin II-induced potentiation of mEPSCs. The effects of mannitol and angiotensin II on the supraoptic MNCs in trpv1 knock-out mice were significantly attenuated compared with those in wild-type mice counterparts. The results suggest that hyperosmotic stimulation and angiotensin II independently modulate mEPSCs through capsaicin-insensitive TRPV1 channel in the presynaptic terminals of the SON.

Topics: Analysis of Variance; Angiotensin II; Anilides; Animals; Capsaicin; Chelating Agents; Cinnamates; Diuretics, Osmotic; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; In Vitro Techniques; Male; Mannitol; Mice; Mice, Inbred C57BL; Mice, Knockout; Miniature Postsynaptic Potentials; Neurons; Piperidines; Pyrazines; Pyrazoles; Pyridines; Rats; Rats, Wistar; Signal Transduction; Supraoptic Nucleus; TRPV Cation Channels; Vasoconstrictor Agents

Cannabinoids and vanilloids are two distinct groups of substances that share some pharmacological targets. Here we report that two cannabinoid type 1 receptor (CB1) agonists, WIN 55212-2 (WIN) and arachidonyl-2'-chloroethylamide (ACEA) have opposing effects on evoked quantal acetylcholine release - WIN decreased quantal content while ACEA increased quantal content. The decrease in quantal content by WIN was blocked by the CB1 antagonist AM 251. The increase in quantal content by ACEA was not blocked by AM 251, indicating it acts through a receptor other than CB1. As ACEA is also an agonist for the vanilloid receptor (TRPV1) we tested the effect of vanilloids on quantal content. Similar to ACEA, the vanilloid agonist capsaicin increased quantal content, and this effect was blocked by capsazepine, a TRPV1 antagonist. Capsazepine also blocked the increase in quantal content by ACEA. Together these data show an inhibitory effect of CB1 activation on evoked acetylcholine release and the first evidence for the presence of a vanilloid receptor at the neuromuscular junction.

Topics: Acetylcholine; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Capsaicin; Morpholines; Naphthalenes; Neuromuscular Junction; Piperidines; Pyrazoles; Rana catesbeiana; Receptor, Cannabinoid, CB1; TRPV Cation Channels

A series of natural and synthetic piperine amides were evaluated for activity on the human TRPV1 expressed in HEK293 cells. The agonistic effect of piperine amides was mainly dependent on the length of the carbon chain. Structural changes of double bonds and stereochemistry in the aliphatic chain of these compounds did not change their potency or efficacy, indicating that increased rigidity or planarity of the piperine structure does not affect the activity. The opening of the methylenedioxy ring or changes in the heterocyclic ring of the piperine molecule reduced or abolished activity. Furthermore, inactive compounds did not display functional antagonistic activity.

Topics: Alkaloids; Amides; Benzodioxoles; Capsaicin; Cell Line; Dose-Response Relationship, Drug; Humans; Molecular Structure; Piperidines; Polyunsaturated Alkamides; Spectrometry, Fluorescence; TRPV Cation Channels

Various volatile organic compounds (VOCs) act as a causative agent of skin inflammation. We investigated the effect of topical application of several VOCs and formalin on microvascular leakage in rat skin. We tested capsaicin, which is a reagent that specifically causes the skin response via endogenously released tachykinins. Evans blue dye extravasation served as an index of the increase in skin vascular permeability. After shaving the abdomen, we applied formalin, m-xylene, toluene, styrene, benzene, ethylbenzene, acetone, diethyl ether, hexane, heptane, cyclohexane and capsaicin to the skin. At 40min after application, skin samples were collected. Among all of the VOCs tested, all of the aromatic compounds significantly produced skin microvascular leakage that was similar to formalin and capsaicin. We also investigated the skin responses seen after the intravenous administration of CP-99,994 (1.5 or 5mg/kg), which is a tachykinin NK1 receptor antagonist, ketotifen (1 or 3mg/kg), which is a histamine H1 receptor antagonist that stabilizes the mast cells, and the topical application of capsazepine (22.5 or 50mM), which is the transient receptor potential vanilloid 1 (TRPV1) antagonist. The response induced by formalin and capsaicin was completely inhibited by CP-99,994. On the other hand, the antagonist partially reduced the response induced by m-xylene, toluene and styrene by 39%, 50% and 46%, respectively. Capsazepine and ketotifen did not alter the response induced by formalin or any of the aromatic compounds. Like capsaicin, formalin and the aromatic compounds at least partially caused skin microvascular leakage, which was due to tachykinin NK1 receptor activation related to the release of tachykinins from the sensory nerve endings. However, it is unlikely that mast cells and TRPV1 play an important role in the skin response.

Topics: Administration, Topical; Animals; Antipruritics; Capillary Leak Syndrome; Capsaicin; Cell Degranulation; Disinfectants; Dose-Response Relationship, Drug; Formaldehyde; Hydrocarbons, Aromatic; Ketotifen; Male; Mast Cells; Neurogenic Inflammation; Neurokinin-1 Receptor Antagonists; Piperidines; Rats; Rats, Wistar; Regional Blood Flow; Skin

Numerous studies, focused on the hypothalamus, have recently implicated endocannabinoids (EC) as orexigenic factors in the central control of food intake. However, the EC system is also highly expressed in the hindbrain autonomic integrator of food intake regulation, i.e. the dorsal vagal complex (DVC). Previous studies have shown that exogenous cannabinoids, by acting on cannabinoid 1 receptor (CB1R), suppress GABAergic and glutamatergic neuronal transmission in adult rat dorsal motor nucleus of the vagus nerve (DMNV), the principal efferent compartment of the DVC. However, no endogenous release of EC has been demonstrated in DVC to date. Using patch-clamp techniques on mouse coronal brainstem slices, we confirmed that both inhibitory and excitatory neurotransmission were depressed by WIN 55,212-2, a CB1R agonist. We demonstrated that DMNV neurons exhibited a rapid and reversible depolarization-induced suppression of electrically evoked GABAergic IPSCs (eIPSCs), classically known as DSI (depolarization-induced suppression of inhibition), while spontaneous or miniature IPSCs activity remained unaltered. Further, no depolarization-induced suppression of glutamatergic eEPSCs (DSE) occurred. Our results indicate that DSI was blocked by SR141716A (Rimonabant), a selective CB1R antagonist, and was dependent on calcium elevation in DMNV neurons, suggesting a release of EC in the DVC. Moreover, the analysis of the paired-pulse ratio, of the coefficient of variation and of the failure rate of eIPSCs support the fact that EC-mediated suppression of GABAergic inhibition takes place at the presynaptic level. These results show for the first time that DMNV neurons release EC in an activity-dependent manner, which in turn differentially regulates their inhibitory and excitatory synaptic inputs.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arachidonic Acids; Benzoxazines; Brain Stem; Calcium Signaling; Cannabinoid Receptor Modulators; Capsaicin; Efferent Pathways; Endocannabinoids; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Inhibitory Postsynaptic Potentials; Kynurenic Acid; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Neurons; Piperidines; Pyrazoles; Pyridazines; Receptor, Cannabinoid, CB1; Receptors, Presynaptic; Rimonabant; Tetrodotoxin; Vagus Nerve

Increasing evidence indicates that endothelin-1 (ET-1) activates nociceptive neurons and sensitizes them to different noxious stimuli, but involvement of TRPV1-dependent mechanisms in mediation of such effects is not yet fully understood. Here we report that intraplantar (i.pl.) injection of ET-1 (10 pmol) into the hind paw of rats induced overt nociceptive behavior over the first hour, followed by a slowly developing thermal hyperalgesia, lasting from 3 to 8h after injection. Both effects were also induced by similar injections of capsaicin (10-1000 pmol), but these responses were shorter lasting than those caused by ET-1. Local pre-treatment with the TRPV1 antagonist capsazepine (30 nmol, i.pl.) reduced only the thermal hyperalgesia induced by ET-1, but fully suppressed both responses to capsaicin (1000 pmol). Injection of a sub-threshold dose of ET-1 (0.1 pmol, i.pl.) prior to capsaicin (1 pmol, i.pl.) markedly sensitized the hind paw to the overt nociceptive and thermal hyperalgesic effects of the later. The potentiation of capsaicin-induced nociception by ET-1 was abolished by prior i.pl. injection of BQ-123 (ET(A) receptor antagonist, 10 nmol), but unaffected by BQ-788 (ET(B) receptors antagonist, 10 nmol), whereas the enhancement of capsaicin-induced hyperalgesia by ET-1 was attenuated by both antagonists. Therefore, differently to what has been reported in mice, in rats TRPV1 receptors contribute selectively to thermal hyperalgesia, but not overt nociception, induced by ET-1. Importantly, although ET-1 augments capsaicin-induced overt nociception and thermal hyperalgesia, potentiation of the former relies solely on ET(A) receptor-mediated signaling mechanisms, whereas both receptors contribute to the latter.

Topics: Analysis of Variance; Animals; Capsaicin; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Hindlimb; Hyperalgesia; Male; Oligopeptides; Pain Measurement; Peptides, Cyclic; Physical Stimulation; Piperidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Sensory System Agents; Temperature; TRPV Cation Channels

Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract may underlie the high concurrence of obstetrical/gynecological inflammation and chronic pelvic pain syndrome characterized by urogenital pain. However, the neural pathway and the neurotransmitters involved are still unknown. We tested the hypothesis that the excitation of capsaicin-sensitive primary afferent fibers arising from the uterus through the stimulation of transient receptor potential vanilloid 1 (TRPV1) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1-1,000 microM, 0.05 ml) was instilled into the uterus to induce cross-organ reflex sensitization. Activation of capsaicin-sensitive primary afferent fibers by capsaicin instillation into the uterine horn sensitized the pelvic-urethra reflex activity that was reversed by an intrauterine pretreatment with capsaizepine, a TRPV1-selective antagonist. Intrathecal injection of AP5, a glutamatergic N-methyl-D-aspartate (NMDA) antagonist, and Co-101244, an NMDA NR2B-selective antagonist, both abolished the cross-organ reflex sensitization caused by capsaicin instillation. These results demonstrated that TRPV1 plays a crucial role in contributing to the capsaicin-sensitive primary afferent fibers mediating the glutamatergic NMDA-dependent cross-organ sensitization between the uterus and the lower urinary tract when there is a tissue injury.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Anesthesia; Animals; Capsaicin; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Muscle, Smooth; Neurons, Afferent; Pelvic Pain; Phosphorylation; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reflex; Spinal Cord; TRPV Cation Channels; Uterus

The present study investigated the central effects of the eCB uptake/metabolism inhibitor AM404 and the phytocannabinoid cannabidiol (CBD) on the extinction of contextual fear memories in rats. Rats were conditioned and 24 h later subjected to three consecutive 9-min non-reinforced exposures to the conditioning context (extinction sessions, 24 h intervals). AM404 or CBD was injected i.c.v. 5 min before each extinction session and a 3-min drug-free test of contextual memory was performed 24 h after the last extinction session. AM404 (1.0 microg/microl, i.c.v.) and CBD (2.0 microg/microl, i.c.v.) facilitated extinction of contextual fear memory, with persistent effects. These responses were antagonized by the CB1-selective antagonist SR141716A (0.2 mg/kg, i.p.), but not by the TRPV1-selective antagonist capsazepine (5.0 microg/microl, i.c.v.). The effect of the anxiolytic drug Diazepam (DZP) on the extinction of contextual fear memory was also investigated. In contrast with the CBD and AM404 results, DZP induced a general reduction in the expression of conditioned freezing. Both AM404 and CBD induced anti-anxiogenic effect in the fear-potentiated plus-maze test, whereas DZP was anxiolytic in conditioned and unconditioned rats. In conclusion, CBD, a non-psychoactive phytocannabinoid could be an interesting pharmacological approach to reduce the anxiogenic effects of stress and promote the extinction of fear memories.

Topics: Analysis of Variance; Animals; Anti-Anxiety Agents; Arachidonic Acids; Behavior, Animal; Cannabidiol; Capsaicin; Conditioning, Psychological; Dose-Response Relationship, Drug; Drug Administration Schedule; Extinction, Psychological; Fear; Freezing Reaction, Cataleptic; Injections, Intraventricular; Male; Maze Learning; Piperidines; Pyrazoles; Rats; Rats, Wistar; Rimonabant; TRPV Cation Channels

Lafutidine is a histamine H(2)-receptor antagonist with gastric antisecretory and gastroprotective activity associated with activation of capsaicin-sensitive nerves. The present study examined the effect of lafutidine on neurotransmission of capsaicin-sensitive calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves) in rat mesenteric resistance arteries. Rat mesenteric vascular beds were perfused with Krebs solution and vascular endothelium was removed by 30-s perfusion with sodium deoxycholate. In preparations preconstricted by continuous perfusion of methoxamine (alpha(1) adrenoceptor agonist), perfusion of lafutidine (0.1 - 10 microM) concentration-dependently augmented vasodilation induced by the periarterial nerve stimulation (PNS, 1 Hz) without affecting vasodilation induced by exogenous CGRP (10 pmol) injection. Perfusion of famotidine (H(2)-receptor antagonist, 1 - 100 microM) had no effect on either PNS-induced or CGRP-induced vasodilation. Perfusion of lafutidine concentration-dependently augmented vasodilation induced by a bolus injection of capsaicin (vanilloid-1 receptor agonist, 30 pmol). The presence of a vanilloid-1 receptor antagonist, ruthenium red (10 microM) or capsazepine (5 microM), abolished capsaicin-induced vasodilation and significantly decreased the PNS-induced vasodilation. The decreased PNS-induced vasodilation by ruthenium red or capsazepine was not affected by perfusion of lafutidine. These results suggest that lafutidine facilitates CGRP nerve-mediated vasodilation by modulating the function of presynaptic vanilloid-1 receptors located in CGRPergic nerves.

Topics: Acetamides; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Drug Synergism; Electric Stimulation; Male; Mesenteric Arteries; Peripheral Nervous System; Piperidines; Pyridines; Rats; Rats, Wistar; Ruthenium Red; TRPV Cation Channels; Vasodilation

Vanilloids like capsaicin and resiniferatoxin (RTX) have been used for more than a decade in the treatment of neurogenic detrusor overactivity. Recently, the vanilloid molecule piperine (PIP) has been shown to have similar pharmacological properties as these drugs. In this study, we looked at PIP-effects on autonomous bladder contractile activity, with particular interest for its selectivity for the transient receptor potential channel 1 (TRPV1) receptor. Additionally, we studied the role of TRPV1 in volume-induced contractile changes using selective and non-selective TRPV1 antagonists.. The acute and prolonged effects of PIP were studied on rat bladders. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. In acute experiments, PIP was added directly to the bathing solution. For prolonged effects, animals were pre-treated intravesically with vehicle (ethanol 5%) or PIP (10(-4) M) and sacrificed 72 hr later. The effects of selective (capsazepine (CZP)) and non-selective (ruthenium red (RR)) TRPV1 antagonists on volume-evoked contractile parameters were also studied.. Acute administration of PIP 10(-4) M significantly increased amplitude of bladder contractions (P < 0.05). These effects were significantly antagonized (P < 0.05) by the TRPV1-selective antagonist CZP (10(-5) M) and the non-selective TRP-antagonist RR (10(-5) M). Intravesical pre-treatment with PIP induced shorter contractions with more periods of non-activity (P < 0.05) compared to controls. Inhibition of TRPV1 with CZP and RR significantly reduced the volume-evoked rise in contractile amplitude in isolated bladders (P < 0.05).. We found evidence for acute and prolonged effects of PIP on bladder contractility, which seem to be mediated through TRPV1. Furthermore, we found evidence for involvement of TRPV1 in afferent signaling of mechanical stimuli.

Topics: Alkaloids; Animals; Benzodioxoles; Capsaicin; Carbachol; Cholinergic Agonists; Dose-Response Relationship, Drug; Female; Indicators and Reagents; Mechanotransduction, Cellular; Muscle Contraction; Piperidines; Polyunsaturated Alkamides; Pressure; Rats; Rats, Wistar; Ruthenium Red; Stress, Mechanical; TRPV Cation Channels; Urinary Bladder

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 microM), the neurokinin NK1 receptor antagonist L-733060 (0.5 microM), the voltage-sensitive calcium channel blocker ruthenium red (100 microM), the TRPV1 receptor antagonist capsazepine (5 microM), the nitric oxide synthetase inhibitor Nomega-nitro-L-arginine methyl ester HCl (L-NAME; 100 microM), the gap junction blocker 18alpha-glycyrrhetinic acid (10 microM), as well as the RhoA kinase inhibitor Y-27632 (1 microM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 microM) + apamin (0.1 microM) and iberiotoxin (0.5 microM) + apamin (0.1 microM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) alpha-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independen

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adolescent; Adult; Aged; Analgesics, Non-Narcotic; Animals; Arteries; Calcitonin Gene-Related Peptide; Capsaicin; Coronary Vessels; Cyclic AMP; Dipeptides; Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Male; Middle Aged; NG-Nitroarginine Methyl Ester; Piperazines; Piperidines; Potassium Channel Blockers; Potassium Chloride; Protein Kinase Inhibitors; Quinazolines; Swine; Vasodilation

Cannabinoids, such as anandamide, are involved in pain transmission. We evaluated the effects of AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), an anandamide reuptake inhibitor, monitoring the expression of c-fos, a marker of activated neurons and the pain-related behaviours using formalin test. The study was carried out in an experimental model of inflammatory pain made by a single injection of formalin in rat hind paws. Formalin test showed that the antinociceptive effect of AM404 was evident in phase I. We found that Fos-positive neurons in dorsal superficial and deep laminae of the lumbar spinal cord increased in formalin-injected animals and that AM404 significantly reduced Fos induction. Co-administration of cannabinoid CB(1) receptor antagonist (AM251), cannabinoid CB(2) receptor antagonist (AM630) and transient receptor potential vanilloid type 1 (TRPV-1) antagonist (capsazepine), attenuate the inhibitory effect of AM404 and this effect was higher using cannabinoid CB(2) and vanilloid TRPV-1 receptor antagonists. These results suggest that AM404 could be a useful drug to reduce inflammatory pain in our experimental model and that cannabinoid CB(2) receptor and vanilloid TRPV-1 receptor, and to a lesser extent, the cannabinoid CB(1) receptor are involved.

Topics: Animals; Arachidonic Acids; Capsaicin; Endocannabinoids; Immunohistochemistry; Indoles; Inflammation; Male; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord; TRPV Cation Channels

To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult.. Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used.. In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively).. The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Cell Count; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Hydrolysis; Intraocular Pressure; Male; Ocular Hypertension; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Thy-1 Antigens; TRPV Cation Channels

Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta.. Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide.. Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide.. Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Topics: Animals; Aorta, Abdominal; Apamin; Arachidonic Acids; Benzamides; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoid Receptor Modulators; Capsaicin; Carbamates; Charybdotoxin; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Indomethacin; Isoindoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sulfonamides; Vasodilation

Calcitonin gene-related peptide (CGRP) is a sensory neurotransmitter in the rat mesenteric arterial bed. Certain cannabinoids can inhibit, via CB(1) receptors, vasorelaxant responses to electrical field stimulation (EFS) of sensory nerves in the rat mesentery, but the mechanism of the inhibitory effect of the cannabinoid delta 9-tetrahydrocannabinol (THC) is unclear. This study assessed directly the effect of THC on EFS-induced release of CGRP from sensory nerves in the rat mesenteric bed and investigated the possible involvement of cannabinoid receptors and transient receptor potential (TRP) ion channels.. Rat mesenteric beds were perfused with physiological salt solution. Sensory nerves were stimulated electrically and perfusate levels of CGRP measured by immunoassay. The effects of THC on EFS-induced CGRP release and vasorelaxant responses to sensory nerve stimulation were investigated in the absence and presence of cannabinoid antagonists and TRP channel blockers.. EFS evoked a release of CGRP and vasodilatation of the mesenteric beds. THC inhibited the electrically-evoked release of CGRP and sensory neurogenic vasorelaxation. The effect of THC was unaffected by the CB1 antagonist AM251, the CB2 antagonist AM630 or the TRPV1 receptor antagonist capsazepine, but was blocked by the TRP channel blocker ruthenium red.. THC inhibits the EFS-induced release of CGRP (and subsequent vasorelaxation), from capsaicin-sensitive sensory nerves in the rat perfused mesentery. The effect of THC was not mediated by CB1, CB2 or TRPV1 receptors, but was sensitive to ruthenium red, suggesting a possible involvement of TRP ion channels.

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Dose-Response Relationship, Drug; Dronabinol; Electric Stimulation; Indoles; Male; Mesenteric Arteries; Methoxamine; Neurons, Afferent; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Ruthenium Red; Time Factors; Vasodilation

This study was designed to test the hypothesis that the systemic administration of N-arachidonoyl-dopamine (NADA), an endovanilloid, causes a depressor effect via activation of transient receptor potential vanilloid type 1 (TRPV1) channels during high-salt intake.. Wistar rats were fed a normal (0.4%) or high (4%) sodium diet for 10 days, and arteries and veins were cannulated for measurement of mean arterial pressure (MAP) or injection of drugs and collection of plasma. Radioimmunoassay and western blot were used to determine the plasma calcitonin gene-related peptide (CGRP) level and TRPV1 protein content, respectively.. The NADA-induced dose-dependent decrease in MAP was greater in high-sodium than normal-treated rats, and was abolished by capsazepine, a selective TRPV1 antagonist, or CGRP8-37, a selective CGRP receptor antagonist, but not by SR141716A, a selective cannabinoid 1 receptor antagonist. Capsaicin, a selective TRPV1 receptor agonist, or CGRP dose-dependently decreased MAP in normal or high-sodium-treated rats, with a greater effect in the latter. Baseline and NADA-induced increases in plasma CGRP levels were higher in high-sodium than normal-treated rats. TRPV1 protein expression in mesenteric arteries was higher in high-sodium than normal-treated rats. In vitro, NADA caused a greater CGRP release from mesenteric arteries of high-sodium than normal-treated rats, which was blocked by capsazepine.. High sodium increases the sensitivity of blood pressure responses to NADA. The enhanced depressor effect induced by NADA during high-sodium intake is prevented by blockade of the TRPV1 or CGRP receptors, but not cannabinoid 1 receptor. High sodium upregulates mesenteric TRPV1 expression, and increases NADA-induced CGRP release in vitro and in vivo.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Capsaicin; Dopamine; Male; Peptide Fragments; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Calcitonin Gene-Related Peptide; Rimonabant; Sodium Chloride, Dietary; TRPV Cation Channels

The mechanisms underlying transient receptor potential vanilloid receptor type 1 (TRPV1)-independent relaxation elicited by capsaicin were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileum longitudinal smooth muscles of guinea-pigs. In acetylcholine-stimulated tissues, capsaicin (1-100 microM) and resiniferatoxin (10 nM-1 microM) produced a concentration-dependent relaxation. The relaxant response was attenuated by 4-aminopyridine and high-KCl solution, but not by capsazepine, tetraethylammonium, Ba(2+), glibenclamide, charybdotoxin plus apamin nor antagonists of cannabinoid receptor type 1 and calcitonin-gene related peptide. A RhoA kinase inhibitor reduced the relaxant effect of capsaicin at 30 microM. Capsaicin and resiniferatoxin reduced acetylcholine- and caffeine-induced transient contractions in a Ca(2+)-free, EGTA solution. Capsaicin at 30 microM for 20 min did not alter basal levels of MLC(20) phosphorylation, but abolished an increase by acetylcholine in MLC(20) phosphorylation. It is suggested that the relaxant effect of capsaicin at concentrations used is not mediated by TRPV1, but by 4-aminopyridine-sensitive K(+) channels, and that capsaicin inhibits contractile mechanisms involving Ca(2+) release from intracellular storage sites. The relaxation could be explained by a decrease in phosphorylation of MLC(20).

Topics: 4-Aminopyridine; Acetylcholine; Animals; Apamin; Barium; Benzopyrans; Caffeine; Calcitonin Gene-Related Peptide; Calcium; Capsaicin; Charybdotoxin; Diclofenac; Dimethyl Sulfoxide; Diterpenes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glyburide; Guanethidine; Guinea Pigs; Ileum; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Muscle Relaxation; Muscle, Smooth; Myosin Light Chains; Nitroarginine; Papaverine; Peptide Fragments; Phosphodiesterase Inhibitors; Phosphorylation; Piperidines; Potassium Channel Blockers; Potassium Channels; Potassium Chloride; Protein Serine-Threonine Kinases; Pyrazoles; Receptor, Cannabinoid, CB1; rho-Associated Kinases; Tetraethylammonium; Vasodilator Agents

Recently, a potential neuroprotective effect of rimonabant, independent of the CB1 receptor interaction, has been proposed. In the present study, the role of transient receptor potential channel vanilloid subfamily member 1, named VR1, on neuroprotective effect of rimonabant, on global cerebral ischemia in gerbils, was investigated. Rimonabant (0.05-3 mg kg-1), given i.p. 5 min after recirculation, dose dependently antagonized the ischemia-induced decrease in electroencephalographic (EEG) total spectral power and restored relative frequency band distribution 7 days after ischemia. Rimonabant (0.125-0.5 mg kg-1) fully prevented ischemia-induced hyperlocomotion 1 day after ischemia and memory impairment evaluated in a passive avoidance task, 3 days after ischemia. At 7 days after ischemia, the survival of pyramidal cells, in the CA1 subfield, was respectively 91 and 96%, in the animals given rimonabant 0.25 and 0.5 mg kg-1, compared to the vehicle group. Higher doses were not protective. The protection induced by rimonabant followed a bell-shaped curve, the maximal active doses being 0.25 and 0.5 mg kg-1. Capsazepine (0.01 mg kg-1), a selective VR1 vanilloid receptor antagonist, completely reversed rimonabant-induced neuroprotective effects against EEG flattening, memory impairment and CA1 hippocampal neuronal loss. These findings suggest that VR1 vanilloid receptors are involved in rimonabant's neuroprotection even if other mechanisms can contribute to this effect.

Topics: Animals; Avoidance Learning; Brain Ischemia; Cannabinoid Receptor Antagonists; Capsaicin; Electroencephalography; Gerbillinae; Male; Motor Activity; Neuroprotective Agents; Piperidines; Pyrazoles; Rimonabant; TRPV Cation Channels

AM 404 inhibits endocannabinoid uptake and enhances the cannabinoid CB(1)-mediated effects of endogenous cannabinoids. Accumulating evidence also suggests that AM 404 acts at sites other than the endocannabinoid system. One site is the transient receptor potential vanilloid 1 cation channel (TRPV1). A useful endpoint for discriminating between TRPV1- or CB(1)-mediated effects of AM 404 is hypothermia. This is because TRPV1 or CB(1) receptor activation produces a significant hypothermia in rats. The present study investigated the effects of AM 404 (1, 5, 10 and 20 mg/kg, i.p.) on body temperature in rats and the involvement of TRPV1 and CB(1) receptors in the effects of AM 404. Doses of 10 and 20 mg/kg of AM 404 produced significant hypothermia. Pre-treatment with capsazepine (30 mg/kg, i.p.) blocked the hypothermia caused by 10 and 20 mg/kg of AM 404. Pre-treatment with SB 366791 (2 mg/kg, i.p.), a new TRPV1 antagonist, also abolished the hypothermia evoked by AM 404 (20 mg/kg, i.p.). In contrast, pre-treatment with SR 141716A (Rimonabant), a CB(1) antagonist, or AA-5-HT, a fatty acid amide hydrolase (FAAH) blocker, did not affect AM 404-evoked hypothermia. The present data demonstrate that AM 404 evokes a significant hypothermia in rats that is dependent on TRPV1 receptor activation.

Topics: Amidohydrolases; Anilides; Animals; Arachidonic Acids; Body Temperature; Capsaicin; Cinnamates; Endocannabinoids; Hypothermia; Male; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; TRPV Cation Channels

Lafutidine, a histamine H2 receptor antagonist, exhibits gastro-protective action mediated by capsaicin-sensitive afferent neurons (CSN). We compared the effect between lafutidine and capsaicin, with respect to the interaction with endogenous prostaglandins (PG), nitric oxide (NO) and the afferent neurons, including transient receptor potential vanilloid subtype 1 (TRPV1).. Male SD rats and C57BL/6 mice, both wild-type and prostacyclin IP receptor knockout animals, were used after 18 h of fasting. Gastric lesions were induced by the po administration of HCl/ethanol (60% in 150 mmol/L HCl) in a volume of 1 mL for rats or 0.3 mL for mice.. Both lafutidine and capsaicin (1-10 mg/kg, po) afforded dose-dependent protection against HCl/ethanol in rats and mice. The effects were attenuated by both the ablation of CSN and pretreatment with N(G)-nitro-L-arginine methyl ester, yet only the effect of capsaicin was mitigated by prior administration of capsazepine, the TRPV1 antagonist, as well as indomethacin. Lafutidine protected the stomach against HCl/ethanol in IP receptor knockout mice, similar to wild-type animals, while capsaicin failed to afford protection in the animals lacking IP receptors. Neither of these agents affected the mucosal PGE2 or 6-keto PGF(1alpha) contents in rat stomachs. Capsaicin evoked an increase in [Ca2+]i in rat TRPV1-transfected HEK293 cells while lafutidine did not.. These results suggest that although both lafutidine and capsaicin exhibit gastro-protective action mediated by CSN, the mode of their effects differs regarding the dependency on endogenous PGs/IP receptors and TRPV1. It is assumed that lafutidine interacts with CSN at yet unidentified sites other than TRPV1.

Topics: 6-Ketoprostaglandin F1 alpha; Acetamides; Animals; Calcium; Capsaicin; Dinoprostone; Dose-Response Relationship, Drug; Gastric Mucosa; Histamine H2 Antagonists; Humans; Indomethacin; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons, Afferent; NG-Nitroarginine Methyl Ester; Piperidines; Prostaglandins; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Epoprostenol; Stomach; Stomach Diseases; TRPV Cation Channels

Hyperdynamic circulation and mesenteric hyperaemia are found in cirrhosis. To delineate the role of endocannabinoids in these changes, we examined the cardiovascular effects of anandamide, AM251 (CB(1) antagonist), AM630 (CB(2) antagonist) and capsazepine (VR1 antagonist), in a rat model of cirrhosis.. Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, diameters of mesenteric arteriole and venule (intravital microscopy), arterial pressure, cardiac output, systemic vascular resistance and superior mesenteric artery (SMA) flow were measured after anandamide, AM251 (with or without anandamide), AM630 and capsazepine administration. CB(1), CB(2) and VR1 receptor expression in SMA was assessed by western blot and RT-PCR.. Anandamide increased mesenteric vessel diameter and flow, and cardiac output in cirrhotic rats, but did not affect controls. Anandamide induced a triphasic arterial pressure response in controls, but this pattern differed markedly in cirrhotic rats. Pre-administration of AM251 blocked the effects of anandamide. AM251 (without anandamide) increased arterial pressure and systemic vascular resistance, constricted mesenteric arterioles, decreased SMA flow and changed cardiac output in a time-dependent fashion in cirrhotic rats. Capsazepine decreased cardiac output and mesenteric arteriolar diameter and flow, and increased systemic vascular resistance in cirrhotic rats, but lacked effect in controls. Expression of CB(1) and VR1 receptor proteins were increased in cirrhotic rats. AM630 did not affect any cardiovascular parameter in either group.. These data suggest that endocannabinoids contribute to hyperdynamic circulation and mesenteric hyperaemia in cirrhosis, via CB(1)- and VR1-mediated mechanisms.

Topics: Animals; Arachidonic Acids; Bile Ducts; Blood Flow Velocity; Blood Pressure; Blotting, Western; Capsaicin; Cardiac Output; Disease Models, Animal; Endocannabinoids; Hyperemia; Indoles; Liver Circulation; Liver Cirrhosis, Biliary; Male; Mesenteric Artery, Superior; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Splanchnic Circulation; Time Factors; TRPV Cation Channels; Vascular Resistance; Vasodilation; Vasodilator Agents

Lafutidine, a histamine H(2) receptor antagonist, exerts gastroprotective effects in addition to gastric antisecretory activity. The gastrointestinal protective effects of lafutidine are mediated by capsaicin-sensitive neurons, where capsaicin excites neurons by opening a member of the transient receptor potential channel family (TRPV1). Since the effect of lafutidine on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells has not been elucidated, we investigated the lafutidine response to [Ca(2+)](i) in rat pheochromocytoma PC12 and human endothelial cells. Lafutidine at pharmacological concentrations greater than 1 mM induced a sustained increase in [Ca(2+)](i) in the presence of extracellular CaCl(2) in PC12 cells, while capsaicin showed dual effects on [Ca(2+)](i) in PC12 cells, where it activated TRPV1 and inhibited store-operated Ca(2+) entry. The thapsigargin (an activator of store-operated Ca(2+) entry)-induced increase in [Ca(2+)](i) in PC12 cells was inhibited by capsaicin and SKF96365, an inhibitor of store-operated Ca(2+) entry, and the lafutidine response was inhibited by capsaicin but not by SKF96365. In endothelial cells, lafutidine induced an increase in [Ca(2+)](i) in a SKF96365-insensitive manner. These results suggest that lafutidine stimulates Ca(2+) entry via the capsaicin-sensitive pathway but not the SKF96365-sensitive pathway. The possible role of store-operated Ca(2+) entry induced by lafutidine on gastrointestinal function is also discussed.

Topics: Acetamides; Animals; Calcium; Calcium Chloride; Capsaicin; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Imidazoles; Immunoblotting; Intracellular Fluid; PC12 Cells; Piperidines; Pyridines; Rats; Thapsigargin; Time Factors

Cannabinoids like anandamide are involved in pain transmission. In this study we evaluated the effects of administrating N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of anandamide reuptake and monitoring the expression of c-fos, a marker of activated neurons in an experimental model of neuropathic pain (sciatic nerve tying). Fos expression was monitored 14 days after tying of sciatic nerve and 2 h after non-noxious stimulation. We showed that non-noxious stimulation increased Fos-positivity in the dorsal superficial laminae of the lumbar spinal cord of tied animals but not in the control animals. AM404 significantly reduced Fos induction in tied animals. Co-administration of cannabinoid CB1 receptor, cannabinoid CB2 receptor and transient receptor potential vanilloid type 1 (TRPV-1) antagonists reduced the effect of AM404 and this reduction was higher using cannabinoid CB1 receptor antagonist. These results suggest that AM404 could be a useful drug to reduce neuropathic pain and that cannabinoid CB1 receptor, cannabinoid CB2 receptor and vanilloid TRPV-1 receptor are involved.

Topics: Animals; Arachidonic Acids; Capsaicin; Constriction; Dose-Response Relationship, Drug; Endocannabinoids; Immunohistochemistry; Indoles; Male; Physical Stimulation; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Spinal Cord

Activation of vanilloid receptors has commonly been used to facilitate neurogenic inflammation and plasma exudation to model components of the pathogenesis of migraine; however, these studies have been performed mainly in species lacking the emetic reflex. In the present studies, therefore, we used Suncus murinus, a species of insectivore capable of emesis, to investigate if the vanilloid receptor agonist resiniferatoxin is capable of modeling the emesis associated with migraine. Resiniferatoxin (100 nmol/kg, s.c.) induced an emetic response that was antagonized significantly (P<0.05) by ruthenium red (1-3 micromol), (2R-trans)-4-[1-[3,5-bis(trifluromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-N-(2,6-dimethylphenyl)-1-acetamide (S)-hydroxybutanedioate (R116301; 10-100 micromol/kg), and scopolamine (1 micromol/kg), but not by dihydroergotamine (0.3-3 micromol/kg), sumatriptan (1-10 micromol/kg), methysergide (1-10 micromol/kg), tropanyl 3,5-dichlorobenzoate (MDL72222; 3-30 micromol/kg), ondansetron (0.3-3 micromol/kg), metoclopramide (3-30 micromol/kg), domperidone (3-30 micromol/kg), diphenhydramine (1-10 micromol/kg), or indomethacin (3-30 micromol/kg). The failure of a wide range of representative anti-migraine drugs to reduce retching and vomiting limits the use of this model to identify/investigate novel treatments for the emesis (and nausea) associated with migraine attacks in humans. However, the results provide further evidence for the involvement of a novel vanilloid receptor in resiniferatoxin-induced emesis and implicate both tachykinins and acetylcholine in the pathway(s) activated by resiniferatoxin in S. murinus.

Topics: Animals; Antiemetics; Butanols; Capsaicin; Cyclooxygenase Inhibitors; Dihydroergotamine; Diphenhydramine; Diterpenes; Domperidone; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Indomethacin; Malates; Methysergide; Metoclopramide; Migraine Disorders; Ondansetron; Piperidines; Ruthenium Red; Scopolamine; Serotonin Antagonists; Serotonin Receptor Agonists; Shrews; Sumatriptan; Time Factors; Tropanes; Vomiting

1. We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. 2. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. 3. The current-voltage relationship of piperine-activated currents showed pronounced outward rectification (25+/-4-fold between -70 and +70 mV) and a reversal potential of 0.0+/-0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. 4. Although piperine was a less potent agonist (EC50=37.9+/-1.9 microM) than capsaicin (EC50=0.29+/-0.05 microM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. 5. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230+/-20%, n=11) or capsaicin (284+/-32%, n=8) upon acidification to pH 6.5. 6. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t(1/2)=9.9+/-0.7 s) than capsaicin (t(1/2)>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. 7. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function.

Topics: Alkaloids; Benzodioxoles; Capsaicin; Cell Line; Humans; Hydrogen-Ion Concentration; Patch-Clamp Techniques; Piper nigrum; Piperidines; Polyunsaturated Alkamides; Receptors, Drug; Ruthenium Red; Structure-Activity Relationship

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Topics: Animals; Apamin; Arachidonic Acids; Azepines; Barium Compounds; Biological Factors; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Charybdotoxin; Chlorides; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Female; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Vasodilation; Verapamil

The neuroprotective effects of Delta(9)-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

Topics: alpha-Tocopherol; Animals; Benzimidazoles; Benzoxazines; Blotting, Northern; Blotting, Western; Calcium Channel Blockers; Calcium Channels; Capsaicin; Cell Count; Cell Death; Cell Lineage; Dizocilpine Maleate; DNA, Single-Stranded; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Excitatory Amino Acid Agonists; Immunohistochemistry; In Situ Nick-End Labeling; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Neuroprotective Agents; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Ruthenium; Tetrazolium Salts; Thiazoles; TRPC Cation Channels

Delta-9 tetrahydrocannabinol (Delta(9)-THC) and (-)-cannabidiol ((-)-CBD) are major constituents of the Cannabis sativa plant with different pharmacological profiles: (Delta(9)-THC activates cannabinoid CB(1) and CB(2) receptors and induces psychoactive and peripheral effects. (-)-CBD possesses no, or very weak affinity for these receptors. We tested a series of (+)- and (-)-CBD derivatives for central and peripheral effects in mice. None of the (-)-CBD derivatives were centrally active, yet most inhibited intestinal motility. Of the five (+)-CBD derivatives, all with CB(1) receptor affinity, only (+)-7-OH-CBD-DMH (DMH=1,1-dimethylheptyl), acted centrally, while all five arrested defecation. The effects of (+)-CBD-DMH and (+)-7-OH-CBD-DMH were inhibited by the CB(1) receptor antagonist SR141716. The CB(2) receptor antagonist SR144528, and the vanilloid TRPV1 receptor antagonist capsazepine, had no influence. Further, the (-)-CBD derivatives (-)-7-COOH-CBD and (-)-7-COOH-CBD-DMH, displayed antiinflammatory activity. We suggest that (+)-CBD analogues have mixed agonist/antagonist activity in the brain. Second, (-)-CBD analogues which are devoid of cannabinoid receptor affinity but which inhibit intestinal motility, suggest the existence of a non-CB(1), non-CB(2) receptor. Therefore, such analogues should be further developed as antidiarrheal and/or antiinflammatory drugs. We propose to study the therapeutic potential of (-)- and (+)-CBD derivatives for complex conditions such as inflammatory bowel disease and cystic fibrosis.

Topics: Animals; Binding, Competitive; Body Temperature; Camphanes; Cannabidiol; Capsaicin; Drug Interactions; Ear, External; Gastrointestinal Motility; Inflammation; Mice; Mice, Inbred ICR; Mice, Inbred Strains; Motor Activity; Pain Measurement; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant

Previous experiments showed that R-(+)-WIN55212-induced inhibition of electrically-evoked contractions of mouse vasa deferentia could be antagonized by cannabidiol in a manner that appeared to be competitive but not to involve direct competition for established cannabinoid receptors. We have now discovered that (-)-7-hydroxy-4'-dimethylheptyl-cannabidiol (7-OH-DMH-CBD) inhibits electrically-evoked contractions of the vas deferens (EC(50)=13.3 nM). This it appeared to do by acting on prejunctional neurones as 100 nM 7-OH-DMH-CBD did not attenuate contractile responses to phenylephrine or beta,gamma-methylene-ATP. Although 7-OH-DMH-CBD was antagonized by SR141716A, it was less susceptible to antagonism by this CB(1) receptor antagonist than R-(+)-WIN55212. 7-OH-DMH-CBD was also antagonized by cannabidiol (1 microM; apparent K(B)=222.2 nM) but not by the CB(2) receptor antagonist, SR144528 (32 nM), or by naloxone (300 nM), ruthenium red (1 microM) or capsazepine (10 microM). Yohimbine (100 nM) enhanced the ability of 7-OH-DMH-CBD to inhibit electrically-evoked contractions. R-(+)-WIN55212 was also potentiated by 100 nM yohimbine, possibly reflecting ongoing sequestration of G(i/o) proteins from CB(1) receptors by alpha(2)-adrenoceptors. Our results suggest that 7-OH-DMH-CBD may activate a neuronal target in the vas deferens that is not a CB(1), CB(2), TRPV1, opioid or alpha(2)-adrenergic receptor but do not exclude the possibility that it also activates CB(1) receptors.

Topics: Adenosine Triphosphate; Adrenergic alpha-2 Receptor Antagonists; Animals; Benzoxazines; Cannabidiol; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Capsaicin; In Vitro Techniques; Isometric Contraction; Male; Mice; Morpholines; Naloxone; Naphthalenes; Phenylephrine; Piperidines; Pyrazoles; Rimonabant; Ruthenium Red; TRPV Cation Channels; Vas Deferens; Yohimbine

We have examined the role of TRPV1 activation in disrupting the blood-brain barrier by measuring the permeability of single pial venular capillaries in anaesthetized rats. Capsaicin application to the brain surface resulted in increased permeability, maximal 2.1+/-0.12 x 10(-6) cm s(-1) (mean+/-s.e.m.) with log EC50 -4.5+/-0.10. Substance P methyl ester gave a similar response (maximal 2.0+/-0.07, n = 6, log EC50 -4.8+/-0.07), but the selective NK2 agonist, beta-Ala8-NKA(4-10) peptide, had no effect. Although CGRP decreased the permeability of venules (log EC50 10.3+/-0.11), its receptor antagonist CGRP(8-37) had no effect on the response to capsaicin. The TRPV1 antagonist capsazepine (1 mM) reduced the response to capsaicin (100 microM), from 1.78+/-0.15 to 0.63+/-0.10 (n = 4). The NK1 receptor antagonists GR205171 (100 microM) and SDZ NKT 376 (1 mM) also reduced the response to capsaicin (from 1.75+/-0.14 to 0.46+/-0.08; n = 6, and from 1.85+/-0.13 to 0.48+/-0.05; n = 5, respectively), indicating that capsaicin acts via TRPV1 in series with NK(1). Starch microspheres were used to produce transient focal ischaemia. Permeability was increased on reperfusion to a greater extent and more rapidly in vessels with diameter greater than 40 microm than those less than 15 microm. Capsazepine given intraperitoneally during ischaemia reduced the permeability increase in small venules from 5.9+/-0.3 to 2.4+/-0.1, and from 11.4+/-0.8 to 5.1+/-0.9 in large venules. In conclusion, the TRPV1 receptor is active in the brain microvasculature and has its permeability-increasing effect via substance P. It also plays a role in the immediate blood-brain barrier disruption following ischaemia-reperfusion.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Capillaries; Capillary Permeability; Capsaicin; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Male; Microspheres; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-1; Starch; Substance P; Tetrazoles; Time Factors; TRPV Cation Channels

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

Topics: Anilides; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Capsaicin; Cinnamates; Cyclic GMP; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Artery, Superior; Nitric Oxide; Nitroarginine; Perfusion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; RNA, Messenger; TRPV Cation Channels; Vasodilation

Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB(1) antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB(1) receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB(1) antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB(1)-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB(1) antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB(1)-dependent manner.

Topics: Amidohydrolases; Analgesia; Animals; Arachidonic Acids; Cannabinoids; Capsaicin; Carrier Proteins; Cytosol; Endocannabinoids; Male; Medulla Oblongata; Mice; Microinjections; Pain Measurement; Periaqueductal Gray; Phospholipases A; Phospholipases A2; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Stress, Psychological; TRPV Cation Channels

This study was designed to test the hypothesis that increased sensitivity of blood pressure to anandamide (AEA), an endocannabinoid compound, occurs during high-salt intake, which can be blocked by a selective vanilloid receptor 1(VR1) antagonist, capsazepine (CAPZ). Intravenous administration of a metabolically stable analog, methanandamide (MethA), dose-dependently decreased mean arterial pressure (MAP) in conscious rats fed a high-sodium diet (HS) for 3 weeks but it had a minimal effect in normal sodium (NS)-treated rats. The MethA-induced decrease in MAP was significantly attenuated but not abolished by CAPZ, or a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, administered separately in HS-treated rats. The MethA-induced depressor effect was prevented by the combined administration of CAPZ and SR141716A in HS-treated rats. Likewise, administration of capsaicin, a selective VR1 receptor agonist, dose-dependently decreased MAP in both HS- and NS-treated rats. The depressor effect of capsaicin was more profound in HS-treated rats, which was prevented by CAPZ. Western blot showed that expression of VR1 but not CB1 in mesenteric arteries was increased in HS-treated compared with NS-treated rats. Therefore, these data show that: (1) HS upregulates mesenteric VR1 expression; (2) HS increases sensitivity of blood pressure to AEA; and (3) HS-induced enhancement of the depressor effect of AEA can be prevented only when both VR1 and CB1 receptors are blocked. These results indicate that AEA contributes to the prevention of salt induced increases in blood pressure via, at least in part, activating the VR1 receptor.

Topics: Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Dose-Response Relationship, Drug; Drug Combinations; Endocannabinoids; Male; Mesenteric Arteries; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Sodium, Dietary; Time Factors; TRPV Cation Channels

1. We have investigated the effects of the endocannabinoid anandamide (AEA) on neuronal excitability and vanilloid TRPV1 receptors in neonatal rat cultured dorsal root ganglion neurones. 2. Using whole-cell patch-clamp electrophysiology, we found that AEA inhibits high-voltage-activated Ca(2+) currents by 33+/-9% (five out of eight neurones) in the absence of the CB(1) receptor antagonist SR141716A (100 nM) and by 32+/-6% (seven out of 10 neurones) in the presence of SR141716A. 3. Fura-2 fluorescence Ca(2+) imaging revealed that AEA produced distinct effects on Ca(2+) transients produced by depolarisation evoked by 30 mM KCl. In a population of neurones of larger somal area (372+/-20 microM(2)), it significantly enhanced Ca(2+) transients (80.26+/-13.12% at 1 microM), an effect that persists after pertussis toxin pretreatment. In a population of neurones of smaller somal area (279+/-18 microM(2)), AEA significantly inhibits Ca(2+) transients (30.75+/-3.54% at 1 microM), an effect that is abolished by PTX pretreatment. 4. Extracellular application of 100 nM AEA failed to evoke TRPV1 receptor inward currents in seven out of eight neurones that responded to capsaicin (1 microM), with a mean inward current of -0.94+/-0.21 nA. In contrast, intracellular application of 100 nM AEA elicited robust inward currents in approximately 62% of neurones, the mean population response was -0.85+/-0.21 nA. When AEA was applied to the intracellular environment with capsazepine (1 microM), the mean population inward current was -0.01+/-0.01 nA. Under control conditions, mean population current fluctuations of -0.09+/-0.05 nA were observed.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Calcium Channels; Calcium Signaling; Capsaicin; Cells, Cultured; Drug Synergism; Endocannabinoids; Fura-2; Ganglia, Spinal; Neurons, Afferent; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Chloride; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; TRPV Cation Channels

1. Our study was undertaken to investigate whether bacterial endotoxin/lipopolysaccharide (LPS) affects the neurogenic vasopressor response in rats in vivo by presynaptic mechanisms and, if so, to characterize the type of presynaptic receptor(s) operating in the initial phase of septic shock. 2. In pithed and vagotomized rats treated with pancuronium, electrical stimulation (ES) (1 Hz, 1 ms, 50 V for 10 s) of the preganglionic sympathetic nerve fibers or intravenous bolus injection of noradrenaline (NA) (1-3 nmol x kg(-1)) increased the diastolic blood pressure (DBP) by about 30 mmHg. Administration of LPS (0.4 and 4 mg x kg(-1)) under continuous infusion of vasopressin inhibited the neurogenic vasopressor response by 25 and 50%, respectively. LPS did not affect the increase in DBP induced by exogenous NA. 3. The LPS-induced inhibition of the neurogenic vasopressor response was counteracted by the cannabinoid CB(1) receptor antagonist SR 141716A (0.1 micromol x kg(-1)), but not by the CB(2) receptor antagonist SR 144528 (3 micromol x kg(-1)), the vanilloid VR1 receptor antagonist capsazepine (1 micromol x kg(-1)) or the histamine H(3) receptor antagonist clobenpropit (0.1 micromol x kg(-1)). The four antagonists by themselves did not affect the increase in DBP induced by ES or by injection of NA in rats not exposed to LPS. 4. We conclude that in the initial phase of septic shock, the activation of presynaptic CB(1) receptors by endogenously formed cannabinoids contributes to the inhibition of the neurogenic vasopressor response.

Topics: Animals; Autonomic Fibers, Postganglionic; Autonomic Fibers, Preganglionic; Blood Pressure; Camphanes; Capsaicin; Decerebrate State; Disease Models, Animal; Electric Stimulation; Germany; Imidazoles; Infusions, Intravenous; Lipopolysaccharides; Male; Norepinephrine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Presynaptic; Rimonabant; Shock, Septic; Solvents; Thiourea; Vagotomy; Vasomotor System; Vasopressins

Anandamide (0.01 to 10 microM) caused greater concentration-dependent reductions of the contractile-induced responses to noradrenaline in female than in male mesenteric vascular beds isolated from adult Sprague-Dawley rats. Greater relaxant responses in females were also induced by the vanilloid TRPV1 receptor agonist capsaicin (0.01 to 10 microM), whereas no sex differences were observed for the relaxations caused by either acetylcholine or sodium nitroprusside. The effect of anandamide in either sex was reduced by the vanilloid TRPV1 receptor antagonist capsazepine but not by the cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A). In males, the anandamide-induced relaxations were potentiated by in vitro exposure during 5 min to 0.5 microM 17beta-oestradiol and unmodified by the protein synthesis inhibitor cycloheximide. The vasorelaxant effects of anandamide in female rats were decreased by ovariectomy. This decrease was prevented by in vivo treatment with 17beta-oestradiol-3-benzoate (450 microg/kg i.m., once a week during 3 weeks) and counteracted by in vitro exposure to oestrogen. In vivo treatment with 17beta-oestradiol also potentiated anandamide-induced responses in males. In conclusion, this study shows an oestrogen-dependent sensitivity to the vanilloid TRPV1 receptor-mediated vasorelaxant effects of anandamide in the mesenteric vasculature of Sprague-Dawley rats, that could be mediated by both genomic and non-genomic mechanisms.

Topics: Acetylcholine; Animals; Arachidonic Acids; Argentina; Capsaicin; Chile; Cycloheximide; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Estradiol; Estrogens; Female; Male; Mesentery; Muscle, Smooth, Vascular; Nitroprusside; Norepinephrine; Ovariectomy; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Sex Characteristics; Time Factors; Vasodilation

N-arachidonoyl-dopamine (NADA) is an endogenous ligand at TRPV1 and CB(1) receptors, which are expressed on primary afferent nociceptors. The aim of this study was to determine contributions of proposed pronociceptive TRPV1 and antinociceptive CB(1) receptors to effects of peripheral NADA on primary afferent fibre function. Effects of NADA on primary afferent nociceptor function, determined by whole cell patch clamp and calcium imaging studies of adult dorsal root ganglion (DRG) neurons, were determined. Application of NADA (1 microm) to DRG neurons depolarized the resting membrane potential (Vm) from -58 +/- 1 to -44 +/- 3 mV (P < 0.00001) and evoked a significant increase (P < 0.0001) in intracellular calcium (74 +/- 11% of response to 60 mm KCl), compared to basal. The TRPV1 receptor antagonist capsazepine abolished NADA-evoked depolarization of Vm (P < 0.0001) and NADA-evoked calcium responses (P < 0.001), which were also blocked by the CB(1) receptor antagonist SR141716A (P < 0.001). Effects of NADA (1.5 microg and 5 microg/50 microL) on mechanically evoked responses of dorsal horn neurons in anaesthetized Sprague-Dawley rats were studied. Intraplantar injection of the higher dose of NADA (5 microg/50 microL) studied significantly inhibited innocuous (8, 10 g) mechanically evoked responses of dorsal horn neurons compared to vehicle, effects blocked by intraplantar injection of SR141716A. Higher weight (26-100 g) noxious-evoked responses of dorsal horn neurons were also significantly inhibited by NADA (5 microg/50 microL), effects blocked by intraplantar injection of the TRPV1 antagonist, iodo-resiniferatoxin. NADA has a complex pattern of effects on DRG neurons and primary afferent fibres, which is likely to reflect its dual site of action at TRPV1 and CB(1) receptors and the differential expression of these receptors by primary afferent fibres.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Calcium; Capsaicin; Cells, Cultured; Dopamine; Drug Interactions; Electrophysiology; Male; Membrane Potentials; Nerve Fibers; Pain Measurement; Physical Stimulation; Piperidines; Posterior Horn Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; Sensory Thresholds; Spinal Cord

To evaluate the effect of St. John's wort (SJW), an effective and safe herbal antidepressant, on rat bladder contractility. Recent data have suggested a strong association between depression and urinary incontinence.. Strips were cut from the bladder body and placed in organ baths containing Krebs solution. Contractions were induced by electrical field stimulation (EFS) and, in some experiments, by exogenous alpha,beta (alpha,beta)-methylene adenosine triphosphate.. St. John's wort was significantly more active in inhibiting the EFS-induced contractions than the alpha,beta-methylene adenosine triphosphate-induced contractions, suggesting both a presynaptic site of action and a direct inhibition of bladder smooth muscle. The inhibitory effect of SJW on EFS-induced contractions was unaffected by methysergide, haloperidol, phentolamine plus propranolol (antagonists that block the action of the neurotransmitters 5-hydroxytriptamine, dopamine, and noradrenaline on their own receptors), the L-type calcium channel antagonist verapamil, capsazepine (which blocks the vanilloid receptor), or cannabinoid CB1 receptor antagonist SR141716A. However, the opioid receptor antagonist naloxone significantly reduced the inhibitory effect of SJW on EFS-induced contractions. Among the chemical constituents of SJW tested, hyperforin and, to a lesser extent, the flavonoid kaempferol showed inhibitory effects.. The results of our study demonstrated that SJW inhibits excitatory transmission of the rat urinary bladder and also directly inhibits smooth muscle contractility. The inhibitory effect on excitatory transmission could involve, at least in part, opioid receptors. SJW may be evaluated for its possible use in treating urinary incontinence in depressed patients.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Anthracenes; Antidepressive Agents; Atropine; Bridged Bicyclo Compounds; Capsaicin; Depression; Electric Stimulation; Female; Haloperidol; Hypericum; Kaempferols; Male; Methysergide; Muscle Contraction; Muscle, Smooth; Naloxone; Perylene; Phentolamine; Phloroglucinol; Piperidines; Plant Extracts; Propranolol; Pyrazoles; Quercetin; Rats; Rats, Wistar; Rimonabant; Rutin; Terpenes; Tetrodotoxin; Urinary Bladder; Urinary Incontinence; Verapamil

Cannabidiol (CBD), a nonpsychoactive marijuana constituent, was recently shown as an oral antihyperalgesic compound in a rat model of acute inflammation. We examined whether the CBD antihyperalgesic effect could be mediated by cannabinoid receptor type 1 (CB1) or cannabinoid receptor type 2 (CB2) and/or by transient receptor potential vanilloid type 1 (TRPV1). Rats received CBD (10 mg kg(-1)) and the selective antagonists: SR141716 (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) for CB1, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3 carboxamide) for CB2 and capsazepine (CPZ) for TRPV1 receptors. The intraplantar injection of carrageenan in rats induced a time-dependent thermal hyperalgesia, which peaked at 3 h and decreased at the following times. CBD, administered 2 h after carrageenan, abolished the hyperalgesia to the thermal stimulus evaluated by plantar test. Neither SR141716 (0.5 mg kg(-1)) nor SR144528 (3 and 10 mg kg(-1)) modified the CBD-induced antihyperalgesia; CPZ partially at the lowest dose (2 mg kg(-1)) and fully at the highest dose (10 mg kg(-1)) reversed this effect. These results demonstrate that TRPV1 receptor could be a molecular target of the CBD antihyperalgesic action.

Topics: Administration, Oral; Animals; Camphanes; Cannabidiol; Capsaicin; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Therapy, Combination; Hyperalgesia; Inflammation; Italy; Male; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Drug; Rimonabant; Time Factors

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8+/-10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9+/-11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1+/-11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3+/-13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE-induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss' myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Capsaicin; Cation Transport Proteins; CHO Cells; Cricetinae; Endothelium, Vascular; Gadolinium; Ganglia, Sympathetic; Guanethidine; Hydroxyeicosatetraenoic Acids; Ion Channels; Male; Mesenteric Arteries; Mice; Mice, Knockout; Models, Cardiovascular; Models, Neurological; Nerve Fibers, Unmyelinated; Nociceptors; Peptides, Cyclic; Piperidines; Pressure; Quinuclidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Ruthenium Red; Sodium Channel Blockers; Splanchnic Circulation; Stress, Mechanical; Sympathectomy, Chemical; Tetrodotoxin; TRPV Cation Channels; Vascular Resistance; Vasoconstriction

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

Topics: Arachidonic Acids; Blood-Brain Barrier; Brain; Camphanes; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Capsaicin; Cell Adhesion Molecules; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Glycerides; Humans; Ion Channels; Microcirculation; Microfilament Proteins; Phosphoproteins; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; TRPV Cation Channels

Most actions of anandamide (AEA) are mediated by the cannabinoid 1 (CB(1)) receptor activation, but on sensory neurones it is also an agonist on the vanilloid subtype 1 receptor (VR(1)). The aim of the present study was to analyse the effect of AEA (10(-6)-10(-4) M) on inhibitory CB(1) and excitatory VR(1) receptors by measuring sensory neuropeptide release such as somatostatin, substance P and calcitonin gene-related peptide, from isolated rat tracheae. AEA (10(-6) M) vas without significant effect, 10(-5) M inhibited neuropeptide release, which was abolished by the G protein-coupled receptor blocker pertussis toxin (100 ng/ml) and the CB(1) receptor antagonist SR141716A (5x10(-7) M). High concentrations of AEA (5x10(-5) M, 10(-4) M) increased the release of the peptides and this inhibition was prevented by the competitive VR(1) antagonist capsazepine (10(-5) M). These results indicate a dual, concentration-dependent action of AEA on CB(1) receptors and VR(1) on peripheral sensory nerve terminals.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Capsaicin; Dose-Response Relationship, Drug; Endocannabinoids; In Vitro Techniques; Male; Neuropeptides; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reference Values; Reproducibility of Results; Rimonabant; Sensitivity and Specificity; Somatostatin; Substance P; Trachea

The effects of cannabinoid drugs on the cholinergic response evoked by electrical field stimulation (0.2 ms pulse width, 20 V amplitude, 10 Hz, 7.5 s train duration) in guinea-pig tracheal preparations were investigated. The stable analogue of the endocannabinoid anandamide, R(+)-methanandamide (10(-7)-10(-4) M), produced a dose-dependent inhibition (up to 27+/-5% of control) of electrical field stimulation-mediated atropine-sensitive response. This effect was not blocked by the selective cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide hydrochloride (SR 141716A; 10(-6) M), and was not reproduced with the cannabinoid CB(1)/CB(2) receptor agonist R(+)-[2,3-dihydro-5-methyl-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate) (WIN 55,212-2; 10(-8)-10(-5) M) or the cannabinoid CB(2) receptor selective agonist 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015; 10(-8)-10(-5) M); it was, on the contrary, antagonized by the vanilloid antagonist 2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-2 benzazepine (capsazepine; 10(-6) M). At the postjunctional level, neither R(+)-methanandamide nor WIN 55,212-2 nor JWH-015 did affect tracheal contractions induced by exogenous acetylcholine (10(-6) M). An inhibitory vanilloid receptor-mediated effect on the cholinergic response evoked by electrical stimulation was confirmed with the vanilloid agonist capsaicin, at doses (3-6 x 10(-8) M) which poorly influenced the basal smooth muscle tone of trachea. In conclusion, our data indicate that in guinea-pig trachea (a) neither CB(1) nor CB(2) cannabinoid receptor-mediated modulation of acetylcholine release occurs; (b) vanilloid VR1-like receptors appear involved in R(+)-methanandamide inhibitory activity on the cholinergic response to electrical field stimulation.

Topics: Acetylcholine; Animals; Arachidonic Acids; Atropine; Benzoxazines; Cannabinoid Receptor Modulators; Capsaicin; Dose-Response Relationship, Drug; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Indoles; Male; Morpholines; Muscle Contraction; Muscle, Smooth; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Trachea

To test the hypothesis that activation of the vanilloid receptor (VR1) contributes to the anandamide-induced depressor effect in spontaneously hypertensive rats (SHR), we used a selective VR1 antagonist capsazepine (CAPZ) and a selective cannabinoid type 1 receptor antagonist SR141716A in conjunction with a VR1 agonist capsaicin in both SHR and Wistar-Kyoto rats (WKY). Mean arterial pressure was increased in SHR compared with WKY (P<0.05). Intravenous administration of capsaicin caused a greater depressor response in SHR compared with WKY (P<0.05), which was blocked by approximately 60% by CAPZ (P<0.05) in SHR only. Methanandamide caused a similar greater depressor response (P<0.05), which was blocked by approximately 50% and 60% by CAPZ and SR141716A, respectively, in SHR (P<0.05) but not in WKY. Radioimmunoassay showed that methanandamide increased plasma calcitonin gene-related peptide (CGRP) levels from baseline in both SHR and WKY (P<0.05), with no difference between 2 strains. Western blot showed that protein expression for the calcitonin receptor-like receptor-but not receptor activity modifying protein 1, VR1, and cannabinoid type 1 receptors-was increased in mesenteric resistance arteries in SHR compared with WKY (P<0.05). These data indicate that in addition to activation of cannabinoid type 1, anandamide may serve as an endogenous compound to stimulate VR1, leading to a decrease in blood pressure via CGRP release from sensory nerve terminals. Increased mesenteric CGRP receptor expression in SHR may account for increased sensitivity of blood pressure to anandamide and may serve as a compensatory response to buffer the increase in blood pressure in SHR.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Capsaicin; Endocannabinoids; Hypertension; Male; Mesenteric Arteries; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B(2) receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8-37), NK(2) or NK(3) receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK(1) antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms.

Topics: Animals; Benzamides; Bradykinin; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Calcium; Calcium Channels; Capsaicin; Carbazoles; Conotoxins; Dipeptides; Dose-Response Relationship, Drug; Egtazic Acid; In Vitro Techniques; Indoles; Iris; Maleimides; Muscle Contraction; Nicardipine; omega-Agatoxin IVA; Peptide Fragments; Piperidines; Protein Kinase Inhibitors; Protein Kinases; Pyrroles; Receptors, Calcitonin Gene-Related Peptide; Receptors, Drug; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Swine; Tachykinins

This study examined the effect of intrathecal (i.t.) injection of the endocannabinoid anandamide in urethane-anesthetized rats. The tip of the i.t. cannula was positioned at the T(12)-L(1) level of the spinal cord. Either anandamide or its metabolically stable analogue methanandamide (25 to 100 nmol) produced dose-dependent decreases in the blood pressure that persisted at least for up to 30 min. The hypotensive responses to 100 nmol anandamide and to 100 nmol methanandamide were -17.7+/-1.6 mmHg ( n=5) and -17.9+/-2.0 mmHg ( n=4), respectively. Hypotensive effects were also obtained with the CB(1) cannabinoid receptor agonist WIN 55212-2 (20 nmol; i.t.) as well as with the vanilloid VR(1) receptor agonist capsaicin (3 nmol; i.t.). Nicotinic ganglionic blockade with hexamethonium bromide [10 mg/kg; intravenous(i.v.)] abolished the responses to both anandamide and capsaicin. The i.t. administration of the CB(1) receptor antagonist, 20 nmol SR 141716A, as well as the VR(1) receptor antagonist, 20 nmol capsazepine, prevented almost completely the hypotensive responses to both anandamide and methanandamide. SR 141716A prevented the hypotension caused by WIN 55212-2 but did not modify the response to the vanilloid receptor agonist capsaicin. On the contrary, capsazepine antagonized the hypotension caused by capsaicin but failed to affect the decrease in blood pressure caused by the CB1 cannabinoid receptor agonist WIN 55212-2. These results suggest that anandamide could modulate the blood pressure through the activation of cannabinoid CB(1) receptors and vanilloid VR(1) receptors localized at the spinal cord.

Topics: Anesthesia, Intravenous; Anesthetics, Intravenous; Animals; Antihypertensive Agents; Arachidonic Acids; Benzoxazines; Blood Pressure; Calcium Channel Blockers; Capsaicin; Dose-Response Relationship, Drug; Endocannabinoids; Ganglionic Blockers; Heart Rate; Hemodynamics; Hexamethonium; Injections, Spinal; Male; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Drug; Rimonabant; Spinal Cord; Urethane

In this study we investigated the effect of cannabinoids on [3H]glutamate release from hippocampal synaptosomes of rat and CB1-null mutant mouse. In the rat, cannabinoid receptor agonists, i.e. CP55,940 (EC50, 0.84 microm), WIN55,212-2 (EC50, 3.47 microm), ACEA (EC50, 17.8 microm), and R-(+)-methanandamide (EC50, 19.8 microm) concentration-dependently inhibited the 25-mm-K+ depolarization-evoked release of [3H]glutamate and, among them, WIN55,212-2 displayed the greatest efficacy. The CB1 receptor antagonists SR141716A (1-5 microm) and AM251 (1 microm) and the VR1 vanilloid receptor antagonist capsazepine (10 microm) did not antagonize the effect of the agonists. SR141716A by itself attenuated the evoked [3H]glutamate release. WIN55,212-2 inhibited the release of [3H]glutamate in CB1 -/- mice as well. These data demonstrate that the action of cannabinoids on glutamate release in the hippocampus is pharmacologically distinct and independent from the cloned CB1 receptor.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Capsaicin; Chromatography, High Pressure Liquid; Cyclohexanols; Dose-Response Relationship, Drug; Drug Interactions; Glutamic Acid; Hippocampus; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Radioactivity; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Synaptosomes; Tritium

Nerve growth factor (NGF) is central to processes involved in an inflammatory hyperalgesia. Administration of exogenous NGF induces a hyperalgesia that is dependent on local neutrophil influx. The effects of administration of the cannabinoid anandamide and the cannabimimetic palmitoylethanolamide on an NGF-induced hyperalgesia and neutrophil accumulation were examined in this study.. Baseline hind limb withdrawal latencies to a noxious heat stimulus were recorded before intraplantar administration of NGF (1 microg in 0.05 ml) to the hind paw of 75 male Wistar rats. Anandamide or palmitoylethanolamide (a substance that has cannabinoid-like actions but little affinity for cannabinoid receptors) at doses of 10 and 25 mg/kg were given (intraperitoneally) immediately after NGF. CB1 (SR141716A) and CB2 (SR144528) receptor antagonists were coadministered with the higher dose of cannabinoids. Withdrawal latencies were expressed as difference from baseline. Seventy rats received intraplantar NGF and intraperitoneal treatments. Neutrophil accumulation in the injected paw was assessed using a myeloperoxidase assay.. Administration of NGF reduced latencies consistent with hyperalgesia. Anandamide and palmitoylethanolamide significantly reduced this hyperalgesia. The action of anandamide was CB1 receptor-mediated. SR144528 abrogated the action of palmitoylethanolamide. NGF also provoked neutrophil accumulation in the injected paw, denoted by an increase in myeloperoxidase. Palmitoylethanolamide significantly reduced neutrophil accumulation by an SR144528-sensitive action, whereas anandamide was without effect.. NGF induced a thermal hyperalgesia that was attenuated by anandamide and palmitoylethanolamide. Only palmitoylethanolamide reduced neutrophil influx. Thus, cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of a proinflammatory neuroimmune process. Such a mechanism suggests a therapeutic site of analgesic action separable from central side effects.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoids; Capsaicin; Cell Movement; Endocannabinoids; Hyperalgesia; Male; Nerve Growth Factor; Neutrophils; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Rimonabant

We have investigated the effect piperine on castor oil-stimulated fluid accumulation in the mouse small intestine. Piperine (2.5-20 mg/kg, i.p.) dose-dependently reduced castor oil-induced intestinal fluid accumulation. The inhibitory effect of piperine (10 mg/kg i.p.) was strongly attenuated in capsaicin (75 mg/kg in total, s.c.)-treated mice but it was not modified by the vanilloid receptor antagonist capsazepine (30 mg/kg i.p.). Pretreatment of mice with hexamethonium (1 mg/kg i.p.), naloxone (2 mg/kg i.p.), yohimbine (1 mg/kg i.p.) or the cannabinoid CB(1) receptor antagonist SR141716A (0.3 mg/kg i.p.) did not modify the inhibitory effect of piperine (10 mg/kg i.p.). These results suggest that piperine reduces castor oil-induced fluid secretion with a mechanism involving capsaicin-sensitive neurons, but not capsazepine-sensitive vanilloid receptors.

Topics: Alkaloids; Animals; Anti-Ulcer Agents; Benzodioxoles; Capsaicin; Castor Oil; Dose-Response Relationship, Drug; Drug Interactions; Injections, Intraperitoneal; Intestinal Secretions; Intestine, Small; Male; Mice; Mice, Inbred ICR; Organ Size; Piperidines; Polyunsaturated Alkamides

1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.

Topics: Aerosols; Animals; Arachidonic Acids; Calcium; Camphanes; Capsaicin; Cells, Cultured; Consciousness; Cough; Dronabinol; Endocannabinoids; Excitatory Amino Acid Antagonists; Guinea Pigs; Male; Nodose Ganglion; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

We have evaluated the effect of the vanilloid receptor agonists resiniferatoxin (RTX), capsaicin and piperine and of the vanilloid receptor antagonist capsazepine on the resting tone in the isolated rat ileum. Capsazepine (10(-8)-3 x 10(-5) M) produced a concentration-related relaxation (8 +/-3%-49 +/-3%) of the rat ileum. By contrast RTX (up to 10(-8) M), capsaicin (up to 10(-6) M) and piperine (up to 10(-5) M) were without effect. Pre-treatment with capsaicin [either in vivo (50 mg/kg s.c.) or in vitro (10(-6) M)] did not modify the inhibitory effect of capsazepine. The L-type Ca2+ channel antagonist nifedipine (10(-6) M), but not the N-type Ca2+ channel antagonist omega-conotoxin GVIA (3 x 10(-8) M) nor the Na+ channel blocker tetrodotoxin (3 x 10(-7) M), counteracted the inhibitory effect of capsazepine. The NK1 receptor antagonist SR 140333 (10(-7) M), the NK2 receptor antagonist SR 48968 (10(-6) M), the NK3 receptor antagonist SR 142801 (10(-7) M), atropine (10(-6) M), hexamethonium (10(-4) M), phentolamine (10(-6) M) plus propranolol (10(-6) M), N(G)-nitro- L-arginine methyl ester ( L-NAME 3 x 10(-4) M), apamin (10(-7) M), methysergide (10(-6) M), the calcitonin gene-related peptide (CGRP) antagonist hCGRP 8-37 (1.5 x 10(-6) M), the VIP antagonist hGRF 1-29 (10(-5) M) did not modify the inhibitory effect of capsazepine. Capsazepine (2.5-40 mg/kg) also decreased upper gastrointestinal transit in vivo. It is concluded that the vanilloid antagonist capsazepine has a direct relaxing effect on rat intestinal smooth muscle which could involve L-type calcium channels. We found no evidence to suggest that capsazepine is antagonizing an endogenous vanilloid.

Topics: Alkaloids; Animals; Benzodioxoles; Calcium Channels, L-Type; Capsaicin; Diterpenes; Dose-Response Relationship, Drug; Gastrointestinal Transit; Ileum; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Drug

This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1-3-6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu(1) and mGlu(5) receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with DL-2-Amino-5-phosphonovaleric acid (DL-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-alpha-Ethylglutamic acid (30 nmol/rat) and (RS)-alpha-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB(1) receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; Animals; Capsaicin; Chromones; Excitatory Amino Acid Antagonists; Male; Pain; Periaqueductal Gray; Phosphoserine; Piperidines; Protein Binding; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Drug; Receptors, Glutamate; Riluzole; Rimonabant; Time Factors

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Biological Transport; Calcium; Calcium Channel Blockers; Cannabinoids; Capsaicin; CHO Cells; Cricetinae; Drug Interactions; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Male; Mice; Morpholines; Muscle Contraction; Naphthalenes; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Structure-Activity Relationship; Transfection; TRPV Cation Channels; Vas Deferens

The possibility that anandamide is an endothelium-derived hyperpolarizing factor was explored in the rat mesenteric vasculature by use of conventional microelectrode techniques. In the main mesenteric artery, anandamide and its more stable analog methanandamide hardly caused a measurable change in membrane potential of the smooth muscle cells, which promptly hyperpolarized to EDHF liberated by acetylcholine. Inhibition of endogenous anandamide breakdown by phenylmethylsulfonyl fluoride did not increase membrane responses to acetylcholine. The CB(1) receptor antagonist SR141716 did not significantly influence EDHF-mediated hyperpolarization except at extremely high concentrations. Smooth muscle cells of third to fourth order branches of the mesenteric artery, which have a more negative resting membrane potential and show smaller responses to acetylcholine, hyperpolarized by about 6 mV to both anandamide and methanandamide, whereas another CB(1) receptor agonist, WIN 55,212-2, had no effect. Mechanical endothelium removal or pre-exposure to SR141716A did not affect anandamide- and methanandamide-induced hyperpolarizations. However, in the presence of capsazepine, a selective vanilloid receptor antagonist, these membrane potential changes were reversed to a small depolarization, whereas EDHF-induced hyperpolarizations were not affected. Pretreating small vessels with capsaicin, causing desensitization of vanilloid receptors and/or depletion of sensory neurotransmitter, completely blocked methanandamide-induced hyperpolarizations. These findings show that anandamide cannot be EDHF. In smooth muscle cells of small arteries, anandamide-induced changes in membrane potential are mediated by vanilloid receptors on capsaicin-sensitive sensory nerves. The different membrane response to the cannabinoids between the main mesenteric artery and its daughter branches might be explained by the different density of perivascular innervation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Biological Factors; Calcium Channel Blockers; Cannabinoids; Capsaicin; Electrophysiology; Endocannabinoids; Endothelium, Vascular; Membrane Potentials; Mesenteric Arteries; Morpholines; Muscle, Smooth, Vascular; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptors, Drug; Rimonabant; Vasodilator Agents

1. In the absence of indomethacin, anandamide did not contract the guinea-pig bronchus at concentrations up to 100 microM. In the presence of indomethacin (10 microM), anandamide induced concentration-related contractions with a pEC(50) value of 5.18+/-0.11. It was significantly less potent than capsaicin (pEC(50) 7.01+/-0.1). The anandamide uptake inhibitor AM404, produced only a 14.1+/-3.22% contraction at 100 microM. All experiments were conducted in the presence of PMSF (20 microM). 2. The vanilloid receptor antagonist, capsazepine (10 microM), significantly attenuated the contractile effect of anandamide, the response to 100 microM anandamide being 40.53+/-7.04% in the presence of vehicle and 1.57+/-8.93% in the presence of 10 microM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan. 3. The log concentration-response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC(50) values for anandamide were 4.88+/-0.08 and 5.17+/-0.19 in the presence of vehicle and SR141716A (1 microM) respectively. 4. The lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 microM anandamide from 34.7+/-1.9% (vehicle) to 7.7+/-5% (ETYA, 10 microM) and from 41.85+/-4.25% (n=6) (vehicle) to 10.31+/-3.54 (n=6) (ETI, 20 microM). Neither inhibitor significantly affected contraction of the tissue by substance P. 5. This study provides evidence that anandamide acts on vanilloid receptors in the guinea-pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acids; Bronchi; Calcium Channel Blockers; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acids, Unsaturated; Guinea Pigs; Hydrazines; In Vitro Techniques; Indomethacin; Lipoxygenase; Muscle Contraction; Oxazepines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Drug; Rimonabant; Thiorphan

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Topics: Acetylcholine; Animals; Arachidonic Acids; Camphanes; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Guinea Pigs; Ileum; In Vitro Techniques; Male; Methacholine Chloride; Muscle Contraction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.

Topics: Amides; Animals; Arachidonic Acids; Capsaicin; Coronary Vessels; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Ethanolamines; Gap Junctions; Glycyrrhetinic Acid; In Vitro Techniques; Indoles; Indomethacin; Male; Palmitic Acids; Peptides; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Serotonin; Tetraethylammonium; Vasodilation

1. The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro. 2. In endothelium-intact rings precontracted to the thromboxane A(2) mimetic, U46619, anandamide (0.01 - 30 microM) induced slowly developing concentration-dependent relaxations (pEC(50) [negative log of EC(50)]=6.1+/-0.1; R(max) [maximum response]=81+/-4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration-relaxation curve without modifying R(max). Methanandamide was without effect on U46619-induced tone. 3. The anandamide-induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 microM), the vanilloid receptor antagonist, capsazepine (3 and 10 microM) or the nitric oxide synthase inhibitor, L-NAME (100 microM). 4. The cyclo-oxygenase inhibitor, indomethacin (3 and 10 microM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 microM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 microM), shifted the anandamide concentration-response curve to the right. 5. Precontraction of endothelium-intact rings with 25 mM KCl attenuated the anandamide-induced relaxations (R(max)=7+/-7%), as did K(+) channel blockade with tetraethylammonium (TEA; 3 microM) or iberiotoxin (100 nM). Blockade of small conductance, Ca(2+)-activated K(+) channels, delayed rectifier K(+) channels, K(ATP) channels or inward rectifier K(+) channels was without effect. 6. These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Arachidonic Acid; Arachidonic Acids; Barium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Coronary Vessels; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glyburide; In Vitro Techniques; Indomethacin; Miconazole; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peptides; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Potassium; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Receptors, Drug; Rimonabant; Sheep; Tetraethylammonium; Vasoconstrictor Agents; Vasodilation

The effects of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) upon rat C6 glioma cell proliferation were examined and compared with a series of synthetic cannabinoids and related compounds. Cells were treated with the compounds each day and cell proliferation was monitored for up to 5 days of exposure. AEA time- and concentration-dependently inhibited C6 cell proliferation. After 4 days of treatment, AEA and 2-AG inhibited C6 cell proliferation with similar potencies (IC(50) values of 1.6 and 1.8 microM, respectively), whereas palmitoylethanolamide showed no significant antiproliferative effects at concentrations up to 10 microM. The antiproliferative effects of both AEA and 2-AG were blocked completely by a combination of antagonists at cannabinoid receptors (SR141716A and SR144528 or AM251 and AM630) and vanilloid receptors (capsazepine) as well as by alpha-tocopherol (0.1 and 10 microM), and reduced by calpeptin (10 microM) and fumonisin B(1) (10 microM), but not by L-cycloserine (1 and 100 microM). CP 55,940, JW015, olvanil, and arachidonoyl-serotonin were all found to affect C6 glioma cell proliferation (IC(50) values of 5.6, 3.2, 5.5, and 1.6 microM, respectively), but the inhibition could not be blocked by cannabinoid + vanilloid receptor antagonists. It is concluded that the antiproliferative effects of the endocannabinoids upon C6 cells are brought about by a mechanism involving combined activation of both vanilloid receptors and to a lesser extent cannabinoid receptors, and leading to oxidative stress and calpain activation. However, there is at present no obvious universal mechanism whereby plant-derived, synthetic, and endogenous cannabinoids affect cell viability and proliferation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cell Division; Glioma; Piperidines; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tumor Cells, Cultured

Intraplantar injection of staphylococcal enterotoxin B induces long-lasting oedema mediated by both cyclooxygenase and lipoxygenase products as well as by neuropeptides from sensory nerves. This study was undertaken to further clarify the role of peripheral primary afferent sensory nerves in staphylococcal enterotoxin B (25 microg/paw)-induced plasma extravasation and oedema formation. The tachykinin NK(1) receptor antagonist (S)-1-[2-[3-(3, 4-dichlorophenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azoniabicyclo [2.2.2]octane cloride (SR140333; 120 nmol/kg, s.c.+120 nmol/kg, i.v.) significantly inhibited plasma exudation and paw oedema evoked by staphylococcal enterotoxin B. The tachykinin NK(2) receptor antagonist (S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3, 4-dichlorophenyl)butyl]-benzamide (SR48968) had no effect on the staphylococcal enterotoxin B-induced responses. The bradykinin B(2) receptor antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]bradykinin (Hoe 140; 400 nmol/kg, i.v.) significantly reduced staphylococcal enterotoxin B-induced responses. The magnitude of the inhibition observed with Hoe 140 alone was similar to that caused by concomitant treatment of animals with SR140333 and Hoe 140, suggesting that there is a final common pathway. Additionally, SR140333 given alone reduced bradykinin (3 nmol/paw)-induced paw oedema. The vanilloid receptor antagonist N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7, 8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine; 100 micromol/kg) significantly reduced staphylococcal enterotoxin B-induced responses. The 5-HT receptor antagonist methysergide (10 mg/kg, i.v.) and the histamine H(1) receptor antagonist mepyramine (10 mg/kg, i.v.) produced a significant reduction in paw oedema whereas plasma exudation was reduced only by methysergide. In diabetic mice, exudation and oedema evoked by staphylococcal enterotoxin B were markedly reduced. Acute administration of insulin (20 UI/kg, s.c., 30 min before) did not restore the increased permeability induced by staphylococcal enterotoxin B. We conclude that plasma exudation and paw oedema in response to staphylococcal enterotoxin B are a consequence of a complex neurogenic response involving direct activation of vanilloid receptors on sensory nerves, release of kinins and subsequent activation of bradykinin B(2) receptors at a prejunctional level, and direct or indirect degranulation of mast cells.

Topics: Animals; Benzamides; Bradykinin; Capillary Permeability; Capsaicin; Diabetes Mellitus, Experimental; Edema; Enterotoxins; Hindlimb; Kinins; Male; Mast Cells; Mice; Neurokinin-1 Receptor Antagonists; Neurons, Afferent; Piperidines; Pyrilamine; Quinuclidines; Receptors, Neurokinin-2

1. The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (VE). The effect was dose-dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). 3. Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). 4. RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. 5. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.

Topics: Anesthesia; Animals; Benzamides; Capsaicin; Dose-Response Relationship, Drug; Indoles; Isoindoles; Male; Microinjections; Neurokinin-1 Receptor Antagonists; Piperidines; Rats; Rats, Wistar; Receptors, Drug; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; Respiratory Mechanics; Solitary Nucleus; Tidal Volume

Contractile and relaxant responses to capsaicin and resiniferatoxin were examined in human isolated bronchus (5-12 mm o.d.). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The contractions averaged 20% of maximal contraction at 1 microM and averaged > 40% maximal contraction at 300 microM (the highest concentration studied). The capsaicin-induced contractions were mimicked by resiniferatoxin (0.1-10 microM) and inhibited by the putative capsaicin receptor antagonist, capsazepine (10 microM). The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968 (0.3 microM), whereas responses to concentrations of neurokinin A (10 nM), neurokinin B (0.1 microM), substance P (1 microM), neuropeptide gamma (10 nM), and neuropeptide K (10 nM) which produced similar-size contractions were almost abolished by 0.1 microM SR 48968. The bronchi isolated from 8 of 16 lungs also exhibited relaxations in response to capsaicin. Capsaicin-induced relaxations were not inhibited by the nitric oxide synthase inhibitor L-nitro-n-arginine (10 microM). In whole-cell patch-clamp experiments on human cultured airway smooth muscle cells, capsaicin was found to enhance outward currents due to the activation of charybdotoxin-sensitive large conductance Ca2+-activated K+ channels. Neither the capsaicin-induced contractions nor the relaxations were mimicked by angiotensin II, bombesin, or calcitonin gene-related peptide at concentrations up to 1 microM. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone, but this response does not appear to involve substance P or related neurokinins. Relaxations to capsaicin may, however, involve the activation of large conductance Ca2+-activated K+ channels.

Topics: Adult; Benzamides; Bronchi; Capsaicin; Cells, Cultured; Diterpenes; Drug Interactions; Female; Humans; Male; Muscle Contraction; Muscle, Smooth; Patch-Clamp Techniques; Piperidines; Tachykinins

Both trigeminal and spinal ganglion neurons show a strong potentiation of responses to the irritant capsaicin in an acidic environment. The present study revealed that there is also a strong interaction between protons and piperine, another vanilloid irritant. We studied the mechanism of the interaction between protons and piperine. Whole-cell patch clamp recordings were performed on cultured adult rat trigeminal ganglion (TG) neurons voltage-clamped near their resting membrane potential (-60 mV). Piperine (10 microM) caused a sustained net inward current associated with either an increase or decrease in membrane conductance. When protons and piperine were co-applied, the membrane currents evoked in piperine-sensitive TG neurons far exceeded the algebraic sum of the responses to the two stimuli applied in isolation. Capsazepine blocked the response of TG neurons to piperine at both physiological and acidic pH. In the presence of capsazepine, responses to the mixture of piperine and protons resembled the response to the low pH stimulus applied alone. Capsazepine had no effect on the sustained proton-induced current. These findings suggest that protons enhance the piperine current by altering the vanilloid receptor/channel complex or increasing the length constant of the space clamp.

Topics: Alkaloids; Animals; Anticonvulsants; Benzodioxoles; Binding, Competitive; Capsaicin; Female; Hydrogen-Ion Concentration; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Protons; Rats; Rats, Sprague-Dawley; Trigeminal Ganglion

We have examined the effects of capsazepine, a selective capsaicin antagonist, and SR 48968, a selective NK2 receptor antagonist, on citric acid inhalation-induced bronchoconstriction in guinea-pigs. Simultaneous inhalation of capsazepine (10 microM) significantly inhibited (by 85%) the bronchoconstriction induced by inhaled citric acid (0.4 M) but not that induced by histamine (2 mM). In capsaicin-pretreated (50 mg/kg s.c. 3 weeks earlier) guinea-pigs, citric acid failed to cause any bronchoconstriction, while the effect of histamine was uninfluenced. Furthermore, citric acid inhalation-induced bronchoconstriction was also markedly inhibited (by 65%) after pretreatment with SR 48968 (0.3 mg/kg i.v.). SR 48968 blocked the bronchoconstriction but not the hypotension evoked by neurokinin A. Therefore, these results suggest that inhalation of a low-pH solution such as citric acid can stimulate sensory neurons through a mechanism similar to that for capsaicin with regard to sensitivity to capsazepine. Tachykinins such as neurokinin A are then locally released from the terminals of sensory nerves and cause bronchoconstriction, mainly by NK2 receptor mechanisms.

Topics: Administration, Inhalation; Analysis of Variance; Animals; Benzamides; Bronchoconstriction; Capsaicin; Citrates; Citric Acid; Female; Guinea Pigs; Histamine; Hydrogen-Ion Concentration; Male; Neurokinin A; Neurons, Afferent; Piperidines

Topics: Animals; Benzamides; Bronchoconstriction; Capsaicin; Citrates; Citric Acid; Electric Stimulation; Female; Guinea Pigs; Male; Neurokinin A; Neurons, Afferent; Piperidines; Receptors, Neurokinin-2; Receptors, Neurotransmitter