olvanil and anandamide

In spite of the rapid advances in our understanding of vanilloid-receptor pharmacology in the PNS, the function of vanilloid receptors in the brain has remained elusive. Recently, the endocannabinoid anandamide has been proposed to function as an endogenous agonist at the vanilloid receptor VR1. This is an exciting hypothesis because the localization of VR1 overlaps with that of anandamide and its preferred cannabinoid receptor CB(1) in various brain areas. The interaction of anandamide and/or related lipid metabolites with these two completely separate receptor systems in the brain clearly places VR1 in a much broader role than pain perception. At a practical level, the overlapping ligand recognition properties of VR1 and CB(1) might be exploited by medicinal chemistry. For example, arvanil, a 'chimeric' ligand that combines structural features of capsaicin and anandamide, promises to be an interesting lead for new drugs that interact at both vanilloid and cannabinoid receptors.

Topics: Animals; Arachidonic Acids; Brain Chemistry; Cannabinoid Receptor Modulators; Capsaicin; Diterpenes; Drug Design; Endocannabinoids; Forecasting; Ganglia, Spinal; Glycerides; Humans; Ligands; Nerve Tissue Proteins; Neurons, Afferent; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship

Nasal transient receptor potential vanilloid 1 (TRPV1) stimulation with capsaicin produces serous and mucinous secretion in the human nasal airway. The primary aim of this study was to examine topical effects of various TRP ion channel agonists on symptoms and secretion of specific mucins: mucin 5 subtype AC (MUC5AC) and B (MUC5B).. Healthy individuals were subjected to nasal challenges with TRPV1 agonists (capsaicin, olvanil and anandamide), TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and mustard oil) and a TRP melastatin 8 (TRPM8) agonist (menthol). Symptoms were monitored, and nasal lavages were analysed for MUC5AC and MUC5B, i.e. specific mucins associated with airway diseases. In separate groups of healthy subjects, nasal biopsies and brush samples were analysed for TRPV1 and MUC5B, using immunohistochemistry and RT-qPCR. Finally, calcium responses and ciliary beat frequency were measured on isolated ciliated epithelial cells.. All TRP agonists induced nasal pain or smart. Capsaicin, olvanil and mustard oil also produced rhinorrhea. Lavage fluids obtained after challenge with capsaicin and mustard oil indicated increased levels of MUC5B, whereas MUC5AC was unaffected. MUC5B and TRPV1 immunoreactivities were primarily localized to submucosal glands and peptidergic nerve fibres, respectively. Although trpv1 transcripts were detected in nasal brush samples, functional responses to capsaicin could not be induced in isolated ciliated epithelial cells.. Agonists of TRPV1 and TRPA1 induced MUC5B release in the human nasal airways in vivo. These findings may be of relevance with regard to the regulation of mucin production under physiological and pathophysiological conditions.

Topics: Acrolein; Administration, Intranasal; Adult; Aged; Arachidonic Acids; Biopsy; Calcium; Calcium Channels; Capsaicin; Cilia; Cross-Over Studies; Double-Blind Method; Endocannabinoids; Humans; Immunohistochemistry; Methanol; Middle Aged; Movement; Mucin 5AC; Mucin-5B; Mustard Plant; Nasal Lavage; Nasal Mucosa; Nerve Tissue Proteins; Pain; Pain Measurement; Plant Oils; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensory System Agents; Sweden; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPM Cation Channels; TRPV Cation Channels; Young Adult

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Topics: Activating Transcription Factor 3; Amidohydrolases; Anilides; Animals; Arachidonic Acids; Benzamides; Capsaicin; Carbamates; Cinnamates; Disease Models, Animal; Endocannabinoids; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Interleukin-6; Lumbar Vertebrae; Male; Osteoarthritis; Pain; Pain Management; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; 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

Patients with allergic rhinitis may be abnormally sensitive to stimulation of the ion channel transient receptor potential vanilloid-1 (TRPV1).. To examine effects of various TRP ion channel activators on sensory symptoms in allergic rhinitis prior to and during seasonal allergen exposure.. Nasal challenges were carried out with the TRPV1-activators capsaicin, anandamide and olvanil. Moreover, challenges were performed with mustard oil (allylisothiocyanate) and cinnamaldehyde as well as menthol, activators of TRPA1 and TRPM8, respectively. Nasal symptoms were monitored after each challenge and compared with symptoms reported following corresponding sham challenges. Symptoms recorded after challenge prior to pollen season were also compared with challenge-induced symptoms during pollen season.. The TRPV1, TRPA1 and TRPM8-activators produced sensory symptoms dominated by pain and smart. During seasonal allergen exposure, but not prior to season, TRPV1-activators also induced itch. Furthermore, the seasonal challenge to the TRPV1-activator olvanil was associated with rhinorrhoea.. Patients with allergic rhinitis feature an increased itch response to TRPV1 stimulation at seasonal allergen exposure. We suggest that this reflects part of the hyperresponsiveness that characterizes on-going allergic rhinitis. Intervention with the TRPV1-signalling pathway may offer potential treatments of this condition.

Topics: Acrolein; Adult; Allergens; Anti-Inflammatory Agents, Non-Steroidal; Antipruritics; Arachidonic Acids; Calcium Channel Blockers; Calcium Channels; Capsaicin; Endocannabinoids; Humans; Menthol; Mustard Plant; Nasal Provocation Tests; Nerve Tissue Proteins; Plant Oils; Polyunsaturated Alkamides; Pruritus; Rhinitis, Allergic, Seasonal; Sensory System Agents; Severity of Illness Index; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPM Cation Channels; TRPV Cation Channels

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.

Topics: Arachidonic Acids; Calcium; Calcium Signaling; Capsaicin; Cell Culture Techniques; Cell Death; Cell Line, Tumor; Cell Proliferation; Diterpenes; Dopamine; Endocannabinoids; Humans; Models, Biological; Neuroblastoma; Neurons; Norepinephrine; Polyunsaturated Alkamides; Recombinant Proteins; Synaptic Transmission; Transfection; TRPV Cation Channels; Up-Regulation

Olvanil (N-9-Z-octadecenoyl-vanillamide) is an agonist of transient receptor potential vanilloid type 1 (TRPV1) channels that lack the pungency of capsaicin and was developed as an oral analgesic. Vanillamides are unmatched in terms of structural simplicity, straightforward synthesis, and safety compared with the more powerful TRPV1 agonists, like the structurally complex phorboid compound resiniferatoxin. We have modified the fatty acyl chain of olvanil to obtain ultra-potent analogs. The insertion of a hydroxyl group at C-12 yielded a compound named rinvanil, after ricinoleic acid, significantly less potent than olvanil (EC(50) = 6 versus 0.7 nM), but more versatile in terms of structural modifications because of the presence of an additional functional group. Acetylation and phenylacetylation of rinvanil re-established and dramatically enhanced, respectively, its potency at hTRPV1. With a two-digit picomolar EC(50) (90 pM), phenylacetylrinvanil (PhAR, IDN5890) is the most potent vanillamide ever described with potency comparable with that of resiniferatoxin (EC(50), 11 pM). Benzoyl- and phenylpropionylrinvanil were as potent and less potent than PhAR, respectively, whereas configurational inversion to ent-PhAR and cyclopropanation (but not hydrogenation or epoxidation) of the double bond were tolerated. Finally, iodination of the aromatic hydroxyl caused a dramatic switch in functional activity, generating compounds that behaved as TRPV1 antagonists rather than agonists. Since the potency of PhAR was maintained in rat dorsal root ganglion neurons and, particularly, in the rat urinary bladder, this compound was investigated in an in vivo rat model of urinary incontinence and proved as effective as resiniferatoxin at reducing bladder detrusor overactivity.

Topics: Amidohydrolases; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Capsaicin; Carrier Proteins; Cell Line, Tumor; Endocannabinoids; Female; Ganglia, Spinal; Humans; In Vitro Techniques; Indicators and Reagents; Ion Channels; Neurons; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Structure-Activity Relationship; TRPV Cation Channels; Urinary Bladder; Urinary Incontinence

The endocannabinoid anandamide is an emerging potential signalling molecule in the cardiovascular system. Anandamide causes vasodilatation, bradycardia and hypotension in animals and has been implicated in the pathophysiology of endotoxic, haemorrhagic and cardiogenic shock, but its vascular effects have not been studied in man. Human forearm blood flow and skin microcirculatory flow were recorded using venous occlusion plethysmography and laser-Doppler perfusion imaging (LDPI), respectively. Each test drug was infused into the brachial artery or applied topically on the skin followed by a standardized pin-prick to disrupt the epidermal barrier. Anandamide failed to affect forearm blood flow when administered intra-arterially at infusion rates of 0.3-300 nmol min(-1). The highest infusion rate led to an anandamide concentration of approximately 1 microM in venous blood as measured by mass spectrometry. Dermal application of anandamide significantly increased skin microcirculatory flow and coapplication of the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine inhibited this effect. The TRPV1 agonists capsaicin, olvanil and arvanil all induced concentration-dependent increases in skin blood flow and burning pain when administered dermally. Coapplication of capsazepine inhibited blood flow and pain responses to all three TRPV1 agonists. This study shows that locally applied anandamide is a vasodilator in the human skin microcirculation. The results are consistent with this lipid being an activator of TRPV1 on primary sensory nerves, but do not support a role for anandamide as a circulating vasoactive hormone in the human forearm vascular bed.

Topics: Adult; Arachidonic Acids; Benzylamines; Capsaicin; Endocannabinoids; Female; Forearm; Humans; Laser-Doppler Flowmetry; Male; Microcirculation; Middle Aged; Muscle, Skeletal; Plethysmography; Polyunsaturated Alkamides; Regional Blood Flow; Skin; TRPV Cation Channels

Certain fatty acid amides such as anandamide (AEA) and olvanil are agonists for the transient receptor potential, vanilloid-1 (TRPV1) receptor, but have been found to activate TRPV1-containing C-fibers in some tissues but not others. We used extracellular recording and whole-cell patch clamp techniques to investigate the effect of olvanil and AEA on different types of vagal C-fibers innervating the same tissue, namely jugular and nodose vagal C-fibers in guinea pig lungs. A 30 s exposure to AEA and olvanil caused action potential discharge in all nodose C-fiber innervating lung but failed to activate jugular C-fibers innervating lung and airways. The activation of nodose C-fibers was blocked by the TRPV1 antagonist iodo-resiniferatoxin. In whole-cell patch clamp recordings of dissociated nodose and jugular capsaicin-sensitive neurons labeled from lungs and airways, olvanil induced large TRPV1-dependent inward currents in cell bodies of both nodose and jugular ganglion neurons. Prolonged exposure (up to 5 min) to olvanil caused action potential discharge in jugular C-fiber innervating lung but the onset latency was four times longer in jugular than in nodose C-fibers. The onsets of capsaicin response in nodose and jugular C-fibers were not different. Decreasing the tissue temperature to 25 degrees C increased the onset latency of olvanil-induced activation of nodose C-fibers 2-3-fold, but did not effect the latency of the capsaicin response. Capsaicin, olvanil, and AEA stimulate jugular C-fibers leading to tachykinergic contractions of isolated bronchi. The time to reach half-maximum is more than four times longer for olvanil and AEA, as compared to capsaicin in evoking contractions. We conclude that brief exposure to certain fatty acid amides, such as AEA and olvanil activate nodose but not jugular C-fiber terminals in the lungs. We hypothesize that this is because the nodose C-fiber terminals are equipped with a temperature-dependent mechanism for effectively and rapidly transporting the TRPV1 agonists so that they gain access to the intracellular binding sites on TRPV1. This transport mechanism may be differently expressed in two distinct subtypes of pulmonary C-fiber terminals innervating the same tissue.

Topics: Action Potentials; Animals; Arachidonic Acids; Bronchi; Capsaicin; Diterpenes; Endocannabinoids; Glomus Jugulare; Guinea Pigs; In Vitro Techniques; Lung; Male; Nerve Fibers, Unmyelinated; Nodose Ganglion; Polyunsaturated Alkamides; Temperature; Time Factors; Trachea; TRPV Cation Channels; Vagus Nerve

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these "transport inhibitors" did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.

Topics: Arachidonic Acids; Astrocytoma; Biological Transport; Cannabinoids; Capsaicin; Carrier Proteins; Endocannabinoids; Humans; Immunohistochemistry; Kinetics; Neuroblastoma; Polyunsaturated Alkamides; Tumor Cells, Cultured

Anandamide, an endogenous lipid, activates both cannabinoid (CB(1)) and vanilloid (VR1) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB(1) and VR1 in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and endogenous VR1 receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB(1) and VR1 receptors to anandamide-induced analgesia.. Using a Flurometric Imaging Plate Reader (FLIPR), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VR1-transfected HEK293 (rVR1-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells.. The VR1 agonists capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)), with similar temporal profiles in both rVR1-HEK and DRG cells, and potency (pEC(50)) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5.5, respectively. These responses were inhibited by the VR1 antagonist capsazepine (1 micro M). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in [Ca(2+)](i). Reapplication of VR1 agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect.. Here we provide evidence that the pharmacology of recombinant rVR1 receptors is similar to those endogenously expressed in DRG cells. Moreover, we have shown that VR1, but not CB(1), receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VR1 activation in DRG cells in vivo.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; Capsaicin; Cells, Cultured; Clone Cells; Endocannabinoids; Ganglia, Spinal; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Topics: Alkaloids; Aniline Compounds; Arachidonic Acids; Benzophenanthridines; Calcium; Capsaicin; Cell Line; Diterpenes; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Fluorescence; Fluorometry; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Phenanthridines; Polycyclic Sesquiterpenes; Polyunsaturated Alkamides; Protein Kinase C; Receptors, Drug; Ruthenium Red; Sesquiterpenes; Time Factors; Xanthenes

To evaluate the effects of topically applied anandamide transport inhibitors, AM404 and olvanil, on the intraocular pressure (IOP) of normotensive rabbits. To determine if the ocular hypotension induced by topical anandamide (AEA) can be potentiated by co-administered AM404.. Test compounds, in either hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or propylene glycol, were administered unilaterally onto rabbit eyes. To determine if AM404 affects the IOP-profile of AEA, AM404 was administered ocularly 15 minutes before topical AEA. Phenylmethylsulfonyl fluoride (PMSF) (24 mg/kg, s.c.) was given 30 min before AEA to prevent its catabolism. IOPs of the treated and untreated eyes were measured. The cannabinoid agonist activities of AM404 and olvanil were studied by using [35S]GTPyS autoradiography.. Topical AM404 (62.5 micirog), in HP-beta-CD vehicle, decreased IOP significantly in treated eyes. AM404 (62.5 microg) induced a significant IOP increase without subsequent decrease when given in propylene glycol vehicle. Olvanil (312.5 microg) caused a significant IOP reduction without provoking an initial hypertensive phase. These compounds did not significantly affect the IOP of untreated eyes. Co-administered AM404 (125 microg in HP-beta-CD) had no significant effect on the IOP profile of AEA (62.5 microg).. Ocular administration of AM404 or olvanil decreased IOP in rabbits, although AM404 can provoke an initial ocular hypertension and did not potentiate the IOP responses induced by exogenous AEA.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Biological Transport; Calcium Channel Blockers; Capsaicin; Endocannabinoids; Female; Intraocular Pressure; Male; Polyunsaturated Alkamides; Rabbits; Rats; Rats, Wistar

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

Topics: Acetylcholine; Animals; Arachidonic Acids; Benzofurans; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Blockers; Camphanes; Cannabinoids; Capsaicin; Cell Line; Dose-Response Relationship, Drug; Endocannabinoids; Humans; In Vitro Techniques; Mesenteric Arteries; Neurons, Afferent; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Ruthenium; Synaptic Transmission; Vasodilation; Vasodilator Agents

The structural similarities between the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonylamide (AM404) and the synthetic vanilloid agonist olvanil [(N-vanillyl)-9-oleamide], prompted us to investigate the possibility that olvanil may interfere with anandamide transport. The intracellular accumulation of [3H]anandamide by human astrocytoma cells was prevented by olvanil with a Ki value of 14.1+/-7.1 microM. By contrast, capsaicin [(8-methyl-N-vanillyl)-6-noneamide], a plant-derived vanilloid agonist, and capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2 H-2-benzazepine-2-carbothioamide), a vanilloid antagonist, had no such effect (Ki > 100 microM). These results indicate that, although less potent than AM404 (Ki 2.1+/-0.2 microM), olvanil may reduce anandamide clearance at concentrations similar to those needed for vanilloid receptor activation.

Topics: Amidohydrolases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Astrocytoma; Biological Transport; Brain; Capsaicin; Depression, Chemical; Endocannabinoids; Humans; Polyunsaturated Alkamides; Rats; Receptors, Drug; Tumor Cells, Cultured

We investigated the effect of changing the length and degree of unsaturation of the fatty acyl chain of N-(3-methoxy-4-hydroxy)-benzyl-cis-9-octadecenoamide (olvanil), a ligand of vanilloid receptors, on its capability to: (i) inhibit anandamide-facilitated transport into cells and enzymatic hydrolysis, (ii) bind to CB1 and CB2 cannabinoid receptors, and (iii) activate the VR1 vanilloid receptor. Potent inhibition of [(14)C]anandamide accumulation into cells was achieved with C20:4 n-6, C18:3 n-6 and n-3, and C18:2 n-6 N-acyl-vanillyl-amides (N-AVAMs). The saturated analogues and Delta(9)-trans-olvanil were inactive. Activity in CB1 binding assays increased when increasing the number of cis-double bonds in a n-6 fatty acyl chain and, in saturated N-AVAMs, was not greatly sensitive to decreasing the chain length. The C20:4 n-6 analogue (arvanil) was a potent inhibitor of anandamide accumulation (IC(50) = 3.6 microM) and was 4-fold more potent than anandamide on CB1 receptors (Ki = 0.25-0.52 microM), whereas the C18:3 n-3 N-AVAM was more selective than arvanil for the uptake (IC(50) = 8.0 microM) vs CB1 receptors (Ki = 3.4 microM). None of the compounds efficiently inhibited [(14)C]anandamide hydrolysis or bound to CB2 receptors. All N-AVAMs activated the cation currents coupled to VR1 receptors overexpressed in Xenopus oocytes. In a simple, intact cell model of both vanilloid- and anandamide-like activity, i.e., the inhibition of human breast cancer cell (HBCC) proliferation, arvanil was shown to behave as a "hybrid" activator of cannabinoid and vanilloid receptors.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Binding Sites; Biological Transport; Capsaicin; Cell Division; Cell Line; Cell Membrane; Diffusion; Electric Conductivity; Endocannabinoids; Fatty Acids, Unsaturated; Humans; Ligands; Mice; Oocytes; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Xenopus laevis

The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N-vanillyl-cis-9-octadecenoamide), and the 'endocannabinoid' anandamide (arachidonoyl-ethanolamide, AEA), suggests possible interactions between the cannabinoid and vanilloid signalling systems. Here we report that olvanil is a stable and potent inhibitor of AEA facilitated transport into rat basophilic leukemia (RBL-2H3) cells. Olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL-2H3 cells (IC50 = 9 microM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50 = 80 microM), AM404 (12.9% inhibition at 10 microM) or oleoylethanolamide (27.5% inhibition at 10 microM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki = 1.64-7.08 microM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 = 1.60 microM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but not capsaicin, also selectively bound to CB1 receptor-containing membranes. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system, and may lead to the design of a novel class of cannabimimetics with potential therapeutic applications as analgesics.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cell Line; Endocannabinoids; Kinetics; Leukemia, Basophilic, Acute; Macrophages; Mice; Neuroblastoma; Polyunsaturated Alkamides; Rats; Receptors, Drug; Tumor Cells, Cultured