tetrodotoxin and Inflammation

Physiological and pharmacological evidence both have demonstrated a critical role for voltage-gated sodium channels (VGSCs) in many types of chronic pain syndromes because these channels play a fundamental role in the excitability of neurons in the central and peripheral nervous systems. Alterations in function of these channels appear to be intimately linked to hyperexcitability of neurons. Many types of pain appear to reflect neuronal hyperexcitability, and importantly, use-dependent sodium channel blockers are effective in the treatment of many types of chronic pain. This review focuses on the role of VGSCs in the hyperexcitability of sensory primary afferent neurons and their contribution to the inflammatory or neuropathic pain states. The discrete localization of the tetrodotoxin (TTX)-resistant channels, in particular NaV1.8, in the peripheral nerves may provide a novel opportunity for the development of a drug targeted at these channels to achieve efficacious pain relief with an acceptable safety profile.

Topics: Analgesics; Anesthetics, Local; Animals; Anticonvulsants; Humans; Hyperalgesia; Inflammation; Ion Channel Gating; Neurons, Afferent; Pain; Peripheral Nervous System Diseases; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Several mechanisms have been identified that may underlie inflammation-induced sensitization of high-threshold primary afferent neurons, including the modulation of voltage- and Ca2+-dependent ion channels and ion channels responsible for the production of generator potentials. One such mechanism that has recently received a lot of attention is the modulation of a tetrodotoxin (TTX)-resistant voltage-gated Na+ current. Evidence supporting a role for TTX-resistant Na+ currents in the sensitization of primary afferent neurons and inflammatory hyperalgesia is reviewed. Such evidence is derived from studies on the distribution of TTX-resistant Na+ currents among primary afferent neurons and other tissues of the body that suggest that these currents are expressed only in a subpopulation of primary afferent neurons that are likely to be involved in nociception. Data from studies on the biophysical properties of these currents suggest that they are ideally suited to mediate the repetitive discharge associated with prolonged membrane depolarizations. Data from studies on the effects of inflammatory mediators and antinociceptive agents on TTX-resistant Na+ currents suggest that modulation of these currents is an underlying mechanism of primary afferent neuron sensitization. In addition, the second-messenger pathways underlying inflammatory mediator-induced modulation of these currents appear to underlie inflammatory mediator-induced hyperalgesia. Finally, recent antisense studies have also yielded data supporting a role for TTX-resistant Na+ currents in inflammatory hyperalgesia. Although data from these studies are compelling, data presented at the Neurobiology of Pain colloquium raised a number of interesting questions regarding the role of TTX-resistant Na+ currents in inflammatory hyperalgesia; implications of three of these questions are discussed.

Topics: Animals; Humans; Hyperalgesia; Inflammation; Pain; Sodium Channels; Tetrodotoxin

Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (I

Topics: Action Potentials; Animals; CA1 Region, Hippocampal; Cell Hypoxia; Culture Media; Delayed Rectifier Potassium Channels; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; In Vitro Techniques; Inflammation; Kinetics; Membrane Potentials; Neuroprotective Agents; Patch-Clamp Techniques; Rats; Reperfusion; Reperfusion Injury; Tetrodotoxin

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2-CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

Topics: Animals; Chemokine CCL2; Drug Synergism; Freund's Adjuvant; Ganglia, Spinal; Gene Expression; Inflammation; Male; Membrane Potentials; Neurons, Afferent; Pain; Patch-Clamp Techniques; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Topics: Animals; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Collagen; Electric Stimulation; Female; Inflammation; Lipopolysaccharides; Lung; Methacholine Chloride; Mice; Mice, Inbred BALB C; Nerve Tissue; Nicotine; Organ Culture Techniques; Respiratory Mechanics; Tetrodotoxin; Time Factors; Toll-Like Receptor 4; Trachea

Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aβ-fiber excitability following persistent inflammation.. Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats.. Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aβ-fibers contributes to inflammatory pain.. Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.

Topics: Ambroxol; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Freund's Adjuvant; Ganglia, Spinal; Gene Expression Regulation; Inflammation; Male; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Nerve Fibers, Myelinated; Neurons; Pain Threshold; Protein Transport; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sodium Channel Blockers; Tetrodotoxin

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage-gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Nav 1.1, Nav 1.2, and Nav 1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS-induced NF-κB activation, expression of TNF-α, IL-1β and inducible nitric oxide synthase, and NO production. LPS-induced p38MAPK activation followed pro-nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS-induced JNK activation was not. TTX also inhibited caspase-3 activation and cell death of primary cortical neurons in neuron/microglia co-cultures by inhibiting LPS-induced microglia activation. Furthermore, TTX attenuated caspase-3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury.

Topics: Animals; Cell Death; Cells, Cultured; Disease Models, Animal; Inflammation; Microglia; Neurons; NF-kappa B; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Spinal Cord Injuries; Tetrodotoxin; Tumor Necrosis Factor-alpha

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

Topics: Animals; Base Sequence; Chronic Pain; CpG Islands; Cyclic AMP-Dependent Protein Kinases; Cystathionine beta-Synthase; Cysteine; Disease Models, Animal; DNA Methylation; Epigenomics; Ganglia, Spinal; Hyperalgesia; Inflammation; Isoquinolines; Molecular Sequence Data; Patch-Clamp Techniques; Promoter Regions, Genetic; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Sulfides; Sulfonamides; Tetrodotoxin

DrTx(1-42) (a carboxyl-terminally truncated version of drosotoxin) is a potent and selective blocker of tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion neurons with analgesic activity. This purpose is to identify key amino acids which are responsible for both blocking and analgesic effects of DrTx(1-42).. On the basis of previous study, we designed five mutants of DrTx(1-42) (delN, D8A, D8K, G9A, and G9R) in the amino-terminal turn (N-turn) region, a proposed functional region located in the amino-terminus of the molecule. All these mutants were expressed in E.coli and purified by RP-HPLC. Electrophysiological properties of these analogues were examined by whole-cell patch-clamp recordings and their antinociceptive effects were investigated by the formalin test and acetic acid induced writhing test.. All the mutants except for G9A possess a similar secondary structure to that of DrTx(1-42), as identified by circular dichroism analysis. Three mutants (delN, D8A and G9A) were found almost inactive to TTX-R Na(+) channels, whereas D8K retains similar activity and G9R showed decreased potency when compared with the wild-type molecule. Consistent with the electrophysiological observations, D8K and G9R exhibited antinociceptive effects in the second phase (inflammatory pain) of the formalin test and the acetic acid induced writhing test, while delN, D8A and G9A lack such effects.. Our results show that the N-turn is closely related to function of DrTx(1-42). The mutant (D8A) as a control peptide further reveals that a charged residue at site 8 of the N-terminus is important for channel blockade and analgesic activity. This study indicates that blocking of voltage-gated TTX-R Na(+) channel in DRG neurons contributes to analgesic effect in rat inflammatory pain. Structural and functional data described here offers support for the development of novel analgesic drugs through targeting TTX-R Na(+) channels.

Topics: Amino Acid Sequence; Analgesics; Animals; Circular Dichroism; Electrophysiological Phenomena; Ganglia, Spinal; Inflammation; Ion Channel Gating; Male; Membrane Potentials; Mice; Mice, Inbred ICR; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Neurons; Pain; Patch-Clamp Techniques; Peptide Fragments; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Systemic carbamazepine, a voltage-gated sodium channel blocker, has been reported to dose-dependently reduce inflammatory hyperalgesia. However, the antinociceptive effects of carbamazepine on the spinal cord in inflammatory conditions are unclear. The aim of the present study was to evaluate the antinociceptive effects of carbamazepine on the spinal cord in a chronic inflammatory condition.. In Sprague-Dawley rats, a chronic inflammatory condition was induced by complete Freund's adjuvant (CFA) inoculation into the tail. Tail flick (TF) latencies were measured following intraperitoneal carbamazepine, or intrathecal carbamazepine or tetrodotoxin injection in intact rats and in the chronic inflammatory rats. From the values of TF latency at 60 min after drug injection, the effective dose required to produce 50% response (ED(50)) of each drug was derived.. Carbamazepine attenuated thermal responses with both systemic and intrathecal administration. The effect was more evident in rats with chronic inflammation than in intact rats; the ED(50s) of intraperitoneal carbamazepine in intact and inflamed rats were 12.39 and 1.54 mg/kg, and those of intrathecal carbamazepine were 0.311 and 0.048 nmol, respectively. Intrathecal tetrodotoxin also clearly inhibited the response, with ED(50s) of 1.006 pmol in intact rats and 0.310 pmol in inflamed rats. The relative potencies of intrathecal carbamazepine versus tetrodotoxin for inhibition were approximately 1:150-1:300 in intact and inflamed rats.. These results indicate that the inhibition of voltage-gated sodium channels, at least tetrodotoxin-sensitive channels, may contribute to the antinociceptive effect of carbamazepine on CFA-induced inflammatory pain, since lower doses of intrathecal carbamazepine and tetrodotoxin attenuated thermal responses to a greater extent in inflamed rats than in intact rats.

Topics: Analgesics, Non-Narcotic; Anesthetics, Local; Animals; Arthritis, Experimental; Carbamazepine; Chronic Disease; Dose-Response Relationship, Drug; Freund's Adjuvant; Hypesthesia; Inflammation; Injections, Intraperitoneal; Injections, Spinal; Rats; Rats, Sprague-Dawley; Reaction Time; Sodium Channel Blockers; Spine; Tail; Tetrodotoxin

Mucosal mast cells (MMCs), epithelial barrier function (EBF) and the enteric nervous system (ENS) are interactive factors in the pathophysiology of functional gastrointestinal disorders. We characterized postinfectious EBF alterations in the Trichinella spiralis infection model of MMC-dependent intestinal dysfunction in rats.. Sprague-Dawley rats were infected with T. spiralis. 30 ± 2 days postinfection, jejunal EBF (electrophysiological parameters, fluorescein isothiocyanate-dextran fluxes and responses to secretagogues and MMC degranulators) was evaluated (Ussing chamber). In some experiments, participation of secretomotor neurons was examined by tetrodotoxin (TTX) pretreatment. Jejunal histology and MMC count and activity were also assessed.. 30 ± 2 days postinfection, when only a low grade inflammation was observed, increased MMC number and activity were associated with altered EBF. EBF alterations were characterized by increased mucosal permeability and ion secretion. In T. spiralis-infected animals, secretory responses to serotonin (5-HT) and immunoglobulin E (IgE)-dependent activation of MMCs were reduced. In contrast, responses to substance P (SP) and capsaicin were similar in infected and noninfected animals. Neuronal blockade with TTX altered secretory responses to SP and capsaicin only in infected rats.. Trichinella spiralis infection in rats, at late stages, results in persistent postinfectious intestinal barrier dysfunctions and mucosal mastocytosis, with other signs suggestive of a low grade inflammation. The altered permeability and the TTX-independent hyporesponsiveness to 5-HT and IgE indicate epithelial alterations. Changes in responses to SP and capsaicin after neuronal blockade suggest an ENS remodeling during this phase. Similar long-lasting neuro-epithelial alterations might contribute to the pathophysiology of functional and postinfectious gastrointestinal disorders.

Topics: Animals; Capsaicin; Chymases; Enteric Nervous System; Gastrointestinal Tract; Inflammation; Intestinal Mucosa; Ion Transport; Male; Mast Cells; Mastocytosis; Neurotransmitter Agents; Permeability; Rats; Rats, Sprague-Dawley; Sensory System Agents; Sodium Channel Blockers; Substance P; Tetrodotoxin; Trichinella spiralis; Trichinellosis

Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor.. In this study we investigated the effect of NK-1 receptor agonist on Na(v)1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly enhanced the Na(v)1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCepsilon, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons.. These data suggest that activation of NK-1 receptor potentiates Na(v)1.8 sodium current via PKCepsilon-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

Topics: Action Potentials; Animals; Electrophysiology; Ganglia, Spinal; Hyperalgesia; Inflammation; Male; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neurons; Patch-Clamp Techniques; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Sodium; Sodium Channels; Tetrodotoxin

Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing.. CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs.. Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation.

Topics: Animals; Behavior, Animal; Colon; Diterpenes; Ganglia, Spinal; Hypersensitivity; Inflammation; NAV1.9 Voltage-Gated Sodium Channel; Neurons; Neurons, Afferent; Neuropeptides; Organ Specificity; Protein Subunits; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Tetrodotoxin; Trinitrobenzenesulfonic Acid; TRPV Cation Channels; Viscera

Inflammation influences several steps of adult neurogenesis, but whether it regulates the functional integration of the new neurons is unknown. Here, we explored, using confocal microscopy and whole-cell patch-clamp recordings, whether a chronic inflammatory environment affects the morphological and electrophysiological properties of new dentate gyrus granule cells, labeled with a retroviral vector encoding green fluorescent protein. Rats were exposed to intrahippocampal injection of lipopolysaccharide, which gave rise to long-lasting microglia activation. Inflammation caused no changes in intrinsic membrane properties, location, dendritic arborization, or spine density and morphology of the new cells. Excitatory synaptic drive increased to the same extent in new and mature cells in the inflammatory environment, suggesting increased network activity in hippocampal neural circuitries of lipopolysaccharide-treated animals. In contrast, inhibitory synaptic drive was more enhanced by inflammation in the new cells. Also, larger clusters of the postsynaptic GABA(A) receptor scaffolding protein gephyrin were found on dendrites of new cells born in the inflammatory environment. We demonstrate for the first time that inflammation influences the functional integration of adult-born hippocampal neurons. Our data indicate a high degree of synaptic plasticity of the new neurons in the inflammatory environment, which enables them to respond to the increase in excitatory input with a compensatory upregulation of activity and efficacy at their afferent inhibitory synapses.

Topics: Analysis of Variance; Animals; Calcium-Binding Proteins; Dendritic Spines; Dose-Response Relationship, Radiation; Ectodysplasins; Electric Stimulation; Electroencephalography; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Inflammation; Inhibitory Postsynaptic Potentials; Lipopolysaccharides; Lysine; Male; Microfilament Proteins; Microscopy, Confocal; Neurogenesis; Neurons; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Seizures; Tetrodotoxin; Time Factors

Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant "persistent" Na+ current called NaN. Nav1.9(-/-) nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.

Topics: Adenosine Triphosphate; Animals; Bradykinin; Dinoprostone; Dose-Response Relationship, Drug; Electrophysiology; Ganglia, Spinal; Gene Expression Regulation; Histamine; Inflammation; Male; Mice; Mice, Knockout; NAV1.9 Voltage-Gated Sodium Channel; Neurons; Neuropeptides; Nociceptors; Norepinephrine; Sodium Channels; Tetrodotoxin; Up-Regulation

Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity.. The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3-5 days) of L4 and L5 ganglia altered voltage-gated K and Na currents.. Tetrodotoxin-sensitive Na currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold.. Neuronal inflammation per se, in the absence of nerve injury, causes large increases in Na channel density and enhanced excitability. The unusual finding of increased K current may reflect regulation of excitability in the face of such large increases in Na current.

Topics: Action Potentials; Anesthetics, Local; Animals; Cells, Cultured; Disease Models, Animal; Electric Conductivity; Electrophysiology; Female; Ganglia, Spinal; Inflammation; Ion Channel Gating; Membrane Potentials; Neural Conduction; Neurons, Afferent; Patch-Clamp Techniques; Potassium Channels, Voltage-Gated; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin

Colitis induced by Trichinella spiralis in rat induces alterations in the spontaneous motor pattern displayed by circular colonic muscle [Auli, M., Fernandez, E., 2005. Characterization of functional and morphological changes in a rat model of colitis induced by T. spiralis. Digestive Diseases and Sciences 50(8), 1432-1443]. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 microM), N-nitro-l-arginine (L-NOARG, 1 mM) and apamin (1 microM) were determined in vitro in the organ bath with circular muscle strips from sham-infected and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission.

Topics: Acetylcholine; Animals; Apamin; Colitis; Disease Models, Animal; Disease Progression; Electric Stimulation; Enteric Nervous System; Gastrointestinal Motility; Inflammation; Intestines; Male; Muscle Contraction; Muscle, Smooth; Nitroarginine; Rats; Rats, Sprague-Dawley; Signal Transduction; Synaptic Transmission; Tetrodotoxin; Time Factors; Trichinella spiralis; Trichinellosis

Antisense (AS) oligodeoxynucleotides (ODNs) targeting the Nav 1.8 sodium channel have been reported to decrease inflammatory hyperalgesia and L5/L6 spinal nerve ligation-induced mechanical allodynia in rats. The present studies were conducted to further characterize Nav 1.8 AS antinociceptive profile in rats to better understand the role of Nav 1.8 in different pain states. Consistent with earlier reports, chronic intrathecal Nav 1.8 AS, but not mismatch (MM), ODN decreased TTX-resistant sodium current density (by 60.5+/-10.2% relative to MM; p<0.05) in neurons from L4 to L5 dorsal root ganglia and significantly attenuated mechanical allodynia following intraplantar complete Freund's adjuvant. In addition, 10 days following chronic constriction injury of the sciatic nerve, Nav 1.8 AS, but not MM, ODN also attenuated mechanical allodynia (54.3+/-8.2% effect, p<0.05 vs. MM) 2 days after initiation of ODN treatment. The anti-allodynic effects remained for the duration of the AS treatment, and CCI rats returned to an allodynic state 4 days after discontinuing AS. In contrast, Nav 1.8 AS ODN failed to reduce mechanical allodynia in the vincristine chemotherapy-induced neuropathic pain model or a skin-incision model of post-operative pain. Finally, Nav 1.8 AS, but not MM, ODN treatment produced a small but significant attenuation of acute noxious mechanical sensitivity in naïve animals (17.6+/-6.2% effect, p<0.05 vs. MM). These data demonstrate a greater involvement of Nav 1.8 in frank nerve injury and inflammatory pain as compared to acute, post-operative or chemotherapy-induced neuropathic pain states.

Topics: Animals; Behavior, Animal; Drug Evaluation, Preclinical; Freund's Adjuvant; Hyperalgesia; Inflammation; Injections, Spinal; Ion Transport; Ligation; Male; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neuralgia; Neurons, Afferent; Oligodeoxyribonucleotides, Antisense; Pain, Postoperative; Patch-Clamp Techniques; Pressure; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sodium; Sodium Channels; Spinal Nerves; Stress, Mechanical; Tetrodotoxin; Vincristine

Local anesthetics exert antiinflammatory actions. To elucidate potential mechanisms, the authors examined effects of bupivacaine or tetrodotoxin, administered to rats by ipsilateral or contralateral sciatic blockade or systemically, on carrageenan-induced hind paw hyperalgesia, edema, and stimulated cytokine production in circulating blood cells.. Twelve groups of rats (n = 9-12) received injections in three sites: (1) right or left hind paw (carrageenan or saline), (2) left sciatic block, and (3) systemically (subcutaneously in the upper back). Sciatic and systemic injections were performed with epinephrine plus bupivacaine, tetrodotoxin, or saline; injections were repeated 6 h later. Fifteen hours later, hyperalgesia and/or sensory and motor block were assessed behaviorally, and paw edema was quantified by magnetic resonance imaging. Stimulated production of tumor necrosis factor alpha, interleukin 10, and interleukin 1beta in whole blood cultures was measured by enzyme-linked immunosorbent assay.. Either ipsilateral or contralateral sciatic blocks using either bupivacaine or tetrodotoxin reduced carrageenan-induced edema and hyperalgesia. Systemic bupivacaine and tetrodotoxin were ineffective in preventing edema and hyperalgesia. Bupivacaine was effective in suppressing systemic tumor necrosis factor alpha and interleukin 1beta by all three routes, whereas tetrodotoxin was ineffective by all three routes.. Bupivacaine and tetrodotoxin, via a contralateral or ipsilateral sciatic block, attenuate local inflammatory edema and hyperalgesia induced by hind paw injection of carrageenan in rats. Mechanisms underlying contralateral effects of sciatic blockade remain unexplained. Bupivacaine inhibits carrageenan-evoked systemic cytokine production by a mechanism not shared by tetrodotoxin; this action may involve tetrodotoxin-resistant sodium channels or a variety of non-sodium-channel targets.

Topics: Animals; Bupivacaine; Carrageenan; Drug Therapy, Combination; Edema; Hindlimb; Inflammation; Male; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The authors previously showed that bupivacaine and tetrodotoxin via contralateral or ipsilateral sciatic block, but not systemically, attenuate local edema and hyperalgesia induced by carrageenan hind paw injection in rats. Bupivacaine, by all three routes, suppressed systemic cytokine activation, whereas tetrodotoxin was ineffective by all three routes. In the current study, the authors examined cytokine and p38 mitogen-activated protein kinase (MAPK) activation in lumbar dorsal root ganglia (DRGs) and spinal cord after carrageenan paw injections and sciatic blocks with either bupivacaine or tetrodotoxin.. Ten groups of rats (n = 3-5) received injections in the following sites: right or left hind paw or right forepaw (carrageenan or saline) and left sciatic block (with epinephrine plus bupivacaine, tetrodotoxin, or saline; repeated 6 h later). Fifteen hours later, tumor necrosis factor alpha, interleukin 1beta, p38 MAPK, and phosphorylated p38 MAPK were measured by enzyme-linked immunosorbent assay in DRGs and in the spinal cord.. Carrageenan-induced hind paw inflammation enhanced tumor necrosis factor-alpha and interleukin-1beta production in bilateral DRGs and spinal cord and enhanced p38 MAPK activation in bilateral DRGs. These pathways were not activated after forepaw injection of carrageenan, suggesting a segmental mechanism. Neither bupivacaine nor tetrodotoxin inhibited cytokine and p38 MAPK activation after carrageenan injection.. Ipsilateral or contralateral sciatic blockade using either bupivacaine or tetrodotoxin does not inhibit carrageenan-induced activation of cytokines and p-38 MAPK in spinal cord and DRGs. Possible explanations may include incomplete degrees of conduction blockade or afferent signaling via saphenous nerves.

Topics: Animals; Bupivacaine; Carrageenan; Cytokines; Drug Therapy, Combination; Edema; Ganglia, Spinal; Hindlimb; Inflammation; Male; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Spinal Cord; Tetrodotoxin

Inflammation-induced changes in voltage-gated sodium currents (I(Na)) in primary afferent neurons may contribute to hyperexcitability and pain. The present study was designed to test the hypothesis that persistent inflammation of the temporomandibular joint (TMJ) increases I(Na) in TMJ afferents. Acutely dissociated retrogradely labeled TMJ afferents were studied using whole-cell patch clamp techniques three days following Complete Freund's Adjuvant-induced inflammation of the TMJ. Inflammation was associated with a decrease in tetrodotoxin (TTX)-sensitive Na+ conductance and no significant change in slowly inactivating TTX-resistant Na+ conductance. However, inflammation increased the excitability of TMJ afferents. These results suggest that changes in ion channels other than those underlying TTX-sensitive and the slowly inactivating TTX-resistant Na+ conductance are likely to account for the inflammation-induced increase in the excitability of TMJ afferents.

Topics: Action Potentials; Afferent Pathways; Animals; Cells, Cultured; Inflammation; Ion Channel Gating; Male; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channels; Temporomandibular Joint; Temporomandibular Joint Disorders; Tetrodotoxin; Trigeminal Ganglion

The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels (NaV1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes NaV1.9. Our results confirm that NaV1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of NaV1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.

Topics: Anesthetics, Local; Animals; Behavior, Animal; DNA Primers; DNA, Complementary; Electrophysiology; Female; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Genetic; NAV1.9 Voltage-Gated Sodium Channel; Neurons; Neuropeptides; Nociceptors; Pain; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sodium; Sodium Channels; Tetrodotoxin

In an attempt to understand mechanisms underlying peripheral sensitization of primary afferent fibers, we investigated the presence of the tetrodotoxin-resistant Na+ channel subunits Nav1.8 (SNS) and Nav1.9 (SNS2) on axons in digital nerves of normal and inflamed rat hindpaws. In normal animals, 14.3% of the unmyelinated and 10.7% of the myelinated axons labeled for the Nav1.8 subunit. These percentages significantly increased in 48 h inflamed animals to 22.0% (1.5-fold increase) and 57.5% (6-fold increase) for unmyelinated and myelinated axons, respectively. In normal animals, Nav1.9 labeled 9.9% of the unmyelinated and 2.1% of the myelinated axons and following inflammation, the proportion of Nav1.9-labeled unmyelinated axons significantly decreased to 3.0% with no change in the proportion of labeled myelinated axons. These data indicate that Nav1.8 and Nav1.9 subunits are transported to the periphery in normal animals and are differentially regulated during inflammation. The massive increase in Nav1.8 expression in myelinated axons suggests that these may contribute to peripheral sensitization and inflammatory hyperalgesia.

Topics: Animals; Freund's Adjuvant; Ganglia, Spinal; Hyperalgesia; Immunohistochemistry; Inflammation; Microscopy, Electron; NAV1.8 Voltage-Gated Sodium Channel; NAV1.9 Voltage-Gated Sodium Channel; Nerve Fibers, Myelinated; Nerve Fibers, Unmyelinated; Nerve Tissue Proteins; Neurons, Afferent; Neuropeptides; Nociceptors; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Up-Regulation

Nociceptive neurons within dorsal root ganglia (DRG) express multiple voltage-gated sodium channels, of which the tetrodotoxin-resistant (TTX-R) channel Na(v)1.8 has been suggested to play a major role in inflammatory pain. Previous work has shown that acute administration of inflammatory mediators, including prostaglandin E2 (PGE2), serotonin, and adenosine, modulates TTX-R current in DRG neurons, producing increased current amplitude and a hyperpolarizing shift of its activation curve. In addition, 4 days following injection of carrageenan into the hind paw, an established model of inflammatory pain, Na(v)1.8 mRNA and slowly-inactivating TTX-R current are increased in DRG neurons projecting to the affected paw. In the present study, the expression of sodium channels Na(v)1.1-Na(v)1.9 in small (< or = 25 micromdiameter) DRG neurons was examined with in situ hybridization, immunocytochemistry, Western blot and whole-cell patch-clamp methods following carrageenan injection into the peripheral projection fields of these cells. The results demonstrate that, following carrageenan injection, there is increased expression of TTX-S channels Na(v)1.3 and Na(v)1.7 and a parallel increase in TTX-S currents. The previously reported upregulation of Na(v)1.8 and slowly-inactivating TTX-R current is not accompanied by upregulation of mRNA or protein for Na(v)1.9, an additional TTX-R channel that is expressed in some DRG neurons. These observations demonstrate that chronic inflammation results in an upregulation in the expression of both TTX-S and TTX-R sodium channels, and suggest that TTX-S sodium channels may also contribute, at least in part, to pain associated with inflammation.

Topics: Anesthetics, Local; Animals; Blotting, Western; Carrageenan; Cells, Cultured; Disease Models, Animal; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Inflammation; Male; Membrane Potentials; Neurons; Pain; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Channels; Tetrodotoxin

Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a + 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.

Topics: Acid Sensing Ion Channels; Animals; Cations, Divalent; Cell Membrane Permeability; Electrophysiology; Ganglia, Spinal; Hydrogen-Ion Concentration; Inflammation; Membrane Potentials; Membrane Proteins; Nerve Tissue Proteins; Neurons, Afferent; Posterior Horn Cells; Rats; Sodium Channels; Spinal Cord; Synaptic Transmission; Tetrodotoxin; Transcription, Genetic

Peripheral inflammation may induce long-lasting sensitization in the central nociceptive system. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the integration and relay of pain-related information. In rats we studied whether changes in passive and active membrane properties and/or alteration of glycine receptor-mediated inhibitory control of spinal lamina I neurons may contribute to central sensitization in a model of peripheral long-lasting inflammation (complete Freund's adjuvant, hindpaw). Spontaneously occurring glycine receptor-mediated miniature inhibitory postsynaptic currents (GlyR-mediated mIPSCs) were recorded in lumbar spinal lamina I neurons. Miniature IPSC rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected by peripheral inflammation. The mean frequency of GlyR-mediated mIPSCs of lamina I neurons ipsilateral to the inflamed hindpaw was, however, significantly reduced by peripheral inflammation when compared with neurons from noninflamed animals. Principal passive and active membrane properties and firing patterns of spinal lamina I neurons were not changed by inflammation. These results indicate that long-lasting peripheral inflammation leads to a reduced glycinergic inhibitory control of spinal lamina I neurons by a presynaptic mechanism.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Anesthetics, Local; Animals; Animals, Newborn; Bicuculline; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Freund's Adjuvant; GABA Antagonists; Glycine Agents; In Vitro Techniques; Inflammation; Male; Membrane Potentials; Neural Inhibition; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Spinal Cord; Strychnine; Synapses; Tetrodotoxin; Valine

We previously demonstrated that activation of a 5HT(4) receptor coupled cAMP-dependent signaling pathway increases tetrodotoxin-resistant Na(+) current (I(Na)) in a nociceptor-like subpopulation of rat dorsal root ganglion cells (type 2). In the present study we used electrophysiology experiments and computer modeling studies to explore the mechanism(s) underlying the increase of I(Na) by 5HT. In electrophysiological experiments with type 2 dorsal root ganglion cells, 5HT increased peak I(Na) and the activation and inactivation rate, without significantly affecting the voltage dependency of activation or availability. Studies on the voltage dependency of channel availability, time course of removal of inactivation, and inactivation of evoked Na(+) currents suggested that there are at least two inactivation states of the Na(+) channel, one (I(fast)) that is induced and retrieved faster than the other (I(slow)). Long (1 s), but not short (60 or 100 ms), inactivating conditioning pulses (CPs) suppressed the 5HT-induced increase in I(Na). Computer modeling studies suggest that 5HT increased I(Na) mainly by decreasing the transition rate (k(OI1)) from an open state to I(fast). Furthermore, 5HT increased I(Na) activation and inactivation rates mainly by increasing the transition rate from closed to open (k(C3O)) and from I(fast) to I(slow) (k(I1I2)), respectively. The antagonism of the 5HT-induced increase in I(Na) by 1-s inactivation CPs may be due an enhancement of transitions from I(fast) to I(slow), via the increase in k(I1I2). This may deplete the pool of channels residing in I(fast), reducing the frequency of reopenings from I(fast), which offsets the increase in I(Na) produced by the reduction in k(OI1). The above findings fit well with previous studies showing that activation of the cAMP/PKA cascade simultaneously increases voltage sensitive tetrodotoxin-resistant Na(+) conductance and inactivation rate in nociceptors. The antagonism of the effects of 5HT by long inactivation CPs suggests that drugs designed to induce and/or stabilize the I(slow) state might be useful for reducing hyperalgesia produced by inflammatory mediators.

Topics: Animals; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Electrophysiology; Inflammation; Kinetics; Models, Chemical; Nociceptors; Rats; Rats, Sprague-Dawley; Serotonin; Signal Transduction; Sodium; Sodium Channels; Software; Temperature; Tetrodotoxin; Time Factors

In inflammatory bowel disease, smooth muscle function reportedly varies with disease duration. The aim of these studies was to determine changes in the control of spontaneous contractions in a model of experimental colitis that included reinflammation of the healed area. The amplitude and frequency of spontaneous contractions in circular smooth muscle were determined after intrarectal administration of trinitrobenzenesulfonic acid in rat distal colon. With the use of a novel paradigm, rats were studied 4 h (acute) or 28 days (healed) after the initial inflammation. At 28 days, rats were studied 4 h after a second inflammation (reinflamed) of the colon. Colitis induced transient increases in the amplitude of spontaneous contractions coincident with a loss of nitric oxide synthase activity. The frequency of contractions was controlled by constitutive nitric oxide in controls. Frequency was increased in healed and reinflamed colon and was associated with a shift in the dominance of neural constitutive nitric oxide synthase control to that of inducible nitric oxide synthase (iNOS). The initial colitis induced a remodeling of the neural control of spontaneous contractions reflecting changes in their regulation by constitutive nitric oxide synthase and iNOS.

Topics: Animals; Colitis; Colon; Disease Models, Animal; Gastrointestinal Motility; Guanidines; In Vitro Techniques; Inflammation; Inflammatory Bowel Diseases; Male; Muscle Contraction; Muscle, Smooth; NADPH Dehydrogenase; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors; Trinitrobenzenesulfonic Acid

Topics: Animals; Dinoprostone; Hyperalgesia; Inflammation; Ion Channel Gating; Neurons, Afferent; Nociceptors; Presynaptic Terminals; Receptors, Opioid, mu; Second Messenger Systems; Sodium Channels; Tetrodotoxin

The effect of potential mediators of mucus secretion was investigated in the isolated vascularly perfused rat colon by using a sandwich enzyme-linked immunosorbent assay for rat colonic mucin and by histochemical analysis. Bethanechol (100-200 microM), bombesin (100 nM), and vasoactive intestinal peptide (VIP, 100 nM) provoked a dramatic mucin discharge (maximal response at 900, 900, and 600% of control loops, respectively). VIP-stimulated mucin secretion was abolished by tetrodotoxin, whereas atropine was without effect. In contrast, both tetrodotoxin and atropine significantly decreased mucin release induced by bombesin. Isoproterenol or calcitonin gene-related peptide was without effect. Serotonin (1-5 microM) and peptide YY (10 nM) evoked mucin discharge, whereas glucagon-like peptide-1 did not release mucin. Finally, bromolasalocid (20 microM), interleukin-1beta (0.25 nM), sodium nitroprusside (1 mM), and dimethyl-PGE2 (2.5 microM) induced mucus discharge. The results demonstrated a good correlation between the immunological method and histological analysis. In conclusion, these findings suggest a role for the enteric nervous system, the enteroendocrine cells, and resident immune cells in mediation of colonic mucus release.

Topics: 16,16-Dimethylprostaglandin E2; Animals; Atropine; Bethanechol; Bombesin; Calcitonin Gene-Related Peptide; Colon; Enteric Nervous System; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Inflammation; Interleukin-1; Intestinal Mucosa; Isoproterenol; Lasalocid; Male; Mucus; Neurotransmitter Agents; Nitroprusside; Peptide Fragments; Peptide YY; Protein Precursors; Rats; Rats, Wistar; Serotonin; Tetrodotoxin; Vasoactive Intestinal Peptide

We report evidence for a contribution of tetrodotoxin-resistant sodium current (TTX-R INa) to prostaglandin E2 (PGE2)-induced hyperalgesia. Behavioral experiments were performed in rats chronically implanted with spinal cannulae. The study employed intrathecal administration of oligodeoxynucleotide (ODN) antisense to the recently cloned channel underlying TTX-R INa (PN3/SNS). The nociceptive flexion reflex was employed to determine changes in mechanical stimulus-induced paw-withdrawal threshold. Administration of antisense but not of sense or mismatch ODN, led to a decrease in PGE2-induced hyperalgesia. PGE2-induced hyperalgesia returned to normal 7 days after the last injection of antisense ODN. Antisense ODN selectively and significantly reduced TTX-R INa current density in cultured sensory neurons. Our observations support the hypothesis that modulation of TTX-R INa, present in peripheral terminals of primary afferent nociceptors, contributes, at least in part, to inflammatory hyperalgesia. Since TTX-R INa is found only in primary afferent nociceptors, our findings suggest TTX-R INa as a promising target for novel therapeutic interventions for the treatment of inflammatory pain.

Topics: Animals; Antisense Elements (Genetics); Dinoprostone; Drug Resistance; Electric Conductivity; Hindlimb; Hyperalgesia; Inflammation; Male; Nociceptors; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Reflex; Sodium Channels; Tetrodotoxin

The role of tachykinins in stimulating phasic and giant migrating contractions (GMCs) in the normal and inflamed colon in conscious dogs was investigated by close-intra-arterial infusions of test substances. At low doses (0.1 nmol), substance P and neurokinin (NK1) receptor agonist ([Sar9,Met(O2)11]substance P] stimulated phasic contractions only. At higher doses (2.0 nmol), they stimulated phasic contractions and GMCs. The phasic contractions were blocked partially but significantly by prior close-intra-arterial infusions of tetrodotoxin and atropine but not by hexamethonium. NK1 receptor antagonist partially but significantly inhibited the phasic contractile response to substance P, whereas NK2 and NK3 receptor antagonists had no significant effect. The contractile response to NK2 receptor agonist was less than one-half of the response to substance P; NK3 receptor agonist did not stimulate any contractile activity. The stimulation of GMCs by higher doses of substance P was not blocked by prior infusions of atropine, tetrodotoxin, or NK1, NK2, and NK3 receptor antagonists, nor was the contractile response to substance P blocked by H1 and H2 receptor antagonists. Inflammation depressed the phasic contractile response but enhanced the stimulation of GMCs by substance P. The ability of substance P to stimulate GMCs is novel and suggests its potential role in increasing the frequency of these contractions during colonic inflammation.

Topics: Animals; Atropine; Colon; Dogs; Female; Hexamethonium; Inflammation; Infusions, Intra-Arterial; Male; Muscle Contraction; Muscle, Smooth; Myoelectric Complex, Migrating; Receptors, Tachykinin; Substance P; Tetrodotoxin

Anti-ganglioside (anti-GM1) antibodies have been implicated in the pathogenesis of Guillain-Barré syndrome, multifocal motor neuropathy and motor neuron diseases. It has been held that they may interfere with saltatory conduction by blocking sodium channels. We tested this hypothesis by analysing action potentials from 140 single nerve fibres in 22 rat ventral roots using external longitudinal current measurement. High-titre anti-GM1 sera from Guillain-Barré syndrome or multifocal motor neuropathy patients, or anti-GM1 rabbit sera were applied to the rat ventral root, where saltatory conduction in single motor fibres was serially observed for 4-12 h (mean 8.2 h). For control experiments, we also tested anti-galactocerebroside (anti-GalC) sera, which causes acute demyelinative conduction block, and tetrodotoxin (TTX), a sodium channel blocker. Conduction block was found in 82% of the fibres treated with anti-GalC sera and 100% treated with TTX, but only in 2% (one out of 44) treated with the patients' sera and 5% (two out of 38) treated with rabbit anti-GM1 sera. All the nodes blocked by anti-GM1 sera revealed intense passive outward membrane current, in the internode just beyond the last active node. This pattern of current flow was similar to that in fibres blocked by demyelination with anti-GalC sera, and quite different from that seen in fibres blocked by reducing sodium currents with TTX. Our findings suggest that anti-GM1 sera neither mediate conduction block nor block sodium channels on their own. We conclude that physiological action of the antibody alone is insufficient to explain clinically observed conduction block in human diseases.

Topics: Action Potentials; Animals; Antibodies; Autoantibodies; Axons; G(M1) Ganglioside; Humans; Inflammation; Membrane Potentials; Motor Neuron Disease; Motor Neurons; Nerve Fibers; Neural Conduction; Polyradiculoneuropathy; Rabbits; Rats; Rats, Wistar; Sodium Channel Blockers; Spinal Nerve Roots; Tetrodotoxin

The present in vitro study was conducted to investigate possible alterations in the control of colonic electrolyte transport in an experimental model of colitis. Intrarectal administration of trinitrobenzenesulfonic acid induced a colitis-like inflammation in the rabbit distal colon. Responses to amiloride and residual short-circuit current after this treatment were unchanged, suggesting that the absorptive and secretory mechanisms remained intact. Electrical field stimulation and vasoactive intestinal polypeptide, a candidate secretomotor neurotransmitter, both elicited similar responses in control and colitic tissue. This suggests that communication at the neuroepithelial junction was unimpaired. In untreated tissue, the effects of prostaglandin E2 (PGE2) and of acetylcholine were attenuated by tetrodotoxin, suggesting, therefore, that both play a role in the modulation of secretomotor neurons. In addition, PGE2 had an appreciable direct epithelial effect. Responses to both of these agonists were absent in colitis. The effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate were unchanged in colitis, suggesting that altered PGE2 responsiveness may involve changes in epithelial receptor number, affinity, or in their ability to mediate an increase in adenosine 3',5'-cyclic monophosphate levels. It is concluded that this rabbit model of colitis exhibits 1) defects in the modulation of secretomotor neurons by acetylcholine and PGE2 and 2) an attenuated epithelial response to PGE2.

Topics: Acetylcholine; Amiloride; Animals; Bucladesine; Colitis; Colon; Dinoprostone; Disease Models, Animal; Epithelium; Inflammation; Male; Membrane Potentials; Motor Neurons; Muscle, Smooth; Rabbits; Tetrodotoxin; Trinitrobenzenesulfonic Acid; Vasoactive Intestinal Peptide

The effects of ruthenium red, an inorganic dye with known capsaicin antagonist properties, was investigated in the rabbit eye. At a dose of 0.24 nmol ruthenium red inhibited the inflammatory effects of capsaicin (1 or 8 nmol). Unexpectedly, when the dye was injected in doses ranging from 0.24 to 7.4 nmol, it caused an inflammatory response with constriction of the pupil (miosis) and a breakdown of the blood-aqueous barrier, leading to a rise intraocular pressure. Tetrodotoxin (30 nmol) inhibited the ruthenium red-induced rise in intraocular pressure but had less effect on the miotic response. The tachykinin antagonist spantide inhibited the miosis but had no effect on the rise in intraocular pressure. Ruthenium red induced an increase in substance P-like immunoreactivity and calcitonin gene-related peptide-like immunoreactivity in the aqueous humor. These levels were positively correlated with the rise in aqueous humor protein concentration. The ruthenium red-induced miosis and, to a less extent, the rise in intraocular pressure were inhibited by the Ca2+ channel-blocking agent omega-conotoxin GVIA (CTX), indicating a partial dependence on an influx of extracellular Ca2+. CTX also attenuated the miotic effect of capsaicin but had no effect on the capsaicin-induced rise in intraocular pressure. It is concluded that, in the rabbit eye, ruthenium red induces a neurogenic inflammatory response besides its capsaicin antagonist effects.

Topics: Animals; Aqueous Humor; Calcitonin Gene-Related Peptide; Calcium Channel Blockers; Capsaicin; Eye; Female; Inflammation; Intraocular Pressure; Male; Miosis; Neurokinin A; Nifedipine; omega-Conotoxin GVIA; Peptides, Cyclic; Rabbits; Ruthenium Red; Substance P; Tetrodotoxin

Bradykinin contracts the isolated rabbit sphincter pupillae muscle. The contraction produced by 10(-8) M bradykinin was resistant to atropine but not to tetrodotoxin, suggesting a non-cholinergic nervous mechanism. The contraction was blocked by specific substance P (SP) antagonists, suggesting the involvement of SP. The SP antagonists tested were [D-Pro2,D-Trp7,9]SP-(1-11) and [Arg5,D-Trp7,9]SP-(5-11). The bradykinin-induced contraction exhibited marked tachyphylaxis in contrast to that induced by SP. It appears that the tachyphylaxis reflects the depletion of a bradykinin-sensitive neuronal pool of SP. Injection of bradykinin into the vitreous chamber of the rabbit eye caused miosis and disruption of the blood-aqueous barrier (manifested as aqueous flare). A second administration of bradykinin a few hours after the first injection evoked a reduced response; the response to SP upon repeated administration was unchanged. Atropine was without effect on the response to bradykinin whereas tetrodotoxin and the SP antagonists reduced the response. The results suggest that bradykinin causes miosis and aqueous flare at least partly through local release of neuronal SP.

Topics: Animals; Atropine; Bradykinin; Eye; In Vitro Techniques; Inflammation; Neurons; Pupil; Rabbits; Substance P; Tetrodotoxin; Time Factors