Origin and colocalization of CGRP- and SP-reactive nerves in cat airway epithelium.

A combination of neuroanatomic techniques was used to examine the origin and neuropeptide content of nerve fibers in the airway epithelium of adult cats. By the use of immunocytochemical methods, the peptides substance P (SP) and calcitonin gene-related peptide (CGRP) were colocalized in airway epithelial nerve fibers. Two days after wheat germ agglutinin (WGA) was injected into the nodose ganglion, fibers containing WGA immunoreactivity (IR) were detected in the airway epithelium. SP-like immunoreactivity (LI) and CGRP-LI were demonstrated separately in the WGA-IR fibers, establishing their origin from nerve cell bodies of nodose ganglion. Vagal transection inferior to the nodose ganglion reduced the number of SP- and CGRP-IR fibers by greater than 90% in ipsilateral airways. In contralateral airways, SP-IR fibers were substantially reduced, whereas the effect on CGRP-IR fibers was not statistically significant. Vagotomy superior to the nodose ganglion did not alter the density of peptide-IR fibers. The results prove that SP- and CGRP-IR nerve fibers of cat airway epithelium originate from nerve cell bodies in the nodose ganglion and that SP- and CGRP-like peptides may be stored together in some nerve fibers of the airway epithelium.     

Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin

The presence of calcitonin-gene related peptide (CGRP)-like immunoreactivity (-LI) in sensory neurons was established by immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-immunoreactive (-IR) nerve fibres were present in many peripheral organs including heart, ureter, uterus and gall bladder of guinea-pig and man. The distribution of CGRP-IR nerves in the dorsal horn of the spinal cord, of positive cell bodies in thoracic spinal and nodose ganglia and nerves in peripheral organs was closely related to that of substance P-LI. Double staining experiments revealed that in most cases peripheral CGRP-IR nerve terminals also contained SP-LI. However, different localization of SP- and CGRP-IR neurons was observed in the nucleus of the solitary tract as well as in the ventral horn of the spinal cord. In the heart, CGRP-IR nerves were associated with myocardial cells (mainly atria), coronary vessels, local parasympathetic ganglia as well as with the epi- and endocardia. Three to 4-fold higher levels of native CGRP-LI were observed in the atria than in the ventricles of the heart. HPLC analysis revealed that the major peak of CGRP-LI in the heart of rat and man had the same retention times as the synthetic equivalents. Systemic capsaicin pretreatment and adult guinea-pigs caused a loss of CGRP-IR terminals in the dorsal horn of the spinal cord as well as in peripheral organs including the heart. After capsaicin treatment, the content of CGRP-IR was reduced by 70% in the heart and by 60% in the dorsal part of the spinal cord. In superfusion experiments with slices from the rat spinal cord, a release of CGRP-LI was induced by 60 mM K+ and 3 microM capsaicin in a calcium-dependent manner.     

Relative sparing of calcitonin gene-related peptide-containing primary sensory neurons following neonatal capsaicin treatment in the rat

Calcitonin gene-related peptide (CGRP)-containing sensory neurons projecting to viscera or skin were detected by immunocytochemistry combined with fluorescent tracer in the dorsal root ganglia (Th9-10) of rats 5-6 weeks old treated neonatally with capsaicin. The number of CGRP-like immunoreactive (IR) cells were reduced by 50-60% with capsaicin treatment. Visceral CGRP-IR sensory neurons were shown to be more sensitive than cutaneous ones, which was also supported by the fact that CGRP-IR fibers in the stomach were completely diminished while epidermal CGRP-IR fibers were spared.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

Distribution of calcitonin gene-related peptide and substance P-immunoreactive nerve fibers and their correlation in the periodontal ligament of the mouse incisor.

The distribution of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) nerve fibers and their correlation in the periodontal ligament of mouse incisors were examined by indirect immunofluorescence. Both CGRP-IR and SP-IR thin nerve fibers were abundant in the apical and middle third of the periodontal ligament. In the lingual portion of the incisal periodontal ligament, these nerve fibers were localized in the alveolar half of the periodontal ligament and were observed as free nerve endings. No CGRP-IR and SP-IR specialized nerve endings, such as Ruffini-like corpuscles, were observed. In the labial periodontal ligament, CGRP-IR and SP-IR nerve fibers ran along the incisal axis. The distribution of CGRP-IR nerve fibers was very similar to that of SP-IR nerve fibers.     

Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures

Effects of docosahexaenoic acid (DHA) on survival and neurite outgrowth were investigated in primary cultures of rat cortical neurons. Cell cultures were prepared from cortex on embryonic day 18 (E-18) for treatment with a series of DHA concentrations (12.5, 25, 50, 75, 100 and 200 microM). Docosahexaenoic acid (25-50 microM) significantly enhanced neuronal viability, but lower concentration of DHA (12.5 microM) did not show an obvious effect. In contrast, higher concentrations of DHA (100-200 microM) exerted the significant opposite effects by decreasing neuronal viability. Furthermore, treatment with 25 microM DHA significantly prevented the neurons from death after different culture days in vitro (DIV). Moreover, measurements from the cultures exposed to 25 microM DHA immediately after plating showed significant increases in the percentage of cells with neurites, the mean number of neurite branches, the total neuritic length per cell and the length of the longest neurite in each cell after 24 and 48 h in vitro (HIV). The DHA-treated neurons had greater growth-associated protein-43 (GAP-43) immunoactivity and higher phosphatidylserine (PS) and phosphatidylethanolamine (PE) contents, but lower phosphatidylcholine (PC) content than control neurons. The significant increased DHA contents were also observed in both PE and PS in the treated neurons. These findings suggest that optimal DHA (25 microM) may have positive effects on the survival and the neurite outgrowth of the cultured fetal rat cortical neurons, and the effects probably are related to DHA-stimulating neuron-specific protein synthesis and its enhancing the discrete phospholipid (PL) content through enrichment of DHA in the PL species.     

Capsaicin-induced activation of erythrocyte membrane sodium/potassium and calcium adenosine triphosphatases

Capsaicin is the pungent ingredient present in hot peppers of the genus Capsicum. Capsaicin's effect on sensory neurons has been well studied; however, its effect on non-neuronal cells is still not fully understood. This study was undertaken to evaluate the effect of capsaicin on erythrocyte membrane enzymes: Na+/K(+)-ATPase and Ca(2+)-ATPase. Treatment with capsaicin (0.01-100 microM) caused a transient increase in the activities of both enzymes; the effect declined at lower concentrations of capsaicin, and no significant effect was observed at 0.01 microM capsaicin. The effect of capsaicin was fast with a significant (p<0.01) activation of enzyme activity observed within minutes of incubation. The findings on the effect of capsaicin on human erythrocyte membrane enzymes Na+/K(+)-ATPase and Ca(2+)-ATPase signify the importance of the non-neuronal effects of capsaicin, and the need for evaluating the physiological impact of high capsaicin (capsicum) consumption in some regions of the world.     

Effect of capsaicin on voltage-gated currents of trigeminal neurones in cell culture and slice preparations

Effects of capsaicin on voltage-gated currents were examined in vitro by whole-cell patch-clamp recordings from small neurones of rat trigeminal ganglia either in slice preparations or in different cell cultures. Cells were classified as sensitive to capsaicin if they responded with inward current and/or conductance change to the agent in nanomolar concentration. Capsaicin (150 to 330 nM) in sensitive cells reduced the mixed inward current evoked by depolarizing step or ramp commands in all preparations. In cultured cells, the inward current was depressed to 32.78 +/- 26.42% (n = 27) of the control. Both the tetrodotoxin-sensitive and -resistant inward currents were affected. The data support the concept that capsaicin besides acting on VR-1 receptors inhibits also some voltage gated channels. In 34 cultured cells, capsaicin increased the slope conductance to 170.5 +/- 68%. Percentage of capsaicin sensitive cells observed in nerve growth factor-treated cultured cell populations was higher (77.8%) than in the two other preparations (14.3 or 38.8%). It is concluded that 1) depression of the voltage-gated currents may play an important role in the functional desensitization of the sensory receptors and in the analgesic effect induced by the agent and 2) cell body of sensory neurones under native condition seems less sensitive to capsaicin then that of cells cultured in the presence of nerve growth factor.     

Analysis of the afferent limb of the vesicovascular reflex using neurotoxins, resiniferatoxin and capsaicin

The afferent limb of the vesicovascular reflex (VV-R) evoked by distension or contraction of the urinary bladder (UB) was studied in urethane-anesthetized female rats by examining the changes in VV-R after administration of C-fiber afferent neurotoxins [capsaicin and resiniferatoxin (RTX)]. Systemic arterial blood pressure increased parallel (5.1 to 53.7 mmHg) with graded increases in UB pressure (20 to 80 cm H(2)O) or during UB contractions. The arterial pressor response to UB distension was significantly reduced (60-85%) by acute or chronic (4 days earlier) intravesical administration of RTX (100-1,000 nM) or by capsaicin (125 mg/kg sc) pretreatment (4 days earlier). Chronic neurotoxin treatments also increased the volume threshold (>100%) for eliciting micturition in anesthetized rats but did not change voiding pressure. Acute RTX treatment (10-50 nM) did not alter the arterial pressor response during reflex UB contractions, whereas higher concentrations of RTX (100-1,000 nM) blocked reflex bladder contractions. It is concluded that VV-R is triggered primarily by distension- and contraction-sensitive C-fiber afferents located, respectively, near the luminal surface and deeper in the muscle layers of the bladder.     

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 microM), ASIC1a+3 (IC50=2 microM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.     

Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

PGE(2) sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli.

Mediators of inflammation, such as PGE(2), are known to sensitize the airways to inhaled irritants and circulating autacoids. Evidence from in vivo studies has shown the involvement of vagal pulmonary C-fiber afferents in the PGE(2)-elicited airway hypersensitivity. However, whether PGE(2) acts directly on these sensory nerves is unclear. The present study aimed to investigate whether PGE(2) has direct potentiating effects on nodose and jugular pulmonary C neurons cultured from adult Sprague-Dawley rats and, if so, determine whether the EP(2) prostanoid receptor is involved. Pulmonary neurons were identified by retrograde labeling with a fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Using perforated patch-clamp technique, our results showed that 1) PGE(2) pretreatment (1 microM) increased the whole cell current density elicited by capsaicin and phenylbiguanide, chemical agents known to stimulate pulmonary C fibers; 2) selective activation of the EP(2) prostanoid receptor by butaprost (3-10 microM) increased the whole cell current density elicited by capsaicin; and 3) PGE(2), as well as butaprost, increased the number of action potentials evoked by current injection. Therefore, we conclude that PGE(2) directly sensitizes vagal pulmonary C neurons to chemical and electrical stimulation. Furthermore, butaprost modulates the neurons in a manner similar to that of PGE(2), suggesting that the effects of PGE(2) are mediated, at least in part, through the EP(2) prostanoid receptor.     

Carbonic anhydrase activity in primary sensory neurons

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.     

Epinephrine enhances the sensitivity of rat vagal chemosensitive neurons: role of beta3-adrenoceptor.

This study was carried out to determine whether epinephrine alters the sensitivity of rat vagal sensory neurons. In anesthetized rats, inhalation of epinephrine aerosol (1 and 5 mg/ml, 3 min) induced an elevated baseline activity of pulmonary C fibers and enhanced their responses to lung inflation (20 cm H(2)O, 10 s) and right atrial injection of capsaicin (0.5 microg/kg). In isolated rat nodose and jugular ganglion neurons, perfusion of epinephrine (3 microM, 5 min) alone did not produce any detectable change of the intracellular Ca(2+) concentration. However, immediately after the pretreatment with epinephrine, the Ca(2+) transients evoked by chemical stimulants (capsaicin, KCl, and ATP) were markedly potentiated; for example, capsaicin (50 nM, 15 s)-evoked Ca(2+) transient was increased by 106% after epinephrine (P < 0.05; n = 11). The effect of epinephrine was mimicked by either BRL 37344 (5 microM, 5 min) or ICI 215,001 (5 microM, 5 min), two selective beta(3)-adrenoceptor agonists, and blocked by SR 59230A (5 microM, 10 min), a selective beta(3)-adrenoceptor antagonist, whereas pretreatment with phenylephrine (alpha(1)-adenoceptor agonist), guanabenz (alpha(2)-adrenoceptor agonist), dobutamine (beta(1)-adrenoceptor agonist), or salbutamol (beta(2)-adrenoceptor agonist) had no significant effect on capsaicin-evoked Ca(2+) transient. Furthermore, pretreatment with SQ 22536 (100-300 microM, 15 min), an adenylate cyclase inhibitor, and H89 (3 microM, 15 min), a PKA inhibitor, completely abolished the potentiating effect of epinephrine. Our results suggest that epinephrine enhances the excitability of rat vagal chemosensitive neurons. This sensitizing effect of epinephrine is likely mediated through the activation of beta(3)-adrenoceptor and intracellular cAMP-PKA signaling cascade.     

Capsaicin-sensitive afferent sensory nerves in modulating gastric mucosal defense against noxious agents.

In the rat stomach, evidence has been provided that capsaicin-sensitive sensory nerves (CSSN) are involved in a local defense mechanism against gastric ulcer. In the present study capsaicin or resiniferatoxin (RTX), a more potent capsaicin analogue, was used to elucidate the role of these sensory nerves in gastric mucosal protection, mucosal permeability, gastric acid secretion and gastrointestinal blood flow in the rat. In the rat stomach and jejunum, intravenous RTX or topical capsaicin or RTX effected a pronounced and long-lasting enhancement of the microcirculation at these sites, measured by laser Doppler flowmetry technique. Introduction of capsaicin into the rat stomach in very low concentrations of ng-microg x mL(-1) range protected the gastric mucosa against damage produced by topical acidified aspirin, indomethacin, ethanol or 0.6 N HCl. Resiniferatoxin exhibited acute gastroprotective effect similar to that of capsaicin and exerted marked protective action on the exogenous HCl, or the secretagogue-induced enhancement of the indomethacin injury. The ulcer preventive effect of both agents was not prevented by atropine or cimetidine treatment. Capsaicin given into the stomach in higher desensitizing concentrations of 6.5 mM markedly enhanced the susceptibility of the gastric mucosa and invariably aggravated gastric mucosal damage evoked by later noxious challenge. Such high desensitizing concentrations of capsaicin, however, did not reduce the cytoprotective effect of prostacyclin (PGI2) or beta-carotene. Capsaicin or RTX had an additive protective effect to that of atropine or cimetidine. In rats pretreated with cysteamine to deplete tissue somatostatin, capsaicin protected against the indomethacin-induced mucosal injury. Gastric acid secretion of the pylorus-ligated rats was inhibited with capsaicin or RTX given in low non-desensitizing concentrations, with the inhibition being most marked in the first hour following pylorus-ligation. Low intragastric concentrations of RTX reduced gastric hydrogen ion back-diffusion evoked by topical acidified salicylates. It is concluded that the gastropotective effect of capsaicin-type agents involves primarily an enhancement of the microcirculation effected through local release of mediator peptides from the sensory nerve terminals. A reduction in gastric acidity may contribute to some degree in the gastric protective action of capsaicin-type agents. The vasodilator and gastroprotective effects of capsaicin-type agents do not depend on vagal efferents or sympathetic neurons, involve prostanoids, histaminergic or cholinergic pathways.     

The response of primary cultured adult mouse sensory neurons to ethanol, propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50's of 600 microM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.     

Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells

AimsXenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions.Main methodsWe studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin.Key findingsXenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection.SignificanceXenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.