Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum

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

Neuropharmacological mechanisms of capsaicin and related substances.

Capsaicin activates poorly myelinated primary afferent neurons, many of which are polymodal nociceptors. Activation is accompanied by membrane depolarization and the opening of a unique, cation-selective, ion channel which can be blocked by the polyvalent dye ruthenium red. The capsaicin-induced activation is mimicked by resiniferatoxin, a potent analogue, and by low pH. Activation is mediated by a specific membrane receptor which can be selectively and competitively antagonized by capsazepine. Repetitive administration of capsaicin produces a desensitization and an inactivation of sensory neurons. Several mechanisms are involved including receptor inactivation, block of voltage activated calcium channels, intracellular accumulation of ions leading to osmotic changes, and activation of proteolytic enzyme processes. Systemic and topical capsaicin produces a reversible antinociceptive and anti-inflammatory action after an initial undesirable algesic effect. Capsaicin analogues, such as olvanil, have similar properties with minimal initial algesic activity. Antinociception produced by capsaicin does not involve neurotoxicity, sensory neuropeptide depletion or activity at peripheral receptors; rather, systemic capsaicin produces antinociception by activating capsaicin receptors on afferent nerve terminals in the spinal cord. Spinal neurotransmission is blocked by a prolonged inactivation of sensory neurotransmitter release. However, local or topical applications of capsaicin block C-fibre conduction and inactive neuropeptide release from peripheral nerve endings. These mechanisms account for localized antinociception and the reduction of neurogenic inflammation, respectively.     

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1. Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2. Capsazepine (10 microM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 microM gamma-aminobutyric acid (GABA) and 5 microM adenosine 5'-triphosphate (ATP) were unaffected. 3. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 +/- 46 nM (mean +/- s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 microM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 +/- 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4. Capsazepine also inhibited the capsaicin- and RTX-evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Capsazepine as a selective antagonist of capsaicin-induced activation of C-fibres in guinea-pig bronchi.

We investigated the action of capsazepine, an antagonist of the actions of capsaicin on sensory neurones, on the contractile responses evoked by capsaicin or by electrical field stimulation (EFS) in guinea-pig bronchi. Capsazepine (10(-5) M) selectively inhibited responses to capsaicin, producing a significant change in EC50 values but not the Hill coefficient (nH), suggesting that capsazepine acts as a competitive antagonist (apparent pKB = 5.12) whereas ruthenium red is a non-competitive antagonist. Capsazepine and ruthenium red were without effect on EFS-induced responses.     

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.     

Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.     

Four motor effects of capsaicin on guinea-pig distal colon.

The motor effects of capsaicin on the guinea-pig distal colon have been investigated in vivo and in vitro. Capsaicin (0.1-10 micrograms kg-1 i.v.) produced a transient relaxation which was reduced by pretreatment with capsaicin itself, atropine, hexamethonium, phentolamine or guanethidine and almost abolished by tetrodotoxin (TTX). Topically applied capsaicin produced a transient inhibition of tone and spontaneous activity prevented by topically applied TTX. In isolated preparations of distal colon, capsaicin produced a transient, TTX- and atropine-sensitive contraction which was followed by a depression of the contractile activity. The depressant effect was unaffected by atropine plus guanethidine but was greatly reduced by TTX, indicating activation of intramural non-adrenergic, non-cholinergic (NANC) mechanisms. The depressant effect on the first exposure to capsaicin (1 microM) was greater than that produced by a second, third or fourth exposure. In preparations excised from capsaicin-pretreated animals, capsaicin (1 microM) only produced an inhibitory effect on spontaneous contractions. Desensitization did not occur to this inhibitory effect. In preparations pre-exposed to capsaicin (1 microM, 1 h before), capsaicin (1-30 microM) produced a concentration-related inhibition of spontaneous contractions (IC50 = 19 microM) and of the high K+-induced tonic contraction (IC50 = 23 microM). A similar effect on spontaneous motility was produced by capsaicin in colonic segments excised from capsaicin-pretreated guinea-pigs (IC50 = 16 microM) or guinea-pigs treated with TTX (IC50 = 20 microM). It is concluded that, in vivo, capsaicin activates inhibitory reflexes, presumably due to stimulation of primary afferent fibres. This effect involves, at least in part, activation of sympathetic nerves to this organ. The contractile effect of capsaicin on the isolated colon involves activation of intramural cholinergic neurones, whereas the TTX-sensitive component of the inhibitory effect involves either release of an inhibitory transmitter through an axon reflex arrangement or activation of NANC neurones. In addition, at high concentrations capsaicin produces a direct depression of smooth muscle contraction.     

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.     

Capsaicin regulates voltage-dependent sodium channels by altering lipid bilayer elasticity

At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.     

Selective antagonism of capsaicin by capsazepine: evidence for a spinal receptor site in capsaicin-induced antinociception.

1. Capsazepine has recently been described as a competitive capsaicin antagonist. We have used this compound to test the hypotheses that the in vitro and in vivo effects of capsaicin are due to interactions with a specific receptor. 2. In an in vitro preparation of the neonatal rat spinal cord with functionally connected tail, the activation of nociceptive afferent fibres by the application of capsaicin, bradykinin or noxious heat (48 degrees C) to the tail could be measured by recording a depolarizing response from a spinal ventral root. Application of capsaicin or substance P to the spinal cord also evoked a depolarizing response which was recorded in a ventral root. 3. When capsazepine (50 nM-20 microM) was administered to the tail or spinal cord it did not evoke any measurable response. However on the tail, capsazepine reversibly antagonized (IC50 = 254 +/- 28 nM) the responses to capsaicin but not to heat or bradykinin administered to the same site. Similarly capsazepine administration to the spinal cord antagonized the responses evoked by capsaicin (IC50 = 230 +/- 20 nM) applied to the cord but not responses evoked by substance P on the cord or by noxious heat and capsaicin on the tail. 4. In halothane anaesthetized rats, C-fibre responses evoked by transcutaneous electrical stimulation of the receptive field were recorded from single wide dynamic range neurones located in the spinal dorsal horn. C-fibre evoked discharges were consistently reduced by the systemic administration of capsaicin (20 mumol kg-1, s.c.) and this action of capsaicin was antagonized by capsazepine (100 mumol kg-1) administered by the same route.(ABSTRACT TRUNCATED AT 250 WORDS)     

CB(1) receptor antagonist SR141716A increases capsaicin-evoked release of Substance P from the adult mouse spinal cord.

Cannabinoids have an antinociceptive action in many pain models. We have investigated a possible modulatory role for Type 1 Cannabinoid receptors (CB(1)) on the release of excitatory transmitter Substance P from the adult mouse spinal cord after stimulation of nociceptor terminals by capsaicin. Capsaicin (0.1 - 10 microM) was applied to superfused cord sections and evoked a dose dependent release of SP above basal outflow of (23.36+/-2.96 fmol 8 ml(-1)). Maximum evoked SP release was obtained with 5 microM Capsaicin (262.4+/-20.8 fmol 8 ml(-1)). Higher capsaicin concentrations (50 - 100 microM) evoked less SP release. Superfusion of CB(1) antagonist SR141716A (5 microM) increased evoked SP release with capsaicin (0.1 - 10 microM) and reversed the reducing effect of high dose capsaicin (100 microM). Antagonism of CB(1) receptors in the spinal cord during capsaicin stimulation, is evidence of tonic CB(1) activity inhibiting the release of excitatory transmitters after activation of nociceptive neurones and is also indicative of endocannabinoid production during noxious stimulation.     

Capsaicin-, Resiniferatoxin-, and Lactic Acid-Evoked Vascular Effects in the Pig Nasal Mucosa in vivo with Reference to Characterization of the Vanilloid Receptor

Abstract: Nasal cavity volume, mucosal and superficial skin blood flow as well as renal splenic vascular effects of capsaicin, resiniferatoxin and lactic acid were investigated, using a novel in vivo pig model. The present results show that locally intraarterially injected capsaicin, resiniferatoxin and lactic acid evoke similar vasodilatory responses, although with different duration, in the nasal mucosa and superficial skin as well as an increase in heart rate and mean arterial blood pressure. Nasal vascular responses evoked by capsaicin, resiniferatoxin and lactic acid were unaffected by the cyclooxygenase inhibitor diclofenac. Moreover, chlorisondamine did not alter the nasal vasodilatory responses evoked by capsaicin and lactic acid. However, chlorisondamine abolished sympathetic reflex-mediated vasoconstrictor effects of capsaicin in the spleen and kidney. Lactic acid-evoked vasodilation in the nasal mucosa and skin was inhibited by the 8-37 fragment of calcitonin gene-related peptide, a calcitonin gene-related peptide-receptor antagonist. Lactic acid-evoked vasoconstriction in the spleen and kidney was reduced but not abolished by chlorisondamine, suggesting that the effects of lactic acid are not exclusively reflex-mediated. Capsazepine did not inhibit the vasodilatation in the nasal mucosa evoked by capsaicin and lactic acid. [³H]Resiniferatoxin bound to pig nasal mucosa membranes with an affinity of 134 pM in a non-cooperative fashion; this binding behaviour contrasted to the apparent positive cooperativity (a Hill coefficient of 2.2) of specific resiniferatoxin binding to pig spinal cord preparations. Specific [³H]resiniferatoxin binding to nasal mucosa membranes was fully inhibited by capsaicin (K_i = 5 μM) and lactic acid (IC₅₀ at pH 5.0) but not by capsazepine (up to 10 μM), in accord with the physiological findings. Capsazepine, by contrast, displaced [³H]resiniferatoxin from spinal vanilloid receptors with an affinity of 3 μM. These findings show the presence of vanilloid receptors in the pig nasal mucosa and suggest heterogeneity in the properties of vanilloid receptors in the pig. Furthermore, lactic acid evokes vascular effects similar to those of capsaicin and resiniferatoxin, possibly via interaction of protons and/or proton-generated substances at vanilloid receptors with a subsequent release of calcitonin gene-related peptide.     

Design of a high-affinity competitive antagonist of the vanilloid receptor selective for the calcium entry-linked receptor population

We describe the synthesis and characterization of N-(4-chlorobenzyl) -N'-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (IBTU), a novel antagonist of the vanilloid receptor 1 (TRPV1 or VR1). IBTU competitively inhibited 45Ca2+ uptake into CHO cells heterologously expressing rat TRPV1, whether induced by capsaicin or resiniferatoxin (Ki = 99 +/- 23 and 93 +/- 34 nM, respectively). IBTU was thus somewhat more potent (5-fold) than capsazepine. In contrast to its antagonism of vanilloid-induced calcium uptake, IBTU (30 microM) inhibited [3H]resiniferatoxin binding to TRPV1 by less than 10%. We hypothesize that these dramatically distinct potencies reflect different fractions of TRPV1 in this system: namely, a minor plasma membrane fraction controlling 45Ca2+ uptake, and the predominant intracellular fraction that dominates the [3H]resiniferatoxin binding measurements. Intracellular Ca2+ imaging supports this explanation. IBTU antagonized the elevation in intracellular Ca2+ in response to 50 nM capsaicin with an IC50 of 106 +/- 35 nM. Likewise, 600 nM IBTU was able to antagonize the elevation in intracellular Ca2+ in response to 100 pM resiniferatoxin in the presence of normal (1.8 mM) extracellular Ca2+, where the increase in intracellular calcium reflects calcium influx. In contrast, in the absence of extracellular Ca2+, where in this system resiniferatoxin induces a modest increase in calcium from intracellular stores, IBTU was unable to block the response to resiniferatoxin, although the TRPV1 antagonist 5-iodoresiniferatoxin was able to do so. In summary, IBTU is a novel, potent TRPV1 antagonist with marked selectivity between subpopulations of TRPV1 and may permit the function of these distinct pools to be explored and potentially exploited.     

辣椒素受体参与骶髓后联合核神经元突触传递

目的研究辣椒素受体对大鼠骶髓后联合核(SDCN)神经元突触传递的影响。方法在脊髓骶段横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs)和γ-氨基丁酸(GABA)能抑制性突触后电流(IPSCs),比较激动辣椒素受体后上述突触电流的变化;观察激动辣椒素受体对SDCN神经元动作电位发放的影响。结果辣椒素受体被其特异性激动剂辣椒素(1μmol.L-1)激动后,自发EPSCs(sEPSCs)的频率和振幅均有明显增加(P<0.05,n=17)。在河豚毒素(0.5μmol.L-1)存在的条件下,辣椒素明显增加微小EPSCs(mEPSCs)的频率(P<0.01,n=13),但对mEPSCs的振幅无影响(P>0.05,n=13),提示辣椒素的作用在突触前。辣椒素也明显增加动作电位发放(P<0.05,n=19)。上述作用均可被辣椒素受体特异性拮抗剂capsazepine(10μmol.L-1)阻断。辣椒素也增加GABA能的自发IPSCs(sIPSCs)的频率(P<0.05,n=20),但对其不依赖动作电位的微小IPSCs(mIPSCs)的频率或振幅均无作用(P>0.05,n=9)。结论在SDCN,辣椒素受体主要表达于兴奋性突触终末;激动辣椒素受体影响兴奋性和抑制性突触活动,并可能参与痛觉信息在脊髓水平的传递和调制。     

Expression of vanilloid receptors in rat gastric epithelial cells: role in cellular protection

Vanilloid receptors subtype 1 (VR1), a nonselective cation channel responsive to capsaicin, protons, and noxious heat, has been recently identified in not only neural but also non-neural cells. In the present study, we demonstrated the peripheral expression of VR1 in gastric mucosal epithelial cells and investigated the role of the receptor in cellular protection. The rat gastric mucosal epithelial cell line was used. The expression of VR1 was examined by Western blotting and RT-PCR. Cell damage was induced by immersion in 10% ethanol or acid (pH 4.0) for 30 min, and cell viability was determined by MTT assay. Capsaicin or resiniferatoxin was added 30 min before the challenge with ethanol or acid, while capsazepine or ruthenium red (a VR1 antagonist) was added simultaneously with capsaicin. The distinct expression of VR1 protein and mRNA was detected in rat gastric mucosal epithelial cell line as well as in the rat stomach and spinal cord by Western blotting and RT-PCR, respectively. The cDNA sequence of the PCR product was found to be almost identical to that of the authentic VR1 (99.8%) when the product was subcloned and sequenced. On the other hand, the cell damage induced by ethanol or acid was dose-dependently prevented by pretreatment with capsaicin. The protective effect of capsaicin was mimicked by resiniferatoxin and almost totally abolished by co-addition of capsazepine or ruthenium red. These findings suggest that VR1 is expressed peripherally in gastric mucosal epithelial cells and plays a cellular protective role.     

Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel.

The interaction of quinidine with a cloned human cardiac potassium channel (HK2) expressed in a stable mouse L cell line was studied using the whole-cell tight-seal voltage-clamp technique. Quinidine (20 microM) did not affect the initial sigmoidal activation time course of the current. However, it reduced the peak current and induced a subsequent decline, with a time constant of 8.2 +/- 0.8 msec, to 28 +/- 6% of control (at +60 mV). The concentration dependence of HK2 block at +60 mV yielded an apparent KD of 6 microM and a Hill coefficient of 0.9. The degree of block was voltage dependent. Block increased from 0.60 +/- 0.09 at 0 mV to 0.72 +/- 0.06 at +60 mV with 20 microM quinidine and from 0.39 +/- 0.20 to 0.48 +/- 0.16 with 6 microM. Paired analysis in seven experiments with 20 microM quinidine indicated that the voltage-dependent increase in block was significant (difference, 12 +/- 4%; p less than 0.001). This voltage dependence was described by an equivalent electrical distance delta of 0.19 +/- 0.02, which suggested that at the binding site quinidine experienced 19% of the applied transmembrane electrical field, referenced to the inner surface. Quinidine reduced the tail current amplitude and slowed the time course relative to control, resulting in a "crossover" phenomenon. These data indicate that 1) the charged form of quinidine blocks the HK2 channel after it opens, 2) binding occurs within the transmembrane electrical field (probably in or near the ion permeation pathway), and 3) unbinding is required before the channel can close.     

Functional mapping of the transient receptor potential vanilloid 1 intracellular binding site

Capsaicin (vanilloid) sensitivity has long served as the functional signature of a subset of nociceptive sensory neurons. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 547 (transmembrane region 4) mediates significant species differences in resiniferatoxin (RTX) sensitivity, and the Ser(512) residue is critical in discriminating between pH and capsaicin gating. In the present study, the pharmacological profiles of a variety of ligands were studied to investigate cross-talk between these two regions. Exchange of residue 547 between species mediated a difference in capsaicin and RTX-dependent gating. Likewise, the potency of iodoresiniferatoxin (I-RTX) and a novel transient receptor potential vanilloid 1 antagonist were also altered. Experiments using the S512Y mutant channel have confirmed the importance of residue 512 for functional interaction of capsaicin and our novel antagonist. In this study, we were surprised to find that the mutation S512Y converted the activity of the antagonist I-RTX into an intrinsic agonist, albeit with a lower potency than its parent compound, RTX. Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the voltage-dependent potassium channel, in which both the 512 and 547 amino acid residues are in close proximity. Our data support the model whereby intracellular ligand interaction occurs within an S3-S4 "sensor" domain, enabling binding of ligands to be transduced to functional gating of the channel. The binding pocket also seems to be exquisitely sensitive to residue-specific interaction with ligands, because subtle changes in either ligand or channel structure can have profound effects on channel activity.     

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1. We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from rat isolated soleus muscle induced by capsaicin (1 microM), by superfusion with low pH medium (pH 5) or by KCl (80 mM). 2. Each one of the three stimuli tested produced a marked CGRP-LI release. Total evoked release (fmol g-1) was 482 +/- 69, 169 +/- 20 and 253 +/- 43 for capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 45 min preincubation) inhibited the evoked CGRP-LI release. Capsaicin-induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) capsazepine, respectively. 5. These findings demonstrate that capsazepine prevents low pH- and capsaicin-induced CGRP-LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP-LI release from peripheral endings of the capsaicin-sensitive primary afferent neurone.