High affinity antagonists of the vanilloid receptor

The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tert-butylbenzyl)-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [(3)H]resiniferatoxin binding to rVR1 with an affinity of 53.5 +/- 6.5 nM and antagonized capsaicin-induced calcium uptake with an EC(50) of 9.2 +/- 1.6 nM, reflecting 25- and 60-fold greater potencies than capsazepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (<5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different activators could thus be differentially affected by different ligands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking response to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine.     

Participation of the spinal TRPV1 receptors in formalin-evoked pain transduction: a study using a selective TRPV1 antagonist, iodo-resiniferatoxin

The involvement of spinal transient receptor potential vanilloid 1 (TRPV1) in formalin-evoked pain has remained unclear, because investigation of this kind of pain with selective antagonists has not been conducted. The purpose of this study is to investigate the participation of spinal TRPV1 in formalin-evoked pain with iodo-resiniferatoxin (I-RTX), a potent TRPV1-selective antagonist. I-RTX given intrathecally dose-dependently and significantly decreased the number of flinching responses in the formalin-evoked 1st and 2nd phase with ID50 values (drug dose producing 50% inhibition of response) of 1.0 and 3.8 microg, respectively, and concentration-dependently suppressed capsaicin-evoked calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release from rat spinal cord slices with an IC50 value (drug concentration producing 50% inhibition of response) of 86 nM. Capsazepine, a classical non-selective TRPV1 antagonist, given intrathecally also inhibited formalin-evoked flinching in both the 1st and 2nd phase with ID50s of 420 and 200 microg, respectively, and CGRP-LI release from rat spinal cord slices with an IC50 of 7.8 microM. Ratios of in-vivo analgesic potencies of I-RTX and capsazepine well reflected their intrinsic in-vitro activity. These findings suggest that spinal TRPV1 participates in the transduction system of formalin-evoked pain.     

Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle

We have investigated the mechanism through which hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H2S, produced concentration-dependent contractile responses (pEC50=3.5+/-0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 microM) and SB 366791 (10 microM) and by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 microM) almost abolished NaHS-induced contractions. Ruthenium red (30 microM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons.     

Lafutidine facilitates calcitonin gene-related peptide (CGRP) nerve-mediated vasodilation via vanilloid-1 receptors in rat mesenteric resistance arteries

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

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.     

Effects of ruthenium red and capsazepine on C-fibres in the rabbit iris.

1. We have investigated the effects of ruthenium red and capsazepine on a C-fibre-smooth muscle preparation (the rabbit isolated iris sphincter muscle). 2. Like capsaicin, ruthenium red and capsazepine were found to produce contractions in a concentration-dependent manner. C-fibre activation was held to be responsible since the contractions could be inhibited by tachykinin receptor blockade. 3. Both ruthenium red and capsazepine inhibited capsaicin-induced contractions concentration-dependently; the pIC50 values were 5.1 and 4.9, respectively. The contractions induced by bradykinin, which, like capsaicin, acts by releasing tachykinins from C-fibres, were also inhibited by ruthenium red and capsazepine in a concentration-dependent manner; the pIC50 values were 4.1 and 4.6, respectively. 4. Electrically evoked, tachykinin-mediated contractions were inhibited by ruthenium red and capsazepine in a concentration-dependent manner; the pIC50 values were 4.3 and 4.5, respectively. 5. The contractile response to neurokinin A (NKA) was inhibited by capsazepine (and by capsaicin), but not by ruthenium red, in a concentration-dependent manner; the pIC50 value was 4.3. 6. The results suggest that, besides their ability to antagonize capsaicin, ruthenium red and capsazepine possess a weak capsaicin-like effect. Conceivably, capsazepine interacts with binding sites for capsaicin, acting as a partial agonist/antagonist, while ruthenium red interacts with capsaicin-operated cation channels. The inhibition of electrically evoked- or bradykinin-induced responses by capsazepine and ruthenium red suggests that capsaicin/capsazepine binding sites and capsaicin-operated cation channels play a role in the process of transmitter release in response not only to capsaicin but also to other C-fibre stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nociceptin inhibits capsaicin-induced bronchoconstriction in isolated guinea pig lung

The isolated perfused guinea pig lung was used to investigate the effect of nociceptin against bronchoconstriction elicited by endogenous and exogenous tachykinins. The opioid receptor-like 1 (ORL1) receptor agonist, nociceptin/orphanin FQ (0.001-1 microM) produced a dose-related inhibition of the capsaicin-induced bronchoconstriction (10(-5)-10(3) microg) in isolated guinea pig lung (P<0.05), a response mediated by the release of endogenous tachykinins from lung sensory nerves. The new ORL1 receptor antagonist 1-[(3R, 4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (J-113397) (0.3 microM) significantly blocked the inhibitory effect of nociceptin/orphanin FQ (0.01 microM) on capsaicin-induced bronchoconstriction, whereas the non-selective opioid receptor antagonist naloxone (1 microM) had no effect. Nociceptin/orphanin FQ (1 microM) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist neurokinin A. In conclusion, the present data provide evidence that nociceptin inhibits capsaicin-evoked tachykinin release from sensory nerve terminals in guinea pig lung by a prejunctional mechanism. This inhibitory action occurs independently from activation of opioid receptors. The present study also indicates that J-113397 is a potent ORL1 receptor antagonist.     

Delta 9-tetrahydrocannabinol inhibits electrically-evoked CGRP release and capsaicin-sensitive sensory neurogenic vasodilatation in the rat mesenteric arterial bed

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

Increased GFR and renal excretory function by activation of TRPV1 in the isolated perfused kidney.

To test the hypothesis that activation of the transient receptor potential vanilloid type 1 (TRPV1) channels leads to natriuresis and diuresis via an increase in glomerular filtration rate (GFR), recirculating Krebs-Henseleit buffer added with inulin was perfused in the isolated perfused kidney of male Wistar rat at a constant flow, and perfusion pressures (PPs) were pre-adjusted to three different levels ( approximately 100, approximately 150, and approximately 190mmHg) with phenylephrine. Capsaicin (Cap), a selective TRPV1 agonist, was perfused in the presence or absence of capsazepine (Capz), a selective TRPV1 antagonist, CGRP(8-37), a selective calcitonin gene-related peptide (CGRP) receptor antagonist, or spantide II (Spa), a selective substance P (SP) receptor antagonist. At the higher (150 and 190mmHg) but not baseline (100mmHg) PP levels, Cap at 10microM significantly decreased PP and increased GFR, urine flow rate (UFR) and Na+ excretion (UNaV). At the highest (190mmHg) PP level, Cap (2, 10, 30microM) dose-dependently decreased PP and increased GFR, UFR, UNaV, and the release of CGRP and SP. Capz or CGRP(8-37) combined with Spa fully blocked the effect of Cap on PP, GFR, UFR, UNaV, and the release of CGRP and SP. In conclusion, activation of TRPV1 in the isolated kidney decreases renal PP and increases GFR and water/sodium excretion possibly via simultaneous activation of CGRP and SP receptors upon their enhanced release, suggesting that TRPV1 plays a key role in modulating renal hemodynamics and excretory function.     

Endothelin-1 affects capsaicin-evoked release of neuropeptides from rat vas deferens

Capsaicin-sensitive neurones release a number of neuropeptides, such as substance P, neurokinin A, somatostatin and calcitonin gene-related peptide (CGRP), which exert a number of effects on smooth muscle tissues. Endothelin-1 was thought to potentiate the capsaicin-evoked release of neuropeptides from sensory neurones of the rat. We have investigated the neuromodulatory effects of endothelin-1 on capsaicin-induced release of neurotransmitters from rat vas deferens. Capsaicin and human α calcitonin gene-related peptide (human αCGRP) reduced the rat vas deferens twitch responses induced by electrical field stimulation. Human β calcitonin gene-related peptide-(8–37) [human βCGRP-(8–37)] (1 μM), a selective αCGRP receptor antagonist, antagonized the inhibitory effects of both drugs. Endothelin-1 concentration dependently evoked an increase in basal tone of the musculature and potentiated the amplitude of the electrically stimulated responses, blocking inhibitory effects of capsaicin but not of human αCGRP. Moreover, endothelin-1 did not markedly change the inhibitory effects of papaverine (0.1–100 μM) or isoprenaline (1 nM–100 μM) on responses to electrical field stimulation. FR 139317 [(N,N-hexamethylene) carbamoyl-Leu-d-Trp(N-Me)-d-2-Pya], a selective endothelin ET_A receptor antagonist, administered 30 min before endothelin-1 restored the capsaicin effects whereas BQ 788 [Dmpc-γ-MeLeu-d-Trp-(1-methoxycarbonyl)-d-Nle], a selective endothelin ET_B receptor antagonist, was completely ineffective. The endothelin-1-induced block of the capsaicin effect was resistant to tetrodotoxin (1 μM) and 30-min pre-treatment with MEN 10.627 (cyclo[(Met-Asp-Trp-Phe-Dap-Leu) cyclo (2β–5β)]), a selective tachykinin NK₂ receptor antagonist, did not abolish the endothelin-1 effect on the inhibitory response to capsaicin. These results suggest that endothelin-1 selectively inhibits the capsaicin-induced release of neurotransmitters from rat vas deferens and these effects are mediated via endothelin ET_A receptors but not by tachykinin release.     

Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channel

Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid-like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin-induced bronchoconstriction in isolated guinea-pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea-pig nodose ganglia cells. Capsaicin increased intracellular Ca(2+) concentration in these cells through activation of vanilloid receptors. Capsaicin-induced Ca(2+) responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 microM). N/OFQ inhibitory effect on the Ca(2+) response in nodose ganglia cells was antagonized by tertiapin (0.5 microM), an inhibitor of inward-rectifier K(+) channels, but not by verapamil, a voltage gated Ca(2+) channel blocker, indicating that an inward-rectifier K(+) channel is involved in N/OFQ inhibitory effect. In isolated guinea-pig bronchus, N/OFQ (1 microM) inhibited capsaicin-induced airway contraction. Tertiapin (0.5 microM) abolished the N/OFQ inhibition of capsaicin-induced bronchial contraction. Capsaicin (10 microg) increased pulmonary inflation pressure in the isolated perfused guinea-pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 microM). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin-induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 microM) blocked the capsaicin-induced tachykinin release. These results indicate that N/OFQ-induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward-rectifier K(+) channel activation, accounts for the inhibition of capsaicin-evoked broncoconstriction.     

The activation of transient receptor potential vanilloid receptor subtype 1 by capsaicin without extracellular Ca2+ is involved in the mechanism of distinct substance P release in cultured rat dorsal root ganglion neurons

Using a highly sensitive substance P (SP) radioimmunoassay, we attempted to elucidate the molecular mechanism of SP release from cultured adult rat dorsal root ganglion (DRG) neurons triggered by capsaicin. As a result, we found that capsaicin can induce SP release in the absence of extracellular Ca(2+) by activating transient receptor potential vanilloid receptor subtype 1 (TRPV1). Therefore, we compared the pharmacological profile of SP release involved in several intracellular effectors (phosphoinositide 3-kinase (PI3K), Ca(2+) release from intracellular stores, and mitogen-activated protein (MAP) kinases) in the presence/absence of extracellular Ca(2+) by stimulating DRG neurons with various concentrations (10 to 1,000 nM) of capsaicin. In the presence of extracellular Ca(2+), a capsaicin-induced maximal release of SP obtained at 100 nM capsaicin was attenuated by either the phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY294002), the inositol 1,4,5-triphosphate (IP(3))-induced Ca(2+) release blocker (2-aminoethyl diphenylborinate, 2-APB) or a specific antagonist of TRPV1 (capsazepine). In contrast, in the absence of extracellular Ca(2+), only a high concentration (1 muM) of capsaicin induced a significant increase in the SP release, which was then completely abolished by either a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or capsazepine, and significantly inhibited by either thapsigargin (a Ca(2+)-ATPase inhibitor) or BAPTA-AM (a rapid Ca(2+) chelator). In summary, the activation of TRPV1 by capsaicin modulates the SP release from DRG neurons via two different mechanisms, one requiring extracellular Ca(2+), the activation of PI3K and the IP(3)-dependent intracellular Ca(2+) release, and the other which is independent of extracellular Ca(2+) but involves the activation of MEK. These data suggest that a distinct SP release mechanism exists on DRG through the activation of TRPV1 without extracellular Ca(2+) by capsaicin to facilitate neuropeptide release.     

Diabetic rats show reduced cardiac-somatic reflex evoked by intrapericardial capsaicin

Painless myocardial infarction is a serious complication of diabetes. The present study examined whether cardiac nociception was altered in the streptozotocin-induced diabetic rat model by assessing intrapericardial capsaicin-evoked electromyography (EMG) responses in the spinotrapezius muscle. Somatic sensitivities to mechanical and thermal stimulation of the skin were also determined. Intrapericardial administration of capsaicin evoked a concentration-dependent EMG response, which was reproducible with repeated administration. However, the capsaicin-induced EMG responses were different in streptozotocin-induced diabetic rats and controls. Intrapericardial capsaicin produced fewer EMG responses, which were delayed and reduced in streptozotocin-treated rats compared to controls. Pretreatment with capsazepine, a TRPV1 antagonist, significantly decreased capsaicin-evoked EMG activity in both streptozotocin-treated and control rats. In addition, streptozotocin-treated rats showed a decreased paw withdrawal threshold in response to mechanical stimulation but no change in response to radiant heat stimulation. These results suggest that streptozotocin-induced diabetic rats develop somatic mechanical hypersensitivity (allodynia), but reduced cardiac nociception. Decreased TRPV1 function may contribute to the reduction of cardiac nociception in the diabetic rat.     

Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist,iodo-resiniferatoxin

(1) Stimulation of the vanilloid receptor-1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. (2) Recently, iodo-resiniferatoxin (I-RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I-RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). (3) We have demonstrated that I-RTX inhibited capsaicin-induced mobilization of intracellular Ca(2+) in rat trigeminal neurons (IC(50) 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC(50) 0.071 nM). (4) Furthermore, I-RTX significantly inhibited both capsaicin-induced CGRP release from slices of rat dorsal spinal cord (IC(50) 0.27 nM) and contraction of isolated guinea-pig and rat urinary bladder (pK(B) of 10.68 and 9.63, respectively), whilst I-RTX failed to alter the response to high KCl or SP. (5) Finally, in vivo I-RTX significantly inhibited acetic acid-induced writhing in mice (ED(50) 0.42 micro mol kg(-1)) and plasma extravasation in mouse urinary bladder (ED(50) 0.41 micro mol kg(-1)). (6) In in vitro and in vivo TRPV1 activated responses I-RTX was approximately 3 log units and approximately 20 times more potent than capsazepine, respectively. This high affinity antagonist, I-RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.     

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function.     

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine

The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.     

SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea) inhibits TNF-alpha production through the activation of capsaicin-sensitive afferent neurons mediated via transient receptor potential vanilloid 1 in vivo

Tumor necrosis factor-alpha (TNF-alpha) is known to play a crucial role in the pathogenesis of rheumatoid arthritis. In the present study, we demonstrate the effects of SA13353 (1-[2-(1-Adamantyl)ethyl]-1-pentyl-3-[3-(4-pyridyl)propyl]urea), a novel orally active inhibitor of TNF-alpha production, in animal models, and its mechanism of action on TNF-alpha production. SA13353 significantly inhibited lipopolysaccharide (LPS)-induced TNF-alpha production in a dose-dependent manner in rats. Moreover, SA13353 exhibited a binding affinity for the rat vanilloid receptor and increased neuropeptide release from the rat dorsal root ganglion neurons. However, its effects were blocked by pretreatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. The ability of SA13353 and capsaicin to inhibit LPS-induced TNF-alpha production was eliminated by sensory denervation or capsazepine pretreatment in vivo. Although they inhibited LPS-induced TNF-alpha production in mice, these effects were not observed in TRPV1 knockout mice. SA13353 provoked the release of neuropeptides without nerve inactivation, even when chronically administered to rats. These results suggest that SA13353 inhibits TNF-alpha production through activation of capsaicin-sensitive afferent neurons mediated via TRPV1 in vivo. Post-onset treatment of SA13353 strongly reduced the hindpaw swelling and joint destruction associated with collagen-induced arthritis in rats. Thus, SA13353 is expected to be a novel anti-arthritic agent with a unique mechanism of action.     

Involvement of P2X receptor subtypes in ATP-induced enhancement of the cough reflex sensitivity

We examined the effect of inhaled ATP on the chemical irritant-induced coughs to clarify the roles of ionotropic purinergic receptors in these modulations. Although inhalation of 0.1 M citric acid by itself produced only a few coughs in guinea pigs, exposure to ATP, at concentrations of 3-10 microM, for 2 min concentration-dependently increased the number of 0.1 M citric acid-induced coughs. ATP-induced enhancement of the number of citric acid-induced coughs was abolished when animals were pretreated with 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), an antagonist of P2X receptor subtypes P2X1-4, at a concentration of 50 microM, for 2 min. However, exposure to pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an antagonist of P2X receptor subtypes P2X1,2,3,5,7, but not of P2X4 receptors, at a concentration of 50 microM, for 2 min, had no effect on the ATP-induced enhancement of the number of citric acid-induced coughs. Furthermore, exposure to reactive blue 2 (RB2, 30 microM, 2 min), an antagonist of P2Y receptors, had no effect on the ATP-induced enhancement of the number of citric acid-induced coughs. Exposure to ATP, at a concentration of 10 microM, for 2 min significantly increased the number of citric acid-induced coughs in capsaicin-pretreated guinea pigs. Furthermore, ATP had no effect on the number of capsaicin-induced coughs in naive animals. These results suggest that although ATP, by itself, does not elicit spontaneous coughs, it likely enhances the cough reflex sensitivity. Furthermore, stimulation of P2X receptors, especially P2X4 receptors, on rapidly adapting receptors may be required for the ATP-induced enhancement of the cough reflex sensitivity.     

Acute myocardial ischemia enhances the vanilloid TRPV1 and serotonin 5-HT3 receptor-mediated Bezold-Jarisch reflex in rats

The Bezold-Jarisch reflex is characterized by a sudden bradycardia associated with hypotension induced by the activation of the vanilloid TRPV1 and serotonin 5-HT(3) receptors. This reflex is associated with several health conditions, including myocardial infarction. The aim of the present study was to elucidate the influence of acute experimental myocardial ischemia on the reflex bradycardia induced by anandamide and phenylbiguanide, agonists of the TRPV1 and 5-HT(3) receptors, respectively. In urethane-anesthetized rats, the rapid iv injection of anandamide (0.6 μmol/kg) or phenylbiguanide (0.03 μmol/kg) decreased heart rate (HR) by about 7-10% of the basal values. Myocardial ischemia (MI) was induced by ligation of the left anterior coronary artery. The agonists were injected 5 min before MI (S(1)) and 10, 20 and 30 min thereafter (S(2)-S(4)). MI potentiated the anandamide-induced reflex bradycardia by approximately 105% at S(2) and 70% at S(3) but had no effect at S(4). This amplificatory effect of MI was virtually abolished by the TRPV1 receptor antagonist capsazepine (1 μmol/kg) and was not modified by the cannabinoid CB(1) receptor antagonist rimonabant (0.1 μmol/kg). MI also amplified the reflex bradycardia elicited by phenylbiguanide by approximately 110, 60 and 90% (S(2), S(3) and S(4), respectively), and this effect was sensitive to the 5-HT(3) receptor antagonist ondansetron (3 μmol/kg). In conclusion, our results suggest that acute myocardial ischemia augments the Bezold-Jarisch reflex induced via activation of TRPV1 and 5-HT(3) receptors located on sensory vagal nerves in the heart.     

Modifications of capsaicin-sensitive neurons in isolated guinea pig ileum by [6]-gingerol and lafutidine

A segment of guinea pig ileum was used to confirm the hypothesis that [6]-gingerol and lafutidine interact with capsaicin-sensitive neurons. Addition of 30 and 100 microM [6]-gingerol (a pungent constituent of ginger) induced contraction of the ileum immediately. Like capsaicin, [6]-gingerol-induced contraction was inhibited by antagonists of the vanilloid receptor (capsazepine and ruthenium red), tetrodotoxin, and atropine. Treatment with [6]-gingerol up to 0.3 microM, which alone had no effect, enhanced 3 microM capsaicin-induced contraction, but greater than 3 microM [6]-gingerol significantly inhibited capsaicin-induced contraction. Treatment with lafutidine (a new type of antagonist of the histamine H(2) receptor), which was suggested to interact with capsaicin-sensitive neurons in vivo, also showed both stimulatory and inhibitory effects on capsaicin-induced contraction depending on the concentrations. Lafutidine alone had no effect. The enhanced contraction induced by capsaicin in the [6]-gingerol- or lafutidine-treated ileum was also inhibited by antagonists of the vanilloid receptor, tetrodotoxin, and atropine. Capsaicin and [6]-gingerol, but not lafutidine, at 30 microM stimulated [(3)H]choline release from the prelabeled slices of the ileum. These findings suggest that [6]-gingerol and lafutidine act on capsaicin-sensitive cholinergic neurons and modulate the contraction in isolated guinea pig ileum.