Capsaicin-induced Relaxation Research Articles

Functional Analysis of TRPA1, TRPM3, and TRPV1 Channels in Human Dermal Arteries and Their Role in Vascular Modulation.

Transient receptor potential (TRP) channels are pivotal in modulating vascular functions. In fact, topical application of cinnamaldehyde or capsaicin (TRPA1 and TRPV1 channel agonists, respectively) induces "local" changes in blood flow by releasing vasodilator neuropeptides. We investigated TRP channels' contributions and the pharmacological mechanisms driving vasodilation in human isolated dermal arteries. Ex vivo studies assessed the vascular function of artery segments and analyzed the effects of different compounds. Concentration-response curves to cinnamaldehyde, pregnenolone sulfate (PregS, TRPM3 agonist), and capsaicin were constructed to evaluate the effect of the antagonists HC030031 (TRPA1); isosakuranetin (TRPM3); and capsazepine (TRPV1). Additionally, the antagonists/inhibitors olcegepant (CGRP receptor); L-NAME (nitric oxide synthase); indomethacin (cyclooxygenase); TRAM-34 plus apamin (K+ channels); and MK-801 (NMDA receptors, only for PregS) were used. Moreover, CGRP release was assessed in the organ bath fluid post-agonist-exposure. In dermal arteries, cinnamaldehyde- and capsaicin-induced relaxation remained unchanged after the aforementioned antagonists, while PregS-induced relaxation was significantly inhibited by isosakuranetin, L-NAME and MK-801. Furthermore, there was a significant increase in CGRP levels post-agonist-exposure. In our experimental model, TRPA1 and TRPV1 channels seem not to be involved in cinnamaldehyde- or capsaicin-induced relaxation, respectively, whereas TRPM3 channels contribute to PregS-induced relaxation, possibly via CGRP-independent mechanisms.

Impaired capsaicin-induced relaxation in diabetic mesenteric arteries

AimsTo investigate capsaicin-induced vasodilation in the diabetic mesenteric arteries. MethodsA diabetic rat model was established by intraperitoneal injection of streptozotocin after a 12-h fast. At 12weeks post-injection, the third branch of the mesenteric artery was dissected out and prepared for vascular reactivity assessment. Capsaicin, capsazepine, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), sodium nitroprusside (SNP), calcitonin gene-related peptide 8-37 (CGRP8-37), calcitonin gene-related peptide (CGRP), and substance P (SP) were added to investigate subsequent alterations in vascular activity. Plasma and physiological salt solution (PSS) levels of CGRP and SP were measured using radioimmunoassay. The expression of transient receptor potential vanilloid 1 (TRPV1) and phospho-eNOS were determined using Western blot analysis, and nitric oxide (NO) production was measured using a fluorescence probe. ResultsThe dilation effect of capsaicin was weaker under the diabetic than control conditions. Capsazepine, L-NAME, and CGRP8-37 attenuated capsaicin-induced vasorelaxation significantly in the diabetic vascular rings. Exogenous CGRP elicited dose-dependent vasodilation in the control arteries, whereas the dilation effect was reduced under diabetic conditions. Plasma and PSS CGRP levels were attenuated and mesenteric artery TRPV1 expression was decreased in the diabetic rats. Phospho-eNOS levels were augmented, and NO production increased following the administration of capsaicin. ConclusionsDecreased expression of TRPV1 and associated neuropeptide release contributed to the impaired capsaicin-induced vasodilation in diabetic mesenteric arteries. Furthermore, an endothelium-dependent NO-related pathway was involved in capsaicin-induced vasodilation.

Vasorelaxant response of coronary arteries to capsaicin and effect of angiotensin II (1057.1)

Vasorelaxant effects and mechanisms of the TRPV1 selective agonist capsaisin and the effect of angiotensin II on capsaicin‐induced relaxation were studied in small porcine coronary arteries (PCAs). In U46619‐constricted PCAs, capsaisin induced dose‐dependent relaxation with significant response starting at 0.1µmol/L and full relaxation at 100µmol/L. Endothelium denudation significantly inhibited relaxant responses to capsaicin at concentrations of 0.1‐10µmol/L. sPCAs pretreated with IKCa and SKCa channel blockers, TRAM‐34 and apamin, showed suppressed relaxation to capsaicin (0.1‐10µmol/L) and the inhibition was comparable to that in PCAs constricted with 40mmol/L KCl (35.5±4.7%, 29.5±3.9% vs. 79.0±4.3% in control, p<0.001). However, relaxation to 100µmol/L capsaicin remained unaffected by endothelium denudation, IKCa and SKCa blockade, or high [K+]o. Exposure of sPCAs to angiotensin II (100nmol/L) for 24h inhibited capsaicin‐induced relaxation (33.3±3.5%, p<0.001). Capsaisin induces relaxation of PCAs through both endothelium‐dependent and ‐independent mechanisms. IKCa and SKCa are involved in the endothelium‐dependent mechanism. Angiotensin II impairs capsaicin‐induced endothelium‐dependent relaxation.Supported by GRF CUHK4774/12M; NSFC 81200123; CUHK Direct Grant 4054015; Key Med. Program of Tianjin Binhai Health Bureau 2011BHKZ001; Tianjin Health Bureau 2012KZ009.Grant Funding Source: Supported by GRF CUHK4774/12M; NSFC 81200123; CUHK Direct Grant 4054015

Influence of Influenza A Infection on Capsaicin-Induced Responses in Murine Airways

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 μM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK₁) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2β-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)₂ and EP₄ receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK₁ receptors, and production of COX products capable of activating relaxant EP₂/EP₄ receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E₂ (PGE₂) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE₂ and the β-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE₂, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE₂.

Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.

Increased Cholesterol is Vital to Impairment of TRPV1 Channel‐Mediated Relaxation of Coronary Artery In Ossabaw Miniature Swine with Metabolic Syndrome

TRPV1 channels are implicated in vascular regulation. Ossabaw swine fed excess high fat/cholesterol diet develop metabolic syndrome (MetS), however studies find high‐fructose diets elicit MetS without dyslipidemia. We tested the hypothesis that dyslipidemia is an important component for eliciting dysfunction of endothelial cell (EC) TRPV1 channels signaling. Coronary arteries were studied from male Ossabaw pigs fed standard chow (lean, n = 8) or excess kcal diets for 28 wk: fructose to induce MetS (n = 6) or high fat/cholesterol/fructose to induce MetS with added dyslipidemia (DMetS, n = 8), defined by increased LDL or total/HDL cholesterol. The TRPV1 channel agonist capsaicin, dose‐dependently relaxed coronary rings from lean pigs (EC50 = 0.12 ± 0.04 μM), which was attenuated in MetS pigs (EC50 = 10 ± 21 μM) and severely attenuated in DMetS pigs (EC50: ~ 1mM). Fura‐2 imaging revealed capsaicin generated calcium influx in healthy EC, which was attenuated in MetS and to a greater extent in DMetS. Coronary TRPV1 protein levels were decreased only in DMetS (lean~MetS>DMetS). Conclusions: Capsaicin‐induced relaxation of coronary conduit artery occurs via EC and TRPV1 channel signaling, which is disrupted in MetS and to a much greater extent in DMetS, suggesting lipids are important regulators of EC TRPV1‐mediated functions. Support: NIH RR013223, HL062552, T32 HL079995.

TRPV1 receptor signaling mediates afferent nerve sensitization during colitis-induced motility disorders in rats

Rats with experimental colitis suffer from impaired gastric emptying (GE). We previously showed that this phenomenon involves afferent neurons within the pelvic nerve. In this study, we aimed to identify the mediators involved in this afferent hyperactivation. Colitis was induced by trinitrobenzene sulfate (TNBS) instillation. We determined GE, distal front, and geometric center (GC) of intestinal transit 30 min after intragastric administration of a semiliquid Evans blue solution. We evaluated the effects of the transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine (5-10 mg/kg) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 1-10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 microg/kg). To determine TRPV1 receptor antagonist sensitivity, we examined their effect on capsaicin-induced relaxations of isolated gastric fundus muscle strips. Immunocytochemical staining of TRPV1 and RT-PCR analysis of TRPV1 mRNA were performed in dorsal root ganglion (DRG) L6-S1. TNBS-induced colitis reduced GE but had no effect on intestinal motility. Capsazepine reduced GE in controls but had no effect in rats with colitis. At doses that had no effects in controls, BCTC and CGRP-(8-37) significantly improved colitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolished them. TNBS-induced colitis increased TRPV1-like immunoreactivity and TRPV1 mRNA content in pelvic afferent neuronal cell bodies in DRG L6-S1. In conclusion, distal colitis in rats impairs GE via sensitized pelvic afferent neurons. We provided pharmacological, immunocytochemical, and molecular biological evidence that this sensitization is mediated by TRPV1 receptors and involves CGRP release.

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 μM), the neurokinin NK1 receptor antagonist L-733060 (0.5 μM), the voltage-sensitive calcium channel blocker ruthenium red (100 μM), the TRPV1 receptor antagonist capsazepine (5 μM), the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl (l-NAME; 100 μM), the gap junction blocker 18α-glycyrrhetinic acid (10 μM), as well as the RhoA kinase inhibitor Y-27632 (1 μM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 μM) + apamin (0.1 μM) and iberiotoxin (0.5 μM) + apamin (0.1 μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC 20) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6±7.5%, n=10 and 97.1±0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca 2+ channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro- l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca 2+-free, EGTA solution. Phosphorylation of MLC 20 (a percentage of phosphorylated to total MLC 20) was increased 1 min after application of 10 μM acetylcholine (7.8±2.0%, n=6 vs. 22.6±3.2%, n=6) and of 65.9 mM KCl (2.2±0.3%, n=8 vs. 10.7±1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC 20 phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC 20 phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC 20 phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca 2+ influx via non-L-type Ca 2+ channels, possibly via store-operated Ca 2+ channels and Ca 2+ release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC 20 phosphorylation.

Actions of endothelin and corticotropin releasing factor in the guinea-pig ileum: no evidence for an interaction with capsaicin-sensitive neurons

Both endothelins and corticotropin releasing factor (CRF) appear in capsaicin-sensitive neurons. We have investigated the effects of human endothelin-1 (ET-1) and CRF in the guinea-pig ileum longitudinal and circular preparations and sought for ways of specific antagonism. With the aid of tachyphylaxis to capsaicin (i.e., rendering capsaicin-sensitive neurons functionally impaired) it was tested if these neurons played a mediating role in the effects of ET-1 or CRF. We also tried to find out whether endogenous endothelin or CRF plays a role in the excitatory and inhibitory effects of capsaicin in the ileum. In preparations at basal tone, both exogenous ET-1 (1–100 nM) and CRF (3–100 nM) caused contraction. These responses were not influenced by capsaicin tachyphylaxis. The contractile effect of ET-1 was not affected by tetrodotoxin (1 μM), atropine (1 μM), methysergide (100 nM), chloropyramine (100 nM) or SR140333 (100 nM) but was significantly inhibited or even abolished by the receptor antagonist BQ123 (3 μM) or BQ788 (3 μM). CRF caused contraction that was fully sensitive to tetrodotoxin (1 μM), tachyphylaxis to CRF or to atropine (1 μM) plus the tachykinin NK 1 receptor antagonist SR140333 (200 nM). Atropine alone had a weak inhibitory effect on the contractile action of CRF. Neither the antagonist BQ123 (3 μM) nor CRF tachyphylaxis inhibited the contractile action of capsaicin (2 μM), even in the presence of a mixture of GR82334 (3 μM) and SR142801 (100 nM), for blocking tachykinin NK 1 and NK 3 receptors, respectively – a treatment that by itself significantly reduced the effect of capsaicin. Exogenous ET-1 (0.3–5 nM), but not CRF (30–100 nM), caused relaxation of the atropine-treated, histamine-precontracted ileum. This effect of ET-1 was significantly inhibited or abolished by BQ123 (10 μM), or BQ788 (3 μM), but was not influenced by capsaicin tachyphylaxis. Likewise, relaxation of the atropine-treated, histamine-precontracted ileum in response to capsaicin was not influenced by the endothelin receptor antagonist BQ788 (3 μM) or BQ788 (3 μM) plus BQ123 (3 μM). Apamin (300 nM) was also without effect on the capsaicin-induced relaxation. In circular muscle strips ET-1 inhibited the indomethacin-induced spontaneous activity. This effect was abolished by BQ123 (3 μM) or BQ788 (3 μM). CRF caused a stimulation of the circular muscle. This stimulatory effect was not influenced by atropine (1 μM) alone, but was inhibited by atropine plus tachykinin NK 1 and NK 2 receptor antagonists (SR140333 (200 nM) and SR48968 (200 nM)) and also by tetrodotoxin (1 μM). It is concluded that capsaicin-sensitive neurons do not play a role in the effects of exogenous ET-1 or CRF in the guinea-pig ileum. ET-1 can both contract and relax the ileal longitudinal smooth muscle directly, probably via both ET A and ET B receptors. CRF acts by specifically stimulating excitatory (but not inhibitory) neurons of the myenteric plexus. Neither endogenous ET-1 nor CRF seems to play a role in the excitatory or inhibitory effects of capsaicin.

Endothelium-independent relaxation to cannabinoids in rat-isolated mesenteric artery and role of Ca2+ influx.

(1) Three cannabinoid receptor agonists, anandamide (CB(1) receptor-selective) and the aminoalkyl-indoles, JWH 015(2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone; (CB(2) receptor-selective), R-(+)-WIN 55,212-2 (R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; slightly CB(2) receptor-selective), as well as the enantiomer S-(-)-WIN 55,212-3(S-(-)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolol[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; inactive at cannabinoid receptors), induced endothelium-independent relaxation of methoxamine-precontracted isolated small mesenteric artery of rat. KCL (60 mM) precontraction did not affect relaxation to the aminoalkylindoles, but reduced that to anandamide. (2) SR14176A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 micro M; CB(1) receptor antagonist) inhibited relaxation only to JWH 015 and anandamide. Neither AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; CB(1) antagonist) nor SR 144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; CB(2) antagonist; both at 3 micro M) affected any of the relaxations. (3) Vanilloid receptor desensitisation with capsaicin reduced anandamide relaxation; addition of SR 141716A (3 micro M) then caused further inhibition. SR 141716A did not affect capsaicin-induced relaxation. (4) The aminoalkylindoles inhibited CaCl(2)-induced contractions in methoxamine-stimulated vessels previously depleted of intracellular Ca(2+). These inhibitory effects were greatly reduced or abolished in ionomycin-(a calcium ionophore) contracted vessels. Anandamide also caused vanilloid receptor-independent, SR 141716A- (3 micro M) insensitive, inhibition of CaCl(2) contractions. (5) In conclusion, the aminoalkylindoles JWH 015, R-(+)-WIN 55,212-2 and S-(-)-WIN 55,212-3 relax rat small mesenteric artery mainly by inhibiting Ca(2+) influx into vascular smooth muscle. Anandamide causes vasorelaxation by activating vanilloid receptors, but may also inhibit Ca(2+) entry. Relaxation to JWH 015 and anandamide was sensitive to SR 141716A, but there is no other evidence for the involvement of CB(1) or CB(2) receptors in responses to these compounds.

Normal perivascular sensory dilator nerve function in arteries of Zucker diabetic fatty rats

Background Type II diabetes in humans is associated with pathology of both the cardiovascular and peripheral sensory nervous systems. Because abnormal vasodilator responses have been reported in animals of type II diabetes and perivascular sensory nerves are a source of vasodilator substances, we tested the hypothesis that sensory nerve-dependent relaxation is abnormal in arteries of the Zucker diabetic fatty (ZDF) rat model of type II diabetes. Methods The ZDF rats and genetic controls were studied at 26 weeks of age. Tail-cuff systolic blood pressure (BP) was measured, serum was obtained for chemical determinations, and mesenteric branch arteries were isolated for wire myograph analysis and confocal-based measurement of calcitonin gene-related peptide (CGRP) positive nerve density. Results No differences in BP were detected. Serum glucose, triglycerides, and cholesterol were significantly elevated in ZDF. Sensory nerve-dependent vasodilation was assessed by measuring relaxation of phenylephrine preconstricted arterial segments to cumulative addition of divalent calcium ion (Ca 2+) or capsaicin. Neither Ca 2+- nor capsaicin-induced relaxation were different in ZDF versus control (maximal ZDF response to Ca 2+ = 64% ± 2% v 59% ± 4%; ED 50 for Ca 2+ = 3.7 ± 0.5 mmol/L v 3.2 ± 0.5 mmol/L; n = 5, P = not significant [NS]; maximal ZDF response to capsaicin = 68% ± 9% v 74% ± 4%; ZDF ED 50 = 3.8 ± 0.5 nmol/L v 9.8 ± 7 nmol/L; n = 5, P = NS). In contrast, the maximal relaxation response to acetylcholine was impaired in ZDF (maximal ZDF response = 83% ± 5% v 94% ± 2%, n = 4, P = .039; ED 50 for acetylcholine = 8.1 ± 2.9 nmol/L for ZDF v 33.5 ± 18.2; n = 4 per group, P = .086). The CGRP positive nerve density was not different between groups. Conclusions Blood pressure, perivascular sensory nerve CGRP content, and dilator function is normal in the ZDF model of type II diabetes, whereas endothelium-dependent relaxation is impaired.

A novel mechanism for the reuptake of CGRP

Unlike classical amine or amino acid transmitters, which are synthesized close to their sites of release, neuropeptides such as the 37-amino-acid calcitonin gene-related peptide (CGRP) are synthesized in the neuronal soma and transported through the axon to sites of release in the nerve terminals. The transport process is time- and energy-consuming, and an intense stimulation could deplete releasable pools of neuropeptides rendering the nerve terminals unresponsive (in terms of release) until this pool has been replenished by newly synthesized and transported peptide. Uptake mechanisms that remove neurotransmitters from the synaptic cleft and spare the synthesis of new molecules of neurotransmitters have been described for small amine or amino acid neurotransmitters. Now, Sams-Nielsen et al. 1xPharmacological evidence for CGRP uptake into perivascular capsaicin sensitive nerve terminals. Sams-Nielsen, A. et al. Br. J. Pharmacol. 2001; 132: 1145–1153Crossref | PubMedSee all References1 provide indirect evidence for a reuptake mechanism for CGRP into capsaicin-sensitive afferent neurones and suggest that this mechanism might have functional relevance for the refilling of a releasable neuropeptide pool in nerve terminals following CGRP release.Isolated segments of the guinea-pig basillary artery, precontracted with prostaglandin F2α (PGF2α), were challenged with capsaicin (10 μm), which caused a CGRP-dependent (blocked by the antagonist CGRP8–37), capsazepine-sensitive relaxation. When the capsaicin challenge was repeated twice again, the relaxation underwent a significant fading (from ∼60% to 20% of PGF2α contraction). At this point, preparations were incubated with CGRP (100 nm) for 20 min and the capsaicin challenges repeated another three times. Following this protocol, the fourth capsaicin challenge (the first after the CGRP incubation) caused a significant relaxation (capsazepine-resistant, but still CGRP-dependent), which showed a significant fading during the fifth and sixth capsaicin challenge. Control experiments showed that repeated challenges with exogenous CGRP induced reproducible relaxations, thus excluding the possibility that desensitization occurs at the smooth-muscle level. In another series of experiments, arterial segments were incubated with [125I]CGRP and [125I] outflow was monitored following relaxation induced by capsaicin in PGF2?-precontracted preparations. Capsaicin induced a significant radioactivity release, which suggests that [125I]CGRP had been taken up by capsaicin-sensitive nerve terminals, even in the absence of a previous capsaicin challenge. Intriguingly, the restoration of capsaicin-induced relaxation was inhibited when CGRP was co-incubated with its antagonist CGRP8–37, suggesting that prejunctional CGRP receptors are involved in the refilling of the neuropeptide in nerve terminals.These results provide indirect evidence for a CGRP reuptake mechanism in capsaicin-sensitive perivascular nerve terminals, possibly mediated through the activation of prejunctional CGRP receptors. Thus, following the activation of prejunctional CGRP receptors and the consequent internalization of the complex peptide ligand–receptor, the peptide ligand might be spared from metabolism and recycled locally for the preservation of physiological functions. In addition, these results highlight a novel role for peptide prejunctional receptors in neurones, as well as in the modulation of neurotransmitter release. Other examples for the transport of relatively large peptides across biological barriers include those mediated by multi-drug resistance protein, other peptide transporters, or specific peptide receptors located at the blood–brain barrier or the choriod plexus epithelium; however, this uptake mechanism for CGRP is the first example of a neuronal uptake of peptides. It will be interesting to assess whether this process represents an exception or similar mechanisms spare other neuropeptides.

Capsaicin-induced relaxation in rabbit coronary artery.

In the present study mechanism of inhibitory effects of capsaicin on the contractility of rabbit coronary artery were studied by measurement of isometric tension and intracellular Ca2+ concentration. Capsaicin (1 microM to 30 microM) relaxed the coronary artery pre-contracted with prostaglandin (PG) F2alpha (1 microM) in a concentration-dependent manner. The PGF2alpha-induced increase in intracellular Ca2+ concentration was also inhibited. The effects of capsaicin were readily reversed by washing capsaicin from the bath. Capsaicin-induced relaxation was not attenuated by pretreatment with capsazepine (1 microM), a blocker of vanilloid receptor or ruthenium red (1 microM), a blocker of non-selective cation channel. Previous exposure to a high concentration of capsaicin (100 microM) or repeated application of capsaicin did not eliminate the relaxation response to subsequent application of capsaicin. Increasing the external K+ concentration to 80 mM significantly attenuated the capsaicin-induced relaxation with simultaneous change in intracellular Ca2+ concentration. Pretreatment with iberiotoxin (100 nM), a blocker of Ca2+-activated K+ channel, only partially inhibited the capsaicin-induced relaxation. However, application of 4-aminopyridine (4-AP, 1 mM), a blocker of delayed rectifier K+ current significantly inhibited the capsaicin-induced relaxation with concomitant attenuation of the effect on intracellular Ca2+ concentration. These results indicate that capsaicin may have a direct relaxing effect on the smooth muscle contractility, and relaxation may be due to activation of the 4-AP-sensitive, delayed rectifier K+ channels in the rabbit coronary artery.

Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres.

1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade. 2. Tityus serrulatus venom (3-100 microg), acetylcholine (ACh; 0.3-30 nmol) and glyceryl trinitrate (GTN; 0.5-10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 microM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 microM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 microM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible. 3. The non-selective NO synthase (NOS) inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME; 10 microM) and NG-iminoethyl-L-ornithine (L-NIO; 30 microM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 microM), but not D-arginine (300 microM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 microM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline. 4. The protease inhibitor aprotinin (Trasylol; 10 microg ml[-1]) and the bradykinin B2 receptor antagonist Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7, Oic8]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K+ channel antagonist glibenclamide (10 microm) and the Ca2+-activated K+ channel antagonists apamin (0.1 microM) and charybdotoxin (0.1 microM) also failed to affect the venom-induced relaxations. Similarly, the K+ channel blocker tetraethylammonium (TEA; 10 microM) had no effect on the venom-induced relaxations. 5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 microM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 microM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum. 6. The sodium channel blocker tetrodotoxin (TTX; 1 microM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 microM) also promptly reversed the response to the venom when infused during the relaxation phase. 7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 microM) or L-NAME (10 microM). 8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.

Mechanism of capsaicin-induced relaxation in equine tracheal smooth muscle.

The effects of capsaicin and neuropeptides were examined in equine tracheal smooth muscle (TSM). Neither capsaicin nor substance P (SP) contracted TSM. Capsaicin (100 microM) elicited relaxation in TSM contracted with methacholine. This relaxation was not mimicked by SP or calcitonin gene-related peptide (CGRP). Relaxation was not attenuated by removal of the epithelium or by pretreatment of tissue with meclofenamate or the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine. Previous exposure of TSM to capsaicin did not eliminate the relaxation responses to subsequent capsaicin. Although vasoactive intestinal peptide (VIP) elicited marked relaxation that was attenuated by alpha-chymotrypsin, alpha-chymotrypsin did not affect the capsaicin-induced relaxation. Capsaicin-induced relaxation was abolished by charybdotoxin, a blocker of large-conductance Ca(2+)-activated K+ channels. These results indicate that capsaicin-induced equine TSM relaxation is not mediated either by neuropeptides such as SP or CGRP released from capsaicin-sensitive sensory nerves or by prostanoids, NO, or VIP. Relaxation is due to the effect of capsaicin on large-conductance Ca(2+)-activated K+ channels. The peptidergic nerves play no important role in the regulation of TSM tone in horse airways.

Capsaicin-induced nitric-oxide-dependent relaxation in isolated dog urethra

Capsaicin (5×10 −8 to 5×10 −5 M) produced a non-adrenergic and non-cholinergic phasic relaxation in a concentration-dependent manner in isolated dog urethral preparations precontracted by noradrenaline. The mode of action of capsaicin was investigated with special reference to the possible involvement of endogenous nitric oxide (NO). A marked tachyphylaxis was observed in the responses to capsaicin. Pretreatment with N G-nitro- l-arginine-methyl-ester ( l-NAME) prevented or markedly reduced the inhibitory effect of l-NAME. Methylene blue inhibited the capsaicin-induced relaxation. In preparations stored at 4°C for 72 h, the reduction in the capsaicin-induced relaxation was significantly greater than that in the relaxation induced by either electrical field stimulation or by sodium nitroprusside. We conclude that capsaicin produces an endogenous-NO-dependent relaxation in the isolated dog urethra via mechanisms that deteriorate during cold storage of the preparations.

Sensory nerve-mediated inhibitory responses in airways of F344 rats

We recently described a sensory nerve inhibitory system that mediates relaxation in the airways of Sprague-Dawley rats. Results of several studies have shown that this system protects the lungs against injury induced by toxic stimuli. Whether a similar inhibitory system exists in the airways of Fischer 344 (F344) rats is unknown. Because this rat strain is used extensively in lung toxicological research, the purpose of this study was to determine whether a sensory nerve inhibitory system exists in intrapulmonary bronchi and tracheae isolated from F344 rats. In intrapulmonary bronchi at resting tone, substance P (1.0 μM) evoked a transient contraction that was inhibited by the 5-HT 2A receptor antagonist, ketanserin. Exposing airway segments to compound 48/80 to degranulate mast cells also abolished substance P-induced contractions. Inhibition of cyclooxygenase with meclofenamate augmented markedly the contraction to substance P in the intrapulmonary bronchi. In intrapulmonary bronchi that were contracted with bethanechol, substance P evoked a biphasic response characterized by an increase in tension above that induced by bethanechol followed by relaxation. Incubation of the airways with ketanserin abolished the contractile portion of the response; relaxation responses were augmented after ketanserin. In contracted intrapulmonary bronchi that had been treated with compound 48/80, substance P and capsaicin caused relaxation responses that were inhibited markedly or were nearly abolished by the NK 1 receptor antagonist, RP67580, by meclofenamate, and by denuding the epithelium. Capsaicin-induced relaxation responses also were abolished by desensitization of C-fibers with capsaicin. Only ketanserin-sensitive contractile responses were observed in response to substance P in tracheal segments. We conclude that a sensory nerve inhibitory system exists in the intrapulmonary airways of F344 rats. The presence of this inhibitory system in F344 rat airways may play a protective role against lung injury induced by inhaled toxicants.

Tachykinin-independent effects of capsaicin on smooth muscle in human isolated bronchi.

Contractile and relaxant responses to capsaicin and resiniferatoxin were examined in human isolated bronchus (5-12 mm o.d.). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The contractions averaged 20% of maximal contraction at 1 microM and averaged > 40% maximal contraction at 300 microM (the highest concentration studied). The capsaicin-induced contractions were mimicked by resiniferatoxin (0.1-10 microM) and inhibited by the putative capsaicin receptor antagonist, capsazepine (10 microM). The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968 (0.3 microM), whereas responses to concentrations of neurokinin A (10 nM), neurokinin B (0.1 microM), substance P (1 microM), neuropeptide gamma (10 nM), and neuropeptide K (10 nM) which produced similar-size contractions were almost abolished by 0.1 microM SR 48968. The bronchi isolated from 8 of 16 lungs also exhibited relaxations in response to capsaicin. Capsaicin-induced relaxations were not inhibited by the nitric oxide synthase inhibitor L-nitro-n-arginine (10 microM). In whole-cell patch-clamp experiments on human cultured airway smooth muscle cells, capsaicin was found to enhance outward currents due to the activation of charybdotoxin-sensitive large conductance Ca2+-activated K+ channels. Neither the capsaicin-induced contractions nor the relaxations were mimicked by angiotensin II, bombesin, or calcitonin gene-related peptide at concentrations up to 1 microM. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone, but this response does not appear to involve substance P or related neurokinins. Relaxations to capsaicin may, however, involve the activation of large conductance Ca2+-activated K+ channels.

Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K + or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba 2+ inward current (I Ba) through the voltage-dependent L-type Ca 2+ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K + currents. These results suggest that inhibition of the voltage-dependent L-type Ca 2+ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K + channels might produced an increase in cell excitability.