The cardioprotective effects of nitroglycerin-induced preconditioning are mediated by calcitonin gene-related peptide

Previous investigations have shown that endogenous calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic preconditioning and that nitroglycerin evokes the release of CGRP. In the present study, we examined whether nitroglycerin provides a preconditioning stimulus, and whether the cardioprotective effects of nitroglycerin-induced preconditioning involve endogenous CGRP. Thirty minutes of global ischemia and 30 min of reperfusion caused a significant impairment of cardiac contractile function and an increased release of creatine kinase. Pretreatment with nitroglycerin at the concentration of 3x10(-7) or 10(-6) M for 5 min produced a significant improvement of cardiac function and a decrease in the release of creatine kinase. The content of CGRP-like immunoreactivity in coronary effluent was increased during nitroglycerin perfusion. However, the cardioprotection afforded by nitroglycerin was abolished by CGRP-(8-37) (10(-7) M), a selective CGRP receptor antagonist. Pretreatment with capsaicin (50 mg/kg, s.c.), which specifically depletes the transmitter content of sensory nerves, also abolished the protective effects of nitroglycerin and markedly reduced the release of CGRP from the heart during nitroglycerin perfusion. These findings suggest that nitroglycerin-induced preconditioning is related to stimulation of CGRP release in rat hearts.     

Role of endogenous hydrogen peroxide in the development of nitrate tolerance

The present study was designed to test the hypothesis that hydrogen peroxide plays a role in the development of nitrate tolerance. Isolated rat aortic rings were suspended in organ chambers for isometric tension recording. The rings were incubated with (tolerant) and without (control) nitroglycerin (10(-4) M) for 90 min, followed by repeated rinsing for 1 h. Hydrogen peroxide release in control and tolerant tissues was measured fluorimetrically using amplex red. Nitroglycerin (10(-9)-10(-4) M) caused concentration-dependent relaxations in control (-logEC50=7.15+/-0.1) and tolerant rings (-logEC50=5.83+/-0.1) contracted with norepinephrine. Nitrate tolerance was evident by a >20-fold rightward shift in the nitroglycerin concentration-response curve in tissues exposed previously to nitroglycerin for 90 min. Incubation of the rings with the superoxide dismutase (SOD)-mimetic, tempol (10(-4) M), during the 90-min exposure period to nitroglycerin caused a leftward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=6.84+/-0.2), but had no effect on the response to nitroglycerin in control rings. Treatment of the rings with catalase (1200 U/ml) or ebselen (1.5x10(-5) M), a glutathione peroxidase-mimetic, during the 90-min exposure period to nitroglycerin resulted in a further rightward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=5.41+/-0.1 and 4.98+/-0.1; catalase and ebselen respectively), without altering the response to nitroglycerin in control rings. In the presence of catalase, the effect of tempol on nitrate tolerance was abolished (-logEC50=5.46+/-0.1). Hydrogen peroxide release was reduced by approximately 64% in nitrate tolerant tissues when compared to control. The decrease in hydrogen peroxide release was completely reversed by treatment with tempol, whereas treatment with ebselen caused a further decrease in hydrogen peroxide release in nitrate tolerant tissues. Addition of hydrogen peroxide (3x10(-5) M) to nitrate tolerant rings caused a leftward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=7.18+/-0.3), but had no effect on the response to nitroglycerin in control rings. These results suggest that nitrate tolerance is associated with decreased endogenous formation of hydrogen peroxide, which attenuates nitrate tolerance development. SOD-mimetics may reduce nitrate tolerance, in part, by increasing the formation of hydrogen peroxide.     

降钙素基因相关肽介导硝酸甘油增强心停搏液的保护作用(英文)

目的:研究硝酸甘油增强心停搏液的保护作用与促进降钙素基因相关肽释放的关系。方法:在StThomas Hospital心停搏液条件下,离体心脏低温缺血4h后再灌40min,记录心率、冠脉流量及心功能,并测定灌注液中降钙素基因相关肽(CGRP)的浓度及肌酸激酶(CK)的释放量。结果:硝酸甘油(0.1或1μmol/L)改善心功能,降低CK释放,同时促进CGRP的释放。CGRP(5或10nmol/L)也改善心功能及降低CK释放。预先用辣椒素耗竭感觉神经递质后。硝酸甘油的心肌保护和升高灌注液中CGRP浓度作用消失。选择性CGRP受体拮抗剂CGRP_(8-37)也能取消硝酸甘油的心肌保护作用。格列苯脲对硝酸甘油和CGRP的心保护作用均无影响。结论:硝酸甘油增强心停搏液的保护作用是通过内源性CGRP所介导,其保护作用与ATP敏感的钾通道无关。     

Reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril: a role of calcitonin gene-related peptide

Previous studies have shown that the development of tolerance to nitroglycerin is related to a decrease in the release of endogenous calcitonin gene-related peptide (CGRP). In the present study, we explored whether endogenous CGRP is involved in reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril in rats in vivo and vitro. Tolerance was induced by exposure to nitroglycerin (4.4 x 10(-6) M) for 10 min in vitro or by pretreatment with nitroglycerin (10 mg/kg, s.c.) three times a day for 8 days in vivo. Nitroglycerin (3 x 10(-9)-10(-6) M) caused a concentration-dependent relaxation in the isolated rat thoracic aorta, an effect that was reduced by CGRP-(8-37) (3 x 10(-7) M) or capsaicin (3 x 10(-7) M). Preincubation with nitroglycerin for 10 min significantly decreased its vasodilation, which was restored in the presence of N-acetylcysteine (10(-5) M) or captopril (10(-5) M). Nitroglycerin (150 microg/kg, i.v.) produced a depressor effect and an increase in concentrations of nitric oxide and CGRP, and the effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, tolerance to nitroglycerin in vivo also was partially restored in the presence of N-acetylcysteine or captopril. The present results suggest that reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril is related to the increased release of CGRP in the rat.     

Relaxant effect of capsaicin in the rat gastric fundus.

The effect of capsaicin was studied in precontracted longitudinal muscle strips of the rat gastric fundus. Capsaicin induced a relaxation in the concentration range 10(-7)-10(-6) M. The relaxation induced by 10(-6) M capsaicin was completely prevented by extrinsic denervation of the stomach. The adrenoceptor antagonists phentolamine and propranolol did not influence the effect of capsaicin while hexamethonium potentiated it; this potentiation was not observed with another nicotinic receptor antagonist trimethaphan. Tetrodotoxin did not have a consistent effect as it reduced the capsaicin-induced relaxation in some but not all tissues. The peptidase trypsin consistently reduced the action of capsaicin but vasoactive intestinal polypeptide (VIP) antiserum, desensitization to calcitonin gene-related peptide (CGRP), and CGRP antiserum had no influence. The neuropeptide involved in the relaxant effect of capsaicin in the rat gastric fundus has thus still to be determined.     

The depressor and vasodilator effects of rutaecarpine are mediated by calcitonin gene-related peptide

Previous studies have shown that rutaecarpine has depressor and vasodilator effects, and activates vanilloid receptors to evoke calcitonin gene-related peptide (CGRP) release. In the present study, we examined whether the depressor and vasodilator effects of rutaecarpine are related to the stimulation of endogenous CGRP release via activation of vanilloid receptors in rats. Rutaecarpine (30, 100, or 300 microg/kg, i. v.) caused a depressor effect concomitantly with an increase in the plasma concentrations of CGRP in a dose-dependent manner, and the effects of rutaecarpine were abolished by pretreatment with capsaicin (50 mg/kg, s. c.) which depletes neurotransmitters in sensory nerves. In aortic and superior mesenteric arterial rings, rutaecarpine (10 (-7)-10(-5) M) or capsaicin (3 x 10(-9)-3 x 10(-6) M) caused a concentration-dependent vasodilator response, which was significantly attenuated by capsazepine (10(-5) M), a competitive vanilloid receptor antagonist, or by CGRP-(8-37) (10(-6) M), a selective CGRP receptor antagonist. After pretreatment with capsaicin (10(-5) M) for 20 min, vasodilator responses to rutaecarpine were also markedly attenuated. Similarly, pretreatment with rutaecarpine (10(-5) M) for 20 min also attenuated vasodilator responses to capsaicin. These results suggest that the depressor and vasodilator effects of rutaecarpine are related to stimulation of endogenous CGRP release via activation of vanilloid receptors in rats.     

Capsazepine inhibits low pH- and lactic acid-evoked release of calcitonin gene-related peptide from sensory nerves in guinea-pig heart.

In the isolated perfused guinea-pig heart low-pH buffer (pH 5), lactic acid (5 mM), capsaicin (10(-7) M) and nicotine (10(-4) M) all evoked a clear-cut release of calcitonin gene-related peptide (CGRP) from sensory nerves. Incubation with the proposed capsaicin receptor antagonist, capsazepine (10(-5) M), significantly reduced the CGRP release evoked not only by capsaicin but also by low pH and lactic acid, indicating a common mechanism of C-fibre activation, while the nicotine response remained unchanged.     

Protective effects of nitroglycerin-induced preconditioning mediated by calcitonin gene-related peptide in rat small intestine

Previous studies of myocardium have shown that ischemic preconditioning could be mimicked by nitroglycerin through stimulating the release of calcitonin gene-related peptide (CGRP). The present study examined whether nitroglycerin could also provide a preconditioning stimulus in the peripheral vascular bed (the anse intestinalis of rat), and whether endogenous CGRP is involved in this process. The model of in situ perfusion was prepared with rat small intestine. One hour of ischemia and 15 min of reperfusion caused a significant impairment of intestinal morphology and an increase in the release of both lactate dehydrogenase and malondialdehyde. Pretreatment with nitroglycerin, 10⁻⁷, 3×10⁻⁷, 10⁻⁶ M for 5 min produced a significant improvement of intestinal tissue morphology and a decrease in the release of both lactate dehydrogenase and malondialdehyde. However, the protection afforded by nitroglycerin was abolished by CGRP-(8-37), a selective CGRP acceptor antagonist. Pretreatment with capsaicin, which specifically depletes the transmitter content of sensory nerves, also abolished the protection by nitroglycerin. In addition, the content of CGRP-like immunoreactivity in the effluent was increased during nitroglycerin perfusion. On the other hand, the results from the in vivo experiment showed that nitroglycerin (i.v. 0.13 mg/kg) injected 5 min before prolonged ischemia could provide significant protection against the injury caused by 30-min ischemia and 1-h reperfusion in the rat small intestine, but would also cause a significant increase in the levels of CGRP in the plasma. All these findings suggest that nitroglycerin-induced preconditioning is related to stimulation of CGRP release in the rat small intestine.     

Differential vasodilator profile of calcitonin gene-related peptide in porcine large and small diameter coronary artery rings.

The vasodilator profile of calcitonin gene-related peptide (CGRP) was compared in large diameter (3-4 mm outer diameter) and small diameter (less than 1 mm outer diameter) rings from porcine left anterior descending coronary arteries (LAD). CGRP relaxed both sized rings in an endothelium-independent manner but was 10 X more potent in small compared to large diameter rings. Repeated administration of CGRP to small diameter rings did not cause the development of tolerance to its effects, whereas in the large diameter rings marked tolerance developed. Pretreatment with the CGRP peptide fragment, CGRP-(8-37) antagonised the vasodilator effects of CGRP in a concentration-dependent manner, but in large diameter rings, the antagonistic potency of CGRP-(8-37) was 10 X less than that seen in the small diameter rings. This differing vasodilator profile of CGRP in small and large diameter rings of pig LADs may be related to a differential CGRP receptor distribution along their length.     

Capsaicin-induced relaxation in the rat isolated external urethral sphincter: characterization of the vanilloid receptor and mediation by CGRP.

1. The potential role of capsaicin-sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2. Capsaicin (1 microM) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mM). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin-pretreated rats. 3. Capsaicin-induced relaxation of NA-precontracted REUS was mimicked by calcitonin gene-related peptide (CGRP, 0.3-10 microM), but not by substance P (1 microM), vasoactive intestinal polypeptide (VIP, 1 microM), alpha-beta methylene ATP (10 microM), gamma-aminobutyric acid (GABA, 3 mM) or galanin (1 microM). A cross-tachyphylaxis between capsaicin (1 microM) and CGRP (1 microM) was observed. Both capsaicin and CGRP-induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8-37) (10 microM). 4. Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 microM) and CGRP (0.01-1 microM) produced an almost complete inhibition of EFS-induced tonic contraction. A cross-tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8-37) (10 microM). 5. [3H]-resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pM and a Bmax of 40 fmol mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of the protective effects of nitroglycerin and nicorandil in cardiac anaphylaxis

Previous investigations have shown that nitric oxide donors and nicorandil can suppress allergic reaction. In the present study, the protective effects of nitroglycerin and nicorandil on cardiac anaphylaxis were examined. Presensitized guinea-pig hearts challenged with specific antigen caused a marked decrease in coronary flow (CF), left ventricular pressure (LVP) and its derivatives (+/-dp/dtmax), increase in heart rate, and prolongation of P-R interval. Nitroglycerin (300 nM) or nicorandil (100 microM) markedly increased the content of calcitonin gene-related peptide (CGRP) concomitant with a significant improvement of the cardiac dysfunction and alleviation of the extension of P-R interval. Nicorandil at a concentration of 100 microM also inhibited the sinus tachycardia and histamine release. The protection afforded by nitroglycerin was abolished by glibenclamide, a blocker of ATP-sensitive potassium channels, or by CGRP8-37, the selective CGRP receptor antagonist, or by pretreatment with capsaicin, which depletes endogenous CGRP. The inhibitory effect of nicorandil on cardiac anaphylaxis was abolished only by glibenclamide but not by pretreatment with capsaicin. These results suggest that nitroglycerin and nicorandil possess a protection of cardiac anaphylactic injury. The present study also suggests that the protective effect of nitroglycerin may be related to stimulation of CGRP release and opening the KATP channel, and that the effect of nicorandil is mainly due to the activation of the KATP channel.     

Transient prevention of ethanol-induced gastric lesion by capsaicin due to release of endogenous calcitonin gene-related peptide in rats

Pre-exposure of the rat gastric mucosa to capsaicin reduced the mucosal lesion by 50% ethanol to 1/4. Treatment with an antagonist of calcitonin gene-related peptide (CGRP), CGRP (8-37), nullified the effect of capsaicin. During constant perfusion of the gastric lumen with physiological saline + pepstatin, the CGRP level was not increased by 50% ethanol, but it showed a peak (802.5 +/- 145.7 pg/2 min) after 1.6 mM capsaicin. Four minutes after capsaicin, the CGRP level was kept at a high level and the gastric lesion was markedly reduced by re-exposure of the mucosa to 50% ethanol. At 20-30 min after capsaicin, the CGRP levels returned to the resting level and the reddened area by 50% ethanol was not reduced. It was concluded that capsaicin transiently prevented the mucosal lesion through CGRP release.     

Involvement of calcitonin gene-related peptide in the development of tolerance to nitroglycerin in the rat

Previous studies have shown that the depressor effect of nitroglycerin is related to stimulation of endogenous calcitonin gene-related peptide (CGRP) release. In the present study, we explored whether endogenous CGRP is involved in the development of tolerance to nitroglycerin in the rat. Tolerance was induced by treatment with nitroglycerin (10 mg/kg, subcutaneous [s.c.]) three times a day for 8 days and confirmed by a reduction in hypotensive responses to intravenous (i.v.) nitroglycerin. Nitroglycerin (30 or 150 microg/kg, i.v.) significantly decreased blood pressure concomitantly with an increase in plasma concentration of nitric oxide (NO) and CGRP, and these effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, the nitroglycerin-induced depressor effect and elevation of NO and CGRP content were restored, partially or completely, 4 or 8 days after nitroglycerin removal in the tolerant rat. The present study suggests that the development of tolerance to nitroglycerin is related to the decreased release of CGRP in the rat.     

Endogenous calcitonin gene-related peptide suppresses vasoconstriction mediated by adrenergic nerves in rat mesenteric resistance blood vessels

The role of perivascular calcitonin gene-related peptide (CGRP)-containing nerves in the modulation of adrenergic nerve-mediated vasoconstrictions was studied in the rat perfused mesenteric vascular bed. A frequency-dependent vasoconstriction induced by periarterial nerve stimulation (1-6 Hz) of the bed was significantly potentiated by perfusion of 1 microM CGRP-(8-37) (CGRP receptor antagonist) or to a similar extent after treatment with 500 nM capsaicin. In the preparations treated with capsaicin, CGRP-(8-37) caused a small potentiation of periarterial nerve stimulation-induced vasoconstriction. Exogenous CGRP (0.1-1 nM) concentration-dependently attenuated the augmented vasoconstriction in response to periarterial nerve stimulation after treatment with capsaicin. However, exogenous CGRP (1 nM) did not attenuate the periarterial nerve stimulation-induced vasoconstriction in the bed untreated with capsaicin. These results suggest that endogenous CGRP, which is released from CGRP-containing nerves, suppresses the adrenergic nerve function involved in mechanisms regulating the tone of resistant blood vessels.     

GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.

1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurogenic insulin resistance in guinea-pigs with cisplatin-induced neuropathy

The aim of the present work was to study whether neurotoxicity produced by cisplatin modified tissue insulin sensitivity in guinea-pigs. One week after selective sensory denervation of the anterior hepatic plexus by means of perineurial 2% capsaicin treatment, hyperinsulinaemic euglycaemic glucose clamp were performed to estimate insulin sensitivity in male guinea-pigs. The guinea-pigs underwent regional sensory denervation of the anterior hepatic plexus exhibited insulin resistance, whereas systemic capsaicin desensitization increased insulin sensitivity. Intraportal administration of L-nitro-arginine methyl ester (L-NAME decreased, whereas capsaicin increased insulin sensitivity. Neither atropine nor acetylcholine produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the pharmacological maneuvers modified the resulting insulin resistant state. Cisplatin pretreatment induced sensory neuropathy and decreased insulin sensitivity. Insulin sensitivity did not change after either regional or systemic capsaicin desensitization in the cisplatin-treated animals. CGRP(8-37), a nonselective calcitonin gene-related peptide (CGRP) antagonist (50 microg/kg i.v.), significantly increased insulin sensitivity in normal animals but only a tendency to insulin sensitization was seen after cisplatin treatment. Cisplatin treatment, similar to regional capsaicin desensitization of the anterior hepatic plexus, produced a significant decrease in insulin-stimulated uptake of 2-deoxy-D [L-14C] glucose in cardiac and gastrocnemius muscle with no effect on percentage suppression of endogenous glucose production by hyperinsulinaemia. We conclude that the majority of cisplatin-induced insulin resistance is related to functional deterioration of the hepatic insulin sensitizing substance (HISS) mechanism.     

Cys2,7EtalphaCGRP is a potent agonist for CGRP1 receptors in SK-N-MC cells

The present study reveals that cystein2,7 ethyl-amidealphaCGRP (Cys2,7EtalphaCGRP), an advertised calcitonin gene-related peptide 2 (CGRP2) receptor subtype-selective agonist, is also a potent agonist for the calcitonin gene-related peptide 1 (CGRP1) receptors natively expressed in the SK-N-MC human neuroblastoma cell line. Cys2,7EtalphaCGRP and alpha calcitonin gene-related peptide (alphaCGRP) promote cyclic AMP accumulation in intact SK-N-MC cells to the same extent with EC50 of 1.6+/-0.2 and 0.4+/-0.08 nM, respectively. The antagonist alpha calcitonin gene-related peptide-8-37 (alphaCGRP-(8-37)) produces a concentration-dependent rightward shift of the alphaCGRP- and Cys2,7EtalphaCGRP concentration-response curves with KB-values (71+/-33 and 47+/-21 nM, respectively). The competitive antagonism by alphaCGRP-(8-37) and the similar KB-values suggests that alphaCGRP and Cys2,7EtalphaCGRP stimulate the same receptor. In competition binding studies with [125I]-alphaCGRP on SK-N-MC cell membranes, Cys2,7EtalphaCGRP and alphaCGRP-(8-37) display high affinity for the majority of the binding sites with Ki-values of 0.030+/-0.013 and 0.60+/-0.013 nM, respectively. The present findings are at odds with the proclaimed utilization of Cys2,7EtalphaCGRP as a CGRP2 receptor-selective pharmacological tool. Differences between the agonistic profile of this ligand in this and other experimental systems might be species--or even cell type--dependent.     

Olvanil: more potent than capsaicin at stimulating the efferent function of sensory nerves.

The capsaicin analogue olvanil stimulated an increase in cutaneous blood flow when injected intradermally into the anaesthetised rabbit, as measured using a 133Xenon clearance method. Olvanil was found to be a 10-fold more potent vasodilator (on a molar basis) than capsaicin. The effect of both vasodilators was significantly inhibited by the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37). These findings suggest that olvanil stimulates the efferent function of cutaneous sensory nerves in a more potent manner than capsaicin. We therefore suggest that olvanil is a useful pharmacological tool for studying the activity of neuropeptides released from sensory nerves.     

Effects of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin.

Piperine (1 microM), a congener of capsaicin, produced an initial contraction blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 microM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 microM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1 microM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross-tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10 microM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 microM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P and CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle.     

Inhibitory effect of capsaicin on the ascending pathway of the guinea-pig ileum and antagonism of this effect by ruthenium red.

A segment of guinea-pig ileum, which was continuous with a strip of longitudinal muscle-myenteric plexus (LM-MP) at the anal end, was used to examine the effect of capsaicin on ascending excitatory pathways. Electrical field stimulation of the LM-MP caused an ascending contraction of the segment. After initially causing contraction capsaicin (3 microM) inhibited the ascending contraction. This inhibitory effect of capsaicin exhibited rapid desensitization and was abolished after extrinsic (mesenteric) denervation. Desensitization to calcitonin gene-related peptide (CGRP) prevented the capsaicin-induced inhibition without affecting the ascending contraction. Neither naloxone nor alpha- and beta-adrenoceptor antagonists affected the capsaicin-induced inhibition. CGRP (25 nM) also inhibited the ascending contraction, mimicking the inhibition induced by capsaicin. Ruthenium red (0.1-3 microM) antagonized the capsaicin-induced inhibition in a concentration-related manner, but did not affect the CGRP-induced inhibition. These findings suggest that the inhibitory effect of capsaicin on the ascending pathways might be mediated via the release of CGRP from extrinsic nerve terminals, and that the site of the antagonism of the action of capsaicin by ruthenium red is prejunctional.