Prejunctional modulatory action of neuropeptide Y on responses due to antidromic activation of peripheral terminals of capsaicin-sensitive sensory nerves in the isolated guinea-pig ileum.

1. The effect of neuropeptide Y (NPY) on motor responses produced by activation of capsaicin-sensitive primary afferents in the guinea-pig isolated ileum was determined by use of capsaicin itself and electrical mesenteric nerve stimulation as stimuli. 2. NPY inhibited or suppressed the cholinergic contractile response produced by electrical mesenteric nerve stimulation while leaving the contractile response to a threshold concentration of capsaicin. 3. NPY had no effect on motor responses produced by a submaximal concentration of substance P, the putative endogenous mediator of the 'efferent' function of sensory fibres in this preparation. 4. It is concluded that NPY exerted a prejunctional inhibitory action on transmitter release from peripheral endings of capsaicin-sensitive nerves at interneuronal synapses.     

The ‘efferent’ function of capsaicin-sensitive nerves: Ruthenium Red discriminates between different mechanisms of activation

We have investigated the ability of Ruthenium Red, an inorganic dye with calcium entry blocking properties, to interfere with the ‘efferent’ function of capsaicin-sensitive sensory nerves. These nerves were activated in the guinea-pig isolated bronchus (atropine in the bath) or left atria (reserpine-pretreated animals, atropine in the bath) by electrical field stimulation or with capsaicin. Both stimuli produced a contraction of the bronchus and a positive inotropic response in the atria, responses which are mediated by endogenous neuropeptides (tachykinins in the bronchus, calcitonin gene-related peptide in the atria) released from sensory nerves. Ruthenium Red (10 μM for 20 min in the cases) selectively inhibited the responses produced by the administration of capsaicin, while leaving the responses to electrical field stimulation unaffected. Likewise, the bronchonconstrictor response to exogenous neurokinin A and the atrial positive inotropic response to calcitonin gene-related peptide were unaffected by Ruthenium Red. A prejunctional site of action Ruthenium Red was confirmed in release experiments where the dye strongly inhibited the capsaicin-evoked outflow of calcitonin gene-related peptide, which is taken as a marker of activation in sensory nerves. Together with other observations, these findings support the concept that there are two independent mechanisms for activating the ‘efferent’ function of sensory nerves, one of which is activated by capsaicin and is Ruthenium Red-sensitive but ω-conotoxin-resistant, while the other is activated by propagated action potentials (field stimulation) and is ω-conotoxin-sensitive and Ruthenium Red-resistant.     

Evidence for two independent modes of activation of the 'efferent' function of capsaicin-sensitive nerves

Field stimulation (10 Hz for 10 s, 0.5 ms pulse width, 60 V) of the guinea-pig isolated main bronchi (atropine plus indomethacin in the bath) produced reproducible contractions which were abolished by tetrodotoxin or in vitro capsaicin desensitization. These responses were almost abolished by omega-conotoxin GVIA (CTX), a peptide modulator of neuronal calcium channels which, however, did not affect the bronchial contraction due to neurokinin A or to capsaicin. Field stimulation (10 Hz for 2.5 s, 1 ms, 60 V) of the electrically driven, isolated guinea-pig left atria excised from reserpine-pretreated animals (atropine in the bath) produced a delayed positive inotropic response which was abolished by tetrodotoxin or in vitro capsaicin desensitization. This response was abolished by CTX, which did not affect the response to exogenous calcitonin gene-related peptide nor that to capsaicin. These findings indicate that CTX-sensitive mechanisms (presumably Ca channels regulating the release of transmitters) are activated upon antidromic invasion of sensory terminals and consequent production of the 'efferent' response while the activation of sensory nerve endings by capsaicin occurs through CTX-resistant mechanisms.     

Nociceptin protects capsaicin-sensitive afferent fibers in the rat urinary bladder from desensitization

Chemical stimulation of primary afferent nerves in the rat urinary bladder in vivo with topical capsaicin (1 microg in 50 microl saline) determines a dual motor response, consisting of a contractile effect mediated by tachykinins released from sensory nerves in the bladder wall and a transient activation of a bladder-to-bladder micturition reflex organized at the supraspinal level (chemoceptive micturition reflex). Both responses undergo complete desensitization upon repeated applications of capsaicin. The i.v. administration of the novel neuropeptide nociceptin (100 nmol/kg) produced a long-lasting protection from capsaicin desensitization of afferent nerves which mediate the chemoceptive micturition reflex. In fact a chemoceptive micturition reflex could be repeatedly evoked by topical capsaicin in nociceptin-pretreated rats. In sharp contrast, nociceptin did not influence the development of desensitization of the local response to capsaicin, corresponding to the 'efferent' function of capsaicin-sensitive afferent neurons. These results suggest that the afferent and 'efferent' function of capsaicin-sensitive primary afferent neurons (CSPANs) in the rat bladder are differentiated by nociceptin. Alternative mechanisms underlying this phenomenon are discussed.     

Modulatory action of galanin on responses due to antidromic activation of peripheral terminals of capsaicin-sensitive sensory nerves

Galanin inhibited, in a concentration-dependent manner (EC50 7.2 nM), the positive inotropic response produced by field stimulation of isolated left atria from reserpine-pretreated guinea-pigs (in the presence of atropine). These responses were shown to involve antidromic activation of capsaicin-sensitive primary afferents. On the other hand, galanin did not affect the inotropic response to capsaicin or calcitonin gene-related peptide, the putative endogenous mediator released from sensory nerves. Galanin-(1-10) was at least 10,000 times less potent than the parent peptide, while galanin-(25-29) was ineffective. Likewise, galanin inhibited the non-cholinergic contraction produced by field stimulation of the isolated guinea-pig bronchus but not the contraction produced by exogenous neurokinin A. These findings indicate a prejunctional neuromodulatory action of galanin on the excitability of peripheral terminals of capsaicin-sensitive sensory nerves.     

Peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) activates capsaicin-sensitive primary afferent nerves in guinea-pig atria and urinary bladder.

1. We have investigated the ability of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) a synthetic analogue of a chemotactic peptide derived from a variety of bacteria, to activate capsaicin-sensitive primary afferents in the guinea-pig atria and urinary bladder. 2. In the isolated, electrically-driven left atria from reserpine-pretreated guinea-pigs (atropine in the bath), FMLP (3 nM-1 microM) produced a biphasic positive inotropic response. The late component of this response was selectively abolished by in vitro capsaicin pretreatment while both the early and late responses were abolished by indomethacin. 3. The inotropic response to FMLP in the guinea-pig atria was unaffected by ruthenium red. The late but not the early response was strongly inhibited or abolished by tetrodotoxin (TTX), omega-conotoxin (CTX) or by the C-terminal fragment (8-37) of human alpha-calcitonin gene-related peptide (hCGRP). hCGRP-(8-37) acts as competitive antagonist at CGRP receptors. 4. In the guinea-pig isolated bladder, FMLP (10 nM-10 microM) produced a concentration-dependent contraction which was unchanged by previous in vitro capsaicin, TTX or CTX pretreatment. The response to low concentrations of FMLP was suppressed by indomethacin, irrespective of the capsaicin pretreatment. 5. FMLP (10 microM) produced a significant increase in the outflow of CGRP-like immunoreactivity (CGRP-LI) from superfused guinea-pig atria or urinary bladder. CGRP-LI outflow induced by FMLP was blocked by indomethacin or in vitro capsaicin pretreatment. 6. These findings indicate that FMLP activates the 'efferent' function of capsaicin-sensitive primary afferents via prostanoid generation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid receptors and prejunctional modulation of capsaicin-sensitive sensory nerves in guinea-pig left atrium

1. In the isolated electrically driven left atria from reserpine-pretreated guinea-pigs and in presence of 1 microM atropine, electrical field stimulation (EFS) at 10 Hz produces a delayed positive inotropic response (DPIR) involving activation of capsaicin-sensitive afferents. 2. Opioids inhibited the DPIR with the following order of potency: dermorphin greater than [D-Ala2,N-MePhe4, Gly5-ol]-enkephalin (DAGO) greater than or equal to [D-Ala2,D-Leu5]-enkephalin (DADLE) greater than morphine greater than dynorphin A (1-13) greater than [D-Pen2,D-Pen5]-enkephalin (DPDPE). U-50488 was ineffective up to 10 microM. 3. Opioids also inhibited resting inotropism (3 Hz) with the following rank order of potency: DADLE greater than DAGO greater than U-50488 = dynorphin A (1-13) = morphine = DPDPE. 4. Both inhibition of the DPIR and inhibition of resting inotropism were prevented by 10 microM naloxone. 5. Neither dermorphin (0.1 microM) nor DAGO (0.3 microM) or DADLE (1 microM) inhibit responses produced by capsaicin (30 nM) or calcitonin gene-related peptide (3 nM). 6. These findings indicate that capsaicin-sensitive nerves in the guinea-pig atrium are endowed with mu opioid receptors which inhibit transmitter release when sensory nerve terminals are activated by EFS but not by capsaicin.     

Evidence that toluene diisocyanate activates the efferent function of capsaicin-sensitive primary afferents

Isocyanates are an important cause of occupational asthma. The mechanism of isocyanate-induced asthma is still unknown. To determine whether toluene diisocyanate stimulates the 'efferent' function of peripheral endings of capsaicin-sensitive sensory nerves, we investigated the effect of toluene diisocyanate in the rat isolated urinary bladder, a preparation in which the action of capsaicin has been well characterized. Toluene diisocyanate (0.03-3 mM) produced a concentration-dependent contraction of the bladder strips. Its maximal effect was about 50% of the response to capsaicin (1 microM). Previous exposure of the strips to capsaicin followed by washing out produced complete unresponsiveness, both to the first exposure to toluene diisocyanate and to a second exposure of capsaicin. Further, the response to both toluene diisocyanate and capsaicin was completely prevented by extrinsic bladder denervation, achieved by bilateral removal of pelvic ganglia (72 h before). Repeated exposure of the rat bladder to toluene diisocyanate reduced the capsaicin-evoked release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI), taken as biochemical marker of activation of these sensory nerves. These experiments provide the first evidence that toluene diisocyanate activates directly or indirectly the efferent function of capsaicin-sensitive primary sensory nerves.     

Effects of opioid drugs on capsaicin-sensitive neurones in guinea-pig atria

Transmural nerve stimulation of isolated guinea-pig atria in the presence of atropine induced a biphasic positive inotropic effect but only a slow increase in contractility (NANC response) in atria obtained from 6-hydroxydopamine-pretreated animals. The latter effect disappeared after exposure of the preparations to capsaicin. The effects of some opioid peptides were investigated on NANC responses. [D-Ala²,D-Leu⁵]enkephalin (DADLE) and [D-Ala²,N-Me-Phe⁴ Gly⁵-ol]enkephalin (DAGO, 0.1–10 μM) inhibited the cardiac response to transmural nerve stimulation in a dose-dependent and naloxone-sensitive manner. Dynorphin-(1–13) and morphine, at 10-fold higher concentrations (1–10 μM), reduced the response in a naloxone-sensitive manner. Naloxone alone however did not affect the response. Opioid peptides were not able to reduce the positive inotropic effect induced by calcitonin gene-related peptide (CGRP), or the increase in cardiac contractility produced by capsaicin. These results suggest that opioid receptors exert a modulatory role on peripheral terminals of capsaicin-sensitive sensory nerves.     

Different effect of antiulcer agents on rat cysteamine-induced duodenal ulcer after sialoadenectomy, but not gastrectomy

An attempt has been made to pharmacologically isolate cholinergic, P(2) purinoceptor-mediated and peptidergic (capsaicin-sensitive, tachykinin-mediated) contraction of the guanethidine-treated rat bladder detrusor preparation, in vitro. The effect of experimental diabetes was assessed on these types of contraction. Responses were evoked by electrical field stimulation (single shocks or 1 Hz for 30 s or 10 Hz for 40 s). Single shocks and 1-Hz stimulation were applied in the presence of (a). atropine (1 microM) or (b). P(2) purinoceptor antagonists (50 microM pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) [PPADS] plus 100 microM suramin. Long-term electrical field stimulation (10 Hz for 40 s) (c). was applied with both atropine and the P(2) purinoceptor antagonists present in the organ bath. The effects of capsaicin (d). and ATP (e). were also studied. Three groups of experimental animals were used: streptozotocin-treated (50 mg.kg(-1) i.p., 8 weeks before the experiment), parallel solvent-treated and untreated rats. (a). Responses to electrical field stimulation in the presence of atropine were reduced by half by PPADS plus suramin, but were resistant to capsaicin tachyphylaxis. They were enhanced in preparations taken from diabetic rats. (b). Contractions to electrical field stimulation in the presence of PPADS plus suramin were reduced by 2/3 by atropine, but were left unchanged by capsaicin or diabetes. (c). Contractions to long-term stimulation had a quick and a sustained phase. Especially the latter was inhibited by capsaicin tachypyhlaxis; it was also strongly reduced in preparations taken from diabetic rats. (d). Contractions to capsaicin (30 nM and 1 microM) were resistant to tetrodotoxin, strongly reduced by a combination of tachykinin NK(1) and NK(2) receptor antagonists, and slightly reduced in preparations from diabetic animals. Capsaicin (1 microM) had no acute inhibitory action on cholinergic or purinergic responses, nor did it cause relaxation in precontracted preparations treated with tachykinin receptor antagonists. (e) ATP-induced contractions were strongly reduced by PPADS plus suramin (50 plus 100 microM) and to a similar degree by 100 plus 200 microM, respectively. It is concluded that experimental diabetes selectively impairs peptidergic, capsaicin-sensitive responses (especially those that involve impulse conduction) in the rat detrusor preparation. The contractile response to electrical field stimulation that remains after atropine plus the P(2) purinoceptor antagonists has a yet unknown transmitter background.     

Cadmium chloride induces contractions of the rat isolated urinary bladder by activation of capsaicin-sensitive sensory nerves

Cadmium chloride (CdCl2)(30 microM-1 mM) produced a concentration-related contraction of the rat isolated urinary bladder which was abolished by tetrodotoxin (1 microM) or extrinsic bladder denervation (72 h before). Complete cross-desensitization was observed between the contractile response to Cd and capsaicin, indicating that, at the peripheral level, this inorganic calcium channel blocker can activate the 'efferent' function of capsaicin-sensitive sensory nerves.     

Pharmacological Characterization of the Autonomous Innervation of the Guinea Pig Tracheobronchial Smooth Muscle

Abstract: From the trachea, main bronchi and hilus bronchi of guinea pigs, preparations were isolated for registration of mechanical tension on electrical field stimulation and drugs. The trachea contracted on short trains of electrical stimulation. Usually these contractions were followed by a relaxant response. The contractions were abolished while the relaxations were potentiated by atropine. In the main bronchi field stimulation induced a contractile response which was abolished by atropine. In the latter preparation relaxant responses were never observed, even not after atropine. In the trachea and main bronchi neither the α₁ blocker prazosin nor the α₂ blocker yohimbine affected the contractile or relaxant responses when used in α-blocking concentrations. The relaxant response in trachea was neither affected by the “P1 blocker” theophylline nor by the “P2 blocker” quinidine but it was partially inhibited by guanethidine or β-blocking agents. The hilus bronchi contracted on field stimulation as well as by histamine. The contractile response on electrical stimulation was only slightly inhibited by atropine or guanethidine. In all three preparations responses on field stimulation were abolished by tetrodotoxin. It is suggested that in both trachea and main bronchi excitatory cholinergic innervation is present. Further, the trachea but not the main bronchi is innervated by both adrenergic and nonadrenergic inhibitory nerves. The hilus bronchi contains a non-adrenergic non-cholinergic excitatory nervous system.     

Connections between P2 purinoceptors and capsaicin-sensitive afferents in the intestine and other tissues.

Relations between P2 purinoceptors and capsaicin-sensitive sensory neurons include an excitatory action of P2 purinoceptor agonists on spinal afferent neurons, as well as release of ATP from afferents at their central and peripheral endings, and a possible participation of ATP in nociception and/or in 'local efferent' responses mediated by sensory nerves at the periphery. The present paper briefly summarizes available evidence on these interrelations. Ample evidence shows that ATP and other P2 purinoceptor agonists can activate primary afferent neurons, through P2X3 receptors and probably other purinoceptors as well, but evidence for an involvement of P2 purinoceptors in nociception or in 'local efferent' responses due to activation of primary afferents is, at best, circumstantial. The possibility is also dealt with that P2 purinoceptor activation may cause small intestinal contraction with the mediation of capsaicin-sensitive sensory neurons and that the motor response to capsaicin in this tissue may involve the release of a P2 purinoceptor stimulant from sensory nerves. Our data show that cholinergic contractions of the guinea-pig ileum in response to the P2 purinoceptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) are blocked by atropine, but not by in vitro capsaicin pretreatment (which completely blocks the contractile action of capsaicin). Cholinergic ileum contractions due to capsaicin (2 microM) are insensitive to suramin (a P2 purinoceptor antagonist; 100 microM). In the presence of antagonists acting at tachykinin NK1 and NK2 receptors, however, suramin (100 microM) causes a significant inhibition of the capsaicin-evoked contraction. These data indicate that capsaicin-sensitive nerves are not involved in the excitatory effect of alpha,beta-methylene ATP on myenteric neurons. On the other hand, ATP is probably involved in the 'non-tachykininergic' component of the capsaicin-induced excitatory response of the small intestine. ATP may originate from sensory neurons and probably acts as activator of myenteric nerves.     

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.     

The effect of thiorphan and epithelium removal on contractions and tachykinin release produced by activation of capsaicin-sensitive afferents in the guinea-pig isolated bronchus

Summary (1) We have studied the effect of epithelium removal (rubbing) and the endopeptidase 24.11 inhibitor, thiorphan, on the contractile response of the guinea-pig isolated bronchi (atropine and indomethacin in the bath) produced by electrical field stimulation, capsaicin or exogenously administered tachykinins (substance P and neurokinin A). (2) The response to field stimulation, thought to involve release of endogenous tachykinins, was potentiated by thiorphan in both epithelium-free and intact bronchi. However, at low frequencies (1–5 Hz), the effect of thiorphan was more evident in intact preparations. (3) The response to capsaicin was enhanced by both epithelium removal and thiorphan administration. (4) The response to exogenous substance P or neurokinin A was potentiated by thiorphan both in epithelium-free and intact bronchi. (5) Capsaicin (1 M) evoked a consistent release of substance P-like immumoreactivity (determined by radioimmunoassay) and tachykinin-like immumoreactivity (determined by a novel immumoenzyme assay), which was enhanced by thiorphan in both epithelium-free and intact bronchi. (6) These findings suggest that a thiorphan-sensitive mechanism, presumably enkephalinase (endopeptidase 24.11), plays a major role in inactivating endogenous tachykinins released from sensory nerves and that this enzymatic activity is still present after removal of the bronchial epithelium. Send offprint requests to C. A. Maggi at the above address     

Differential effects of neuropeptide Y and opioids on neurogenic responses of the perfused rat mesentery

In perfused rat mesentery transmural nerve stimulation activates both adrenergic and capsaicin-sensitive sensory nerves. When adrenergic nerves were blocked with guanethidine and smooth muscle tone was increased, transmural nerve stimulation caused a dilator response which was attenuated by tetrodotoxin and abolished by capsaicin. Indomethacin increased the vasodilator response to transmural nerve stimulation, but did not affect the dilation to calcitonin gene-related peptide. Neuropeptide Y (NPY) potentiated vasoconstrictor responses to transmural nerve stimulation, but suppressed capsaicin-sensitive vasodilation, an effect which was unaltered by indomethacin. Opioid agonists selective for mu, delta or kappa receptors, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin), DPDPE ([D-Pen2,D-Pen5]enkephalin) and ethylketocyclazocine, had no effect on the vasoconstrictor response to transmural nerve stimulation. DAMGO and DPDPE significantly inhibited vasodilator responses to transmural nerve stimulation, but ethylketocyclazocine was without effect. After treatment with indomethacin, DAMGO still inhibited the vasodilator response, but DPDPE was no longer effective. Prejunctional control of transmitter release by NPY or opioids is dependent on the specific nerve type as well as, in some cases, the participation of endogenous prostaglandins.     

Effects of capsaicin on nonadrenergic noncholinergic nerves in the guinea pig atria: role of calcitonin gene-related peptide as cardiac neurotransmitter

Transmural nerve stimulation (TNS) in vitro produced positive chronotropic and inotropic responses in the spontaneously beating right atria and electrically driven left atria of guinea pigs, respectively. Both the positive responses of the atria were mediated not only by adrenergic but also by nonadrenergic noncholinergic (NANC) nerves. Numerous calcitonin gene-related peptide (CGRP)-like immunoreactive nerves were found in the sinus node and the muscle layers of both atria of guinea pigs. Exogenously applied CGRP produced positive chronotropic and inotropic responses in a dose-dependent manner. In both the right and left atria, capsaicin (10(-5) M) induced positive chronotropic and inotropic responses. The second application of capsaicin did not cause any responses in the atria, indicating a rapid development of tachyphylaxis. When the tissues developed tachyphylaxis to capsaicin, the intensity of CGRP-like immunoreactivity greatly decreased in the sinus node and in both atria. In the atria that developed tachyphylaxis to capsaicin, the positive chronotropic and inotropic responses to NANC nerve stimulation diminished markedly, but the responses to exogenous CGRP and those attributed to adrenergic nerves were not affected. These results suggest that CGRP is the neurotransmitter of intracardiac NANC nerves and that capsaicin produces positive chronotropic and inotropic responses through the release of CGRP from NANC nerves in the guinea pig atria. It is also suggested that capsaicin depletes endogenous CGRP, thereby accounting for the rapid development of tachyphylaxis to capsaicin and the marked and selective diminution of TNS-induced NANC responses following application of capsaicin.     

Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder

Capsaicin (0.03-3 microM) induces contractions of the rat isolated bladder which are unaffected by either atropine (3 microM) or tetrodotoxin (0.5 microM). In the presence of capsaicin (0.1 microM) an enhancement of field stimulation-induced contractions was observed. Capsaicin-desensitization did not modify the height of these. The neurogenic nature of the capsaicin-induced contractions was proved by the observation that 'chronic' (48 h) denervation prevented, while 'acute' (2 h) denervation did not modify the effect of capsaicin. Denervated bladders maintained their responsiveness to acetylcholine but not to field stimulation. Isolated bladders from rat pups (1-2 days old) did not respond to capsaicin while strong contractile responses to acetylcholine or field stimulation were obtained in these preparations. In bladders from two week old animals, capsaicin produced similar contractions to those observed in preparations from adult animals. The bladders from rats receiving a high dose of capsaicin (50 mg kg-1 s.c.) at birth were heavier than those of their age-matched, vehicle-treated controls. Isolated bladders from 2 month old animals pretreated with capsaicin at birth were unresponsive to capsaicin while responsiveness to acetylcholine, substance P or field stimulation was unaffected compared with that of vehicle-treated controls. These experiments provide evidence that a capsaicin-sensitive innervation exists in the rat urinary bladder which undergoes a postnatal development at end organ level.     

Neurochemical evidence of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release from capsaicin-sensitive nerves in rat mesenteric arteries and veins

1. The ability of capsaicin and antidromic stimulation of perivascular nerve fibers to release sensory neuropeptides (SP-LI and CGRP-LI) have been investigated in rat mesenteric arteries and veins. 2. Both in mesenteric arteries and veins substantial SP-LI and CGRP-LI tissue levels were measured. A significant reduction in sensory neuropeptides levels was observed in tissues obtained from capsaicin-pretreated animals. 3. Superfusion of isolated vessels with capsaicin (1 microM) produced a prompt and remarkable release of both SP-LI and CGRP-LI, which can be evoked only once in each preparation. 4. Electrical field stimulation (EFS, 20 Hz, 50 V, 0.5 msec, trains of 10 sec every 20 sec for 15 min) of isolated vessels resulted in a significant release of CGRP-LI. This release was significantly greater in veins as compared to arteries. EFS-induced CGRP-LI release was unaffected by atropine or guanethidine and absent in preparations obtained from capsaicin-pretreated rats. 5. These neurochemical findings further suggest that the local release of sensory neuropeptides from capsaicin-sensitive nerve endings might be important in the regulation of mesenteric circulation.     

Suppression by neuropeptide Y of capsaicin-sensitive sensory nerve-mediated contraction in guinea-pig airways.

1. In the present study we have examined whether neuropeptide Y (NPY) interferes with non-adrenergic, non-cholinergic nerve-mediated contractions and relaxations in the guinea-pig airways. In these experiments we have used ring preparations of bronchi and trachea, incubated in the presence of atropine, propranolol and indomethacin (each 1 microM). 2. The contractile response to electrical stimulation of non-adrenergic, non-cholinergic nerve fibres was suppressed by NPY and NPY 13-36 in a concentration-dependent manner, these agents having similar inhibitory potencies. NPY caused a more complete inhibition than the C terminal fragment. 3. NPY affected neither the basal tension nor the substance P-evoked contraction in the bronchi and trachea and did not interfere with nerve-mediated, non-adrenergic relaxation in the trachea. 4. On the basis of these results, it is suggested that NPY may act on the terminals of sensory neurones in the airways to prevent antidromic, excitatory neurotransmission by inhibiting transmitter release.