Adenosine receptors involved in the inhibitory control of non-adrenergic non-cholinergic neurotransmission in guinea-pig atria belong to the A1 subtype

Summary We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N⁶cyclopentyladenosine (CPA), 5 N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA.The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC₅₀ value of 2.8 nM; RPIA, NECA and 2-CADO showed IC₅₀ values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC₅₀ value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRR Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA₂ values (6.77±0.01 and 6.63±0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype. Send offprint requests to A. Rubino at the above address     

Actions of calcitonin gene-related peptide and tachykinins in relation to the contractile effects of capsaicin in the guinea-pig and rat heart in vitro

Summary 1. The effects of calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide K (NPK) and substance P (SP) on contractility of the guinea-pig and rat heart were studied in vitro in relation to the response to capsaicin. 2. Human (h) CGRP alpha (a) and beta () were equipotent in stimulating the contractile force and rate of the spontaneously beating guinea-pig right atrium. 3. Both NKA and NPK inhibited contractile force and rate in the guinea-pig atrium whilst a mainly negative chronotropic effect was observed in the whole heart. SP did not influence cardiac contractility. 4. Human CGRP and mimicked the contractile effects of capsaicin in the guinea-pig atrium. In the whole heart preparation, hCGRP and capsaicin increased heart rate whereas capsaicin also evoked an atropine-resistant inhibition of contractile force. The stimulatory effect of capsaicin on heart rate was absent after systemic capsaicin pretreatment, while the inhibition of ventricular contractile tension remained unchanged. 5. Extended incubation with hCGRP or was associated with development of cross-tachyphylaxis between these two agents in the guinea-pig atrium. However, CGRP tachyphylaxis did not change the atrial response to noradrenaline, forskolin or NKA. The stimulatory effects of capsaicin on atrial contractility were absent after tachyphylaxis to hCGRP or . 6. There was no detectable supersensitivity to the stimulatory effects of rat (r) CGRP on the spontaneously beating atrium after capsaicin pretreatment of adult or neonatal rats. In conclusion the present data provide further evidence that the capsaicin-induced stimulation of atrial contractility is due to local release of CGRP. CGRP tachyphylaxis seems to be a specific, receptor-mediated event and is not related to down-regulation of the adenylate cyclase system. The inhibition of the contractile force in whole heart preparations by capsaicin is most likely due to a non-cholinergic effect independent of tachykinin release. Send offprint requests to A. Franco-Cereceda at the above address     

Adenosine receptor modulation of sympathetic neurotransmission in rat isolated kidney

In the present study we set out to define, using discriminatory agonists and antagonists, the adenosine receptors modulating sympathetic neurotransmission in the rat kidney. Isolated kidneys from male Wistar rats were perfused with modified Krebs-Henseleit buffer solution at constant flow. The neuronal noradrenaline stores were labeled with ³H-noradrenaline and the renal nerves stimulated electrically (2 Hz, 3 msec, 9 mA, during 20 sec at intervals of 6 min). ³H overflow was taken as an index of ³H-noradrenaline release. The A₁ receptor selective agonists N⁶-cyclopentyladenosine (CPA), N⁶-cyclohexyladenosine (CHA), and N⁶-[R(−)-1-phenyl-2-propyl]adenosine (R-PIA), and the mixed A₁/A_2A receptor agonists 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) inhibited evoked ³H outflow concentration-dependently. The selective A_2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), at concentrations selective for A_2A receptors, failed to modify ³H outflow, whereas at higher concentrations it induced inhibition. The rank order of potency of agonists, CPA > CHA = R-PIA > NECA > CADO >> CGS 21680, is typical for an interaction with the A₁ receptor. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), at concentrations selective for blockade of A₁ receptors, blocked concentration-dependently the inhibitory effects of CPA and NECA; no evidence of an increase in outflow was seen with NECA in the presence of DPCPX. The selective A_2A receptor antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazol[1,5-c] quinazoline-5-amine (CGS 15943) did not influence the agonist effects at concentrations interacting selectively with A_2A receptors but antagonized them concentration-dependently at higher, non-selective concentrations. Taken together, our data establish the presence of inhibitory adenosine A₁ receptors on the terminal sympathetic neurons of rat kidney. No evidence was obtained for the presence of functional A_2A receptors in this preparation. © 1996 Wiley-Liss, Inc.     

Theophylline-sensitive modulation of non-cholinergic excitatory neurotransmission in the guinea-pig ileum.

Atropine-resistant longitudinal contractions of the guinea-pig ileum due to field stimulation were greatly augmented by theophylline (150 microM). Adenosine exerted an opposite effect. Theophylline did not potentiate contractions evoked by exogenous substance P. It is suggested that a theophylline-sensitive inhibitory mechanism (possibly mediated by adenosine or a related substance) controls transmitter release from enteric non-cholinergic excitatory neurones.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea

BACKGROUND AND PURPOSE: Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. EXPERIMENTAL APPROACH: NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method.Key results:alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. CONCLUSIONS AND IMPLICATIONS: In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.     

Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01–1 μM) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05).
2. The μ-, δ- and κ-opioid receptor antagonists, naloxone (0.3 μM), naltrindole (3 μM) and nor-binaltorphimine (1 μM), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 μM; P>0.05).
3. The novel nociceptin antagonist, [Phe¹ψ(CH₂-NH)Gly²]nociceptin(1–13)NH₂ (0.03–1 μM); the σ ligands, carbetapentane (30 μM), 3-phenylpiperidine (30–100 μM) and (+)-cyclazocine (10–100 μM) significantly reversed the inhibitory effect of nociceptin (0.03 μM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 μM) at any concentration tested (P>0.05).
4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 μM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59±11% (n=5) inhibition of EFS-induced release of SP-LI.
5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.

     

Selective sensory denervation by capsaicin aggravates adriamycin-induced cardiomyopathy in rats

Capsaicin-sensitive sensory nerves that contain calcitonin gene-related peptide (CGRP) contribute significantly to cardioprotective mechanisms. In this study, the possible role of capsaicin-sensitive afferent nerves in the development of congestive heart failure was examined in an established model of adriamycin-induced experimental cardiomyopathy in rats. Systemic treatment with capsaicin was utilized to deplete sensory neuropeptides from cardiac afferent nerves. Echocardiography was applied to assess the cardiac function in adriamycin-treated rats pretreated with capsaicin or its vehicle. In control rats, adriamycin treatment produced a reduction in the fractional shortening of the left ventricle and an increase in the ratio of the left atrial diameter and the aortic diameter, indicative of a decreased myocardial contractility and heart failure only at 3–4 weeks post-treatment. In contrast, in capsaicin-pretreated rats, a deterioration of the cardiac function was already evident 1 week after the cessation of adriamycin administration, while the clinical signs associated with cardiomyopathy were more severe and displayed a significantly more rapid progression. Immunohistochemistry revealed a complete depletion of calcitonin gene-related peptide from cardiac sensory nerves after systemic capsaicin treatment. This study has demonstrated that elimination of capsaicin-sensitive afferent nerves promotes the development and progression of adriamycin-induced myocardial dysfunction. The results suggest that interfering with capsaicin/vanilloid receptor function and/or perturbation of the myocardial CGRP metabolism may open up new perspectives concerning prevention and/or alleviation of the pathological changes that follow adriamycin treatment.     

Adenosine deaminase antagonizes inhibitory responses to adenosine and non-adrenergic, non-cholinergic inhibitory nerve stimulation in isolated preparations of guinea-pig trachea.

In guinea-pig isolated tracheal tube preparations treated with dipyridamole, adenosine deaminase antagonized inhibitory responses to adenosine and non-adrenergic, non-cholinergic nerve stimulation. Inhibitory responses to vasoactive intestinal peptide (VIP) were unaffected and hyoscine-sensitive excitatory responses to field stimulation were not reduced. The evidence supports a role of adenosine as an inhibitory neurotransmitter in non-adrenergic, non-cholinergic nerves in trachea.     

Evidence for the involvement of calcitonin gene-related peptide in the epithelium-dependent contraction of guinea-pig trachea in response to capsaicin

Summary Capsaicin (10^–9 to 10^–5 M) contracted guinea-pig tracheal strips. Epithelium-containing tracheal strips developed a maximum active tension which was significantly higher than that observed in epithelium-free strips. Anti-CGRP (calcitonin gene-related peptide) serum blocked the epithelium-dependent potentiation of the capsaicin-induced contraction in the intact tracheal strips, without affecting the response of the epithelium-free strips. This result suggests the occurrence of an epithelium-dependent release of CGRP. This same serum markedly reduced the contraction induced by exogenous rat CGRP in both intact and epithelium-free tracheal strips. In epithelium-free tracheal strips, capsaicin-induced contraction was abolished by spantide (10^–6 and 10^–5 M), a substance P antagonist, but, in intact tracheal strips, spantide did not abolish the capsaicin-induced contraction, showing that both CGRP and substance P release are directly induced by capsaicin. Moreover, the contractile responses to rat CGRP of intact tracheal strips from guinea pig suggest that CGRP itself might be able to release a contracting factor from the airway epithelium. Therefore, CGRP originating from the airway epithelium may play a major role in the control of airway smooth muscle tone.Send offprint request to E. Tschirhart at the above address     

Regulation by phosphodiesterase isoenzymes of non-adrenergic non-cholinergic contraction in guinea-pig isolated main bronchus.

1. We have investigated the role of phosphodiesterase isoenzymes in modulating electric field stimulation (EFS), substance P and capsaicin-induced contraction of the guinea-pig isolated main bronchus. 2. Non-adrenergic non-cholinergic contractile responses were elicited by EFS (3 Hz, 20 s) in the guinea-pig isolated main bronchus in the presence of the non-selective muscarinic antagonist, atropine (0.1 microM), the non-selective beta-adrenoceptor antagonist, propranolol (1 microM), the neutral endopeptidase inhibitor, thiorphan (10 microM) and the cyclo-oxygenase inhibitor, indomethacin (5 microM). The type III, type III/IV, type IV and type V phosphodiesterase isoenzyme inhibitor, SKF 94836, benzafentrine, Ro-20-1724 and zaprinast respectively, significantly attenuated the contractile response to EFS. The IC50 (95% confidence limits) value for SKF 94836, benzafentrine, Ro-20-1724 and zaprinast was 8.3 microM (0.89-78); 0.7 microM (0.1-4.5); 0.5 microM (0.2-1.2) and 13 microM (2-87) respectively. 3. The phosphodiesterase isoenzyme inhibitors, SKF 94836, Ro-20-1724 and zaprinast, partially attenuated the contractile response to substance P (10 nM). Benzafentrine significantly inhibited the contractile response to substance P, yielding an IC50 value of 1.9 microM (0.9-3.8). 4. The phosphodiesterase isoenzyme inhibitor, Ro-20-1724 (0.1-100 microM) failed to reduce significantly the contractile potency of capsaicin (P > 0.05). In contrast, SKF 94836 (1 microM), benzafentrine (10 microM) and zaprinast (100 microM) significantly reduced the contractile potency of capsaicin (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple mechanisms in the smooth muscle relaxant action of calcitonin gene-related peptide (CGRP) in the guinea-pig ureter

We have investigated the ability of human CGRP (CGRP) to inhibit the electrically-evoked myogenic contractions of the guinea-pig ureter, in comparison with the K channel opener, cromakalim, and the adenylate cyclase activator, forskolin. CGRP (0.1 nM -0.1 M) produced a concentration-dependent inhibition of the evoked contractions; its action was prevented by the CGRP receptor antagonist, CGRP(8–37) (1 M), while it was unaffected by the nitric oxide (NO) synthase inhibitor, L-nitroarginine (30 M). The effect of CGRP was antagonized in a noncompetitive manner (depression of E _max, no change in EC₅₀) by glibenclamide (1–10 M), a blocker of ATP-sensitive potassium channels (K_ATP). A substantial fraction of the inhibitory effect of CGRP was glibenclamide-resistant, however. Glibenclamide also blocked the inhibitory action of cromakalim (0.1–10 M) without affecting the inhibition produced by forskolin (0.1–30 M). When tested in a low-K medium (extracellular K reduced from 5.9 to 1.2 mM), the inhibitory effects of CGRP, cromakalim and forskolin were enhanced. The inhibitory effect of forskolin was partly antagonized by glibenclamide when tested in a low-K medium.CGRP (0.1 M), cromakalim (3 M) and forskolin (10 M) inhibited the contractile response to KCl (80 M), which is characterized by a distinct phasic and tonic component: cromakalim selectively inhibited the phasic response to KCl with CGRP and forskolin inhibited both components. The inhibitory effect of CGRP on the phasic contraction to KCl was partly glibenclamide (1 M) sensitive, while that on the tonic contraction was glibenclamide-resistant. The inhibitory action of forskolin on both components of the response to KCl was unchanged by glibenclamide. The inhibitory effect of cromakalim on the phasic response was prevented by glibenclamide.In sucrose gap electrophysiological experiments, CGRP (0.3 M) produced hyperpolarization of the membrane, increased the delay between stimulus application and onset of the action potential, shortened the action potential duration and eventually suppressed both action potential and phasic contractions in 4 out 8 cases tested. The membrane hyperpolarization and the ability of CGRP to suppress the evoked action potential were completely prevented by glibenclamide (1 M), but an inhibitory effect on the action potential duration and contractility persisted in the presence of glibenclamide.We conclude that activation of glibenclamide-sensitive K channels mediates part of the smooth muscle relaxant action of CGRP, and accounts for the ability of CGRP to suppress phasic contractions evoked through electromechanical coupling. However, a glibenclamide-resistant component also exists in the smooth muscle relaxant activity of CGRP. The possible involvement of cAMP accumulation as an intermediate link between CGRP receptor occupation and activation of K channels by CGRP remains speculative. Correspondence to: C. A. Maggi at the above address     

Role of Ca2+ in non-cholinergic,non-adrenergic responses to nerve stimulation of the guinea-pig small intestine

Summary The effects of calcium channel blockers (D-600, verapamil), sodium nitroprusside, papaverine, indomethacin, local anaesthetics and blockade of sodium pump activity on the non-cholinergic, non-adrenergic transmission in the guinea-pig duodenum, jejunum, proximal and terminal ileum were analysed in the presence of atropine and guanethidine.A decrease of the extracellular Ca²⁺ concentration inhibited the primary and rebound contractions but only in Ca²⁺-free solution was the primary relaxation diminished. D-600, verapamil, sodium nitroprusside and papaverine inhibited both the primary and rebound contractions to the same degree and their effects on the primary relaxation were less pronounced than on the contractions.Indomethacin dissolved in alkaline solution did not depress the non-cholinergic, non-adrenergic responses in any region of the small intestine, whereas indomethacin dissolved in ethanol antagonized both the primary and rebound contractions in the muscles.Local anaesthetics (procaine, trimecaine) in low concentrations inhibited only the primary contraction. Higher concentrations also inhibited both the rebound contraction and primary relaxation. Procaine in low concentrations did not markedly affect the non-cholinergic, non-adrenergic i.j.p.s and e.j.p.s., but did block the action potentials induced by e.j.p.s.Our findings indicate that the primary relaxation, and primary and rebound contractions are probably induced by different mechanisms and are not mediated by ATP. We confirmed that prostaglandins did not participate in the generation of the rebound contraction.This study was supported in part by JSPS Japan     

L-NG-nitro arginine inhibits non-adrenergic, non-cholinergic relaxations of guinea-pig isolated tracheal smooth muscle.

The effects of L-NG-nitro arginine (L-NOARG) on alpha-chymotrypsin-resistant, non-adrenergic, non-cholinergic (NANC) relaxations of guinea-pig tracheal smooth muscle have been examined. L-NOARG (1-100 microM), but not D-NOARG (100 microM), inhibited the NANC relaxations in a concentration-related manner. The effects of L-NOARG were partially reversed by L-arginine but not D-arginine. L-NOARG was without effect on acetylcholine-induced contractile responses of the trachea or on relaxations produced by vasoactive intestinal peptide, sodium nitroprusside or isoprenaline. These results suggest that an endogenous nitrate may contribute to NANC relaxations of tracheal smooth muscle.     

Characterization of histamine-H3 receptors controlling non-adrenergic non-cholinergic contractions of the guinea-pig isolated ileum.

1. In the presence of atropine, mepyramine and ranitidine, electric field stimulation of the guinea-pig isolated ileum longitudinal muscle-myenteric plexus preparation resulted in a two component non-adrenergic non-cholinergic contraction. The initial contraction had a duration of approximately 1 s whereas the second contraction lasted approximately 10 s. The second contraction was completely inhibited by tetrodotoxin (0.2 x 10(-6) M) with minimal effect on the initial contraction. Phentolamine (3 x 10(-6) M), propranolol (3 x 10(-6) M) and hexamethonium (10(-4) M), did not significantly reduce either component of the contractile response. 2. The neurokinin NK1 receptor antagonists, GR82334 and GR71251, produced concentration-related (EC50 = 564 and 173 nM respectively) inhibitions of the second contraction with no effect on the initial contraction. The neurokinin NK2 receptor antagonists MEN 10207 and Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 (R 396), 1 x 10(-9)-10(-5) M, were without effect on either component of the contractile response. 3. Concentration-related inhibitions of the second contraction, with no effect on the initial contraction, were observed after inclusion of the histamine H3 receptor agonists (R)-alpha-methylhistamine (pD2 = 7.6), N alpha-methylhistamine (pD2 = 7.7) and N alpha,N alpha-dimethylhistamine (pD2 = 6.3). Histamine also inhibited the second contraction (pD2 = 6.2) in a concentration-related manner but produced a lower maximum inhibitory effect than the other agonists tested. 4. Inclusion of the H3 receptor antagonists, thioperamide, burimamide, impromidine and phenylbutanoylhistamine, caused parallel concentration-related rightward shifts in the concentration-response curve to (R)-alpha-methylhistamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sildenafil on non-adrenergic non-cholinergic neurotransmission in bovine penile small arteries

The purpose of the present study was to investigate the effect of the phosphodiesterase isoenzyme V inhibitor, sildenafil, on non-adrenergic non-cholinergic neurogenic relaxations of intracavernous isolated penile small arteries. Dense plexes of nerve fibres immunoreactive for neural nitric oxide (NO) synthase were observed in the adventitia-media junction of the penile small arteries. In 5-hydroxytryptamine-contracted preparations, the inhibitor of NO synthase, N(G)-nitro-L-arginine (L-NOARG), and of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), reduced the electrical field stimulation-induced relaxations. Sildenafil and exogenous NO induced relaxations of penile small arteries. Sildenafil enhanced NO and vasoactive intestinal peptide-induced relaxations. Moreover, sildenafil increased the duration of the relaxations elicited by electrical field stimulation in penile small arteries and corpus cavernosum tissue. In the presence of L-NOARG, sildenafil only at supratherapeutic concentrations reduced the prazosin-sensitive contractions elicited by EFS in penile small arteries. Neurogenic NO-mediated and guanylyl cyclase-dependent relaxations of penile small arteries and corpus cavernosum tissue, considered to be associated with the vasodilatation leading to erection, are selectively enhanced by an inhibitor of phosphodiesterase V.     

Role of N-type calcium channels in autonomic neurotransmission in guinea-pig isolated left atria.

1. Calcium entry via neuronal calcium channels is essential for the process of neurotransmission. We investigated the calcium channel subtypes involved in the operation of cardiac autonomic neurotransmission by examining the effects of selective calcium channel blockers on the inotropic responses to electrical field stimulation (EFS) of driven (4 Hz) guinea-pig isolated left atria. In this tissue, a previous report (Hong & Chang, 1995) found no evidence for N-type channels involved in the vagal negative inotropic response and only weak involvement in sympathetic responses. 2. The effects of cumulative concentrations of the selective N-type calcium channel blocker, omega-conotoxin GVIA (GVIA; 0.1-10 nM) and the non-selective N-, P/Q-type calcium channel blocker, omega-conotoxin MVIIC (MVIIC; 0.01-10 nM) were examined on the positive (with atropine, 1 microM present) and negative (with propranolol, 1 microM and clonidine, 1 microM present) inotropic responses to EFS (eight trains, each train four pulses per punctate stimulus). 3. GVIA caused complete inhibition of both cardiac vagal and sympathetic inotropic responses to EFS. GVIA was equipotent at inhibiting positive (pIC50 9.29+/-0.08) and negative (pIC50 9.13+/-0.17) inotropic responses. MVIIC also mediated complete inhibition of inotropic responses to EFS and was 160 and 85 fold less potent than GVIA at inhibiting positive (pIC50 7.08+/-0.10) and negative (pIC50 7.20+/-0.14) inotropic responses, respectively. MVIIC was also equipotent at inhibiting both sympathetic and vagal responses. 4. Our data demonstrates that N-type calcium channels account for all the calcium current required for cardiac autonomic neurotransmission in the guinea-pig isolated left atrium.     

Mediators of adenosine- and ovalbumen-induced bronchoconstriction of sensitized guinea-pig isolated airways

The mediators of bronchoconstriction of isolated lungs and trachea from ovalbumen sensitized guinea-pigs to adenosine and ovalbumen were examined using relevant antagonists. Changes in perfusion pressure and tension of paired lung halves and tracheal spiral strips, respectively, were recorded in response to adenosine (1 mM lung, 300 microM trachea), histamine (10 microM), methacholine (10 microM) and ovalbumen (10 microg). One half was perfused with antagonist while the other received vehicle. Tracheal strips were superfused throughout with the P(1) receptor antagonist 8-phenyltheophylline, to examine 8-phenyltheophylline-resistant responses. The histamine H(1) receptor antagonist, mepyramine (1.5 mM), the cyclooxygenase inhibitors, indomethacin (5 mM) and diclofenac (5 mM), the leukotriene receptor antagonist, zafirlukast (1 mM), and the lipoxygenase inhibitor, zileuton (20 mM), alone failed to inhibit bronchoconstriction by adenosine and ovalbumen of the lung and trachea. When two antagonists were combined, only mepyramine and zafirlukast significantly reduced the lung responses to adenosine and ovalbumen. The tracheal adenosine response was substantially reduced, although not significantly, while ovalbumen was significantly reduced. When mepyramine, indomethacin and zafirlukast were combined, the lung constriction by adenosine and ovalbumen were virtually abolished. Similarly, the combination of mepyramine, diclofenac and zafirlukast significantly attenuated the lung responses to adenosine and ovalbumen. Thus, histamine, cyclooxygenase products and leukotrienes alone are not responsible for the bronchoconstriction of isolated sensitized lung tissues to adenosine or ovalbumen, which appears to be due to the release of all three mediators.     

Adenosine-modulation of cholinergic and non-adrenergic non-cholinergic neurotransmission in the rabbit iris sphincter.

The characteristics of smooth muscle responses to transmural nerve stimulation in the rabbit iris sphincter were examined. Transmural stimulation elicited a composite contractile response that could be divided in two phases. Atropine abolished the phase I contraction and inhibited the phase II contraction. The atropine-resistant component of the phase II contraction which was unaltered by sympathetic denervation, was mimicked by substance P and abolished by capsaicin. Adenosine inhibited the phase I contraction. The adenosine analogue L-N6-phenylisopropyladenosine (L-PIA) was more potent than 5'-N-ethylcarboxamideadenosine (NECA) in mimicking this adenosine effect. By contrast, adenosine enhanced the phase II contraction in non-pretreated preparations, as well as the atropine-resistant capsaicin-sensitive part of this contraction. Here, NECA was more potent than L-PIA. Adenosine, NECA, L-PIA and D-PIA also enhanced the atropine-sensitive component of the phase II contraction, as well as the contractile response to exogenous acetylcholine or carbachol, but not to exogenous substance P. In this respect, L-PIA was the most powerful adenosine analogue with at least 10 fold higher potency than D-PIA. The adenosine antagonist 8-p-sulphophenyltheophylline enhanced the phase I contraction and decreased the capsaicin-sensitive non-adrenergic non-cholinergic component of the phase II contraction. We conclude that adenosine inhibited the nerve-induced cholinergic twitch (phase I) responses by action at prejunctional A1-receptors. Furthermore, adenosine enhanced the phase II contractile responses via postjunctional enhancement of the cholinergic transmission by action at A1-receptors, and via enhancement of the non-adrenergic non-cholinergic transmission by action at presumably prejunctional A2 receptors.     

Metabolism of endogenous and exogenous noradrenaline in guinea-pig atria

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from guinea-pig isolated atria was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs, the outflow of endogenous noradrenaline over a period of 3 h averaged 1.6 pmol×g^–1×min^–1 and the outflow of DOPEG 17 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.31 pmol×g^–1×min^–1). Tyramine greatly increased the outflow of noradrenaline and DOPEG, and the reserpine-like compound Ro 4-1284 selectively increased the outflow of DOPEG; DOMA remained below the detection limit. When atria were exposed to (–)-noradrenaline 1.7 or 17 M, the subsequent outflow of noradrenaline and DOPEG was enhanced. Moreover, substantial amounts of DOMA were now found. This outflow of DOMA was prevented when atria were exposed to (–)-noradrenaline in the presence of cocaine or after an initial incubation with amezinium. Exposure to (–)-noradrenaline 1.7 M mainly enhanced the formation of DOPGE, while exposure to (+)-noradrenaline 1.7 M mainly enhanced the formation of DOMA.Our experiments confirm some and qualify other conclusions drawn from studies in which exogenous ³H-noradrenaline had been used to examine the metabolism of noradrenaline in guinea-pig atria. In agreement with the isotope studies, DOPEG is a major metabolite of endogenous noradrenaline. In contrast to what the isotope studies had suggested, however, endogenous DOMA is a very minor product, at least as long as the neurones are at rest. DOMA is only formed when the tissue is exposed to high concentrations of exogenous noradrenaline. In further contrast to previous conclusions, DOMA is then formed intra- and not extraneuronally.