M. J. Rand

Nitric oxide and vasoactive intestinal polypeptide mediate non-adrenergic, non-cholinergic inhibitory transmission to smooth muscle of the rat gastric fundus

The nitric oxide (NO) synthesis inhibitor NG-monomethyl L-arginine (L-NMMA) reduced NANC-mediated relaxations of isolated strips of the rat gastric fundus elicited by low frequencies or short periods of field stimulation, but D-NMMA had no effect. The inhibitory effect of L-NMMA on NANC-mediated relaxations was partially reversed by L-arginine but was not affected by D-arginine. A VIP antibody abolished the relaxant response to VIP and reduced the responses to stimulation. Residual responses to stimulation in the presence of VIP antibody were further reduced by L-NMMA. The tone of the fundus strip was slightly increased by L-NMMA and slightly reduced by L-arginine. The relaxation produced by VIP was slightly reduced by L-NMMA and enhanced by L-arginine. Relaxations produced by peptide histidine isoleucine, sodium nitroprusside or isoprenaline were not affected by L-NMMA or L-arginine. The results suggest that NO as well as VIP is involved in NANC-mediated relaxations of the rat gastric fundus.

NITRERGIC TRANSMISSION: NITRIC OXIDE AS A MEDIATOR OF NON‐ADRENERGIC, NON‐CHOLINERGIC NEURO‐EFFECTOR TRANSMISSION

1. The possibility that transmission at some non‐adrenergic, non‐cholinergic (NANC) neuro‐effector junctions is mediated by nitric oxide (NO) arose from the discoveries that NO mediated the effects of nitrovasodilator drugs and that endothelium‐derived relaxing factor (EDRF) was NO or a NO‐yielding substance.

Evidence that part of the NANC relaxant response of guinea-pig trachea to electrical field stimulation is mediated by nitric oxide.

1 The nitric oxide (NO) synthesis inhibitors NG‐monomethyl l‐arginine (l‐NMMA) and l‐nitroarginine methyl ester (l‐NAME) reduced relaxations of guinea‐pig tracheal smooth muscle elicited by stimulation of intramural non‐adrenergic, non‐cholinergic (NANC) nerves, but d‐NMMA had no effect. l‐NAME was 10–30 times more potent than l‐NMMA. Relaxations produced by sodium nitroprusside and vasoactive intestinal polypeptide (VIP) were not affected by l‐NMMA or l‐NAME. 2 The inhibitory effect of l‐NMMA on NANC‐mediated relaxations was partially reversed by l‐arginine but was not affected by d‐arginine. 3 VIP antibody and α‐chymotrypsin abolished or greatly reduced the relaxant action of VIP and reduced relaxations elicited by stimulation of NANC nerves; the residual NANC relaxation was further reduced by l‐NAME. 4 The results suggest that NO and VIP are mediators of NANC‐induced relaxations of guinea‐pig tracheal smooth muscle. We propose the term ‘nitrergic’ to describe transmission processes which are mediated by NO.

EVIDENCE FOR A ROLE OF NITRIC OXIDE IN THE NEUROTRANSMITTER SYSTEM MEDIATING RELAXATION OF THE RAT ANOCOCCYGEUS MUSCLEC

1. The nitric oxide (NO) synthesis inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA), but not d‐NMMA, inhibited the NANC‐mediated relaxations of the rat anococcygeus muscle, but did not affect the relaxation produced by sodium nitroprusside.

ACTIVATION OF PREJUNCTIONAL β‐ADRENOCEPTORS IN RAT ATRIA BY ADRENALINE APPLIED EXOGENOUSLY OR RELEASED AS A CO‐TRANSMITTER

1 Adrenaline (10 nM) significantly enhanced the stimulation‐induced efflux of radioactivity from rat atria previously incubated with [3H]‐noradrenaline ([3H]‐NA). This effect was abolished by metoprolol (0.1 μm). 2 Adrenaline in a higher concentration (1μm) and NA (1μm) significantly reduced the stimulation‐induced efflux of radioactivity. However, in the presence of phenoxybenzamine (10μm), adrenaline (1μm) enhanced the efflux, whereas NA (1μm) had no effect. 3 In rat isolated atria pre‐incubated with adrenaline and then incubated with NA, both catecholamines were taken up and were released by field stimulation. When pre‐incubation was with adrenaline and incubation was with [3H]‐NA, metoprolol decreased the stimulation‐induced efflux of radioactivity. This effect did not occur if the atria were pre‐incubated with NA instead of adrenaline, suggesting that neuronally released adrenaline activates prejunctional β‐adrenoceptors. 4 In conscious rats, intravenously administered adrenaline (6.0 and 0.6 nmol/kg) was taken up and retained in the atria and could be released by field stimulation. The release was calcium‐dependent from these rats up to 24 h after administration.

Partial agonist action of clonidine on prejunctional and postjunctional α-adrenoceptors

SummaryThe effects of clonidine on prejunctional α-adrenoceptors in guinea-pig atria and postjunctional α-adrenoceptors in rabbit aorta were studied and compared.1.In isolated aortic strips, clonidine acted as a partial agonist on postjunctional α-adrenoceptors, causing smooth muscle contraction. There was no indirect component to the effect of clonidine. The contractile response to clondine was competitively antagonized by phentolamine. Clonidine competitively antagonized the contractile effect of noradrenaline.2.In isolated guinea-pig atria, the effect of clonidine on stimulation-induced release of transmitter noradrenaline was inversely dependent on the frequency of electrical stimulation. Using stimulation of 5 pulses (1 Hz for 5 s) and 20 pulses (2 Hz for 10 s), clonidine (10 μM) decreased release; using 50 pulses (2.5 Hz for 20 s) or 90 pulses (3 Hz for 30 s), release was unaffected; using 150 pulses (5 Hz for 30 s), release was enhanced.3.The inhibitory effect of clonidine on transmitter release in guinea-pig atria using 5 pulses (1 Hz for 5 s) was concentration-dependent.4.The enhancement of release in atria with clonidine (10 μM), using stimulation of 150 pulses (5 Hz for 30 s) was unaffected by cocaine (30 μM) but was reversed by phentolamine (3 μM).5.The results in guinea-pig atria are consistent with a partial agonistic effect of clonidine on prejunctional α-adrenoceptors; as an agonist clonidine is approximately 500 times more potent on these receptors than on postjunctional α-adrenoceptors in rabbit aorta.

Effects of ω‐conotoxin GVIA on autonomic neuroeffector transmission in various tissues

1 The effects of ω‐conotoxin GVIA (conotoxin), a potent inhibitor of neuronal N‐type Ca2+ channels, have been examined on responses to stimulation of noradrenergic, cholinergic and non‐adrenergic, non‐cholinergic (NANC) nerves in a range of isolated tissues to investigate the role of conotoxin‐sensitive Ca2+ channels in neurotransmission. 2 Contractions elicited by field stimulation of noradrenergic nerves in rat and mouse anococcygeus muscles, rabbit ear artery and rat vas deferens (epididymal portion) were inhibited by conotoxin. Responses to noradrenaline, and to adenosine triphosphate in the vas deferens, were not affected. 3 Positive chronotropic responses to field stimulation of noradrenergic nerves were inhibited by conotoxin in rat and mouse atria, but responses to noradrenaline and tyramine were not affected. 4 The stimulation‐induced release of noradrenaline was inhibited by conotoxin in the rabbit ear artery and in rat and mouse atria. 5 Relaxations in response to stimulation of the noradrenergic perivascular mesenteric nerves were reduced or abolished by conotoxin in rat and rabbit jejunum. The response to noradrenaline in rat jejunum was not affected. 6 Contractions elicited by stimulation of cholinergic nerves were inhibited by conotoxin in rat jejunum and mouse ileum (perivascular mesenteric nerves), and in guinea‐pig taenia caeci (field stimulation). Responses to acetylcholine in rat jejunum and mouse ileum were not affected. 7 Contractions elicted by stimulation of the cholinergic plus NANC pelvic nerves were inhibited by conotoxin in rabbit colon, and to a lesser extent in guinea‐pig colon. The stimulation‐induced contraction of the guinea‐pig colon was inhibited by conotoxin by a greater proportion in the presence than in the absence of atropine. Responses to acetylcholine were not affected in the rabbit colon but were slightly reduced in the guinea‐pig colon. 8 Relaxations in response to field stimulation of NANC nerves were inhibited by conotoxin in guinea‐pig taenia caeci and rat gastric fundus strips, and in rat anococcygeus muscle when the tone was raised by guanethidine but not when it was raised by carbachol. The relaxations produced by sodium nitroprusside in the rat gastric fundus and anococcygeus were not affected. 9 Contractions of the rat bladder elicited by stimulation of the peri‐urethral nerves, which are NANC‐and cholinergically mediated, were relatively insensitive to inhibition by conotoxin. The responses were almost completely abolished by tetrodotoxin. 10 The conotoxin‐induced inhibitions of responses to nerve stimulation developed slowly and persisted after removal of conotoxin. 11 The inhibitory effect of conotoxin was inversely proportional to the frequency of stimulation (in several preparations) and to the Ca2+ concentration in the bathing solution (in rat vas deferens). These observations suggest that the inhibition by conotoxin of the Ca2+ influx required for excitation‐secretion coupling in autonomic nerve terminals is not absolute, and can be overcome by repeated stimulation or by raising the Ca2+ concentration.

Differential effects of hydroxocobalamin on NO‐mediated relaxations in rat aorta and anococcygeus muscle

In rat aortic rings, hydroxocobalamin (10–30 μm) produced concentration‐dependent reductions of the relaxant action of nitric oxide (NO) and the endothelium‐dependent, NO‐mediated, relaxant action of acetylcholine. In anococcygeus muscles, hydroxocobalamin (10–30 μm) reduced but also prolonged, NO‐induced relaxations, but had no effect on non‐adrenergic, non‐cholinergic‐mediated relaxations. Hydroxocobalamin had no effect on the NO‐independent relaxant action of papaverine in either tissue. It is suggested that hydroxocobalamin sequesters NO by forming nitrosocobalamin. Nitrosocobalamin did not relax aortic rings, but produced a slowly developing and prolonged relaxation of anococcygeus muscles.

Differential actions of L‐cysteine on responses to nitric oxide, nitroxyl anions and EDRF in the rat aorta

The effects of L‐cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO•), nitroxyl (NO−) derived from Angelis salt and endothelium‐derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 μM or less of L‐cysteine had no effect on responses. Relaxations produced by exogenous NO• (0.25–2.5 μM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001–0.3 μM) were enhanced by 1 and 3 mM L‐cysteine. The enhancements by L‐cysteine of responses to NO• and sodium nitroprusside may be attributed to the formation of S‐nitrosocysteine. Relaxations mediated by the nitroxyl anion (0.3 μM) donated from Angelis salt were more prolonged than those produced by NO•, and nitroxyl‐induced relaxations were reduced by L‐cysteine (1 and 3 mM). EDRF‐mediated relaxations produced by acetylcholine (0.01–10 μM), ATP (3–100 μM) and the calcium ionophore A23187 (0.1 μM) were significantly reduced by 3 mM L‐cysteine. The similarity between the inhibitory effects of L‐cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.

ADRENALINE ACTIVATION OF PREJUNCTIONAL β-ADRENOCEPTORS IN GUINEA-PIG ATRIA

1 Adrenaline in a concentration of 1.0 μm depressed the stimulation‐induced efflux of tritium from the guinea‐pig atria incubated with [3H]‐noradrenaline, whereas adrenaline in a concentration of 0.5 nm significantly enhanced the stimulation‐induced efflux of tritium. This enhancement was blocked by metoprolol (0.1 μm) and thus appears to be mediated by β‐adrenoceptors. 2 In guinea‐pig atria incubated with unlabelled adrenaline and then with [3H]‐noradrenaline, both catecholamines were released by field stimulation. In such atria metoprolol, practolol, oxprenolol or propranolol decreased the stimulation‐induced efflux of tritium. These effects did not occur if the atria were incubated with unlabelled noradrenaline and then with [3H]‐noradrenaline, suggesting that neuronally released adrenaline activates prejunctional β‐adrenoceptors. 3 The effect of oxprenolol in decreasing the release of tritium from guinea‐pig atria, incubated with unlabelled adrenaline and then with [3H]‐noradrenaline was greater in the presence of phentolamine. This may reflect the α‐adrenoceptor blocking activity of oxprenolol.