John S. Gillespie

The effects of L-arginine and NG-monomethyl L-arginine on the response of the rat anococcygeus muscle to NANC nerve stimulation

The effect of the competitive inhibitor of L‐arginine, NG‐monomethyl L‐arginine (L‐NMMA) on the response of the rat anococcygeus muscle to non‐adrenergic, non‐cholinergic (NANC) inhibitory nerve stimulation has been examined. L‐NMMA causes a rise in muscle tone and inhibition of the response to nerve stimulation. The stereoisomer D‐NMMA is without effect. The rise in tone and inhibition of the nerve response is reversed by L‐arginine. Another analogue, L‐canavanine, which is effective against L‐arginine utilization in the macrophage, was without effect on the rat anococcygeus. These results provide indirect evidence for nitric oxide (NO) or a substance releasing NO as the transmitter of the NANC nerves in this tissue.

The rat anococcygeus muscle and its response to nerve stimulation and to some drugs

1 . A new smooth muscle preparation, the rat anococcygeus muscle, is described. The muscle is paired, thin, consists of smooth muscle only and the muscle cells are organized in parallel bundles. It has a dense adrenergic innervation distributed throughout the muscle but apparently no cholinergic innervation. The muscles are easily isolated. 2 . The muscle contracts to noradrenaline, acetylcholine, furmethide, 5‐hydroxytryptamine, but not to histamine. Isoprenaline produces contraction at high concentrations. The effects of noradrenaline and acetylcholine are blocked by phentolamine and atropine respectively. The response to isoprenaline is little affected by propranolol. 3 . The muscle contracts in response to field stimulation or stimulation of extrinsic nerves. This response is completely blocked by phentolamine but unaffected by hexamethonium or atropine. 4 . Guanethidine 10−6–5 × 10−6m blocks the motor response to nerve stimulation and potentiates that to noradrenaline. Higher concentrations of guanethidine raise tone. In the presence of raised tone, field stimulation produces an inhibitory response insensitive to hexamethonium but abolished by tetrodotoxin 2 × 10−7 g/ml. This inhibitory response to stimulation can also be shown after other drugs which raise tone. 5 . The inhibitory response to nerve stimulation is not mimicked by acetylcholine, isoprenaline or ATP, nor blocked by atropine, phentolamine, phenoxybenzamine, propranolol, hexamethonium or lysergic acid diethylamide.

The effects of pyrogallol and hydroquinone on the response to NANC nerve stimulation in the rat anococcygeus and the bovine retractor penis muscles

1 The effects of pyrogallol and hydroquinone on the bovine retractor penis (BRP) and rat anococcygeus muscles to non‐adrenergic, non‐cholinergic (NANC) nerve stimulation have been examined. Both drugs at a concentration of 10−4 m significantly reduced the response in the rat anococcygeus muscle but had no effect in the BRP muscle. 2 The inhibition of the NANC response in the rat anococcygeus muscle by pyrogallol was completely reversed by superoxide dismutase suggesting it was due to the generation of superoxide anions. 3 Pyrogallol inhibited the response to nitric oxide (NO) in the rat anococcygeus muscle but not that to 3‐isobutyl‐1‐methyl‐xanthine (IBMX) which confirmed a selective action. 4 These results suggest that the NANC neurotransmitter in the rat anococcygeus muscle is susceptible to superoxide anions and may be NO or a substance that can liberate NO.

Influence of haemoglobin and erythrocytes on the effects of EDRF, a smooth muscle inhibitory factor, and nitric oxide on vascular and non-vascular smooth muscle

1 The relaxant action of endothelium‐derived relaxing factor (EDRF), the smooth muscle inhibitory factor (IF) isolated from the bovine retractor penis (BRP), nitric oxide (NO) and sodium nitroprusside (NaNP) on four vascular and non‐vascular smooth muscle preparations has been examined. Their sensitivity to EDRF, the IF and NO was the same, suggesting all might be NO. Sodium nitroprusside produced complete relaxation of the rat anococcygeus at low doses, suggesting an action additional to the intracellular release of NO. 2 Haemoglobin added to solutions of EDRF, activated IF or NO completely removed their relaxant properties, consistent with all three acting by virtue of NO. 3 Suspensions of red blood cells with a haemoglobin concentration equivalent to that used in the previous experiments were as effective as haemoglobin in abolishing the relaxant effect of EDRF or NO but were ineffective against the activated IF. 4 The similarity in sensitivity of a series of smooth muscles and the binding by haemoglobin are consistent with NO being the active principle of both EDRF and the acid activated IF. The abolition of the effect of EDRF by red blood cells (RBCs) is further confirmation for this hypothesis, but the ineffectiveness of RBCs against acid‐activated IF suggests that either the latter is not NO or that it is bound in a way which makes it unable to diffuse through cell membranes.

Effects of NG-substituted analogues of L-arginine on NANC relaxation of the rat anococcygeus and bovine retractor penis muscles and the bovine penile artery

1 The effects of two inhibitors of nitric oxide synthase, NG‐monomethyl l‐arginine (l‐NMMA) and NG‐nitro l‐arginine (l‐NOARG), were examined on non‐adrenergic non‐cholinergic (NANC) inhibitory transmission in the rat anococcygeus, bovine retractor penis (BRP) and bovine penile artery. 2 In the rat anococcygeus, l‐NMMA (10–1000 μm) produced a concentration‐dependent augmentation of guanethidine (30 μm)‐induced tone and inhibited NANC relaxation at all frequencies tested (0.1–20 Hz): the maximum inhibition obtained was 56 ± 6% (n = 6). l‐NOARG (0.3–30 μm) also augmented tone and inhibited NANC relaxation in a concentration‐dependent manner, but unlike l‐NMMA the maximum inhibition was 100%. 3 In the BRP, l‐NMMA (10–100 μm) had no effect on tone or NANC‐induced relaxation, but at 1000 μm tone was increased and NANC relaxation inhibited by 25 ± 7% (n = 6). l‐NOARG (0.3–30 μm) produced a concentration‐dependent increase in tone and inhibition of NANC relaxation. As in the rat anococcygeus, inhibition of NANC relaxation was complete. 4 The effects of l‐NMMA and l‐NOARG were stereospecific since d‐NMMA (10–1000 μm) and d‐NOARG (1–1000 μm) had no effect on tone or NANC relaxation of the rat anococcygeus or BRP. 5 l‐Arginine (10–300 μm) had no effect by itself on NANC‐induced relaxation of the rat anococcygeus or BRP. It did, however, reverse the ability of l‐NMMA (10–1000 μm) to augment tone and inhibit NANC relaxation in the rat anococcygeus and BRP. The actions of low concentrations l‐NOARG (0.3–10 μm) were also reversed by l‐arginine (300 μm), but those of higher concentrations were not. d‐Arginine (1000 μm) had no effect on the ability of l‐NMMA or l‐NOARG to augment tone and inhibit NANC relaxation in the anococcygeus and BRP. 6 On the bovine penile artery, both l‐NMMA (100μm) and l‐NOARG (30 μm) augmented the tone induced by guanethidine (30 μm) and 5‐hydroxytryptamine (0.2 μm) in an endothelium‐dependent manner. l‐NMMA had no effect on NANC‐induced relaxation, but inhibited acetylcholine‐induced endothelium‐dependent relaxation. l‐NOARG abolished NANC relaxation at all frequencies tested and inhibited acetylcholine‐induced relaxation. d‐NOARG (30 μm) had no effect on NANC or acetylcholine‐induced relaxation. 7 The ability of l‐NOARG to abolish NANC‐induced relaxation in the rat anococcygeus, BRP and bovine penile artery suggests that the l‐arginine‐nitric oxide pathway mediates neurotransmission in all three tissues. The effectiveness of l‐NMMA in blocking NANC relaxation in the rat anococcygeus but not the BRP and bovine penile artery suggests a species difference in the neuronal nitric oxide synthase. The neuronal and endothelial nitric oxide synthases in the penile artery also appear to differ.

Block of some non-adrenergic inhibitory responses of smooth muscle by a substance from haemolysed erythrocytes

1. A preparation of haemolysed rat erythrocytes (the haemolysate) blocked the relaxations of both the bovine retractor penis and the rat anococcygeus muscles in response to field stimulation of their non‐adrenergic inhibitory nerves. The effective concentration range was 5‐20 μl./ml. of haemolysate, equivalent to 0·25‐1·0 μl./ml. of blood. The active principle in the haemolysate was a non‐dialysable, heat‐labile material of molecular weight between 50,000 and 100,000 daltons. If, as appeared probable, the active component of the haemolysate was oxyhaemoglobin, its effective blocking concentration was 0·5‐2 μM.

Oxyhaemoglobin blocks non-adrenergic non-cholinergic inhibition in the bovine retractor penis muscle.

Abstract The bovine retractor penis muscle is innervated by motor nerves which are adrenergic, and by inhibitory nerves whose transmitter is unknown. Relaxations in response to inhibitory nerve stimulation, or to the inhibitory factor extracted from the retractor penis, are blocked by oxyhaemoglobin.

The response of the cat anococcygeus muscle to nerve or drug stimulation and a comparison with the rat anococcygeus

1 The cat anococcygeus muscle is shown to possess a dual innervation similar to the rat anococcygeus, with a motor adrenergic innervation and an inhibitory innervation whose transmitter is unknown. The pharmacological properties of the cat muscle were investigated and compared with those of the rat muscle. 2 The cat muscle contracts to noradrenaline, 5‐hydroxytryptamine, tyramine, amphetamine, guanethidine, cocaine and lysergic acid diethylamide (LSD). The effects of noradrenaline and 5‐hydroxytryptamine are blocked by phentolamine and methysergide respectively. 3 The cat anococcygeus is relaxed by acetylcholine, carbachol, isoprenaline, ATP, prostaglandins E1, E2 and F2α and vasopressin, all of which contract the rat muscle. The effects of acetylcholine and carbachol are blocked by atropine and those of isoprenaline by propranolol. 4 Field stimulation produces contraction of the cat anococcygeus, which is blocked by phentolamine and guanethidine but unaffected by hexamethonium, atropine or neostigmine. 5 In the presence of guanethidine (10−5m), the tone of the muscle is raised and field stimulation produces relaxation of the muscle. These inhibitory responses are unaffected by phentolamine, hexamethonium, atropine or neostigmine. 6 Neostigmine potentiates the effects of acetylcholine, but not of carbachol in relaxing the cat anococcygeus and in contracting the rat anococcygeus, but has no effect on either motor or inhibitory responses to field stimulation. 7 Cold storage for up to eight days had little effect on either the motor response to noradrenaline or the motor or inhibitory response to field stimulation of the cat anococcygeus. Beyond eight days, the response to field stimulation diminishes more rapidly than the response to noradrenaline.

Uptake kinetics and ion requirements for extraneuronal uptake of noradrenaline by arterial smooth muscle and collagen

1 The ionic requirements for noradrenaline uptake into vascular smooth muscle cells were studied by perfusing rabbit isolated ear arteries with noradrenaline (10−3M) either in Krebs solution or in Krebs solution modified by altering the concentration of one or more ion. Noradrenaline uptake was measured by quantitative microphotometry. 2 Some uptake into smooth muscle continued in isotonic sucrose in the absence of all ions. Omission of Na+ from the Krebs solution partially inhibited uptake as did high (100 mm) K+. Omission of K+, Ca++ or Mg++ had no effect on uptake. Lithium was able completely to substitute for Na+. 3 Alteration in ion concentration did not affect the binding of noradrenaline to collagen. 4 The kinetics of uptake of noradrenaline into smooth muscle were analysed and found to be saturable with a Km of 4·9 × 10−4M. 5 It is concluded that the ionic requirements of the transport mechanism for the uptake of noradrenaline by vascular smooth muscle show a relatively low specificity.

Non-adrenergic, non-cholinergic relaxation of the bovine retractor penis muscle: role of S-nitrosothiols.

1 This study examined the possibility that an S‐nitrosothiol, rather than nitric oxide, functions as the non‐adrenergic, non‐cholinergic (NANC) inhibitory neurotransmitter in the bovine retractor penis (BRP) muscle. 2 Treatment of BRP muscle with either of two sulphydryl inactivating agents, diamide (1 mm) and N‐ethylmaleimide (0.3 mm), inhibited NANC relaxation and this was prevented by pretreating tissues with l‐cysteine (3 mm), l‐glutathione (3 mm) or dithiothreitol (3 mm). Inhibition was not specific, however, since the inactivating agents also inhibited the relaxant actions of authentic nitric oxide (0.3 μm), glyceryl trinitrate (0.001–1 μm) and isoprenaline (0.01–1 μm). 3 Reacting nitric oxide with l‐cysteine in nominally oxygen‐free solution at pH 3, followed by purging to remove free nitric oxide and neutralisation, produced greater and more prolonged relaxant activity when assayed on rabbit aortic rings than could be attributed to nitric oxide alone. H.p.l.c. analysis of the mixture identified a new peak distinct from either l‐cysteine or nitric oxide which was responsible for the relaxant activity. The spectral absorption of this new compound had two bands with peaks at 218 and 335 nm. 4 Using a series of structural analogues of l‐cysteine (all at 15 mm) it was found that removal of the carboxyl group (l‐cysteamine), replacement of the carboxyl with an ester function (l‐cysteine methyl ester) or substitution at the amino group (N‐acetyl‐l‐cysteine) had no effect on the ability to generate relaxant activity upon reaction with nitric oxide (0.1 mm). In contrast, substitution at the sulphydryl group (S‐methyl‐l‐cysteine, l‐cysteinesulfinic acid and l‐cysteic acid), or formation of disulphides (l‐cystine and l‐cystamine) led to a complete loss of ability to generate relaxant activity. l‐Glutathione was also able to react with nitric oxide to produce relaxant activity, and this too was blocked upon substitution of the free sulphydryl group (S‐methyl‐l‐glutathione). A free sulphydryl group was therefore required to generate relaxant activity following reaction with nitric oxide. 5 Reacting l‐cysteine (10 mm) with nitric oxide (∼3 mm) under more stringent oxygen‐free conditions followed by purging to remove free nitric oxide resulted in the generation of low relaxant activity and small absorption peaks at 218 and 335 nm and these were unaffected upon exposure to the air. In contrast, admitting air to the reaction chamber before purging enhanced both relaxant activity and the absorption peaks at 218 and 335 nm by some 40 fold and the solution turned pink due to the appearance of another absorption peak at 543 nm. This enhanced relaxant activity was not due to nitrogen dioxide being the reactive species, since at 0.1 mm this gas failed to react with l‐cysteine to generate relaxant activity, and at 1 mm generated less activity than the equivalent concentration of nitric oxide. 6 The relaxant activity generated by reacting nitric oxide with l‐cysteine or l‐glutathione was abolished following treatment with haemoglobin (3 μm), methylene blue (10 μm) or N‐methylhydroxylamine (100 μm), but was unaffected by NG‐nitro‐l‐arginine (30 μm). Furthermore, two agents that generate superoxide anion, pyrogallol (0.1 mm) and hydroquinone (0.1 mm), also inhibited this relaxant activity as well as that induced by authentic nitric oxide (0.3 μm) but as previously reported, had no effect on relaxation induced by NANC nerve stimulation. Superoxide dismutase (100 u ml−1) reversed the actions of pyrogallol and hydroquinone but had no effect on NANC relaxation. 7 In conclusion, the reaction of nitric oxide with l‐cysteine or l‐glutathione generates relaxant activity which exceeds that of nitric oxide alone and probably results from formation of S‐nitrosocysteine and S‐nitrosoglutathione, respectively. The effects of pyrogallol and hydroquinone suggest that the NANC neurotransmitter is a superoxide anion‐resistant, nitric oxide‐releasing molecule and that neither S‐nitrocysteine nor S‐nitrosoglutathione is a suitable candidate for this.