THE INHIBITION OF NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE BY DIPHENYLENE IODONIUM

1. The effects of diphenylene iodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate-dependent oxidases (which generate superoxide anions), were studied on nitric oxide (NO)-mediated responses in isolated preparations of the rat aorta and anococcygeus muscle. 2. In aortic rings, the endothelium-dependent relaxant action of acetylcholine was reduced by DPI (0.3–10 μmol/L) in a concentration-dependent manner and abolished by the NO synthase (NOS) inhibitor L-nitro-N^G-arginine methylester (l-NAME; 100 μmol/L). Relaxations induced by sodium nitroprusside (SNP) or NO were not affected by DPI or l-NAME. 3. In anococcygeus muscles, DPI (0.3–10 μmol/ L) as well as l-NAME (5–100 μmol/L) produced concentration-dependent reductions of relaxations produced by nitrergic nerve stimulation. Relaxations induced by NO and SNP were not affected by either DPI or l-NAME. l-Arginine (1 mmol/L) prevented the reduction of nitrergic relaxations by l-NAME but not by DPI. 4. Contractions of anococcygeus muscles elicited by exogenous noradrenaline (1 μmol/ L) were not affected or were inhibited by DPI (0.3–10 μmol/L), but the contractions elicited by noradrenergic nerve stimulation were significantly enhanced by DPI and l-NAME. When noradrenergic contractions had already been maximally enhanced by l-NAME (100 μmol/L), DPI produced no further enhancement. l-Arginine (1 mmol/L) prevented the enhancement of noradrenergic contractions by l-NAME but not by DPI. 5. The efflux of radioactivity induced by field stimulation from anococcygeus muscles previously incubated with [³H]-noradrenaline was not affected by either DPI or l-NAME. 6. Superoxide dismutase (SOD, 100 U/mL) had no significant effects on noradrenergic contractions, nitrergic relaxations, relaxations induced by NO or the actions of DPI in the rat anococcygeus muscle. 7. The results suggest that the effects of DPI in reducing the NO-mediated relaxations produced by acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle are due to the inhibition of NOS in these tissues. The effects of DPI were not sensitive to l-arginine, and thus the mechanism of inhibition of NOS differs from that of l-NAME.     

Effects of omega-conotoxin GVIA on autonomic neuroeffector transmission in various tissues.

1. The effects of omega-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 elicited 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 response were almost completely abolished by tetrodotoxin. 10. The conotoxin-induced inhibitions of responses to nerve stimulation developed slowly and persisted after removal of conotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

RESPONSES OF THE ISOLATED ANOCOCCYGEUS MUSCLE OF THE MOUSE TO DRUGS AND TO FIELD STIMULATION

• 1 The responses of the isolated anococcygeus muscle of the mouse to drugs and to field stimulation were investigated.
• 2 Fluorescence micrographs of transverse sections of the muscle revealed fluorescent nerve fibres in control tissues, but not in tissues from mice pretreated with 6-hydroxydopamine.
• 3 The isolated muscle displayed no spontaneous tone or rhythmic activity. Field stimulation produced frequency dependent contractions which were blocked by phentolamine and by low concentrations of guanethidine (500 nM). Higher concentrations of guanethidine (30 juM) raised muscle tone and, subsequently, field stimulation produced frequency dependent relaxations. In muscles from 6-hydroxydopamine pretreated mice neither field stimulation nor guanethidine produced contractions, but when muscle tone was raised by carbachol inhibitory responses to field stimulation were still observed.
• 4 Contractions of the muscle were produced by noradrenaline (α-adrenoreceptors), acetylcholine (muscarinic receptors), 5-hydroxytryptamine, and prostaglandins E₁, E₂, and F_2a.
• 5 Adenosine 5′-triphosphate (ATP) normally produced muscle contraction, but following incubation of the tissue with indomethacin, ATP produced relaxations of carbachol-induced tone.
• 6 Vasoactive intestinal polypeptide, but not bradykinin, produced relaxations of tissues in which tone had been raised by carbachol.
• 7 The isolated anococcygeus muscle of the mouse provides a useful preparation for the study of various aspects of autonomic neurotransmission in smooth muscle.

     

Effects of hydroxocobalamin and carboxy-PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles.

The effects of carboxy-PTIO and hydroxocobalamin were studied on nitrergic transmission in anococcygeus and retractor penis muscles taken during post mortem examination from young male pigs. In both muscles under resting conditions, electrical field stimulation (EFS) caused contractions that were sensitive to tetrodotoxin (1 microM) and were greatly inhibited by prazosin (1 microM) and guanethidine (10-30 microM), but were not significantly affected by atropine (1 microM). In the anococcygeus muscle, but not in the retractor penis muscle, guanethidine produced a prolonged contraction. After tone was raised by guanethidine in the anococcygeus or by phenylephrine (1 microM) in the presence of guanethidine in the retractor penis, EFS caused tetrodotoxin-sensitive relaxations. The EFS-induced relaxations were abolished by the NO synthase inhibitor N(G)-L-nitro-arginine methyl ester (L-NAME; 100 microM) and its effect was partly overcome by L-arginine (1 mM), indicating it was mediated by nitrergic nerves. Carboxy-PTIO (0.1-1 mM) had no significant effect in reducing stimulation-induced nitrergic relaxations in either muscle. However, hydroxocobalamin (0.1-1 mM) caused concentration-dependent reductions of nitrergic relaxations in both muscles. Relaxations to exogenous nitric oxide (1 microM) in both muscles were abolished by carboxy-PTIO (0.3 mM) and hydroxocobalamin (0.1 mM). There were no differences in reactivity to carboxy-PTIO or hydroxocobalamin between anococcygeus and retractor penis muscles from the same species (pig). The finding also confirms earlier observations that the nitrergic transmitter is generally resistant to the NO-scavenger carboxy-PTIO.     

EFFECTS OF ARGININOSUCCINIC ACID ON NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE

1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.     

INHIBITION BY ETHACRYNIC ACID OF NO-MEDIATED RELAXATIONS OF THE RAT ANOCOCCYGEUS MUSCLE

1. The effects of ethacrynic acid were studied on relaxations elicited by nitric oxide (NO), the NO-donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), nitrergic nerve stimulation and the NO-independent agent papaverine in isolated preparations of rat anococcygeus muscles. 2. Ethacrynic acid (100 μmol/L) produced complete relaxation of partially contracted anococcygeus muscles, but the tone recovered after the ethacrynic acid was washed out. Following exposure to ethacrynic acid, the relaxant responses to NO, SNP, GTN and nitrergic nerve stimulation were abolished or markedly reduced; however, the response to papaverine was only slightly reduced. 3. The presence of 3 mmol/L l-cysteine during the period of exposure to ethacrynic acid prevented the inhibition of the relaxing effects of SNP, GTN and nitrergic nerve stimulation almost completely, but did not affect the slight reduction in responses to papaverine. 4. The addition of l-cysteine (3 mmol/L) after incubation with ethacrynic acid did not significantly affect the inhibited responses to SNP and GTN; however, the inhibited responses to nitrergic nerve stimulation were slightly but significantly increased. 5. The results suggest that endogenous sulphydryl groups are required for the actions of NO, NO-donating drugs and the nitrergic transmitter in the rat anococcygeus muscle and possibly for the synthesis or release of the nitrergic transmitter.     

RANITIDINE INHIBITS ADRENERGIC TRANSMISSION IN THE RAT ISOLATED ANOCOCCYGEUS MUSCLE

1. The effects of ranitidine on adrenergic transmission in the rat isolated anococcygeus muscle were investigated. 2. Cumulative doses (2-8 mmol/L) or ranitidine produced a concentration-dependent inhibition of motor responses of the rat isolated anococcygeus muscle evoked by field stimulation (20-25 V, 10 Hz for 10 s, 1 ms pulse width) every 2 min, but also potentiated the contractile response to exogenous noradrenaline (5 mumol/L). The inhibited motor responses recovered rapidly and completely after washing out ranitidine. 3. 4-Aminopyridine (100 mumol/L) effectively reversed the partially inhibited (55% or greater) motor responses. 4. The results strongly suggest that ranitidine can inhibit adrenergic transmission in the anococcygeus muscle by a prejunctional mechanism with, presumably, consequent development of supersensitivity of the effector cells to noradrenaline.     

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. 2. The inhibitory effect of L-NMMA was reversed by L-arginine but not by D-arginine, and prior exposure to L-arginine blocked the effect of L-NMMA. 3. The noradrenergically mediated contractions of the anococcygeus elicited by field stimulation were slightly enhanced by L-NMMA, but the response to noradrenaline was not affected. 4. The results suggest that NANC transmission in the rat anococcygeus muscle involves the generation of NO from arginine.     

Modulation of acetylcholine-induced contractions of the rat anococcygeus muscle by activation of nitrergic nerves.

1. Acetylcholine-induced contractions of the rat isolated anococcygeus muscle were blocked by atropine (0.1 microM), slightly enhanced by hexamethonium (0.1 mM) and tetrodotoxin (1 microM), but little affected by prazosin (0.1 microM). 2. In the presence of the alpha 2-adrenoceptor agonist, UK14304, which raised the tone of the muscle, acetylcholine had a biphasic effect consisting of an initial relaxation followed by a contraction. 3. Atropine (0.1 microM) enhanced the relaxant component and abolished the contractile component of the response, whereas tetrodotoxin, omega-conotoxin GVIA or hexamethonium abolished or greatly reduced the relaxant component. 4. The nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) increased acetylcholine-induced contractions in the absence of UK14304 and markedly reduced the relaxant component to acetylcholine in the presence of UK14304. The effects of L-NAME were annulled by L-arginine (300 microM). 5. The results suggest that acetylcholine acts concurrently on muscarinic receptors of the smooth muscle to cause contraction and nicotinic receptors of nitrergic nerves to cause relaxation. The observed response is the resultant of these two opposing effects and depends also on the prevailing tone.     

MEDIATORS OF NICOTINE-INDUCED RELAXATIONS OF THE RAT GASTRIC FUNDUS

1. Relaxations of strips of rat gastric fundus were elicited with nicotine (100 μmol/L), nitric oxide (NO; 30 μmol/L), sodium nitroprusside (SNP; 100 nmol/L) and vasoactive intestinal polypeptide (VIP; 1 nmol/L). 2. Methylene blue (30 μmol/L), an inhibitor of soluble guanylate cyclase, reduced relaxations elicited by NO and nicotine, but not those elicited by VIP. 3. Chymotrypsin (1 U/mL) abolished VIP-induced relaxations and reduced nicotine-induced relaxations, but had no effect on SNP-induced relaxations. 4. N^G-nitro-l-arginine methyl ester (l-NAME; 100 μmol/L), an inhibitor of NO synthase, reduced relaxations elicited by nicotine, but not those elicited by SNP or VIP. 5. When nicotine-induced relaxations had been reduced by either l-NAME or chymotrypsin, the addition of the other agent produced a greater reduction. However, the relaxations were not abolished. 6. Nicotine-induced relaxations were abolished by tetrodotoxin (1 μmol/L) or hexamethonium (100 μmol/L), indicating that they were due to activation of neuronal nicotinic receptors. Their reduction by methylene blue and l-NAME indicates that an NO-like mediator was involved. Their reduction by chymotrypsin indicates that a VIP-like peptide was involved. However, since they were not abolished by a combination of l-NAME and chymotrypsin, it appears that at least one more as yet unidentified mediator may be involved.     

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 E(1), E(2) and F(2alpha) 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(-5)M), 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.     

PHARMACOLOGICAL ACTIONS OF THE COENZYMES NAD(H) AND NADP(H) ON THE RAT ANOCOCCYGEUS MUSCLE

1. The pharmacological actions of the oxidized and reduced forms of nicotinamide-adenosine dinucleotide (NAD, NADH) and nicotinamide-adenosine dinucleotide phosphate (NADP, NADPH) were studied on rat isolated anococcygeus muscles. 2. The actions of the two nucleotides were different, but there were no apparent qualitative differences between the oxidized and reduced forms of each. 3. In fully relaxed anococcygeus muscles, NADP(H) produced transient contractions that were subject to desensitization, but NAD(H) had no effect. 4. NADP(H) slightly enhanced contractions elicited by noradrenergic nerve stimulation. In contrast, noradrenergic contractions were inhibited by NAD(H). NADH reduced the stimulation-induced release of noradrenaline, but enhanced contractions elicited by exogenous noradrenaline. 5. In anococcygeus muscles partly contracted with guanethidine, NAD(H) produced a further sustained increase in tone; in contrast, NADP(H) mainly produced transient relaxations to which there was immediate desensitization. 6. Relaxations of anococcygeus muscle elicited by nitrergic nerve stimulation were not affected by NAD. In contrast, NADP(H) reduced them. 7. The actions of NAD(H) were generally the same as those of adenosine and can be attributed to activation of P₁-purinoceptors since they were blocked by the selective antagonist 8-sulphophenyl-theophylline. 8. The actions of NADP resembled those of the P₂-purinoceptor agonist ATP to some extent, but there were some differences. As suggested by others, NADP may act on a unique receptor.     

Role of copper in the relaxant action of S-nitrosothiols in the rat anococcygeus muscle

1. The effects of copper chelators were investigated on the relaxant actions of the S-nitrosothiols S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-d,l-penicillamine (SNAP), the non-S-nitrosothiol nitric oxide (NO) donor sodium nitroprusside (SNP), free radical NO (NO.) and the nitrergic neurotransmitter in rat isolated anococcygeus muscle. 2. Cumulative additions of GSNO (0.01-100 micro mol/L), SNAP (0.001-10 micro mol/L), SNP (0.001-1 micro mol/L) and NO. (0.5-5 micro mol/L) and electrical field stimulation (EFS; 1-5 Hz, 10 s) of nitrergic nerves in preparations precontracted with guanethidine (10-30 micro mol/L) and clonidine (0.01-0.3 micro mol/L) produced concentration-dependent relaxations. 3. The Cu[I] chelator neocuproine (10-30 micro mol/L) produced concentration-dependent inhibitions of the relaxations to GSNO and SNAP. At 30 micro mol/L, neocuprinone had no effect on relaxations to SNP (0.001-1 micro mol/L), NO. (0.5-5 micro mol/L) or EFS (1-5 Hz, 10 s). 4. The Cu[II] chelator cuprizone (30 micro mol/L) slightly and significantly enhanced relaxations to GSNO and NO., but had no effect on relaxations to SNAP, SNP or EFS. 5. In conclusion, the results indicate that Cu[I], but not Cu[II], may be involved in the relaxant actions of GSNO and SNAP in the rat anococcygeus muscle.     

CHOLINORECEPTORS IN THE CORPUS CAVERNOSUM MUSCLE OF THE HUMAN PENIS

• 1 Acetylcholine produced contraction, relaxation or a biphasic effect on corpus cavernosum muscle strips of human penis. These effects were antagonized by atropine but were modified by hexamethonium or tetrodotoxin suggesting the presence of excitatory and inhibitory muscarinic receptors in the human penis.
• 2 Nicotine and DMPP also produced contraction or relaxation of the corpus cavernosum muscle strips. These effects were presumably indirect as they were abolished by hexamethonium and tetrodotoxin.
• 3 Contractions produced by nicotine or DMPP were abolished by atropine whereas the relaxations produced by these agents were only partially blocked by atropine. Hence, the receptors mediating these relaxations are unlikely to be entirely muscarinic.

     

Cholinergic prejunctional inhibition of nitrergic neurotransmission in the guinea-pig isolated basilar artery

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 mumol/L prostaglandin F2 alpha and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 mmol/L) and physostigmine (10 mumol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 mumol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 mumol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha 2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.     

The effects of lysergic acid diethylamide on the response to field stimulation of the rat vas deferens and the rat and cat anococcygeus muscles.

1. The effect of lysergic acid diethylamid (LSD) on the response to field stimulation in vitro of the rat vas deferens and anococcygeus muscle was examined. 2. LSD in concentrations from 10(-9) to 10(-6) M caused an increase in tone or rhythmic activity in both tissues, effects identical to those produced by guanethidine or tyramine. The motor effects of all three drugs were abolished by phentolamine 2 x 10(-6) M. Methysergide 2 x 10(-7) M given before LSD reduced the motor effect but was ineffective once the LSD contraction had developed. 3. LSD 10(-9) to 10(-6) M reduced and eventually abolished the response to motor adrenergic nerve stimulation in the anococcygeus muscle with no effect on the response to noradrenaline (NA) and no evidence of differential sensitivity according to the number of stimulating pulses. In the vas deferens LSD abolished the initial twitch component with no effect on the secondary slow contraction. LSD had no effect on the response to inhibitory nerve stimulation in the anococcygeus. 4. These results suggest that in the anococcygeus LSD closely resembles guanethidine in its effects as an adrenergic neurone blocking drug with indirect sympathomimetic actions. In the vas deferens these properties would explain the block of the initial twitch component in the motor response to field stimulation and the increase in rhythmic activity but do not explain the resistance of the secondary slow component of the motor response.     

Relaxations of the isolated portal vein of the rabbit induced by nicotine and electrical stimulation

1. A pharmacological analysis of the inhibitory innervation of the isolated portal vein of the rabbit has been made.2. In untreated preparations, transmural stimulation elicited a long-lasting relaxation at low frequencies (0.2-1 Hz); at higher frequencies a contraction followed by a prolonged after-relaxation occurred. Tetrodotoxin abolished the contractions but a higher dose was required to abolish the relaxations. Veratrine lowered the threshold of stimulation for producing relaxations in the untreated vein. The relaxations were unaffected by hyoscine or hexamethonium. They were reduced or altered by antagonists of alpha-adrenoceptors for catecholamines and by adrenergic neurone blockade. They were sometimes slightly reduced by antagonists of beta-adrenoceptors.3. In the presence of antagonists of alpha-adrenoceptors, electrical stimulation elicited relaxations which increased with frequency of stimulation and became maximal at 20-30 Hz. These relaxations were partially reduced by antagonists of beta-adrenoceptors, or by adrenergic neurone block; the antagonisms were more pronounced at the higher frequencies of stimulation. Noradrenaline also caused relaxations which were abolished by beta-adrenoceptor blocking drugs. Cocaine increased the sensitivity to noradrenaline by 7-8 fold after alpha-adrenoceptor blockade but had little or no effect on the relaxations induced by electrical stimulation at high frequencies.4. In the presence of antagonists of alpha- and beta-adrenoceptors, or adrenergic neurone blocking agents, or in veins taken from rabbits pretreated with reserpine, electrical stimulation elicited rapid relaxations which were greatest at 20-30 Hz. These relaxations were increased by veratrine and abolished by tetrodotoxin or by storing the vein for 9 days at 4 degrees C. They were unaffected by antagonists of acetylcholine, or by dipyridamole.5. Prostaglandins E(1), E(2) and F(2alpha) inhibited contractions elicited by electrical stimulation and noradrenaline, but in higher doses caused contractions themselves.6. Nicotine (10(-6)-10(-5) g/ml) relaxed the portal vein; higher concentrations elicited mixed inhibitory and excitatory effects. All these effects were abolished by tetrodotoxin, cocaine, hexamethonium or storage. The contractor effects were abolished by drugs or procedures that blocked adrenergic mechanisms.7. The relaxations produced by nicotine in untreated preparations and in veins from rabbits pretreated with reserpine were mediated mainly by a non-adrenergic non-cholinergic nervous mechanism. Relaxations induced by nicotine in the presence of antagonists of a-adrenoceptors were only partially antagonized by antagonists of f3-adrenoceptors.8. It was concluded that all the effects of nicotine and transmural stimulation were mediated by nerves. Part of the inhibitory effects was mediated by non-adrenergic, non-cholinergic nerves.     

EFFECTS OF HYDROXOCOBALAMIN AND HAEMOGLOBIN ON NO-MEDIATED RELAXATIONS IN THE RAT ANOCOCCYGEUS MUSCLE

1. The effects of hydroxocobalamin (Vitamin B_12a) on relaxations produced by nitric oxide (NO), some NO-donating compounds and nitrergic nerve stimulation in isolated preparations of the rat anococcygeus muscle were compared with the effects of haemoglobin. 2. Hydroxocobalamin (30 μmol/L) significantly reduced relaxations induced by NO (0.1–3 μmol/L) and sodium nitroprusside (SNP; 0.01–0.3 μmol/L) but did not affect relaxations induced by glyceryl trinitrate (GTN; 0.01–1 μmol/L), S-nitrosocysteine (0.1–0.3 μmol/L) or stimulation of nitrergic nerves. A higher concentration of hydroxocobalamin (100 μmol/L) slightly reduced nitrergic nerve stimulation-induced relaxations. 3. Haemoglobin (10 μmol/L) blocked relaxation induced by NO and reduced relaxations induced by SNP, GTN, S-nitrosocysteine and nitrergic nerve stimulation. 4. When nitrergic nerve stimulation-induced relaxations had been partially reduced by the NO synthase inhibitor l-NAME (5–10 μmol/L), hydroxocobalamin had only a weak and transient inhibitory effect. 5. Noradrenergic contractions induced by field stimulation were not affected by hydroxocobalamin (30 μmol/L), but were enhanced by haemoglobin (10 μmol/L). 6. The results suggest that the transmitter released from nitrergic nerves in anococcygeus muscles resembles NO-releasing compounds such as S-nitrosocysteine and GTN but not SNP or free NO.     

Evidence for a Postsynaptic Action of Neostigmine on Muscarinic Receptors in the Anococcygeus Muscle of the Rat

Abstract— In the rat anococcygeus muscle neostigmine induces atropine-sensitive, significant leftward displacements of the log concentration-response curves to both noradrenaline and 5-hydroxytryptamine. Responses to high K+ were also potentiated by neostigmine. However, high K+ elicited only small and irregular overflows of tritium from [³H]noradrenaline pre-incubated tissues, in contrast to the large overflow elicited by field stimulation. In addition guanethidine blocked responses to field stimulation but not those to high K+. This is consistent with the dose-related tension responses to high K+ being elicited postsynaptically. These results indicate that postsynaptic muscarinic receptors are involved in the potentiation by neostigmine of rat anococcygeus muscle responses to field stimulation or exogenous agonists, possibly by an action on receptor operated ion channels. Additional support for a postsynaptic site of action comes from the failure of neostigmine to potentiate tension responses to nerve or field stimulation in the chick expansor secundariorum, a muscle which is devoid of postsynaptic muscarinic receptors.     

The effects of vasoactive intestinal polypeptide and of adenosine 5'-triphosphate on the isolated anococcygeus muscle of the mouse.

1 Vasoactive intestinal polypeptide (VIP, 0.01- MicroM) produced dose-related relaxations of the mouse anococcygeus muscle. 2 Following incubation with indomethacin (2.8 microM 1 h) adenosine 5'-triphosphate (ATP, 0.5-10 mM) produced dose-related relaxations of the mouse anococcygeus. 3 Haemolysed blood reduced inhibitory responses of the mouse anococcygeus to field stimulation but had no effect on relaxations to VIP or ATP. 4 Apamin (0.5 microM) had no effect on the relaxation of mouse anococcygeus to field stimulation, VIP, or ATP. 5 2-2'-Pyridylisatogen tosylate (PIT, 50 microM) itself reduced muscle tone but it did not abolish inhibitory responses to field stimulation, VIP, or ATP. 6 During prolonged inhibitory nerve stimulation the relaxation of the mouse anococcygeus in response to VIP was reduced greatly while that to ATP was unaffected. 7 Bundles of VIP-immunoreactive sites were detected in sections of the mouse anococcygeus treated by the peroxidase-antiperoxidase (PAP) immunocytochemical technique. 8 The results suggest that the mechanisms underlying non-adrenergic, non-cholinergic inhibitory transmission in the mouse anococcygeus are similar to those in the bovine retractor penis and unlike those in the guinea-pig taenia caeci. 9 The possibility that VIP or ATP might be involved in inhibitory neurotransmission in the mouse anococcygeus is discussed.