Membrane hyperpolarization, cyclic nucleotide levels and relaxation in the guinea-pig internal anal sphincter.

1. Changes in membrane potential (measured with an intracellular microelectrode) and in cyclic nucleotide (adenosine 3':5'-cyclic monophosphate, cyclic AMP and guanosine 3':5'-cyclic monophosphate, cyclic GMP) levels (measured by radioimmunoassay) in response to inhibitory non-adrenergic non-cholinergic (NANC) field stimulation and drugs were investigated in the guinea-pig internal and anal sphincter (gpIAS) in the presence of phentolamine and atropine (each 10(-6) M). 2. Inhibitory NANC nerve stimulation (single pulse, 5 pulses at 5, 10 and 20 Hz, 0.5 ms supramaximal voltage) and adenosine triphosphate (ATP, 10(-7)-10(-3) M) inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of inhibitory nerve stimulation were blocked by tetrodotoxin (TTX, 10(-6) M) and, with those of ATP, were blocked by apamin (5 x 10(-6) M). 3. Isoprenaline (10(-9)-10(-4) M), cromakalim (10(-9)-10(-5) M), sodium nitroprusside (NaNP 10(-5) M), M&B 22948 (10(-4) M) and 8-bromocyclic GMP (8-Br-cyclic GMP, 10(-4) M) also inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of isoprenaline were blocked by propranolol (10(-6) M). However, forskolin (10(-9)-10(-7) M), M&B 22948 (10(-9)-10(-5) M) and lower concentrations of NaNP (10(-9)-10(-6) M) relaxed the sphincter without affecting the membrane potential. 4. The characteristics of the membrane potential changes in response to different inhibitory stimuli in the gpIAS differed. Hyperpolarization produced by inhibitory NANC nerve stimulation and ATP were rapid in onset, of brief duration and of comparable amplitude. Isoprenaline and direct electrical stimulation also hyperpolarized the membrane and relaxed the muscle although the extent of the relaxation in these two cases was much less than that with nerve stimulation and ATP. In each case, the membrane potential change preceded relaxation and probably accounted for it. 5. Both inhibitory NANC nerve stimulation (80 pulses 8Hz supramaximal voltage 0.5 ms) and ATP (10-aM) raised levels of cyclic GMP significantly and to a comparable degree and relaxed the sphincter. The effect of nerve stimulation was prevented by TTX (10- 6M) but not by apamin (5 x 10- 6M). Isoprenaline (10-s M), cromakalim (10 5 M) and forskolin (10 5M) were ineffective. 6. Inhibitory NANC nerve stimulation (80 pulses 8Hz 0.5ms supramaximal voltage) and ATP (10-4M) raised levels of cyclic AMP significantly to a comparable degree and relaxed the sphincter. The increase produced by nerve stimulation was abolished by TTX (10-6M) and apamin (5 x 10-6M). Isoprenaline (10-4M), cromakalim (10-5 M) and forskolin (10-5 M) raised levels of this nucleotide significantly.

Effects of end-tidal concentrations of cyclopropane, halothane and diethyl ether on peripheral autonomic neuroeffector systems in the rat.

The effects of the inhalation anaesthetics, cyclopropane, halothane and diethyl ether were examined on peripheral neuroeffector systems in the pithed and in the conscious rat. 2 In the absence of a suitable means of accurately quantifying doses of inhalation anaesthetics given to small animals, an apparatus was constructed whereby end-tidal gas samples were collected semi-automatically from the mechanically ventilated rat. 3 Cyclopropane (15.3 and 29.3% end-tidal), halothane (0.20, 0.52 and 0.83% end-tidal) and diethyl ether (2% and 4% end-tidal) lowered the arterial pressure of the pithed rat. Heart rate was increased by diethyl ether 4%, decreased by halothane and unchanged by cyclopropane. 4 While each anaesthetic depressed the pressor responses to sympathetic nerve stimulation, cyclopropane increased and halothane and diethyl ether depressed the pressor responses to exogenous noradrenaline. 5 Each anaesthetic reduced the motor responses of the smooth muscle of the colon to parasympathetic stimulation. 6 The significance of the effects on peripheral neuroeffector systems is discussed in relation to the overall circulatory changes produced by these anaesthetics in the whole animal.

An investigation of the role of ganglia in the innervation of the rat anococcygeus muscle: an electrical and mechanical study.

1 A preparation is described which allows the rat anococcygeus muscle to be stimulated via its two extrinsic nerves. Each nerve contains both excitatory and inhibitory fibres. A ganglionated nerve plexus lies on the surface of the muscle. 2 The possibility that at least part of the excitatory pathway was interrupted as a ganglion synapse lying in one of the nodes of plexus close to the muscle was suggested by the observations that (a) the excitatory response to extrinsic nerve stimulation was reduced by the nicotinic antagonists tubocurarine (0.13 to 0.26 mM) and dihydro beta-erythroidine (0.1 to 0.14 mM). (b) Fibres from one extrinsic nerve were shown to synapse on a ganglion cell from which intracellular recordings were made while the output from this ganglion cell was traced microscopically to the muscle. 3 Intracellular recording from ganglion cells in this plexus indicated that cholinergic synaptic transmission occurred in these ganglia. Tubocurarine (0.13 mM) and hexamethonium (1.3 mM) reversibly abolished intracellularly-recorded synaptic potentials. 4 Hexamethonium (0.1 to 1 mM) initially enhanced the motor response to nerve stimulation and raised muscle tone, probably by an action involving pre- and postsynaptic sites. Subsequently, hexamethonium inhibited the response to extrinsic nerve stimulation presumably by an effect at ganglia lying along the excitatory pathway. Hexamethonium enhanced, without subsequently inhibiting, the response to exogenously added noradrenaline in both untreated and 6-hydroxydopamine-treated rats. These results suggest that the initial enhancement produced by hexamethonium involved sites at postganglionic nerve endings and on smooth muscle receptors. 5 Inhibitory responses were obtained following extrinsic nerve stimulation when the tone of the muscle was raised and the excitatory response abolished by either guanethidine (3 micron) alone or by carbachol (10 micron) followed by phentolamine (3 micron). The inhibitory response was not reduced by hexamethonium (up to 2.8 mM) tubocurarine (up to 1.3 mM) or by atropine (up to 1 micron).

The response of the rabbit rectococcygeus muscle to stimulation of extrinsic inhibitory nerves and to sympathomimetic drugs.

1 The effects of stimulating sympathetic or non-adrenergic non-cholinergic (NANC) nerves or of the addition of noradrenaline (NA) or isoprenaline (Iso) were investigated on carbachol-induced tone and on contractions produced by acetylcholine (ACh) and by pelvic nerve stimulation, in the rabbit rectococcygeus muscle.2 Each procedure reduced carbachol-induced tone; sympathetic and NANC nerve stimulation were equipotent but both were less effective than sympathomimetic drugs, of which Iso was the better. Both Iso and NA, but not sympathetic nerve stimulation, inhibited the contractions produced by pelvic nerve stimulation in a concentration-dependent manner. Against ACh-induced contractions, only Iso was effective. The effects of NANC nerve stimulation on the motor responses to pelvic nerve stimulation or to ACh were not investigated.3 The inhibitory effects of sympathetic nerve stimulation, of Iso and of NA were reduced by propranolol (3 x 10(-6) M) but unaffected by phentolamine (3 x 10(-5) M).4 In the presence of high (45 mM) concentrations of KCl, Iso and NA produced a concentration-dependent inhibition of tone that was antagonized by propranolol (3 x 10(-6) M).5 Methoxamine (4 x 10(-7) to 4 x 10(-5) M) and phenylephrine (5 x 10(-7) to 5 x 10(-5) M) which interact mainly with alpha(1)-adrenoceptors, produced only small, transient reductions in carbachol-induced tone which were subject to tachyphylaxis, unlike those produced by Iso and NA. These inhibitory effects were antagonized by phentolamine (3 x 10(-6) M) or azapetine (3 x 10(-6) M).6 Phenylephrine (5 x 10(-4) M) and high doses (3 x 10(-5) M or greater) of NA enhanced the contractile response to pelvic nerve stimulation and, on occasion, produced muscle contraction. These effects were antagonized by phentolamine (3 x 10(-6) M).7 These results suggest that inhibition of the rectococcygeus, a muscle which has no intramural nerve plexus, can be inhibited by stimulation of extrinsic NANC nerves, the transmitter for which is unknown and by sympathetic nerve stimulation via alpha- and beta-adrenoceptors located postsynaptically on the muscle. Excitatory alpha-adrenoceptors may also be present.

Microelectrode recording of the effects of agonists and antagonists on alpha-adrenoceptors on rat somatic nerve terminals.

The effects of apomorphine, catechol, clonidine, isoprenaline, (-)-and (+/-)-noradrenaline, phenylephrine, pyrogallol and xylazine were investigated on the frequency and amplitude of miniature endplate potentials (m.e.p.ps) and, with the exception of apomorphine, catechol and pyrogallol, on the amplitude of endplate potentials (e.p.ps) in the rat phrenic nerve diaphragm preparation. Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each at 1.5 X 10(-5)M) increased m.e.p.p. frequency but not amplitude. The other drugs were ineffective, except isoprenaline (1.5 X 10(-5)M) which enhanced m.e.p.p. amplitude but not frequency. The increase in m.e.p.p. frequency was inhibited by phentolamine, prazosin and yohimbine (each 1.5 X 10(-9)M). Prazosin and yohimbine alone each reduced m.e.p.p. frequency but failed to abolish m.e.p.ps even at high concentrations (10(-3)M). Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each 3 X 10(-6)M) enhanced e.p.p. amplitude; this enhancement was blocked by prazosin and by yohimbine (each 3 X 10(-6)M). In preparations fatigued by prolonged continuous nerve stimulation (5 Hz, 0.05 ms for 30 min), (-)-noradrenaline (3.3 X 10(-4)M) restored m.e.p.p. frequency. The results indicate that adrenoceptors on somatic nerve terminals interact with both alpha 1- and alpha 2-agonists and antagonists and show different characteristics from those at autonomic neuroeffector junctions. The alpha-adrenoceptors on somatic nerve terminals may have an ancilliary physiological role in influencing but not controlling transmitter release.

Mechanisms underlying the electrical and mechanical responses of the guinea-pig internal anal sphincter to field stimulation and to drugs.

The electrical membrane characteristics and the response of the circular muscle of the guinea-pig internal anal sphincter (i.a.s.) to field stimulation were studied in vitro using intracellular microelectrodes and conventional mechanical recording techniques. The i.a.s. developed its own tone (3-4 g), following initial stretch (1 g) and spontaneous spike potentials were evident. In the absence of spike potentials, tone declined and disappeared. Tone was not significantly reduced by phentolamine (1 X 10(-6)M). The resting membrane potential, measured between spontaneous spike potentials, was -45 +/- 3.0 mV (n = 224); the space constant (lambda) was 1.13 +/- 0.1 mm (n = 13). Spikes usually overshot by approximately 15 mV. The frequency of spike potential discharge (1-3 Hz) varied with the degree of membrane depolarization, being increased in K+-rich and decreased in K+-deficient solutions or by the presence of Mn2+. It was not significantly affected by C1-withdrawal but was increased in Na+-deficient solutions with or without tetrodotoxin (TTX; 1 X 10(-6)M). Field stimulation (1-20 Hz, 0.5 ms, supramaximal voltage) produced inhibitory junction potentials (i.j.ps) and relaxed tone; at high frequencies (50 Hz or greater), contractions were observed but excitatory junction potentials (e.j.ps) were not. I.j.ps and relaxations were inhibited by apamin (1 X 10(-6)M), TTX (1 X 10(-6)M) but not by atropine (1 X 10(-6)M), phentolamine (1 X 10(-6)M) or hexamethonium (1 X 10(-6)M). I.j.ps were reduced by hyperpolarization and enhanced by depolarization of the membrane by current pulses (15s). The mean equilibrium potential for the i.j.p. was -94 mV (correlation coefficient, gamma = 0.71, n = 5, p less than 0.001). I.j.ps were enhanced in K+-deficient solutions and reduced in K+-rich solutions. Together these results suggest that the i.j.p. is mediated by an increased GK. The absence of [Ca2+]o or the presence of Mn2+ (2 mM) abolished the i.j.p.; in contrast Na+-deficient or C1-free solutions were ineffective in this respect. Tetraethylammonium (5-50mM) abolished the i.j.p.; the accompanying relaxation was reduced by about 80%. The major aspect of the relaxation to nerve stimulation is mediated by membrane hyperpolarization.

Mechanisms underlying electrical and mechanical responses of the bovine retractor penis to inhibitory nerve stimulation and to an inhibitory extract.

The response of the bovine retractor penis (BRP) to stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves and to an inhibitory extract prepared from this muscle have been studied using intracellular microelectrode, sucrose gap and conventional mechanical recording techniques. Both inhibitory nerve stimulation and inhibitory extract hyperpolarized the membrane potential and relaxed spontaneous or guanethidine (3 X 10(-5) M)-induced tone. These effects were accompanied by an increase in membrane resistance. Following membrane potential displacement from an average value of -53 +/- 7 mV (n = 184; Byrne & Muir, 1984) inhibitory potentials to nerve stimulation were abolished at approximately -30 mV; there was no evidence of reversal. Displacement by inward hyperpolarizing current over the range -45 to -60 mV increased the inhibitory response to nerve stimulation and to inhibitory extract; at more negative potential values (above approximately -60 mV) the inhibitory potential decreased and was abolished (approximately -103 mV). There was no evidence of reversal. Removal of [K+]o reversibly reduced hyperpolarization to nerve stimulation and inhibitory extract. No enhancement was observed. Increasing the [K+]o to 20 mM reduced the inhibitory potential to nerve stimulation but this was restored by passive membrane hyperpolarization. Inhibitory potentials were obtained at membrane potential values exceeding that of the estimated EK (-49 mV). [Cl-]o-free or [Cl-]o-deficient solutions reduced and abolished (after some 20-25 min) the hyperpolarization produced by inhibitory nerve stimulation or inhibitory extract. The inhibitory potential amplitude following nerve stimulation was not restored by passive displacement of the membrane potential from -26 to -104 mV approximately. Ouabain (1-5 X 10(-5) M) reduced then (45-60 min later) abolished the inhibitory potential to nerve stimulation. The effects of this drug on the extract were not investigated. It is concluded that the inhibitory response to nerve stimulation and extract in the BRP may involve several ionic species. However, unlike that in gastrointestinal muscles the NANC response in the BRP is accompanied by an increased membrane resistance and does not primarily involve K+. The underlying mechanisms for the inhibitory response to both NANC nerve stimulation and inhibitory extract appear to be similar, compatible with the view that the latter may contain the inhibitory transmitter released from these nerves in this tissue.

The electrical and mechanical responses of the rabbit saphenous artery to nerve stimulation and drugs.

1. Electrical and mechanical responses to field stimulation (1-64 Hz, 0.5 ms supramaximal voltage) were recorded simultaneously in the rabbit saphenous artery. The electrical response consisted entirely of excitatory junction potentials (e.j.ps) which were abolished by alpha, beta methylene ATP (alpha, beta MeATP, 10(-6) M) and by tetrodotoxin (TTX, 10(-6) M) but were unaffected by the alpha 1-adrenoceptor antagonist, prazosin (10(-6) M). No additional electrical response was evoked by field stimulation, even in the presence of normetanephrine (NMN) and desmethylimipramine (DMI, each 10(-6) M), which block neuronal and extraneuronal uptake of noradrenaline (NA) respectively. Action potentials to field stimulation were produced only in the presence of tetraethylammonium (10(-3) M) which also enhanced the contraction. 2. Contractions to field stimulation were reduced (by some 50%) by prazosin (10(-6) M) and abolished by the additional presence of alpha, beta MeATP (10(-6) M), which blocks purinoceptors by desensitization, suggesting the involvement of both NA and an ATP-like substance in the contractile response. 3. Idazoxan (10(-6) M) which blocks prejunctional alpha 2-adrenoceptors, significantly increased the amplitude of both e.j.ps and the contraction to field stimulation (10 pulses, 1-4 Hz, 0.5 ms, supramaximal voltage). 4. NA (10(-2) M by pressure ejection) did not alter membrane potential even in the presence of NMN and DMI (each 10(-6) M). ATP (10(-2) M by pressure ejection) produced a concentration-dependent, alpha, beta MeATP-sensitive depolarization. 5. In tissues desensitized by constant infusion of alpha, beta MeATP (10(-6) M contractions to NA (10(-7) - 3 x 10(-5) M), histamine (10(-7) - 3 x 10(-5) M) and KCl (1-1.6 x 10(-2) M) were unaffected.6. Following restoration of the membrane potential, after an initial depolarization, alpha,beta MeATP (4 x 10-6M) did not change the amplitude of electrotonic hyperpolarizing current pulses significantly but abolished evoked ej.ps. The rates of recovery of evoked ej.ps and the depolarization to ATP (10-2M by pressure ejection) following desensitization to alpha,beta MeATP (10- 6 M) were comparable. These results suggest that the effects of a,fl MeATP are mediated selectively via receptors (purinoceptors).7. Suramin (10-3M) abolished ej.ps and the prazosin (10-6M), insenstive component of the contractile response and antagonized contractions to xfl MeATP (10-7_1i-5M), ATP (10-5_1i-3M), histamine (10- 7-3 x 10-SM) and 5-hydroxytryptamine (10-7-10-SM) but those to NA (10-7-10-SM) and KCI (1-1.6 x 10-2 M) were unaffected. Suramin is insufficiently selective, under these conditions, as a purinoceptor antagonist.

A technique for the study of muscle relaxants by stimulating the spinal motor nerve outflow in the pithed rat.

The motor nerve outflow in the pithed rat was stimulated from the spinal column and contractions of individual skeletal muscles recorded. The preparation is anaesthetic-free and well suited to a study of muscle relaxants.

Noradrenaline-stimulated inositol phosphate accumulation in arteries from spontaneously-hypertensive rats.

1. The effects of noradrenaline upon polyphosphoinositide (PPI) breakdown was investigated by measuring the accumulation of inositol phosphates (IPs) in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. 2. Noradrenaline (10(-7)-10(-3) M) evoked a concentration-dependent increase in total IP accumulation in both WKY and SHR rats but no significant differences between the populations were detected. 3. In contrast, significant differences in the accumulation of the individual IPs, which contributed to the total IP, occurred. A significantly greater noradrenaline-stimulated accumulation of inositol trisphosphate (IP3) was observed in tissues from SHR compared with those from WKY rats at each effective concentration of noradrenaline. This was paralleled by an equivalent reduction in inositol monophosphate (IP1) accumulation, consistent with the lack of a significant difference in noradrenaline-stimulated total IP accumulation between the two populations. 4. In time course studies, an enhanced noradrenaline-induced accumulation of IP3, in SHR compared to WKY rats, occurred from the earliest time point studied after the addition of the catecholamine both in the presence and absence of LiCL (10 mM). In the presence of LiCl (10 mM) no significant difference in noradrenaline-evoked total IP accumulation between SHR and WKY rats was observed; in the absence of LiCl noradrenaline-evoked a greater total IP accumulation in SHR than in WKY rats at all time points investigated. 5. These studies suggest that the main reason for the enhanced noradrenaline-induced accumulation of IP3 in arteries from SHR rats is a reduced rate of dephosphorylation of both IP3 and inositol bisphosphate (IP2) rather than a greater formation of IP3 from PPIs.6. This enhanced accumulation of IP3 may result in an increased calcium mobilisation accounting for the increased contractility to noradrenaline of tail arteries from SHR as compared with those from WKY rats.

Is co-transmission involved in the excitatory responses of the rat anococcygeus muscle?

1 Electrical and mechanical responses to field (transmural) and extrinsic nerve stimulation were recorded simultaneously in the rat anococcygeus muscle. Membrane potential changes recorded intracellularly following either method of stimulation were indistinguishable. Single stimuli usually produced a slow depolarization; trains of pulses produced a fast excitatory junction potential (e.j.p.) initially, followed by a slow depolarization similar to that produced by single pulses. The fast e.j.ps, the slow depolarizations and the accompanying contractions were abolished by the alpha-adrenoceptor antagonists, phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M) and by tetrodotoxin (TTX, 1 X 10(-6)M) but unaffected by alpha, beta-methylene adenosine triphosphate (alpha, beta-MeATP, 1(-10) X 10(-6)M), an agent known to desensitize purinoceptors. 2 Application of noradrenaline (NA, 1 X 10(-8)-1 X 10(-6)M), by pressure ejection from a micropipette, depolarized the membrane and produced a localized contraction, both of which were abolished by phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M). 3 Application of adenosine-5'-triphosphate (ATP, 1 X 10(-4)-1 X 10(-3)M), by pressure ejection from a micropipette, produced a small membrane depolarization and localized contraction which were unaffected by phentolamine (1 X 10(-6)M) or prazosin (1 X 10(-7)M) but abolished by alpha, beta-MeATP (1 X 10(-6)M). 4 The results show that, in the rat annococcygeus muscle, (1) field or extrinsic nerve stimulation released only one excitatory transmitter, namely NA, although receptors for both NA and ATP were present on the muscle, (2) alpha, beta-MeATP was selective for purinoceptors and (3) there was no evidence for excitatory co-transmission in this tissue.

Effects of calcium channel antagonists on action potential conduction and transmitter release in the guinea-pig vas deferens.

The effects of the Ca2+ channel antagonists amlodipine, cobalt, diltiazem, nifedipine and verapamil and the local anaesthetic lignocaine were investigated on action potential conduction in and on evoked transmitter release from sympathetic nerves in the guinea-pig isolated vas deferens. Transmitter release was investigated by measurement of evoked (trains of pulses at 1 and 2 Hz, 0.1-0.5 ms supramaximal voltage) excitatory junction potentials (e.j.ps) using microelectrodes; tension was recorded simultaneously; tritium [3H] overflow from vasa preincubated (37 degrees C, 30 min) in Krebs solution containing either [3H]-noradrenaline (NA, 25 microCi ml-1, 2 X 10(-6) M NA) or [3H]-adenosine (50 microCi ml-1, 1 X 10(-6) M adenosine). Amlodipine (0.5-2 X 10(-4) M), verapamil (0.5-2 X 10(-4) M), diltiazem (1-8 X 10(-4) M), lignocaine (0.1-2 X 10(-3) M) and cobalt (2-6 X 10(-2) M) in descending order of potency, but not nifedipine (1-5 X 10(-3) M), increased the latency and inhibited, then abolished, the amplitude and number of action potentials in a concentration-dependent manner. Amlodipine (0.5-1 X 10(-4) M), verapamil (1-2 X 10(-4) M), diltiazem (1-5 X 10(-4) M) and cobalt (1 X 10(-3) M), in descending order of potency, but not nifedipine (5 X 10(-4) M), inhibited then abolished evoked e.j.ps in a concentration-dependent manner. Cobalt inhibited e.j.ps at a lower concentration than that (2-6 X 10(-2) M) required to block action potential conduction. In unstimulated tissues, the resting [3H] overflow following preincubation with [3H]-NA consisted largely of 4-hydroxy 3-methoxymandelic acid (VMA), 4-hydroxy 3-methoxy phenylglycol (MOPEG), 3,4 dihydroxyphenylglycol (DOPEG) and NA; stimulated tissues (300 pulses at 20 Hz, 0.5 ms supramaximal voltage) released mainly NA. Verapamil (0.1-1 X 10(-4) M), amlodipine (0.05-1 X 10(-4) M) and nifedipine (1-5 X 10(-4) M), but not cobalt (2 X 10(-3) M), increased, significantly, the resting overflow of 3H comprising mainly DOPEG. Cobalt (2 X 10(-3) M) inhibited, significantly, the stimulation-evoked overflow of 3H. Verapamil (1 X 10(-4) M) had little effect on the resting overflow of 3H following preincubation with [3H]-adenosine. Diltiazem (5 X 10(-4) M) and cobalt (2 X 10(-3) M) both inhibited evoked 3H overflow. Nifedipine (5 X 10(-4) M) was ineffective. 6 The effectiveness of Ca2+ channel antagonists at pre- and postjunctional sites differ; the results are discussed in terms of the selectivity of these drugs for each site and their differential effects on transmitter release.

The effects of noradrenaline and adenosine 5'-triphosphate on polyphosphoinositide and phosphatidylcholine hydrolysis in arterial smooth muscle.

1. The effects of noradrenaline and alpha,beta,methylene adenosine 5'-triphosphate (alpha,beta,methylene ATP) on polyphosphoinositide metabolism, phosphatidylcholine hydrolysis and contraction in rabbit saphenous arteries were investigated. The effect of noradrenaline upon polyphosphoinositide metabolism was also investigated in the rat tail artery. 2. Noradrenaline (10(-7)-10(-4) M) evoked a concentration-dependent increase in total inositol phosphate accumulation in the rat tail but not in the rabbit saphenous artery. Propranolol (3 x 10(-6) M) did not alter this result in the rabbit saphenous artery. In addition, alpha,beta,methylene ATP (10(-6) M) significantly increased total inositol phosphate accumulation in the rabbit saphenous artery, while potassium chloride (8 x 10(-2) M) was ineffective. 3. Phorbol 1,2-myristate 1,3-acetate (3 x 10(-8) M) enhanced noradrenaline (10(-2)-10(-4) M)-evoked contractions in rabbit saphenous artery. The contractile responses to potassium chloride (1- 16 x 10(-2) M) in tissues treated with 6-hydroxydopamine (5 x 10(-4) M), in vitro, were unaffected by these concentrations of the phorbol ester. 4. Noradrenaline (10(-6)-10(-4) M) evoked a concentration-dependent increase in the levels of choline and choline phosphate, but not in those of glycerophosphocholine, in the rabbit saphenous artery. Choline levels increased significantly over the first 15-30 s then declined to control levels within 2 min of addition of noradrenaline (10(-5) M). A smaller initial rise in choline phosphate levels (15-30 s) was followed by a larger secondary rise at 2-4 min.5. alpha, beta, methylene ATP (10-1_ 0-4 M) also evoked a concentration-dependent increase in the levels of both choline and choline phosphate, but not those of glycerophosphocholine, in the rabbit saphenous artery. alpha, beta, methylene ATP (10-4 M) significantly increased levels of both of these products within the first 15-30 s of addition of the drug; these levels reached a stable plateau 1 min after addition.6. The maximum accumulation of choline or choline phosphate evoked by either noradrenaline or alpha, beta, methylene ATP, acting alone or in combination, was not significantly different. No evidence of synergism between noradrenaline and alpha, beta, methylene ATP was observed.7. This study demonstrates that each of the co-transmitters in the rabbit saphenous artery, noradrenaline and adenosine 5'-triphosphate (ATP), promote phosphatidylcholine hydrolysis. Noradrenaline seems to rely on phosphatidylcholine hydrolysis to mediate its contractile effects, whilst ATP promotes both polyphosphoinositide and phosphatidylcholine metabolism suggesting that multiple signal-transduction mechanisms are involved in stimulus-contraction coupling in this artery.

Effect of iontophoretic application of cholinergic agonists and their antagonists to guinea-pig pelvic ganglia.

1. The electrical activity of guinea-pig pelvic ganglion cells following iontophoretically applied cholinergic drugs, alone and during orthodromic nerve stimulation via the hypogastric nerve, has been recorded intracellularly.2. Iontophoretic application of nicotine (Nic) and acetylcholine (ACh) reduced membrane resistance and caused a depolarization which in approximately 80% of cells led to the firing of action potentials. In the remainder, depolarization was unaccompanied by firing.3. Iontophoretic application of Nic and ACh reduced or abolished the amplitude of successively evoked orthodromic responses.4. ACh-induced depolarization, unlike that caused by tetanic stimulation, was not followed by a subsequent increase in the frequency of synaptic potentials.5. Di-hydrobetaerythroidine (DHbetaE) and atropine (Atr) inhibited the response to both orthodromic stimulation and iontophoretic application of Nic and ACh.6. There was no evidence for the existence of muscarinic receptors in guinea-pig pelvic ganglia. Iontophoretic application of muscarinic agonists alone and after tetanic stimulation of the hypogastric nerve produced no significant depolarization of the ganglion cell membrane.

Neuroeffector transmission in the guinea-pig internal anal sphincter: an electrical and mechanical study.

ATP (10(-7)-10(-4) M), ADP (10(-7)-10(-4) M), AMP (10(-7)-10(-4) M) and adenosine (10(-6)-10(-4) M) each hyperpolarized the membrane, inhibited spontaneous spike discharge and relaxed the guinea-pig internal anal sphincter. All experiments were carried out using intracellular microelectrode and simultaneous tension recording techniques in the presence of phentolamine (10(-6) M) and atropine (10(-6) M). ATP was the most effective and produced a concentration-dependent membrane potential change comparable in amplitude to that produced by field stimulation of non-adrenergic non-cholinergic (NANC) nerves. Inhibitory junction potentials, the accompanying relaxations and the responses to ATP (5 X 10(-6)-5 X 10(-5) M) were additive and were increased in K+-deficient and decreased in K+-rich solutions and inhibited by apamin (10(-7) M). A proteolytic enzyme, alpha-chymotrypsin (0.5 U/ml) preferentially antagonized the ability of vasoactive intestinal polypeptide (10(-7) M) to hyperpolarize the membrane and relax the sphincter. The electrical and mechanical responses to ATP (10(-5) M) and inhibitory nerve stimulation were only slightly reduced. The results are consistent with the view that ATP or a related adenine nucleotide may have a transmitter role in the guinea-pig internal anal sphincter.

The effect of clonidine on the response to stimulation of non-adrenergic non-cholinergic nerves in the guinea-pig urinary bladder in-vitro.

The effects of clonidine on the response of the guinea-pig urinary bladder detrusor muscle to stimulation of non-adrenergic non-cholinergic (NANC) nerves were investigated in-vitro. In tissues from both treated and chemically sympathectomized animals, in the presence of atropine (10(-5) M) to inhibit cholinergic responses, clonidine (10(-10)-10(-6) M) invariably enhanced the contractile response to NANC nerve stimulation at 2, 5, 10 and 20 Hz. The enhancement was not inhibited by yohimbine (10(-5) M), phentolamine (10(-5) M), or the histamine H1- or H2-receptor antagonists mepyramine (10(-6) M) or cimetidine (10(-5) M) respectively. Phentolamine (10(-5) M), like clonidine, enhanced the response to stimulation of NANC nerves at 2, 5, 10 and 20 Hz. Xylazine, another alpha 2-agonist, which, unlike both clonidine and phentolamine, is not a substituted 2-imidazoline compound, failed to enhance the response in the bladder to NANC nerve stimulation. The results suggest that clonidine enhances the response to NANC nerve stimulation, independently of its effects on alpha-adrenoceptors, by increasing the amount of NANC transmitter available.

The interaction between noradrenaline and ATP upon polyphosphoinositide metabolism and contraction in tail arteries from normo- and hypertensive rats.

The effects of, and interaction between, noradrenaline and alpha,beta-methylene ATP upon polyphosphoinositide (PPI) breakdown, investigated by measuring the accumulation of inositol phosphates, and contraction, were studied in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. Noradrenaline (10(-7)-10(-3) M) evoked a prazosin (10(-6) M)-sensitive, concentration-dependent increase in total inositol phosphate accumulation in both WKY and SHR rats. No significant differences were observed in either the maximal response or in the concentration range over which noradrenaline evoked this response, between these two populations. Noradrenaline (5 x 10(-7)-5 x 10(-5) M) evoked a concentration-dependent contraction of arteries from both SHR and WKY rats. The responses to noradrenaline were about 2-fold greater at all effective concentrations of noradrenaline in SHR compared with WKY rats. alpha,beta-Methylene ATP (10(-6) M) did not alter noradrenaline-stimulated total inositol phosphate accumulation, in arteries from either SHR or WKY rats, measured either as the maximal response or as the EC50. alpha,beta-Methylene ATP (5 x 10(-6) M), by itself, evoked a contractile response, which was quantitatively similar in SHR and WKY rats, and was additive with the contractile responses to noradrenaline (5 x 10(-7)-5 x 10(-5) M). The maximum response produced by a combination of noradrenaline and alpha,beta-Methylene ATP was quantitatively similar to that produced by noradrenaline alone. No evidence of synergism between alpha,beta-Methylene ATP and noradrenaline upon contraction was observed.(ABSTRACT TRUNCATED AT 250 WORDS)