Sarcolemma agonist-induced interactions between InsP3 and ryanodine receptors in Ca2+ oscillations and waves in smooth muscle.

Smooth muscle cells respond to InsP(3)-generating (sarcolemma-acting) neurotransmitters and hormones by releasing Ca(2+) from the internal store. However, the release of Ca(2+) does not occur uniformly throughout the cytoplasm but often into a localized area before being transmitted to other regions of the cell in the form of Ca(2+) waves and oscillations to actively spread information within and between cells. Yet, despite their significance, our understanding of the generation of oscillations to waves is incomplete. A major aspect of controversy centres on whether or not Ca(2+) released from the InsP(3) receptor activates RyRs (ryanodine receptors) to generate further release by Ca(2+)-induced Ca(2+) release and propagate waves or whether the entire process arises from InsP(3) receptor activity alone. Under normal physiological conditions the [Ca(2+)] required to activate RyR (approx. 15 microM) exceeds the bulk average [Ca(2+)](c) (cytoplasmic Ca(2+) concentration) generated by InsP(3) receptor activity (<1 microM). Progression of waves and oscillations by RyR activity would require a loss of control of RyR activity and an unrestrained positive feedback on Ca(2+) release. Under store-overload conditions, RyR Ca(2+) sensitivity is increased and this enables waves to be induced by RyR activity. However, the relevance of these Ca(2+)-release events to normal physiological functioning is unclear. The InsP(3) receptor, on the other hand, is activated by Ca(2+) over the physiological range (up to 300 nM) and deactivated by higher [Ca(2+)](c) (>300 nM), features that favour intermittent activity of the receptor as occurs in waves and oscillations. Experimental evidence for the involvement of RyR relies mainly on pharmacological approaches in the intact cell where poor drug specificity could have led to ambiguous results. In this brief review the possible interactions between InsP(3) receptors and RyR in the generation of oscillations and waves will be discussed. Evidence is presented that RyRs are not required for InsP(3)-mediated Ca(2+) transients. Notwithstanding, ryanodine can inhibit InsP(3)-mediated Ca(2+) responses after RyR activity has been induced by caffeine or by steady depolarization which evokes spontaneous transient outward currents (a sarcolemmal manifestation of RyR activity). Ryanodine inhibits InsP(3)-mediated Ca(2+) transients by depleting the store of Ca(2+) rather than by RyR involvement in the InsP(3)-mediated Ca(2+) increase.

Functionally separate intracellular Ca2+ stores in smooth muscle.

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca(2+) concentrations ([Ca(2+)](c)). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca(2+) signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca(2+) signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P(3)R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P(3), suggesting that Ins(1,4,5)P(3)R and RyR share a common Ca(2+) store. Ca(2+) release from the Ins(1,4,5)P(3)R did not activate Ca(2+)-induced Ca(2+) release at the RyR. Depletion of the Ins(1,4,5)P(3)-sensitive store, by the removal of external Ca(2+), on the other hand, caused only a small decrease ( approximately 26%) in caffeine-evoked Ca(2+) transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P(3)R. Dependence of the stores on external Ca(2+) for replenishment also differed; removal of external Ca(2+) depleted the Ins(1,4,5)P(3)-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P(3)-sensitive and caffeine-sensitive Ca(2+) stores was inhibited by each of the SR Ca(2+) ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca(2+) stores; the first expressing only RyR and refilled from [Ca(2+)](c), the second expressing both Ins(1,4,5)P(3)R and RyR and dependent upon external Ca(2+) for refilling.

Two Ca2+ entry pathways mediate InsP3-sensitive store refilling in guinea-pig colonic smooth muscle.

Sarcolemma Ca2+ influx, necessary for store refilling, was well maintained, over a wide range (-70 to + 40 mV) of membrane voltages, in guinea-pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP3-evoked Ca2+ transients. This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca2+ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. -70 mV), Ca2+ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage-gated Ca2+ channels were largely responsible. Sarcolemma voltage-gated Ca2+ currents were not activated following store depletion. Removal of external Ca2+ or the addition of the Ca2+ channel blocker nimodipine (1 microM) inhibited store refilling, as assessed by the magnitude of InsP3-evoked Ca2+ transients, with little or no change in bulk average cytoplasmic Ca2+ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca2+ gradient. Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca2+ gradient. Store access to the gradient allows InsP3-evoked Ca2+ signalling to be maintained over a wide voltage range in colonic smooth muscle.

Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle.

1. Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+]c) in guinea-pig single colonic myocytes has been examined, using whole-cell recording, flash photolysis of caged InsP3 and microfluorimetry. 2. Depolarization increased [Ca2+]c and triggered contraction. Resting [Ca2+]c was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50 % of its fully relaxed length. The muscle then slowly relaxed (t = 17 s). 3. The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+]c. The extent of the overshoot or undershoot increased with increasing peak [Ca2+]c. 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 microM), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high ( > 300 nM) but not at lower [Ca2+]c and abolished [Ca2+]c overshoots. Oligomycin B (5 microM), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5. During depolarization, the global rhod-2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+]m) rose slowly in a CCCP-sensitive manner during and for about 3 s after depolarization had ended. [Ca2+]m then slowly decreased over tens of seconds. 6. Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7. Flash photolysis of caged InsP3 (20 microM) evoked reproducible increases in [Ca2+]c. CCCP (5 microM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 microM) did not reduce the inhibition of the InsP3-induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8. While CCCP reduced the magnitude of the InsP3-evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin-evoked Ca2+ release, did not decrease significantly. 9. [Ca2+]c decline in smooth muscle, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.

Neuronal mediators of inhibitory junction potentials and relaxation in the guinea-pig internal anal sphincter.

1. Inhibitory junction potentials (IJPs) and relaxations evoked in response to field stimulation (supramaximal voltage, 0.1 ms, single stimulus and 5 stimuli at 5-40 Hz) of non-adrenergic non-cholinergic (NANC) nerves with atropine and phentolamine (each 1 microM) were measured in the guinea-pig internal anal sphincter (gpIAS). The mean resting membrane potential was -44.2 +/- 0.2 mV (n = 1119 cells from 260 preparations). 2. NANC nerve stimulation evoked frequency-dependent IJPs (19.7 +/- 1.1 mV, n = 165, 33 tissues to a single stimulus) and relaxations. IJPs consisted of two tetrodotoxin (1 microM)-sensitive components: one was abolished by apamin (0.3 microM) and the P2-purinoceptor antagonist suramin (100 microM); the other, smaller in amplitude, was sensitive to inhibitors of nitric oxide synthase (NOS, e.g. L-NAME, 100 microM) and the nitric oxide (NO) scavenger oxyhaemoglobin (HbO, 10 microM). 3. ATP (1 mM), vasoactive intestinal polypeptide (VIP, 0.01-0.25 microM) and pituitary adenylate cyclase-activating peptide (PACAP(1-27), 0.84 microM) each hyperpolarized and relaxed the gpIAS; only ATP responses resembled the evoked IJPs in time course. 4. The guanylyl cyclase inhibitor LY83583 (10 microM) abolished apamin-insensitive IJPs and relaxations. The cGMP phosphodiesterase inhibitor M&B 22948 (30 microM) and 8-Br-cGMP (100 microM) each hyperpolarized the gpIAS. 5. Two components comprise the IJP and relaxation evoked in response to NANC nerve stimulation in the gpIAS. One, sensitive to apamin, resembles the response to ATP and is modulated by purinoceptor antagonists; the other, apamin and suramin insensitive, is inhibited by NO antagonists.

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)

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.

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.

Mechanical and biochemical effects of individual co-transmitters in rat tail arteries.

1. The effects of (1) nerve stimulation (NS, 0.5 ms, 1-16 Hz, supramaximal voltage, 20 s) upon vasoconstriction, as measured by changes in perfusion pressure and (2) exogenously added co-transmitters noradrenaline (NA) and alpha, beta,-methylene ATP (alpha, beta, MeATP) upon polyphosphoinositide (PPI) breakdown, and vasoconstriction, were studied in rat tail perfused arteries or arterial rings. The interaction between NA and ATP upon contraction of artery rings and inositol phosphate (IP) accumulation was also studied. 2. Nerve stimulation evoked vasoconstriction in rat tail arteries. These pressor responses were largely (approximately 80%) blocked by the alpha 1-adrenoceptor antagonist, prazosin (10(-6) M), but not significantly affected by desensitization of P2x-purinoceptors by prior, repeated (five times) addition of alpha, beta, MeATP (10(-6) M). 3. Noradrenaline evoked a prazosin (10(-6) M)-sensitive, concentration-dependent, increase in both perfusion pressure and total inositol phosphate (IP) accumulation over the same concentration range (10(-6)-10(-4) M). The amplitude of the pressor responses to NA were about 80% of those obtained to nerve stimulation (0.5 ms, 1-16HZ 20s supramaximal voltage). 4. alpha, beta,-methylene ATP (10(-7)-10(-5) M) evoked a concentration-dependent increase in perfusion pressure which, at maximum, was equivalent in amplitude to approximately 20% of that obtained to nerve stimulation. Concentrations of the nucleotide greater than 10(-5) M were required to stimulate total IP accumulation. 5. The effects of NA (10(-6) M) and alpha, beta, MeATP (10(-5) M) upon contraction of artery rings and IP accumulation were additive and no evidence of synergism between them, on either parameter, was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Intracellular recordings from gallbladder ganglia of opossums.

Postganglionic parasympathetic neurons of the opossum gallbladder were studied using morphological and intracellular electrophysiological recording techniques. On average there were 17 ganglia/cm2 with 8 neurons/ganglion arranged as loosely or densely packed clusters. Intracellular injection of horseradish peroxidase identified two types of neurons. Most of the neurons (approximately 80%) had a single long process that extended beyond the ganglion border with few or no short additional neurites. The other type was multipolar, having several processes that did not appear to extend beyond the ganglion border. Gallbladder neurons had passive and active electrical properties similar to other parasympathetic ganglion cells. Most of neurons studied (71%) responded to intracellular injection of suprathreshold depolarizing current with a short burst of action potentials. The remaining neurons (29%) responded with only an initial action potential. Seventy percent of the neurons tested received nicotinic fast excitatory postsynaptic potentials (EPSPs). Nineteen percent of these neurons also exhibited a slow depolarizing response following repetitive orthodromic nerve stimulation. Cholecystokinin octapeptide sulfate (CCK-8, 0.1-500 nM) had no effect on resting membrane potential or membrane input resistance. However, CCK-8 increased the amplitude of fast EPSPs evoked by orthodromic nerve stimulation by facilitating release of acetylcholine because CCK-8 had no effect on the postsynaptic response to exogenously applied acetylcholine. These data suggest that the ganglionated plexus of the gallbladder may mediate some of the observed effects of CCK-8 on gallbladder motility.

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.

Vascular smooth muscle responses in normo- and hypertensive rats to sympathetic nerve stimulation and putative transmitters.

1. Using changes in perfusion pressure as a measure of end organ response, the effects of field stimulation (0.5 ms supramaximal voltage), noradrenaline (NA, 10(-5)-10(-3) M), adrenaline (ADR, 10(-6)-10(-4) M) and adenosine triphosphate (ATP, 10(-4)-10(-2) M) on the tail artery and mesenteric bed preparations in both normotensive (WKY) and spontaneously hypertensive (SHR) rats were examined. 2. The pressor responses in both preparations from SHR rats to field stimulation, NA and ADR were significantly (P less than 0.05) greater than those from age-matched WKY controls. Responses of both preparations to ATP in normo- and hypertensive rats did not differ significantly. 3. In both preparations from either WKY or SHR rats, pressor responses to ATP (10(-4)-10(-2) M) were inhibited by alpha,beta-methylene ATP (alpha,beta MeATP, 1 x 10(-6) M) while those to field stimulation were not. Phentolamine (2 x 10(-6) M) and prazosin (1 x 10(-7) M) each inhibited the pressor responses to both field stimulation, NA and ADR. 4. [3H] was released by field stimulation from tail arteries pre-incubated with either [3H]-NA or [3H]-adenosine in both normotensive and hypertensive rats. Release in each case was abolished by tetrodotoxin (1 x 10(-6) M). 5. There was no significant difference in the stimulation-evoked [3H]-NA overflow between SHR & WKY rats, alpha,beta MeATP had no significant inhibitory effect on the overflow of [3H] following incubation with [3H]-NA from either group of animals. 6. In the presence of diltiazem (2 x 10(-6) M) and prazosin (5 x 10(-7) M) to abolish any squeezing effect of muscle contractions on ATP release, there was no significant difference in the [3H] overflow between tail arteries from SHR and WKY rats following incubation with [3H]-adenosine. 7. The results confirm the increased response to nerve stimulation in hypertensive animals, an effect probably mediated postsynaptically via alpha-adrenoreceptors. There was no evidence for the involvement of ATP in the hypertensive state.

The electrical and mechanical basis of co-transmission in some vascular and non-vascular smooth muscles.

1. The intracellularly-recorded electrical and mechanical responses to field stimulation of intramural nerves in three sympathetically-innervated smooth muscles--the mouse vas deferens, the rabbit ear artery and the rabbit mesenteric bed preparation were investigated. 2. In each tissue there was evidence for co-transmission involving noradrenaline (NA) and adenosine 5'-triphosphate (ATP) or a closely related nucleotide. 3. The electrical response in each tissue consisted of excitatory junction potentials (ejps) which were abolished by alpha, beta-methylene ATP (alpha, beta MeATP, 1-10 X 10(-6) M), suggesting that they were mediated by ATP. Only in the rabbit ear artery was there an additional electrical event mediated by NA. This took the form of a small slow membrane depolarization which followed the ejps and which was antagonized by either of the alpha-adrenoreceptor blocking agents phentolamine (1 X 10(-6) M) or prazosin (1 X 10(-7) M). 4. In the mouse vas deferens and rabbit mesenteric artery, both transmitter substances (NA and ATP) played a role in the contractile response to field stimulation. In the rabbit ear artery, NA alone appeared to mediate the contractile event. 5. Contractile responses to nerve-released ATP were accompanied by a membrane potential change, whereas those to NA appeared to be mediated largely by a voltage-independent mechanism. 6. In the mouse vas deferens, the ejps and action potentials evoked by field stimulation appeared to be mediated by a discrete increase in permeability to Na+ and K+. 7. In the mouse vas deferens, local application of bradykinin (1-100 X 10(-7) M) produced a small, slow membrane hyperpolarization. VIP (1-100 X 10(-7) M), neuropeptide Y (1-100 X 10(-7) M), substance P (1-100 X 10(-7) M), somatostatin (1-100 X 10(-7) M), leu-enkephalin (1-100 X 10(-7) M), metenkephalin (1-100 X 10(-7) M) and bombesin (1-100 X 10(-7) M), similarly applied, each produced no significant change in membrane potential. None of these peptides appear to be the transmitter mediating the ejp in this tissue.

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.

Excitatory innervation of the rabbit rectococcygeus muscle by enteric nerves from the terminal large intestine.

The rectococcygeus muscle extends from the longitudinal layer of the external muscles of the terminal colon, anchoring the large intestine to the coccygeal vertebrae. The muscle is thought to assist the colon during defaecation, drawing the large intestine partly into the pelvic cavity. In the rabbit, many fine nerves run between the wall of the colon and a ganglionated nerve plexus on the ventral face of the rectococcygeus muscle. Extracellular recordings of evoked and spontaneously occurring multiunit discharges from these nerve bundles indicated that they may take their origin from the enteric nervous system of the large intestine. Enteric nerves (as we have called them) conducted bursts of spontaneously occurring multiunit discharges, each burst associated with a twisting movement of the rectococcygeus and the colon. Electrical stimulation of enteric nerves elicited cholinergically mediated contractions of the rectococcygeus; however, nerve stimulation failed to elicit relaxation of muscle tone induced by carbachol. Therefore, enteric nerves appeared to supply the rectococcygeus with an excitatory innervation only. These excitatory nerves appeared to remain uninterrupted by a ganglion synapse as they travelled to the rectococcygeus. Stimulation of enteric nerves caused the left and right hemirectococcygeal muscle strips to twist and deform the colo-rectal region to which they were attached. From these results, it is suggested that the rectococcygeus may delay the movement of luminal contents from colon to rectum by forming a weak valve which partially occludes the colo-rectal region. The enteric nerves to the rectococcygeus may assist in the maintenance of continence.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.