Verapamil intensifies neuromuscular blockade produced by gallamine and pancuronium at the chick neuromuscular junction

Experiments were performed to study the effect of verapamil on neuromuscular transmission and muscle contraction at a chick skeletal muscle-nerve preparation. In addition, the effects and interactions of verapamil with some muscle relaxants were studied in the same preparation. These effects were explored by studying the effects of verapamil on: directly-and indirectly-elicited twitch contractions, and neuromuscular blockade produced by gallamine and pancuronium. The results showed that verapamil (2-200 microM) had a differential effect on the twitch responses; more reductions occurred in the indirectly-elicited twitch tension, whereas the directly-elicited twitch response was reduced only by 20-30% of maximum indirectly-elicited twitch tension. Furthermore, in low concentrations (1-20 microM), verapamil significantly increased the neuromuscular blockade produced by gallamine (28-1280 nM) and pancuronium (18-573 nM). In high concentrations (greater than 200 microM), verapamil completely blocked the indirectly-elicited twitch response and produced a marked contracture in the chick skeletal muscle (1.0 +/- 0.1 g, n = 6). It was concluded that by reducing twitch tension and inhibiting neuromuscular transmission, verapamil increases (intensifies) neuromuscular blockade produced by muscle relaxants, e.g. gallamine and pancuronium.     

Actions of polypeptides at the neuromuscular junction

The effects of several polypeptides, e.g. angiotensin II, substance P, oxytocin and vasopressin, on the isolated frog gastrocnemius, chick biventer cervicis and rat hemodiaphragm preparations were studied using electrophysiological and neurochemical techniques. The effects of angiotensin II, substance P, oxytocin and vasopressin on neuromuscular transmission and muscle contraction were investigated by studying the following parameters: the directly and indirectly-elicited twitch and tetanic contractions, nerve compound action potential, uptake of 3H-methylcholine into nerve-muscle preparations, the contractures produced by depolarizing drugs, e.g. ACh or TEA. The results showed that angiotensin II (10(-10)-10(-6) M) and substance P (10(-7)-10(-6) M) enhanced neuromuscular transmission and muscle contraction by increasing the amplitudes of the indirectly-elicited twitch and tetanic contractions. Oxytocin and vasopressin (1-100 mU/ml-1) both depressed neuromuscular transmission by reducing the contractile and electrical response in the frog, chick and rat skeletal muscle. It was concluded that, like their effects on ganglionic transmission, the peptides can modify neuromuscular transmission. The mechanism by which these peptides produce their effects may be dependent on external calcium concentration. These peptides may affect both pre- and postjunctional mechanisms; prejunctionally by increasing/decreasing the release of ACh, and postjunctionally by affecting the sensitivity of the postjunctional membrane to depolarizing drugs and/or producing a contracture in the skeletal muscle.     

The neuromuscular effects of lignocaine

The local anesthetic drug lignocaine, in concentrations which completely block both nerve and muscle action potentials, produces concentration-dependent contractures in the vertebrate skeletal and smooth muscle fibres. The contractures produced by lignocaine were not related to membrane depolarization. Although lignocaine greatly reduced the depolarizations and contractures produced by depolarizing agents, such ACh or carbachol, it had a differential effect on the responses produced by tetraethylammonium (TEA) at the chick neuromuscular junction. Lignocaine reduced the TEA-induced depolarizations while it markedly enhanced the contracture responses. In the absence of membrane depolarization, the contractures produced by lignocaine may be attributed to an effect on release of intracellular calcium (Ca2+) from the sarcoplasmic reticulum (SR). The results suggested a multiple site of action of lignocaine at the vertebrate neuromuscular junction.     

Effects of some intravenous anaesthetics on the contractile responses produced in the chick biventer cervicis skeletal muscle

The effects of some intravenous anaesthetics, in low and high concentrations, on the contractile responses produced in the chick biventer cervicis skeletal muscle were studied using electrophysiological and pharmacological techniques. The anaesthetics used were methohexitone sodium, thiopentone, ICI 35 868 (propofol, diprivan), althesin and etomidate. The results showed that all these anaesthetics had a common mode of action at the chick neuromuscular junction. In low concentrations, they increased the amplitude of the indirectly-elicited twitch and tetanic contractions, whereas in high concentrations they reduced the evoked contractions and the contractures produced by depolarizing agents such as acetylcholine or tetraethylammonium. The results indicated that, in low concentrations, anaesthetics may stimulate skeletal muscle whereas in high concentrations they reduce the sensitivity of the postjunctional membrane to depolarizing drugs. The possibility that anaesthetics may block endplate ion channels was also discussed.     

Trichloroethylene interactions with muscle cells

The toxic effect of trichlorethylene (TCE) was investigated on isolated muscles prepared from frog and rats. Twitch and tetanic contractions as well as caffeine-induced contractures, were recorded. Trichloroethylene at a concentration of 0.25-4.0 mM depressed the force development of both twitch and tetanic tension in a dose-dependent manner. This effect was not influenced by the type of muscle. As TCE shortened the time to peak of twitch contractions, it may alter the Ca2+ binding kinetics. Subthreshold caffeine concentrations applied after pre-exposure to TCE (1 or 2mM) induced contractures. The same TCE exposure enhanced regular caffeine contractures through increasing the speed of tension development and the absolute force. Exposure to 5 or 10 mM TCE did not affect the first caffeine-induced contracture but enhanced the potency of the second caffeine dose given 15 min after the first. The results suggest that the interaction of TCE with membrane sites is responsible for Ca2+ release for contractile processes.     

The effect of carbamazepine on the post-tetanic twitch tension in the diaphragm of the mouse

The effects of carbamazepine (0.084-0.25 mM) on the post-tetanic potentiation of the twitch tension, were studied on the isolated phrenic nerve diaphragm preparation of the mouse. Carbamazepine decreased the post-tetanic potentiation of the twitch tension. The maximal depressant effect was found after higher frequencies and longer durations of stimulation. After repetitive stimulation, the amplitude of the endplate potential and the frequency of miniature endplate potentials were potentiated. Carbamazepine decreased the post-tetanic potentiation of the amplitude of endplate potential and the frequency of miniature endplate potentials. The directly-elicited muscle action potential was not affected when post-tetanic potentiation of the twitch tension was decreased. It is concluded that carbamazepine suppressed the post-tetanic potentiation of the indirectly-elicited twitch tension, mainly due to its pre-synaptic inhibitory effect.     

Blockade of acetylcholine release at the motor endplate by a polypeptide from the venom of Phoneutria nigriventer.

1. The mechanisms underlying the muscle relaxation effect of a fraction (PF3) isolated from the Phoneutria nigriventer spider venom were assessed on mouse diaphragm and chick biventer cervicis muscle preparations. 2. PF3 (0.25-4 micrograms ml-1) produced a concentration-dependent blockade of the nerve-elicited muscle twitch of the mouse diaphragm (IC50 = 0.8 micrograms ml-1) without affecting the directly induced muscle twitch. In similar preparations, the crude venom (1-10 micrograms ml-1) produced muscle contracture and blocked both the direct and indirectly induced muscle twitches. 3. In the chick biventer cervicis muscle, PF3 (1-5 micrograms ml-1) blocked the nerve stimulated muscle twitch (IC50 = 1.26 micrograms ml-1), but did not alter the postjunctional response to exogenous acetylcholine (ACh, 10 microM-10 mM). 4. PF3 (2-8 micrograms ml-1) reduced the frequency of miniature endplate potentials (m.e.p.ps) recorded intracellularly from the mouse diaphragm muscle fibers by 58 to 64%, and diminished the amplitude of m.e.p.ps by 20 to 40% of control. The relationship between log m.e.p.p. frequency and log [Ca2+]o was shifted rightwards in the presence of 4 micrograms ml-1 PF3. 5. Raising the frequency of m.e.p.ps with high K+ medium or theophylline (3 mM) did not prevent the toxin-induced depression of spontaneous ACh release. 6. The quantal content of e.p.ps (m), determined in cut-diaphragm muscle fibres, was reduced by 53% and 77% of control by 1 and 4 micrograms ml-1 PF3, respectively. At 1 microgram ml-1 the toxin shifted the relationship between log m and log [Ca2+]o towards higher values without apparent change of the slope. 7. E.p.p. trains elicited at 10 to 50 Hz in the presence of PF3 (1 microgram ml-1) exhibited irregular amplitudes and facilitation related to the frequency of nerve stimulation. 8. It is concluded that PF3 blocks neuromuscular transmission by acting prejunctionally and reducing the nerve-evoked transmitter release. The effect was related to a diminished Ca2+ entry into the nerve terminal associated with inhibition of exocytosis.     

Assessment of neuromuscular blockade produced by atracurium in frog sartorius muscle

The neuromuscular effects of atracurium in frog sartorius muscle were studied and the results were compared with those obtained by tubocurarine in the same preparation. Single twitch (at 1 per 5 s) and tetanic stimulation (at 1-100 per s) of the motor nerve, stimulated with 5-10 V (maximum) and 0.2 ms pulse duration, were used to assess the neuromuscular blockade produced by atracurium and tubocurarine in the frog sartorius muscle. The results showed that atracurium was twice as potent as tubocurarine in reducing the amplitude of the indirectly-elicited twitch contractions in the frog sartorius muscle. The mean IC50 values (concentration to produce 50% inhibition of twitch tension) of atracurium and tubocurarine-induced depression of the indirectly-elicited twitch tension were 0.64 +/- 0.1 microM and 1.2 +/- 0.2 microM respectively (means +/- s.e., n = 6, P less than 0.001). Furthermore, atracurium had a shorter time of onset, time to 50% block and time to 95% recovery than tubocurarine.     

Effect of lignocaine on arginine-vasopressin plasma levels: baseline or induced by frusemide.

1. To assess whether or not lignocaine influences baseline and frusemide-induced (5 mg kg-1) plasma concentrations of arginine-vasopressin (AVP), 2 groups of rabbits received an infusion of lignocaine (130 micrograms min-1 kg-1) for 6 h. Lignocaine-induced changes in AVP plasma concentrations were substantiated by measurement of diuresis and natriuresis and hepatic plasma flow, by means of an infusion of indocyanine green (ICG) (249 micrograms min-1 kg-1). 2. Baseline plasma AVP levels were 4.9 +/- 0.9 pg ml-1 (+/- s.e.), and following lignocaine, these values were reduced to 0.7 +/- 0.1 pg ml-1 (P less than 0.01). Frusemide increased AVP levels to 134.1 +/- 73.6 pg ml-1 (P less than 0.05) and lignocaine totally prevented this increase, e.g. mean AVP levels of 2.7 pg ml-1. 3. Lignocaine enhanced baseline diuresis secondary to an increase in free water clearance; none of the experimental conditions affected the diuresis and natriuresis induced by frusemide. 4. Frusemide reduced the hepatic plasma flow and this decrease was not reversed by the infusion of lignocaine. 5. It is concluded that in healthy rabbits lignocaine reduces baseline secretion of AVP and its antidiuretic effect; in addition, lignocaine prevents the rise in AVP induced by frusemide.     

Mode of inhibitory actions of acute and chronic chloroquine administration on the electrically stimulated mouse diaphragm in vitro.

1. The effects of bath applied chloroquine (Chlo) and of acute and chronic Chlo administration on skeletal muscle reactivity to electrical stimulation and to drugs have been studied on mouse hemidiaphragm preparations in vitro. 2. Chlo (0.15-150 micrograms) produced a concentration-dependent inhibition twitch and tetanic contractions due to direct muscle stimulation (MS). Acute and chronic administration of Chlo (45 mg kg-1, i.p. daily, for 3-28 days) progressively shifted the concentration-response curve to bath-applied Chlo to the right, with maximum effect occurring from day 14 of Chlo pretreatment. 3. Acute and chronic administration of Chlo decreased the twitch and tetanus tension, raised the minimal fusion frequency (MFR) for tetanic contraction to occur and did not alter the twitch/tetanus tension ratio. Tetanus tension unlike twitch tension was not significantly decreased on day 3. 4. Caffeine (5-500 microM)--and isoprenaline (0.001-0.8 microM)-induced potentiations of twitch contraction were attenuated in a concentration-dependent manner by bath-applied Chlo and by acute and chronic administration of Chlo. Higher concentrations of caffeine (0.1-5 microM) and KCl (10 mM-130 mM) produced contracture of the muscle which was sensitive to inhibition by Chlo (50-150 microM). Moreover, the spike contractions superimposed on caffeine contracture were more sensitive to the inhibitory effect of Chlo than the contracture. 5. The inhibitory effects of dantrolene sodium and (+)-tubocurarine on MS and on indirectly stimulated hemidiaphragm respectively were not significantly altered by acute and chronic administration of Chlo. In contrast, the inhibitory concentration-response curve to procaine was shifted to the right.(ABSTRACT TRUNCATED AT 250 WORDS)     

STUDIES ON NORMAL HUMAN SKELETAL MUSCLE IN RELATION TO THE PATHOPHARMACOLOGY OF MALIGNANT HYPERPYREXIA

1. The effects of dantrolene on pharmacologically-induced contractures and potentiated isometric twitches in normal human skeletal muscle have been studied in vitro. 2. Dantrolene sodium, at concentrations of 3 μmol/l or less, attenuates basal twitch, inhibits halothane potentiation of basal twitch and inhibits halothane-potentiated potassium contractures, but has less effect on twitch potentiation by 2 mmol/l caffeine. 3. Caffeine contractures are attenuated by dantrolene concentrations of 12 μmol/l or greater. The effect of dantrolene on caffeine contracture is characterized by decreased contracture tension and by prolonged time to peak contracture. 4. The results indicate that halothane and 2 mmol/l caffeine have agonistic effects on the excitation-contraction (E-C) coupling mechanism, and suggest that they may act at separate E-C coupling sites. The relationships of these findings to the pathopharmacology of malignant hyperpyrexia are discussed.     

Neuromuscular and lethal effects of phospholipase A from Vipera ammodytes venom

—Four homogeneous proteins having phospholipase A activity were separated and studied for their i.v. lethal effects in mice and nerve-muscle activity in the guinea pig diaphragm preparation. Fraction “j” had an ld₅₀ of 0.30 mg/kg, with 3.6 μg/ml of bath solution causing a decrease in the indirectly-elicited nerve-muscle contractions to 20% of control, without significantly changing the directly-elicited muscle contractions. Fraction “k₂” had an ld₅₀ of 0.021 mg/kg and caused a similar nerve or nerve-muscle block at 5 μg/ml of bath solution, without altering the directly-elicited contractions. Fraction “k₁” had an ld₅₀ of 0.58 mg/kg and produced less distinct but dose-related changes in the nerve or in nerve-muscle transmission, as well as weakening directly-elicited muscle contractions to within 60% of control. Fraction “l” had an ld₅₀ of 3.6 mg/kg but high doses of the fraction were required to produce changes in diaphragm contractility. High doses (60 and 195 μg/ml) produced marked effects on both the directly- and indirectly-elicited contractions, suggesting that this fraction affects the muscle directly.     

The effect of substance P on the mechanical and electrical responses of frog, chick and rat skeletal muscle

The effect of substance P (SP) on the electrical and contractile responses of skeletal muscles of frog, chick and rat were studied using electrophysiological techniques. In low concentrations, SP increased the amplitude of twitch and tetanic contractions in response to motor nerve stimulation of frog, chick and rat preparations, increased the amplitude and duration of frog sciatic nerve compound action potential and reduced the contracture responses produced by acetylcholine (ACh) or tetraethylammonium (TEA) in the chick skeletal muscle. In all these actions, the effect of SP was calcium-dependent. The results provide evidence that SP had a dual action; a prejunctional facilitatory effect by causing a further release of ACh and/or Ca2+ entry or release, and postjunctional effect by reducing the sensitivity of the postjunctional membrane to depolarizing drugs.     

Separate sites for the dantrolene-induced inhibition of contracture of the rat diaphragm preparation due to depolarization or to caffeine.

Addition of dantrolene 8.5 x 10(-5) M caused a mono-exponential decay of the depolarization contractures caused by inhibition of the sarcolemmal Na,K-ATPase with propranolol 1 mM or by depolarization of the sarcolemma and T tubular membranes with KCl 100 mM. The half-times of the inhibitory effects were 6 s for the propranolol contracture and 11 s for the KCl contracture. The inhibition of both contractures was complete. Inhibition of the caffeine (10 mM) contracture was bi-exponential with half-times of 45 s and 9.5 min. Inhibition was incomplete; 29.6 +/- 5.0% of the contracture tension could not be inhibited. The inhibition of twitch contractions was similar to that of the caffeine contracture, with half-times of 48 s and 9.1 min, and 20.6 +/- 1.2% of the initial twitch tension could not be inhibited. The contracture tensions induced by release of Ca from the mitochondria with dicumarol, and by actin-myosin binding with the sulfhydryl inhibitor, N-ethyl-maleimide, could not be inhibited by dantrolene. The present results indicate that dantrolene inhibits depolarization signals from the sarcolemma and the T tubular membranes, in addition to inhibition of the coupling between the T tubules and the sarcoplasmic reticulum, and of the release of Ca from the sarcoplasmic reticulum. All these effects of dantrolene may contribute to its therapeutic effect in malignant hyperthermia.     

The role of acetylcholine in tetraethylammonium induced contractures of the chick biventer cervicis muscle in the presence of lidocaine

Lidocaine (0.92 mM) potentiated tetraethylammonium (TEA, 0.6-6 mM) induced contractures of the chick biventer cervicis muscle (BVC) in vitro. The dose ratio for TEA (EC50 in lidocaine/EC50 control) was 1.16 X 10(-3). Lidocaine (0.92 mM) blocked nerve and muscle action potentials in the BVC preparation, blocked slow fibre MEPPS, and blocked indirectly elicited muscle contraction. Lidocaine (0.92 mM) non-competitively blocked acetylcholine (ACh) induced contractures of the chick BVC. Gallamine competitively blocked ACh contractures (pA2 6.53) but produced only a relatively weak non-competitive block of TEA induced contractures in lidocaine. TEA induced contractures of the chick BVC in the presence of lidocaine probably do not involve ACh.     

Three different ways in which 5-hydroxytryptamine can affect cholinergic activity in guinea-pig isolated ileum

The effects of 5-hydroxytryptamine (5-HT) have been studied on electrically-evoked contractions mediated by cholinergic nerves in guinea-pig isolated ileum. Low concentrations of 5-HT (0.0001-0.01 microgram ml-1) caused a sustained increase in submaximal, electrically-evoked contractions. Higher concentrations of 5-HT (0.1-10 micrograms ml-1) initially evoked a fast, rapidly-fading contraction of the muscle. Subsequently, 5-HT 0.1-10 micrograms ml-1 caused a sustained reduction in the height of the electrically-evoked contractions. The effects of 5-HT 0.01 and 0.1 microgram ml-1 on the electrically-evoked contractions were not blocked by methysergide 0.1 microgram ml-1 or by hexamethonium 10 micrograms ml-1, and may be due to changes in neuronal acetylcholine (ACh) release, since contractions evoked by exogenous ACh were unaffected by 5-HT. The results therefore imply that 5-HT can affect gut cholinergic activity in at least three different ways, two of which may modulate evoked ACh release by mechanisms which may be insensitive to tachyphylaxis.     

Effects of atropine and glycopyrrolate on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation

1. The effects of atropine and glycopyrrolate on neuromuscular transmission and on muscle contraction, were studied, in the rat diaphragm preparation, by analyzing their effects on the indirectly (and directly)-elicited twitch (0.2 Hz), tetanic (50 Hz for 20 sec duration), post-tetanic twitch responses (at 5 sec after the tetanus), and on the phenomenon of post-tetanic twitch potentiation (PTP), which is thought to be of a presynaptic origin, i.e. due to increased transmitter release. 2. Atropine (0.001-10 microM) increased the indirectly-elicited twitch tension by 22 +/- 2.1% (control 0.9 +/- 0.1 g, P less than 0.02), the tetanus by 15 +/- 1.1% (control 3.9 +/- 0.7 g, P less than 0.05), the post-tetanic twitch response by 33 +/- 3.1% (control 1.2 +/- 0.1 g, P less than 0.01) and the PTP value by 36 +/- 1.9% (control 33 +/- 2.3%, P less than 0.01, means +/- SEM = 6). 3. Atropine (0.001-10 microM) had little effect on the directly-elicited twitch tension, but in high concentrations (e.g. 20 microM), it blocked the twitch tension. 4. In contrast, glycopyrrolate (0.1-100 microM) had little effect on the twitch tension (direct or indirect), but it significantly reduced the tetanus (by 38 +/- 3.5%, P less than 0.01), the post-tetanic twitch response (by 17 +/- 1.2%, P less than 0.05) and the PTP values (by 24 +/- 3.1% P less than 0.02). 5. In the presence of hemicholinium (1.3 microM) the responses to atropine and glycopyrrolate were altered (decreased), indicating a possible action on presynaptic mechanism of transmission. 6. It is concluded that atropine and glycopyrrolate produce different (opposite) effects at the rat neuromuscular junction, atropine enhances whereas glycopyrrolate depresses neuromuscular transmission. The effects of these two antimuscarinic drugs may be exerted at the presynaptic nerve terminals, i.e. on presynaptic muscarinic receptors, which are involved in the feedback mechanism of transmitter release.     

Ca antagonistic and non-specific effects of diltiazem on the rat phrenic nerve diaphragm preparation

The calcium antagonist diltiazem (2.8 X 10(-4) M) blocked the twitches of a rat phrenic nerve diaphragm preparation after a period of twitch potentiation. Its ability to block twitches was greater during indirect than direct stimulation. Experiments on the isolated phrenic nerve indicated that the excitability of the nerve was blocked. Diltiazem (2.3-9.0 X 10(-5) M) caused a similar inhibition of indirectly and directly elicited tetanic contractions and EMG. Experiments with d-tubocurarine and lowered temperature disclosed a separate inhibition at the neuromuscular junction. High Ca2+ did not reverse the diltiazem-affected twitch or tetanic contractions, which suggests that they are non-specific effects. KCl (100 mM)-induced contractures were antagonized at low (2.3-4.5 X 10(-5) M) but not at high (1 mM) concentrations of diltiazem. Diltiazem depressed the initial phase of the two-phasic caffeine (10 mM) contracture and increased and accelerated the slow phase. Diltiazem greatly reduced the amplitude and duration of the caffeine-potentiated KCl contracture, and reduced and delayed the slow phase of the KCl-potentiated caffeine contracture. The effects on the combined contractures (caffeine-induced, KCl-potentiated) were partly antagonized by a high Ca2+ (2.2 X 10(-5) M) solution, which suggests that diltiazem has calcium antagonistic effects.     

A pharmacological study of the effect of carbamazepine on neuromuscular transmission in the rat diaphragm

The effects of carbamazepine (0.042-0.42 mM) on neuromuscular transmission were studied on the isolated rat phrenic nerve diaphragm preparation using standard pharmacological and electrophysiological methods. Carbamazepine decreased (1) the antidromic activity of the phrenic nerve, (2) the amplitude of the endplate potential (EPP) and miniature endplate potential (MEPP), (3) the quantal content of the endplate potential, (4) the indirectly-elicited twitch tension, (5) the muscle contracture in chronically denervated muscle induced by acetylcholine (ACh) and (6) the amplitude of the compound phrenic nerve action potential, in a concentration-dependent manner. The antidromic activity of the phrenic nerve was the most affected, while the phrenic nerve compound action potential was least affected. However, the IC50 for carbamazepine (the concentration of carbamazepine that inhibited 50% of the response) was in the same order of concentration, i.e. 0.11-0.3 mM. Compared with the effect of carbamazepine on the indirectly-elicited twitch tension with its actions described above, it is concluded that carbamazepine interfered with the neuromuscular activity by inhibiting pre- and postsynaptic process and conduction in the phrenic nerve.     

Cyclo-oxygenase inhibitors antagonize indirectly evoked contractions of the guinea-pig isolated ileum by inhibiting acetylcholine release

The effects of indomethacin, sodium meclofenamate and ketoprofen on the contractile responses of the guinea-pig isolated ileum to directly and indirectly evoked stimuli were investigated. The effects of the cyclo-oxygenase inhibitors on acetylcholine (ACh) release from plexus containing longitudinal muscle strips were also studied. The cyclo-oxygenase inhibitors reduced contractile responses to transmural stimulation (TMS) and nicotine at concentrations which had no effect on ACh-induced contractions. In whole ileum preparations (WIP) indomethacin and ketoprofen (40 micrograms ml-1) reduced TMS responses by 17 +/- 1.8% and 12 +/- 1.8% (n = 6), respectively (30 min incubation). In longitudinal muscle strips (LMS) in which Auerbach's plexus is exposed, indomethacin and ketoprofen (1 microgram ml-1) reduced TMS responses by 28 +/- 2.3% and 34 +/- 2.7% (n = 6), respectively (10 min incubation). Thus the cyclo-oxygenase inhibitors were up to 80 times more effective in LMS than in WIP. The drugs were similarly more effective in blocking nicotine contractions in LMS than in WIP. The cyclo-oxygenase inhibitors reduced basal and stimulated ACh release from LMS. For example, indomethacin (1 microgram ml-1) reduced stimulated ACh release by 35% after 10 min incubation. The percentage inhibition increased to 79% after 40 min incubation (n = 6). Prostaglandin E2 (PGE2) (0.1-2.5 ng ml-1) restored the contractile responses and ACh release depressed by the cyclo-oxygenase inhibitors but not the contractile responses depressed by atropine. PGF2 alpha had no effect on mechanical responses or ACh release depressed by the cyclo-oxygenase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)