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1. Whole-cell patch-clamp recordings were made from smooth muscle cells isolated from the longitudinal muscle layer of guinea-pig ileum. Carbachol (acting at muscarinic receptors) or histamine (acting at H1 histamine receptors) suppressed Ca2+ channel current. The effect of either agonist had an initial transient component followed by a sustained component. 2. Wortmannin inhibited transient and sustained components of carbachol-induced Ca2+ channel current suppression: half-effective inhibitory concentrations (IC50) were 1.1 microM and 0.6 microM for the two components respectively. Wortmannin also inhibited the transient phase of carbachol-induced cationic current (IC50 1.6 microM) and Ca2+-dependent K+-current (IC50 1.7 microM). Wortmannin did not appear to produce any direct block of cationic channels or Ca2+ channels. 3. Intracellular application of the phospholipase inhibitor D609 (tricyclodecan-9-ylxanthogenate) inhibited transient and sustained components of histamine action on the Ca2+ channel current: the IC50 was about 130 microM for both components. Carbachol action on Ca2+ channels was also inhibited by D609. D609 had no significant direct blocking effect on Ca2+ channels, cationic channels activated by carbachol, or Ca2+-activated K+-current in response to flash-photolysis of caged-inositol 1,4,5-trisphosphate. 4. Micromolar concentrations of wortmannin and D609 are inhibitors of both components of spasmogen-induced Ca2+ channel suppression. The data suggest that both components are mediated by a common, or similar, signal transduction element which is a phospholipase C (PLC) or phospholipase D (PLD) isoform.

1. To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude. 3. Nicardipine (1 microM) and La3+ (10 microM), blockers of voltage-dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient. 4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+-activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients. 5. Thapsigargin (1 microM), an inhibitor of SR Ca2+-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca2+]i transients without affecting action potentials. In addition. in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential-triggered [Ca2+]i transient. 6. Similar action potential-triggered [Ca2+]i transients were induced by brief exposures to high-K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 microM) and caffeine (10 mM) through an open-lock of Ca2+-induced Ca2+ release (CICR)-related channels. 7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca2+]i transient.

1. The effect of trimebutine on the voltage-dependent inward Ca2+ current was investigated by the whole-cell voltage-clamp technique in single smooth muscle cells from rabbit ileum. 2. Trimebutine (3-100 microM) reduced the Ca2+ current in a concentration-dependent manner. The inhibitory effect on the Ca2+ current was also dependent on the holding potential. The Ca2+ current after a low holding potential was inhibited to a greater extent than that after a high membrane potential: the IC50 values were 7 microM and 36 microM at holding potentials of -40 mV and -60 mV, respectively. The Ca2+ current elicited from a holding potential of -80 mV could not be reduced by as much as 50% of the control by trimebutine at concentrations as high as 100 microM. 3. Trimebutine (30 microM) shifted the voltage-dependent inactivation curve for the Ca2+ current by 18 mV in the negative direction. The affinity of the drug for Ca2+ channels was calculated to be 36 times higher in the inactivated state than in the closed-available state. 4. Blockade of the Ca2+ current by trimebutine, unlike verapamil, was not use-dependent. 5. The results suggest that trimebutine inhibits the voltage-dependent inward Ca2+ current through a preferential binding to Ca2+ channels in the inactivated state in the smooth muscle cell from rabbit ileum. The inhibitory effect of trimebutine on gastrointestinal motility is discussed in the light of the present findings.

1. The effects of neurotensin (NT) on membrane potential and membrane current of the longitudinal smooth muscle of chicken rectum were investigated by intracellular recording and whole-cell voltage clamp. 2. NT (3 nM-1.2 microM), when applied via the bathing medium, produced a concentration-dependent membrane depolarization with an EC50 of 18 +/- 2 nM (n = 7) which was accompanied by an increase in the membrane conductance. The effect was biphasic: an initial, rapid depolarization reached a peak within 2-3 min and then declined to a lower but still elevated level which was sustained until washout. 3. Excitatory junction potentials (e.j.ps), which were non-adrenergic non-cholinergic (NANC) in nature, were decreased in amplitude and total duration in the presence of NT (0.6 microM). The depression of the e.j.p. was due mainly to the reduction of the membrane resistance. 4. When NT was applied locally by means of pressure ejection from a micropipette containing NT, some cells responded with a membrane depolarization and some failed to respond, whereas e.j.ps could invariably be elicited from all of them. 5. In single muscle cells enzymatically isolated from the muscle and dialyzed under voltage clamp at -50 mV with a CsCl-rich solution, NT (5 or 10 microM) produced an inward current. NT-induced inward currents were obtained with inclusion of 10 mM EGTA in the pipette solution and their reversal potential was around 0 mV. In cells dialyzed under voltage clamp at 0 mV with a KCl-rich solution, NT (5 microM) produced a brief outward current followed by abolition of spontaneous transient outward currents.6. The present results suggest that the membrane depolarization, which may arise from activation of non-selective cation channels, and release of calcium from internal stores produced by neurotensin are responsible for its contractile activity in the longitudinal smooth muscle of chicken rectum. Further, the depolarizing effect may provide support for the involvement of NT in the NANC transmission in this preparation.

1. The effects of cromakalim on mechanical and electrical activities of the circular smooth muscles of guinea-pig stomach antrum were observed. 2. Cromakalim (greater than 1 x 10(-7) M) decreased the amplitude of spontaneous rhythmic contractions and also the acetylcholine-enhanced spontaneous contractions. Cromakalim was less effective against the 25.9 mM and 35.9 mM K(+)-induced tonic contractions. 3. Glibenclamide (1 x 10(-6) M) itself caused no detectable change in the spontaneous contractions, those potentiated by acetylcholine or tonic contractions induced by high K+ solutions, but attenuated the actions of cromakalim. On the other hand, charybdotoxin (3 x 10(-8) M) increased the amplitude of spontaneous contractions but failed to affect the actions of cromakalim. 4. Cromakalim (greater than 1 x 10(-6) M) decreased the amplitude and duration of slow waves, and hyperpolarized the membrane. These actions of cromakalim were completely antagonized by 1 x 10(-6) M glibenclamide, whereas part of the effects of cromakalim on mechanical activity was resistant to glibenclamide. 5. The results suggest that the inhibition by cromakalim of the electrical activity and the hyperpolarization, which may be associated with the opening of glibenclamide-sensitive K+ channel, are responsible for its inhibitory action on circular smooth muscle of guinea-pig stomach. Further, some effects independent of glibenclamide-sensitive K+ channel may also be responsible for the mechanical effect.

1. The times of appearance and the concentrations of noradrenaline and adrenaline were determined fluorimetrically in the gut of the embryonic and developing chick. 2. In the duodenum and jejunum, noradrenaline was detected in embryos on the 12th day of incubation. The concentration continued to increase throughout the embryonic stage and attained a maximum within 3 days of hatching. Afterwards, it decreased to about one-third of the peak level. No adrenaline was detectable in most stages of development. 3. In the rectum, noradrenaline was detected in embryos on the 12th day of incubation; both noradrenaline and adrenaline were invariably found on the 15th day of incubation. The concentrations of both amines fluctuated after hatching, but the amount of adrenaline was always approximately 40% of the amount of noradrenaline, except in the adult.

1. Changes in membrane potential in response to local application of ATP by pressure ejection from a micropipette were recorded intracellularly from smooth muscle cells of the longitudinal muscle strip of chicken rectum. 2. The local application of ATP produced a membrane depolarization. The depolarizing response increased to a plateau of 33 mV with prolongation of the duration of pressure pulses which determines the amount of ATP ejected. The latency and the time required to reach a peak depolarization were not related to the pulse duration, and the shortest latency was 50 ms. 3. When the application of ATP was repeated at a short interval, the second and subsequent depolarizing responses were suppressed, and their latency and time to reach a peak were also increased; the muscle cells became desensitized to ATP. Recovery from the desensitization occurred slowly over a period of 60 s. 4. Electrotonic potentials decreased in amplitude and time course during an ATP-induced depolarization, indicating a decrease in membrane resistance. 5. The ATP-induced depolarization was longer in the latency than an excitatory junction potential (e.j.p.) elicited by electrical field stimulation of the intramural nerves. The other variables such as amplitude, time to reach the peak and duration could not be matched with those of the e.j.p. at the same time. 6. The e.j.p. decreased in amplitude and duration during the ATP-induced depolarization, and its initial amplitude and duration were restored immediately after termination of the ATP-induced depolarization, as in the case of electrotonic potentials. The ej.ps were recorded in the same manner as the control from the muscle cell which was in a state of desensitization to ATP. 7. The present results provide an argument against the hypothesis that ATP mediates the ej.p. in the rectum of the chicken.

1. Effects of prolonged exposure to alpha,beta-methylene ATP (alpha,beta-Me ATP) on contractions and excitatory junction potentials (e.j.ps) evoked by non-adrenergic, non-cholinergic (NANC) excitatory nerve stimulation have been investigated in the chicken isolated rectum and longitudinal muscle strip from chicken rectum pretreated with atropine (0.5 microM), methysergide (2 microM) and pyrilamine (3 microM). 2. Alpha,beta-Me ATP (20 nM-4 microM) caused a rapid rise in tension of the longitudinal muscle of the isolated rectum preparation which returned to the baseline levels after a few minutes. The magnitude of the contractile response to NANC nerve stimulation was reduced after exposure to the drug. The inhibitory effect was related to the drug concentration; at 4 microM the nerve-mediated contraction was abolished and frequently converted to a relaxation. 3. Adenosine 5'-triphosphate (ATP, 100 microM), bovine neurotensin (2.5 nM) and K+-rich solutions (30 nM and 60 nM) all produced a transient contraction of the isolated rectum preparation. The exposure to alpha,beta-Me ATP (0.2 and 4 microM) also rendered the preparation less sensitive to these stimulant substances. 4. Alpha,beta-Me ATP (0.2 and 4 microM) caused a membrane depolarization in cells of the longitudinal muscle strip. The depolarization reached a peak within 2-3 min after application and then decayed to a steady level that was still more positive than the baseline level. The electrotonic potentials were reduced in amplitude to 44 +/- 8% (n = 7) of the normal amplitude if measured at the peak depolarization produced with 0.2 microM alpha,beta-Me ATP, and to 62 +/- 10% (n = 7) if measured at the steady-state depolarization. With 4 microM, the corresponding percentages were 33 +/- 7% (n = 8) and 55 +/- 7% (n = 8), indicating a decrease in membrane resistance. 5. The e.j.ps in response to field stimulation of the intramural nerves and Remak's nerve stimulation were decreased in amplitude and duration during exposure to alpha,beta-Me ATP (0.2 and 4 microM). 6. The smooth muscle cells regained normal membrane resistance and sensitivity to ATP on washout of alpha,beta-Me ATP (4 microM) more rapidly than the responses to NANC nerve stimulation. 7. It can be argued from the results that the suppression by alpha,beta-Me ATP of the contraction and e.j.p. evoked by NANC nerve stimulation in the chicken rectum, unlike the mammalian preparation described previously, is due mainly to a change in the electrical properties of the membrane of the smooth muscle cells, rather than being due, or only partly due, to desensitization of the purine receptor.

1. Development of the excitatory innervation to the rectal region of the intestine via Remak's nerve has been investigated in the rectum with Remak's nerve supply isolated from chicken embryos and young chicks aged less than two weeks. 2. Electrical stimulation of Remak's nerve produced a small contraction of the rectum isolated from chicken embryos on the 14th day of incubation (the earliest time examined). The contractile response was inhibited partially or totally by atropine (0.1 micrograms ml-1) but enhanced by physostigmine (0.01 to 0.05 micrograms ml-1), indicating its cholinergic nature. 3. During the embryonic stage, the proportion of the atropine-resistant component in the contractile response increased, and the contractile response became almost entirely atropine-resistant within the first week after hatching. 4. Later after hatching, the contractile response was increased in magnitude by atropine and reduced by physostigmine. 5. It is concluded that the excitatory innervation to the chicken rectum via Remak's nerve is cholinergic at the 14-16th day of incubation and is gradually replaced by a non-cholinergic innervation during embryonic development.

1. Isolated longitudinal muscle strips from the chicken rectum responded to isoprenaline, adrenaline and noradrenaline with a prolonged relaxation. The concentrations required to produce 50% of the maximum relaxation were 1.3 x 10(-8) M for isoprenaline, 1.7 x 10(-8) M for adrenaline and 10(-6) M for noradrenaline. The relaxing potency of isoprenaline is about equal to that of adrenaline, but more than 50 times that of noradrenaline. 2. Propranolol, 3.4 x 10(-6) M, blocked the isoprenaline-induced relaxation, and in the presence of this drug the responses to adrenaline and noradrenaline were converted into small, transient relaxations. The residual relaxation was blocked by phentolamine, 2.6 x 10(-6) M. 3. These catecholamines suppressed spontaneous spike discharge and produced membrane hyperpolarization. Propranolol, 3.4 x 10(-6) M, prevented the inhibitory effects of isoprenaline, and reduced but did not completely abolish those of adrenaline and noradrenaline. 4. Adrenaline and noradrenaline, but not isoprenaline, reduced membrane resistance in some preparations. 5. In the rectal muscle of the chicken, the beta-adrenoceptor mediates a prolonged relaxation and the alpha-adrenoceptor a fast and short-lasting relaxation which is usually obscured by the beta-response and unmasked only after blockade of the beta-adrenoceptors. The alpha- and beta-mediated relaxations are each associated with the suppression of spontaneous spike activity.

Peripheral nerve pathways responsible for adrenergic inhibition of mechanical and electrical activities in the chicken rectum and receptors mediating the adrenergic inhibition were investigated in isolated extrinsically-innervated rectum of the chicken. Electrical stimulation of the anal end (Ra) or the ileal cut end (Ri) of Remak's nerve, or perivascular nerves (P) elicited relaxation of the rectum pretreated with atropine (0.5 microM) and hexamethonium (0.3 mM) to block the cholinergic and non-cholinergic, non-adrenergic excitatory innervations. Ri stimulation was much less effective than Ra and P stimulations. The relaxation was shown to be related to cessation of spontaneous spike discharge of the longitudinal muscle which was accompanied by membrane hyperpolarization. The inhibitory effects elicited by Ra and P stimulations, which were prolonged beyond the period of the stimulation, were converted to transient ones by propranolol (3.4 microM). Phentolamine (2.6 microM) reduced effectively the residual effects. In contrast, the effects of Ri stimulation were little affected by these drugs. The present results provide evidence for the existence of two nerve pathways responsible for direct adrenergic inhibitory innervation to the chicken rectum, one running orally in Remak's nerve trunk, leaving it and descending in the branches to the rectum, and the other running as the perivascular nerves along the arterial supplies of the rectum. The direct innervation is mediated predominantly by beta-adrenoceptors.