Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca(2+)](i) and Cl(-) channels.

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca(2+)](i) and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca(2+)](i), with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca(2+)](i) was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca(2+)](i) were abolished by loading the preparations with the Ca(2+) chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP(3) induced Ca(2+) release, by N-ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl(-))-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP(3) formation. This causes Ca(2+) release from intracellular stores which activates Ca(2+)-dependent Cl(-) channels.

Parallel metabotropic pathways in the heart of the toad, Bufo marinus.

This study examined the transduction pathways activated by epinephrine in the pacemaker region of the toad heart. Recordings of membrane potential, force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were made from arrested toad sinus venosus. Sympathetic nerve stimulation activated non-alpha-, non-beta-adrenoceptors to evoke a membrane depolarization and a transient increase in [Ca(2+)](i). In contrast, the beta-adrenoceptor agonist isoprenaline (10 microM) caused membrane hyperpolarization and decreased [Ca(2+)](i). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the isoprenaline-evoked membrane hyperpolarization. Epinephrine (10-50 microM) caused an initial membrane depolarization and an increase in [Ca(2+)](i) followed by membrane hyperpolarization and decreased [Ca(2+)](i). The membrane depolarizations evoked by sympathetic nerve stimulation or epinephrine were abolished either by the phospholipase C inhibitor U-73122 (20 microM) or by the blocker of D-myo-inositol 1,4,5,-trisphosphate-induced Ca(2+) release, 2-aminoethoxydiphenyl borate (2-APB, 60 microM). Neither U-73122 nor 2-APB had an affect on the membrane hyperpolarization evoked by beta-adrenoceptor activation. These results suggest that in the toad sinus venosus, two distinct transduction pathways can be activated by epinephrine to cause an increase in heart rate.

Potentiation by neostigmine of responses to vagal nerve stimulation in the sinus venosus of the toad.

The effects of the cholinesterase inhibitor neostigmine on the responses to vagus nerve stimulation of isolated sinus venosus/atrial preparations of the toad, Bufo marinus, were examined. In control solutions, trains of stimuli applied to the vagus nerve led to a decrease in heart rate that was susceptible to muscarinic receptor blockade. Membrane potential recordings made from sinus venosus cells showed that the responses to trains of stimuli, delivered at frequencies of less than 10 Hz, were little changed by the addition of neostigmine. However, the responses to longer trains of stimuli at 10 Hz (30 versus 10 s) were potentiated and the nature of the membrane potential changes was altered. The results suggest that, due to the activity of cholinesterases, acetylcholine (ACh) released from parasympathetic nerves normally has little access to the muscarinic receptors in the pacemaker region, which are linked to potassium channels.

Mechanisms of excitatory neuromuscular transmission in the guinea-pig urinary bladder.

1. In smooth muscle of the guinea-pig bladder, either membrane potential recordings or [Ca2+]i measurements were made simultaneously with isometric tension recordings. 2. Single transmural stimuli initiated excitatory junction potentials (EJPs) which triggered action potentials, transient increases in [Ca2+]i and associated contractions. These responses were abolished by alpha, beta-methylene ATP, suggesting that they resulted from the activation of purinoceptors by neurally released ATP. 3. Nifedipine abolished action potentials leaving the underlying EJPs and reduced the amplitude of both nerve-evoked increases in [Ca2+]i and associated contractions. The subsequent co-application of caffeine and ryanodine inhibited the residual responses without inhibiting EJPs. These results indicate that stimulation of purinoceptors activates both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular Ca2+ stores. 4. In the presence of alpha, beta-methylene ATP, trains of stimuli failed to initiate EJPs but increased the frequency of action potentials. Trains of stimuli also initiated oscillatory increases in [Ca2+]i and associated contractions. These responses were abolished by hyoscine, indicating that they resulted from the activation of muscarinic receptors by neurally released ACh. 5. Oscillatory increases in [Ca2+]i and associated contractions were inhibited by either nifedipine or caffeine, indicating that the stimulation of muscarinic receptors activates both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular Ca2+ stores.

Electrical behavior of guinea pig tracheal smooth muscle.

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.

Sympathetic neuroeffector transmission in the rat anococcygeus muscle.

1. When intracellular recordings were made from preparations of rat anococcygeus muscle, transmural nerve stimulation evoked noradrenergic excitatory junction potentials (EJPs) made up of two distinct components. Both components were abolished by either guanethidine or alpha-adrenoceptor antagonists, indicating that they resulted from the release of transmitter from sympathetic nerves and the subsequent activation of alpha-adrenoceptors. 2. The first component was associated with a transient increase in the intracellular concentration of calcium ions ([Ca2+]i) and a contraction. Although the second component was often associated with a long lasting increase in [Ca2+]i it was not associated with a contraction unless the second component initiated an action potential. 3. The increase in [Ca2+]i associated with the first component resulted from Ca2+ release from an intracellular store and from entry of Ca2+ through voltage-dependent Ca2+ channels. The increase in [Ca2+]i associated with the second component resulted only from the entry of Ca2+ through L-type Ca2+ channels (CaL channels). The depolarization associated with the initial increase in [Ca2+]i was abolished by reducing the external concentration of chloride ions ([Cl-]o), suggesting that it involved the activation of a Cl- conductance. 4. When the relationships between changes in [Ca2+]i, membrane depolarization and contraction produced by an increasing number of sympathetic nerve stimuli were determined in control, and caffeine- and nifedipine-containing solutions, it was found that an increase in [Ca2+]i recorded in nifedipine produced a larger contraction and larger membrane depolarization than did a similar increase in [Ca2+]i recorded in either control or caffeine-containing solutions. These observations indicate that Ca2+ released from stores more readily triggers contraction and membrane depolarization than does Ca2+ entry via CaL channels.

Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i, and force in the toad sinus venosus.

The effects of sympathetic nerve stimulation on beat rate, force, intracellular Ca2+ concentration ([Ca2+]i) measured using fura 2, and membrane potential were recorded from the spontaneously beating toad sinus venosus. Short trains of stimuli evoked an increase in the beat rate and force. During this tachycardia the amplitude of pacemaker action potentials was not changed, but there was an increase in the basal level of [Ca2+]i with little change in peak [Ca2+]i measured during each action potential. Depletion of intracellular Ca2+ stores with caffeine (3 mM) abolished all responses to sympathetic nerve stimulation. The effects of caffeine were fully reversible. Caffeine (3 mM), in the presence of the Ca2+-ATPase inhibitor thapsigargin (30 microM), abolished irreversibly the chronotropic and inotropic responses evoked by sympathetic nerve stimulation. Ryanodine (10 microM) attenuated, but did not abolish, these responses. These results suggest that, in the toad sinus venosus, increases in force and beat rate evoked by sympathetic nerve stimulation result from the release of Ca2+ from intracellular Ca2+ stores.

Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i and force in the arrested sinus venosus of the toad, Bufo marinus.

1. The effects of sympathetic nerve stimulation on membrane potential and on the intracellular concentration of calcium ions, [Ca2+]i, were recorded concurrently from the sinus venosus of the toad, Bufo marinus, in preparations where beating had been abolished by adding an organic calcium antagonist to the physiological saline. In a separate set of experiments the effects of sympathetic nerve stimulation on force production were examined. 2. Stimulation of the sympathetic nerves caused a membrane depolarization and a simultaneous increase in [Ca2+]i. Both responses were reduced by dihydroergotamine (20 microM). 3. The membrane depolarization and increase in [Ca2+]i evoked by sympathetic nerve stimulation were abolished by ryanodine (10 microM), or caffeine (3 mM). The effects of caffeine, but not those of ryanodine, were fully reversible. 4. Although the Ca(2+)-ATPase inhibitor thapsigargin (30 microM) itself had little effect on the responses to sympathetic nerve stimulation, in its presence caffeine (3 mM) irreversibly abolished the responses. 5. In the presence of nifedipine (10 microM), sympathetic nerve stimulation caused contractions of the sinus venosus. These responses were abolished by either ryanodine (10 microM) or caffeine (3 mM). 6. The results suggest that neuronally released transmitter activates a complex biochemical pathway which triggers the release of Ca2+ from internal stores.

Electrical properties of smooth muscle in the guinea-pig urinary bladder.

1. The effects of transmural nerve stimulation were examined on preparations of detrusor smooth muscle from guinea-pig urinary bladder using intracellular recording techniques. Most recordings were made from preparations in which spontaneous and evoked action potentials had been inhibited by nifedipine (10 microM), a dihydropyridine that blocks L-type Ca2+ channels. 2. Supramaximal stimuli evoked excitatory junction potentials (EJPs) which could be divided into three basic types. Type 1 EJPs had short latencies (< 30 ms) and fast rise times (< 60 ms). Type 2 EJPs consisted of two components: a small depolarization that was followed by a second depolarization with a faster rise time. In a third type of cell, at high strengths of stimulation, EJPs resembled type 1 EJPs but at lower strengths of stimulation were similar in time course to type 2 EJPs. 3. All EJPs were abolished by tetrodotoxin (1 microM) and reduced by omega-conotoxin (0.1 microM), but were unaffected by hexamethonium (0.1 mM), suggesting that they result from the release of transmitter from post-ganglionic nerve fibres. All responses persisted in the presence of atropine (1 microM) but were abolished following the desensitization of P2-purinoceptors with alpha, beta-methylene ATP (m-ATP; 10 microM). 4. Spontaneous excitatory junction potentials (SEJPs) were also recorded from most cells. SEJPs were similar in appearance to fast single-component EJPs; however, in general they had a briefer time course. SEJPs persisted in the presence of tetrodotoxin (1 microM). 5. The electrical properties of urinary bladder smooth muscle were also examined. Voltage changes induced by point current injection into cells had fast rates of rise and decay (time constant, 5-20 ms). The input resistance of cells ranged between 12 and 108 M omega. When recordings were taken from cells near the point of current injection, resultant electrotonic potentials could be detected in only a small proportions of cells. 6. The results are discussed in relation to the idea that transmural nerve stimulation in the guinea-pig urinary bladder causes the activation of at least two different membrane conductances. Cells appear to be electrically coupled with one another. However, it is likely that coupling exists within discrete bundles of the smooth muscle.

Ionophoretically applied acetylcholine and vagal stimulation in the arrested sinus venosus of the toad, Bufo marinus.

1. The effects of acetylcholine (ACh), applied by ionophoresis, on the isolated arrested sinus venosus of the toad, Bufo marinus, were examined. 2. At each position where ACh was applied across the surface of sinus venosus preparations, a hyperpolarization was produced. These responses were abolished by hyoscine, indicating that muscarinic cholinoceptors are widely distributed over the surface of these muscle cells. 3. Vagal stimulation produced hyperpolarizations which were mimicked, to some extent, by ionophoretically applied ACh. 4. The responses to ionophoretically applied ACh were abolished by adding barium ions to the perfusion fluid, whereas responses to vagal stimulation persisted. 5. The responses to ionophoretically applied ACh were consistently slower than those to vagal stimulation. It is argued that the pathways activated by neural and applied ACh have different kinetics of activation.