Electrical coupling between the myenteric interstitial cells of Cajal and adjacent muscle layers in the guinea-pig gastric antrum.

Intracellular recordings were made from short segments of the muscular wall of the guinea-pig gastric antrum. Preparations were impaled using two independent microelectrodes, one positioned in the circular layer and the other either in the longitudinal layer, in the network of myenteric interstitial cells of Cajal (ICCMY) or in the circular layer. Cells in each layer displayed characteristic patterns of rhythmical activity, with the largest signals being generated by ICCMY. Current pulses injected into the circular muscle layer produced electrotonic potentials in each cell layer, indicating that the layers are electrically interconnected. The amplitudes of these electrotonic potentials were largest in the circular layer and smallest in the longitudinal layer. An analysis of electrical coupling between the three layers suggests that although the cells in each layer are well coupled to neighbouring cells, the coupling between either muscle layer and the network of ICCMY is relatively poor. The electrical connections between ICCMY and the circular layer did not rectify. In parallel immunohistochemical studies, the distribution of the connexins Cx40, Cx43 and Cx45 within the antral wall was determined. Only Cx43 was detected; it was widely distributed on ICCMY and throughout the circular smooth muscle layer, being concentrated around ICCIM, but was less abundant in the circular muscle layer immediately adjacent to ICCMY. Although the electrophysiological studies indicate that smooth muscle cells in the longitudinal muscle layer are electrically coupled to each other, none of the connexins examined were detected in this layer.

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.

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.

Neuronally released and applied acetylcholine on the longitudinal muscle of the guinea-pig ileum.

Brief transmural stimuli, which selectively excited cholinergic fibres, initiated contractions and excitatory junction potentials in preparations of longitudinal muscle isolated from the guinea-pig ileum: these responses were associated with an increase in the internal concentration of calcium ions. When muscle voltage-dependent calcium channels were blocked using the organic calcium antagonist nifedipine, brief stimuli continued to initiate contractions, evoke excitatory junction potentials and cause an increase in the intracellular calcium concentration. Ionophoretically applied acetylcholine caused depolarizations which resembled the excitatory junction potentials evoked by cholinergic nerve stimulation. Both responses had slow time courses and were abolished by muscarinic receptor antagonists. However, the depolarizations produced by ionophoretically applied acetylcholine, unlike those produced by nerve stimulation, were frequently interrupted by transient hyperpolarizations. The transient hyperpolarizations were abolished by barium ions or charybdotoxin. High concentrations of the calcium antagonists nicardipine, verapamil or diltiazem had a tendency to preferentially abolish the excitatory junction potential. When the effects of the cholinesterase inhibitor, eserine, on excitatory junction potentials were examined, it became apparent that when the destruction of acetylcholine was prevented it initiated an additional conductance change to that initiated by acetylcholine in untreated tissues. The results are discussed in relation to the idea that neuronally released acetylcholine and applied acetylcholine might activate different subsets of muscarinic receptors on longitudinal ileal smooth muscle.

Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea-pig ileum.

1. Brief transmural stimuli, 0.5-1 ms, initiated contractions of the longitudinal muscle taken from the guinea-pig ileum that were recorded isometrically. In separate preparations similar stimuli were found to initiate excitatory junction potentials which were recorded using intracellular recording electrodes. All of these responses were abolished by either tetrodotoxin, omega-conotoxin or hyoscine. 2. The contractions produced by increasing [K+]o were blocked by nifedipine, 1 x 10(-7) M; nicardipine, 1 x 10(-7) M; verapamil, 1 x 10(-5) M or diltiazem, 1 x 10(-5) M. In these solutions brief stimuli continued to initiate contractions: this indicates that neuronally released acetylcholine continues to trigger a contraction when muscle voltage-dependent calcium channels appear to have been blocked. 3. When membrane potential recordings were made from the smooth muscle layer, brief transmural stimuli initiated excitatory junction potentials that triggered muscle action potentials. Although muscle action potentials were abolished by low concentrations of a range of organic calcium antagonists, excitatory junction potentials persisted and continued to initiate contractions of reduced amplitude. 4. When the internal concentration of calcium ions, [Ca2+]i, was measured using fura-2, brief transmural stimuli caused an increase in [Ca2+]i. Part of this response, which occurred at a time corresponding to the unblocked excitatory junction potential, persisted in the presence of the organic calcium antagonist nifedipine. 5. Two explanations appear possible. Neuronally released acetylcholine may simultaneously activate non-selective cation channels and cause the release of Ca2+ from an internal store. Alternatively, neuronally released acetylcholine may cause an increase in [Ca2+]i which is separate from that which accompanies the activation of voltage-dependent calcium channels. At this stage there is little other anatomical or electrophysiological evidence to support this view.