Cellular mechanism of the voltage-dependent change in slow potentials generated in circular smooth muscle of the guinea-pig gastric corpus.

The cellular mechanism of the voltage-dependent properties of slow potentials were investigated in single bundles of circular smooth muscle isolated from the gastric corpus of guinea-pig using conventional microelectrode recordings. Hyperpolarization of the membrane by current injection decreased the frequency and increased the amplitude of slow potentials linearly. At potentials negative of -80 mV, slow potential generation was abolished and a periodic generation of clustered unitary potentials was evident. Application of cyclopiazonic acid (CPA, 20 microM) or thapsigargin (1 microM; inhibitors of Ca(2+)-ATPase), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 0.1 microM; mitochondrial protonophore) or 2-aminoethoxydiphenyl borate (2-APB, 20 microM; inhibitor of IP(3) receptor-mediated Ca(2+) release) depolarized the membrane and reduced or inhibited the amplitude and frequency of slow potentials: repolarization of the membrane to the resting level by current injection resulted in a recovery of the amplitude of slow potentials in the presence of CPA or CCCP, but not 2-APB. The slow potentials abolished by thapsigargin did not recover upon membrane repolarization. The altered frequency of slow potentials by 2-APB, CPA or CCCP was not reversed by membrane repolarization to control potentials. Depolarization of the membrane by about 10 mV with high-potassium solution also reduced the amplitude and increased the frequency of slow potentials in a manner restored by repolarization to control potentials upon current injection, suggesting that membrane depolarization did not affect the voltage dependency of pacemaker activity. The results indicate that in corpus circular muscles the voltage dependency of the frequency and amplitude of slow potentials requires a functional Ca(2+) store and mitochondria.

Transmission at autonomic neuroeffector junctions.

For organs innervated by the autonomic nervous system, it is generally held that neuroeffector transmission is achieved by varicosities releasing transmitters some distance from the membranes of target cells. Transmitters are thought to diffuse through the extracellular space and interact with post-junctional receptors that are widely distributed over the cell membranes. This article presents an alternative view, suggesting that transmission can occur at organized neuroeffector contacts, that transmitters interact with restricted pools of specialized junctional receptors, and that many receptors on target cells are not involved in neuroeffector transmission.

An ultrastructural analysis of the sympathetic neuromuscular junctions on arterioles of the submucosa of the guinea pig ileum.

The relationship of the varicosities of sympathetic postganglionic nerve terminals to the smooth muscle cells of arterioles in the submucosa of the guinea pig ileum has been investigated quantitatively by electron microscopy. Longitudinal sections were cut through arterioles about 50 micron in diameter after fixation in vitro or in situ under pressure. About 13% of the varicosities in individual ultrathin sections made contact with the outer surface of the smooth muscle cells. The neuromuscular junctions resembled those in skeletal muscle: the basal laminae of the axon bundle and of the smooth muscle were fused, and synaptic vesicles were accumulated close to the region of fusion. When individual varicosities were examined in serial sections, 92% and 83% in two preparations were found to form junctions of this kind. Most of the noncontacting varicosities were bare of Schwann cell toward the arteriolar surface and separated from it by less than 200 nm. Almost all axon profiles contained synaptic vesicles with electron dense cores after exposure to 5-hydroxydopamine. In electrophysiological experiments, ionophoretic application of noradrenaline to the arteriolar surface along the nerve bundles (demonstrated subsequently by fluorescence histochemistry) produced responses resembling those evoked by nerve stimulation. These anatomical and physiological data, taken together with the evidence for quantal release in this preparation (see Hirst et al., '85), suggest that neuromuscular transmission involves the rare release of a quantum of noradrenaline at discrete points on the smooth muscle membrane.

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.

Lack of involvement of alpha-adrenoceptors in sympathetic neural vasoconstriction in the hindquarters of the rabbit.

The hypothesis that sympathetic nerves in arterial blood vessels activate excitatory receptors distinct from alpha-adrenoceptors was investigated in vivo in the rabbit. In anaesthetized, ganglion-blocked rabbits, graded stimulation of the lumbar sympathetic nerve chains caused graded hind limb vasoconstriction. The responses to single pulses and short trains of stimuli were unaffected by benextramine (10 mg kg-1) and the longer trains were enhanced. Phenoxybenzamine (5 mg kg-1) slightly reduced the responses to short trains of stimuli and did not affect the responses to long trains. The dose-response curve to intra-arterial noradrenaline (after beta-adrenoceptor blockade) was shifted rightwards about ten fold by benextramine (10 mg kg-1) and by phenoxybenzamine (5 mg kg-1). In conscious rabbits the vasoconstriction caused by the nasopharyngeal reflex initiated by smoke inhalation was unaffected by benextramine (10 mg kg-1). Small mesenteric arteries (less than 250 microns) taken from untreated rabbits responded to noradrenaline with a threshold concentration of about 1 microM. Similar tissues from benextramine (10 mg kg-1)-treated rabbits were unresponsive to noradrenaline at concentrations up to 300 microM. However, these tissues were able to respond to potassium and angiotensin II. Aortic ring segments taken from the same rabbits were only about ten fold less sensitive to noradrenaline than segments from control rabbits. These results are in accord with the hypothesis that sympathetic nerves activate non-alpha-receptors in the vasculature of the rabbit.

Prostaglandin E1 and noradrenaline at the neuromuscular junction.

The effects of prostaglandin E(1) (PGE(1)) on neuromuscular transmission in the frog nerve-sartorius muscle preparation were studied with intracellular recording techniques. PGE(1) was inactive in all concentrations examined; both spontaneous miniature end-plate potentials and evoked end-plate potentials were unchanged after preparations were bathed in up to 2.2x10(-5)M (8 mug/ml) PGE(1). PGE(1) did not prevent the increase in end-plate potential amplitude which frequently occurred on addition of noradrenaline to the bathing solution.

Changes in the time course of transmitter action produced by procaine.

1. In addition to reducing the sensitivity of the end-plate to transmitter, procaine produced changes in the time course of the end-plate potential. The time to peak potential was reduced by procaine, whether recording from fibres paralysed by tubocurarine or from fibres paralysed by high magnesium concentration. The rate of decay of potential was also markedly reduced. Similar observations were made recording spontaneous miniature end-plate potentials.2. Procaine did not produce any marked change in the electrical constants of muscle fibres.3. In the presence of procaine the accumulation of charge at the end-plate was prolonged; the rate of decay of charge was reduced. It is suggested that the prolonged end-plate potentials arise from a prolongation of the end-plate current.

On the neuromuscular paralysis produced by procaine.

1. The effects of procaine on neuromuscular transmission in the rat phrenic nerve diaphragm preparation have been examined using intracellular recording techniques.2. The paralysis produced by procaine resembled that produced by tubocurarine and it is concluded that this effect of procaine results from a change in post-junctional sensitivity to transmitter; at low rates of stimulation procaine did not depress the transmitter release.3. During high frequency stimulation of the phrenic nerve a distinct form of prejunctional failure was observed. It is suggested that this depression results from a combination of local anaesthesia and anoxia and that it would explain reports of a reduction in the amount of transmitter released when assayed in a conventional manner.

The distribution of gamma-adrenoceptors and P2 purinoceptors in mesenteric arteries and veins of the guinea-pig.

1. Membrane potential changes and contractions were recorded from mesenteric arteries and veins of the guinea-pig, during perivascular nerve stimulation or application of noradrenaline or adenosine triphosphate (ATP). 2. After alpha-adrenoceptor blockade, noradrenaline activated low affinity adrenoceptors (gamma-adrenoceptors) causing depolarization and arterial contraction only in the presence of an inhibitor of catecholamine uptake. 3. Noradrenaline did not cause depolarization or contraction of the vein after alpha-adrenoceptor blockade even after catecholamine uptake was blocked. 4. Adenosine triphosphate caused depolarization and contraction of both arteries and veins. These responses were abolished by alpha-,beta-,methylene adenosine triphosphate (Me-ATP). 5. Me-ATP abolished rapid excitatory junction potentials (e.j.ps) caused by perivascular nerve stimulation of arteries but had no effect on arterial responses mediated by gamma-adrenoceptors. 6. In veins, perivascular nerve stimulation evoked slow e.j.ps which persisted in the presence of Me-ATP but were abolished after blockade of alpha-adrenoceptors. 7. The observations indicate that P2 purinoceptors are present on both mesenteric artery and vein whilst gamma-adrenoceptors are localized near the neuromuscular junction of the artery. However gamma-adrenoceptors do not appear to be directly involved in the generation of arterial e.j.ps.

Electrophysiological analysis of the nature of adrenoceptors in the rat basilar artery during development.

The nature of adrenoceptors in basilar arteries of neonatal rats was investigated by means of electrophysiological techniques. In immature (2-6 day postnatal) rats, micro-injection of noradrenaline elicited a depolarization which consisted of two components. The initial 'fast' component (time to peak of 0.3-4s) was slightly reduced by phentolamine and was not antagonized by propranolol. The second 'slow' component (time to peak of about 50s) was not blocked by phentolamine but was antagonized by low concentrations (10(-7) M) of propranolol. In immature rats, micro-injection of isoprenaline was more potent than noradrenaline in evoking the 'slow' depolarization but less effective in eliciting the 'fast' response. The pharmacology with respect to adrenoceptor antagonists of both components of the isoprenaline- and noradrenaline-induced depolarizations was similar. There was some evidence of inhibitory beta-adrenoceptors in immature rat basilar vessels. In adult rats (6 week old) noradrenaline produced a large 'fast' depolarization which was followed by a 'slow' tail response. Both components were not antagonized by phentolamine or propranolol. It appears that in the basilar artery of neonatal rats there are excitatory alpha- and inhibitory beta-adrenoceptors but the major responses to noradrenaline and isoprenaline are mediated by gamma- and excitatory beta-receptors. In adult animals the gamma-adrenoceptor predominates. Experiments were carried out in which agonists were applied by ionophoresis. These results confirm the presence of excitatory beta-receptors in neonatal basilar vessels and show the response has slow kinetics and it is likely that the beta-receptors are distributed uniformly over the smooth muscle surface. In adult animals it was not possible to elicit an excitatory beta-receptor-mediated response. The ionophoretic application of noradrenaline never evoked a perceptible depolarization which could be attributed to gamma-adrenoceptor stimulation. This result is discussed in terms of receptor distribution with respect to synaptic function in a syncytium.

Noradrenaline (gamma) and ATP responses of innervated and non-innervated rat cerebral arteries.

1. The distribution of sympathetic adrenergic nerves on the rat middle cerebral artery and on the arterioles which originated from it was determined by use of gloxylic histochemistry. 2. Whereas the middle cerebral artery and proximal arterioles arising from this artery received a sympathetic innervation, the distal regions of the same arterioles were devoid of innervation. 3. The arteries and arterioles which were innervated were depolarized by noradrenaline in the combined presence of alpha- and beta-adrenoceptor antagonists. Those which were not innervated were not depolarized by noradrenaline. 4. ATP depolarized all arteries and arterioles examined. 5. These observations are discussed with respect to the similarities and differences between gamma-adrenoceptors and P2 purinoceptors.

Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles.

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal tract and serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. Recent morphological studies have provided evidence that motor neurotransmission in the gut does not occur through loosely defined synaptic structures between nerves and smooth muscle, but rather via synaptic-like contacts that exist between varicose nerve terminals and intramuscular ICC (ICC-IM). ICC-IM are coupled to smooth muscle cells via gap junctions and electrical responses elicited in ICC are conducted to muscle cells. Electrophysiological studies of the stomach of wild-type and mutant animals that lack ICC-IM have provided functional evidence for the importance of ICC in cholinergic and nitrergic motor neurotransmission. The synaptic-like contacts between nerve terminals and ICC-IM facilitate rapid diffusion of transmitters to specific receptors on ICC. ICC-IM also play a role in generating unitary potentials in the stomach that contribute to the excitability of the gastric fundus and antrum.

Failure of Ba2+ and Cs+ to block the effects of vagal nerve stimulation in sinoatrial node cells of the guinea-pig heart.

The aim of the study was to evaluate which ionic currents are modified in the sinoatrial node of guinea pigs when the vagus is stimulated. Responses of isolated atrial preparations to bilateral vagus nerve stimulation were examined. In bath-mounted preparations, 10-s trains of vagal stimulation (1-50 Hz) slowed the rate at which atrial contractions occurred. After the trains of stimuli, the force generated by each contraction was reduced. Both vago-inhibitory responses persisted in the presence of caesium (2 mM) and barium ions (1 mM). Vagal stimulation evoked a similar bradycardia in superperfused preparations in which intracellular recordings were made from pacemaker cells in the sinoatrial node. When pacemaking activity was abolished by adding the organic calcium channel antagonist nifedipine (1 microM) to the perfusate, vagal stimulation generated an inhibitory junction potential (IJP). Both the bradycardia and the amplitude of the inhibitory junction potential increased as the frequency of vagal stimulation was increased. The ability of vagal stimulation to produce inhibitory junction potentials was unaffected by the addition of caesium and barium ions to the perfusate. These observations suggest that the negative chronotropic and inotropic responses to vagal stimulation only minimally involve a muscarinically activated potassium current (I(KACh)) or changes in the hyperpolarization-activated pacemaker current Ih.

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.

An electrical description of the generation of slow waves in the antrum of the guinea-pig.

This paper provides an electrical description of the generation of slow waves in the guinea-pig gastric antrum. A short segment of a circular smooth muscle bundle with an attached network of myenteric interstitial cells of Cajal (ICC-MY) and longitudinal muscle sheet was modelled as three electrical compartments with resistive connexions between the ICC-MY compartment and each of the smooth muscle compartments. The circular smooth muscle layer contains a proportion of intramuscular interstitial cells of Cajal (ICC-IM), responsible for the regenerative component of the slow wave. Hence the equivalent cell representing the circular muscle layer incorporated a mechanism, modelled as a two stage reaction, which produces an intracellular messenger. The first stage of the reaction is proposed to be activated in a voltage-dependent manner as described by Hodgkin and Huxley. A similar mechanism was incorporated into the equivalent cell describing the ICC-MY network. Spontaneous discrete transient depolarizations, termed unitary potentials, are detected in records taken from either bundles of circular smooth muscle containing ICC-IM or from ICC-MY. In the simulation the mean rate of discharge of unitary potentials was allowed to vary with the concentration of messenger according to a conventional dose-effect relationship. Such a mechanism, which describes regenerative potentials generated by the circular muscle layer, also simulated the plateau component of the pacemaker potential in the ICC-MY network. A voltage-sensitive membrane conductance was included in the ICC-MY compartment; this was used to describe the primary component of the pacemaker potential. The model generates a range of membrane potential changes with properties similar to those generated by the three cell types present in the intact tissue.

Neuromuscular transmission in arterioles of guinea-pig submucosa.

1. Intracellular recordings were made from arterioles lying in the submucosa of guinea-pig small intestine. 2. Low frequency perivascular nerve stimulation evoked subthreshold excitatory junction potentials which facilitated. 3. Higher frequency stimulation caused summation of excitatory junction potentials and the initiation of muscle action potentials. 4. Arteriolar constriction was only observed following the initiation of a muscle action potential.