Narelle J. Bramich

Regenerative component of slow waves in the guinea‐pig gastric antrum involves a delayed increase in [Ca2+]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 [Ca2+]i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca2+]i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca2+]i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca2+]i were abolished by loading the preparations with the Ca2+ chelator MAPTA‐AM. Regenerative potentials were abolished by 2‐aminoethoxydiphenyl borate (2APB), an inhibitor of IP3 induced Ca2+ 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 IP3 formation. This causes Ca2+ release from intracellular stores which activates Ca2+‐dependent Cl− 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 α,β‐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 α,β‐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.

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.

Sympathetic nerve stimulation and applied transmitters on the sinus venosus of the toad.

1. The effect of sympathetic nerve stimulation on pacemaker cells of the isolated sinus venosus of the toad, Bufo marinus, were examined using intracellular recording techniques. 2. Train of stimuli applied to the sympathetic outflow led to a two‐component increase in heart rate. Shortly after the onset of stimulation the rate of discharge of pacemaker action potentials increased. After the end of the train of stimuli, the heart rate fell and then again increased to remain high for several minutes. 3. During the early tachycardia, the peak diastolic potential was reduced and the rate of diastolic depolarization increased. During the late tachycardia, the peak diastolic potential and rate of diastolic depolarization were increased; both the amplitude and the rate of repolarization of the action potentials were increased. 4. When membrane potential recordings were made from sinus venosus cells in which beating had been abolished by adding the organic calcium antagonist nicardipine, sympathetic nerve stimulation caused membrane depolarization. 5. The responses to sympathetic nerve stimulation, recorded from beating or arrested hearts, were abolished by bretylium but persisted in the presence of a number of beta‐adrenoceptor antagonists. 6. Bath‐applied adrenaline caused a tachycardia which was associated with a large increase in the amplitudes of pacemaker action potentials. These effects were largely mediated by the activation of beta 2‐adrenoceptors. 7. In the presence of high concentrations of beta‐adrenoceptor antagonists, applied adrenaline produced membrane potential changes that although slower in time course were similar to those produced by sympathetic nerve stimulation. 8. Many aspects of the responses to nerve stimulation could be mimicked by applied ATP. 9. The early phase of sympathetic tachycardia was abolished after P2‐purinoceptor desensitization; this phase was also inhibited by dihydroergotamine. 10. The results are discussed in relation to the idea that sympathetic nerve stimulation causes the early tachycardia by increasing inward current flow during diastole, a response involving activation of specialized adrenoceptors and perhaps ATP receptors.

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.

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 α‐adrenoceptor antagonists, indicating that they resulted from the release of transmitter from sympathetic nerves and the subsequent activation of α‐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 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 μm). 3 The membrane depolarization and increase in [Ca2+]i evoked by sympathetic nerve stimulation were abolished by ryanodine (10 μm), or caffeine (3 mm). The effects of caffeine, but not those of ryanodine, were fully reversible. 4 Although the Ca2+‐ATPase inhibitor thapsigargin (30 μm) itself had little effect on the responses to sympathetic nerve stimulation, in its presence caffeine (3 him) irreversibly abolished the responses. 5 In the presence of nifedipine (10 μm), sympathetic nerve stimulation caused contractions of the sinus venosus. These responses were abolished by either ryanodine (10 μm) 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.

Responses to sympathetic nerve stimulation of the sinus venosus of the toad.

1. The changes in membrane potential produced by sympathetic nerve stimulation were recorded from sinus venosus preparations of the toad, Bufo marinus, in which beating had been prevented by the dihydropyridine calcium antagonist, nifedipine. 2. Supramaximal sympathetic stimuli initiated long‐lasting excitatory junction potentials which started with the same latencies, some 1 to 2 s, as did sympathetic tachycardias recorded from beating preparations. 3. Brief trains of stimuli increased the amplitude of excitatory junction potentials and shortened their latency of onset. Similarly when excitatory junction potentials were facilitated their latency of onset was shortened. 4. The time courses of excitatory junction potentials were prolonged by cooling the preparation but unchanged when the neuronal uptake of catecholamines was inhibited. 5. In arrested preparations, beta‐adrenoceptor activation causes a hyperpolarization, as did the inhibition of phosphodiesterases or the activation of adenylate cyclase. This contrasts with the depolarization produced by sympathetic nerve stimulation which could be mimicked by the rapid application of either adrenaline or noradrenaline but not by beta‐adrenoceptor activation, phosphodiesterase inhibition or by adenylate cyclase activation. 6. The results are discussed in relation to the idea that neuronally released adrenaline activates a set of adrenoceptors which are linked to a set of channels by a pathway that does not involve cyclic AMP.

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+]iwith little change in peak [Ca2+]imeasured 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 μM), abolished irreversibly the chronotropic and inotropic responses evoked by sympathetic nerve stimulation. Ryanodine (10 μM) 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.

Re-innervation of smooth muscle that is transplanted to provide urethral sphincter augmentation

A number of methods to augment the resistance of the outlet of the urinary bladder and to improve continence have been developed, including the artificial urinary sphincter and the placement of skeletal muscle around the urethra. It has been recently shown in a rabbit model that transplantation of smooth muscle around the proximal urethra reduces incontinence caused by internal sphincter deficiency. In the present work we have investigated the re-innervation of a peri-urethral smooth muscle transplant, and whether re-innervating axons have an appropriate effect when they are stimulated. Detrusor muscle from the dome of the bladder was transplanted to encircle the proximal urethras of rats. Rats tolerated the surgery and transplantation without any signs of compromised health. At 8 weeks the new sphincter was intact and easily recognised. The transplant contracted in response to transmural stimulation (1-5Hz for up to 5min) in a similar way to freshly removed detrusor strips. Contractions were graded with stimulus frequency, they peaked at about 10s and faded to a lower tension that was maintained. The amplitudes of sustained contractions of the transplants were reduced to about 10% by hyoscine and were almost abolished by tetrodotoxin. Histological examination revealed healthy, vascularised smooth muscle in the transplants, similar in appearance to freshly dissected detrusor. Re-innervation was confirmed immunohistochemically for transplanted detrusor muscle and transplants of dartos muscle. We conclude that smooth muscle transplanted to form a new sphincter around the urethra becomes functionally re-innervated and has potential to be used for sphincter augmentation.