F. R. Edwards

Inward rectification in rat cerebral arterioles; involvement of potassium ions in autoregulation.

1. The resting membrane potentials of proximal and distal segments of the arterioles which arise from the rat middle cerebral artery were determined. Proximal segments had stable membrane potentials with a mean value of ‐69 mV. The membrane potentials of distal segments were less negative and often unstable. 2. When the extracellular concentration of potassium ions [( K+]o) was increased proximal segments of arteriole were depolarized whereas distal ones were hyperpolarized. When [K+]o was decreased both proximal and distal segments were depolarized, the changes being more marked in proximal arterioles. 3. The membranes of proximal segments of arteriole displayed inward rectification at potentials near rest; inward rectification in distal segments of arteriole, when detected, was less pronounced. 4. The activation curve for inward rectification in proximal segments of arteriole was changed by changing the extracellular concentration of K+. A reduction in [K+]o caused the activation curve to move to such negative potentials that the inward rectifier no longer contributed to the resting conductance. 5. Increasing [K+]o changed the activation curve for inward rectification in distal segments of arteriole so that more K+ current flowed at potentials near resting. At the same time the membrane potential hyperpolarized. 6. The results are discussed in relation to autoregulatory changes which occur following changes in the K+ concentration of cerebrospinal fluid.

Selective knockout of intramuscular interstitial cells reveals their role in the generation of slow waves in mouse stomach

1 Intracellular recording techniques were used to compare the patterns of electrical activity generated in the antral region of the stomachs of wild‐type and W/WV mutant mice. Immunohistochemical techniques were used to determine the distribution of c‐kit‐positive interstitial cells of Cajal (ICC) within the same region of the stomach. 2 In wild‐type mice interstitial cells were found at the level of the myenteric plexus (ICCMY) and distributed within the smooth muscle bundles (ICCIM). In these preparations slow waves, which consisted of initial and secondary components, were detected. 3 In W/WV mutant mice ICCMY could be identified at the level of the myenteric plexus but ICCIM were not detected within smooth muscle bundles. Intracellular recordings revealed that smooth muscle cells generated waves of depolarization; these lacked a secondary component. 4 These results indicate that the secondary regenerative component of a slow wave is generated by ICCIM. Thus the depolarization arising from the pacemaker cells, ICCMY, is augmented by ICCIM, so causing a substantial membrane depolarization in the circular muscle layer. Rather than contributing directly to rhythmical electrical activity, smooth muscle cells appear to depolarize at the command of the two subpopulations of ICC.

Intercellular electrical communication among smooth muscle and endothelial cells in guinea‐pig mesenteric arterioles

1 Current clamp studies using two patch electrodes and morphological observations have been performed in guinea‐pig mesenteric arterioles to evaluate intercellular electrical couplings. 2 In electron micrographs, preparations were found to have a single layer of smooth muscle cells. Typical gap junctions were readily observed between endothelial cells only. 3 While immunoreactivity to connexin 40 was strongly expressed on the membranes of endothelial cells only, that to connexin 43 was expressed on both smooth muscle and endothelial cell membranes. 4 Neurobiotin injected into a smooth muscle cell diffused into several neighbouring smooth muscle cells while that injected into an endothelial cell diffused into many endothelial cells. 5 Acetylcholine‐induced hyperpolarizations were conducted from endothelial cells to smooth muscle cells with a relative amplitude of 80.1 %. Ba2+‐induced action potentials were conducted in the opposite direction with a relative amplitude of 92.4 %. 6 An electrotonic potential produced in a smooth muscle cell by current injection diminished steeply with distance as it spread along the muscle layer, plateauing at distances beyond 25 μm. An electrotonic potential produced in an endothelial cell spread within the intima with virtually no reduction. Electrotonic potentials could conduct through myoendothelial couplings, which seemed to behave as ohmic resistors without rectification. 7 The coupling resistance between adjacent smooth muscle cells was estimated to be at least 90 MΩ and that between a smooth muscle cell and the whole endothelial layer to be 0.9 GΩ. 8 The results indicate that although the resistance of myoendothelial couplings is appreciable, the endothelium may be important as a low resistance path connecting many smooth muscle cells.

Unitary nature of regenerative potentials recorded from circular smooth muscle of guinea-pig antrum.

1 When short segments of single bundles of circular muscle of guinea‐pig antrum were isolated and impaled with two microelectrodes, the membrane potential recordings displayed an ongoing discharge of noise. 2 Treating the preparations with acetoxymethyl ester form of BAPTA (BAPTA AM) reduced the membrane noise and revealed discrete depolarizing unitary potentials. The spectral densities determined from control preparations and ones loaded with BAPTA had similar shapes but those from control preparations had higher amplitudes, suggesting that membrane noise results from a high frequency discharge of unitary potentials. 3 Depolarization of isolated segments of antrum initiated regenerative responses. These responses, along with membrane noise and unitary potentials, were inhibited by a low concentration of caffeine (1 mM). 4 Loading the preparations with BAPTA decreased the amplitudes of regenerative responses. Depolarization was now seen to increase the frequency and mean amplitude of unitary potentials over a time course similar to that of a regenerative potential. 5 Noise spectra determined during periods of rest, during regenerative potentials triggered by direct depolarization and during slow waves, recorded from preparations containing interstitial cells of Cajal (ICC), had very similar shapes but different amplitudes. 6 The observations suggest that a regenerative potential, the secondary component of a slow wave, is made up of a cluster of several discrete unitary potentials rather than from the activation of voltage‐dependent ion channels.

Generation of slow waves in the antral region of guinea‐pig stomach ‐ a stochastic process

1 Slow waves were recorded from the circular muscle layer of the antral region of guinea‐pig stomach. Slow waves were abolished by 2APB, an inhibitor of IP3‐induced Ca2+ release. 2 When the rate of generation of slow waves was monitored it was found to vary from cycle to cycle around a mean value. The variation persisted after abolishing neuronal activity with tetrodotoxin. 3 When simultaneous recordings were made from interstitial cells in the myenteric region (ICCMY) and smooth muscle cells of the circular layer, variations in the rate of generation of slow waves were found to be linked with variations in the rate of generation of driving potentials by ICCMY. 4 A preparation was devised which consisted of the longitudinal muscle layer and ICCMY. In this preparation ICCMY and smooth muscle cells lying in the longitudinal muscle layer generated driving potentials and follower potentials, synchronously. 6 Driving potentials had two components, a rapid primary component that was followed by a prolonged plateau component. Caffeine (3 mM) abolished the plateau component; conversely reducing the external concentration of calcium ions [Ca2+]o mainly affected the primary component. 7 Analysis of the variations in the rate of generation of driving potentials indicated that this arose because both the duration of individual driving potentials and the interval between successive driving potentials varied. 8 It is suggested that the initiation of pacemaker activity in a network of ICCMY is a stochastic process, with the probability of initiating a driving potential slowly increasing, after a delay, from a low to a higher value following the previous driving potential.

Involvement of intramuscular interstitial cells in nitrergic inhibition in the mouse gastric antrum

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse gastric antrum and the responses evoked by stimulating intrinsic nerve fibres were examined. Transmural nerve stimulation evoked a fast inhibitory junction potential (fast‐IJP) which was followed initially by a smaller amplitude long lasting inhibitory junction potential (slow‐IJP) and a period of excitation. The excitatory component of the response was abolished by atropine, suggesting that it resulted from the release of acetylcholine and activation of muscarinic receptors. Fast‐IJPs were selectively reduced in amplitude by apamin and slow‐IJPs were abolished by Nω‐nitro‐l‐arginine. Slow‐IJPs were associated with a drop in membrane noise, suggesting that inhibition resulted from a reduced discharge of unitary potentials by intramuscular interstitial cells of Cajal (ICCIM). The chloride channel blocker, anthracene‐9‐carboxylic acid, reduced the discharge of membrane noise in a manner similar to that detected during the slow‐IJP. When recordings were made from the antrum of W/WV mice, which lack ICCIM, the cholinergic and nitrergic components were absent, with only fast‐IJPs being detected. The observations suggest that neurally released nitric oxide selectively targets ICCIM causing a hyperpolarization by suppressing the discharge of unitary potentials.

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.

Different types of ganglion cell in the cardiac plexus of guinea‐pigs.

1. Intracellular recordings were made from the parasympathetic ganglion cells that lie in the epicardium of the left atrium of guinea‐pig heart near the interatrial septum. 2. Three distinct types of neurone were identified on the basis of their electrophysiological properties. In one group of neurones, S cells, somatic action potentials were followed by brief after‐hyperpolarizations. In the other two sets of neurones, somatic action potentials were followed by prolonged after‐hyperpolarizations. The neurones with prominent after‐hyperpolarization were further subdivided: one group of neurones, P cells, showed inward rectification at membrane potentials near the resting membrane potential whilst neurones in the other group, SAH cells, did so only at more negative potentials. 3. In the group of neurones that displayed inward rectification at potentials near rest, rectification resulted from the activation of an inward current, which resembled the hyperpolarization‐activated inward current present in cardiac muscle pacemaker cells. 4. The three different types of neurone received different patterns of synaptic input. Each SAH cell received a synaptic excitatory connection from the vagus which in most cells released sufficient transmitter to initiate an action potential in that cell; several SAH cells also received a separate connection, which could be activated by local stimulation. Although most S cells failed to receive a synaptic input from the vagus, all of those tested received an excitatory synaptic input which could be activated by local stimulation. Virtually all P cells failed to receive a synaptic input from the vagus; in addition, local stimulation failed to initiate synaptic potentials in P cells. 5. When the structure of cardiac ganglion cells was determined, by loading the cells with either biocytin or neurobiotin, it was found that most cells lacked extensive dendritic processes. S cells were invariably monopolar, most P cells were dipolar or pseudodipolar, whereas many SAH cells were multipolar. 6. In many neurones an on‐going discharge of action potentials was detected in the absence of obvious stimulation. In S and SAH cells, the action potentials resulted from an on‐going discharge of excitatory synaptic potentials. However, when a spontaneous discharge of action potentials was detected in P cells a discharge of excitatory synaptic potentials was not detected. 7. The results are discussed in relation to the idea that the three different types of cell may have different functions and that some of the cells may be organized in such a way as to permit the local handling of neuronal information within the heart.

Inward rectification in submucosal arterioles of guinea‐pig ileum.

1. The current‐voltage relationships of short segments of submucosal arteriole of guinea‐pig have been determined using constant current and voltage clamp techniques. 2. The current‐voltage relationships were non‐linear over the membrane potential range ‐60 to ‐90 mV, the conductance increasing with hyperpolarization. 3. The membrane potential ranges over which the membrane conductance increased were changed by changing the external concentration of potassium ions. 4. Changing the external concentrations of sodium and chloride ions had no effect on the arteriolar current‐voltage relationships. 5. The hyperpolarization‐activated conductance increase was prevented by low concentrations of barium ions. 6. It is suggested that these arterioles display potassium‐selective inward rectification and that the rectifier supplies the dominant resting potassium conductance. The properties of this rectifier are compared with those of other tissues.

Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.

1 Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea‐pig urethra using conventional microelectrode techniques. 2 Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3 STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4 When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5 Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either α,β‐methylene‐ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co‐application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6 In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+‐activated chloride channels. STDs sum to activate L‐type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.