Otto F. Schanne

Electrical properties of glass microelectrodes

The electrical characteristics of a glass microelectrode can be designated as a parallel resistance and capacitance in series with a source of dc voltage. Measurement of these parameters depends upon the following factors: concentration, ionic species, pH, and the hydrostatic pressures of the electrolyte solutions inside the microelectrode and surrounding its tip. The physicochemical mechanism underlying microelectrode resistance and tip potential is interpreted as interaction of the surface charges of the glass and the conductivity of the solution inside the microelectrode. Methods for recording microelectrode resistance and tip potential, a method for filling microelectrodes at room temperature, and the automatic recording of microelectrode and tip potential are descried.

Frequency Dependence of the Ionic Currents Determining the Action Potential Repolarization in Rat Ventricular Muscle

Abstract In rat ventricular muscle, the action potential configuration and the ionic currents were studied as a function of frequency under current and voltage clamp conditions. Increase of stimulation frequency produced (a) a progressive loss of the plateau accompanied by a shortening of the action potential; (b) no significant change in the amplitude of the instantaneous outward current in [Ca 2+ ] o between 2.2 and 10.0 m m , and (c) an increase in the amplitude of the slow inward current, I si and a marked reduction of its inactivation time constant τ f up to frequency of about 75 stimuli/min. Above this frequency, the amplitude of I si decreased and τ f increased again.

Determinants of electrical activity in clusters of cultured cardiac cells from neonatal rats

Cell clusters derived from ventricles of newborn rats were found to have action potentials insensitive to TTX but which were abolished by 2 mM manganese. Phase plane trajectories indicated that there was only one current component responsible for the upstroke of the action potential. The mean values for the electrical parameters of these cells were: maximum diastolic potential: −41.6 mV; resting potential: −38.2 m V; firing level: −26.0 m V; action potential amplitude: 59.8 mV; overshoot: 18.5 mV; maximum rate of depolarization 6.56 Vs −1 ; time to peak: 16.2 ms. The maximum inward current was estimated as 15 μA/cm 2 . Circumstantial evidence suggests that in these cells pacemaker activity depends on a slow inward current sensitive to manganese. This preparation could serve as model to study the properties of ectopic pacemakers in ventricular cells.

Effects of K+ channel blockers on the action potential of hypoxic rabbit myocardium

1 In order to assess the role of different ionic currents in hypoxia‐induced action potential shortening, we investigated the effects of blockers of voltage‐dependent and ATP‐sensitive K+‐channels on the membrane potential of hypoxic rabbit hearts and papillary muscles. The response to blocking of the inward rectifier was studied at three external K+ concentrations: 2.5, 5, and 7.5 mm. 2 Hypoxia produced a progressive decline in action potential duration (APD) that levelled off after 15 to 20 min. Steady state APD values at 25% and 95% repolarization (APD25 and APD95) were 26.0 ± 1.9% and 42.2 ± 2.4% of controls respectively. 3 Tetraethylammonium (TEA, 10 mm) delayed but did not reduce APD shortening at the steady state. 4 Blocking of IK1 with a mixture of 0.2 mm Ba2+ and 4 mm Cs+ lengthened APD in normoxia and prevented APD95 shortening in hypoxia. The APD25 shortening was significantly attenuated at all [K]o. 5 Glibenclamide (Glib, 30 μm) did not prevent APD shortening, but produced a progressive action potential (AP) lengthening after 15 min of hypoxia. Steady levels of 48 ± 3.5% and 62 ± 5.0% of controls for APD25 and APD95 respectively were reached after 45 min. 6 The relation between APD25 and pacing rate was determined in normoxic and hypoxic papillary muscles and the effects of 2 mm 4‐aminopyridine (4‐AP) were examined. Hypoxia attenuated the APD25 shortening currently observed when the stimulation rate was lowered from 1 to 0.1 Hz without altering the plateau reduction occurring at frequencies above 2 Hz. These effects were potentiated by 4‐AP. 7 Our data suggest that the accelerated AP repolarization in hypoxic rabbit myocardium represents a delicate balance of several outward currents: IK1, IK‐ATP, and at least one yet unidentified current component rather insensitive to changes in [K]o and to K+ channel blockers.

Effects of TTX and verapamil on the upstroke components of the action potential from the atrioventricular node of the rabbit

Abstract The effects of tetrodotoxin (TTX) and verapamil on atrial, atrionodal and nodal action potentials were studied in the perfused rabbit heart. Control recordings from atrionodal and nodal fibers showed that the upstroke was composed of a fast depolarization (phase I) followed by a slow depolarization (phase II). The amplitude of these phases and their maximum rate of rise ( V max ), as well as action potential amplitude and resting potential, were determined under control conditions and in the presence of TTX (0.3 μ m ) or verapamil (1.1 μ m . TTX selectively depressed phase I in the action potentials from all fiber types. Concomitantly, the amplitude of phase II increased, but its V max did not change. In contrast, the effects of verapamil were limited to phase II, its V max , and the plateau. The slow depolarization was greatly depressed or abolished in the nodal action potential under verapamil. These results support the hypothesis that the fast sodium channel is present in the membrane of nodal cells and that the upstroke of the nodal action potential results from the activation of two inward currents, but the fast inward current contributes less to depolarization in nodal than in atrial fibers because of the low resting potential of the former. It is estimated that the slow inward current contributes about 40% of the total depolarization in nodal fibers.

Electrophysiological responses to ischemia and reperfusion

Myocardial ischemia, defined as an imbalance between energetic demands of the heart and supply of metabolic substrates (inclusive of O2), produces profound changes in cardiac electrical activity which can eventually lead to the development of severe arrhythmias and sudden death. While reperfusion of ischemic myocardium reduces or prevents myocardial necrosis, this procedure may also lead to arrhythmia development, contractile failure and the precipitation of cell death. Although both processes, ischemic and reperfusion injury may share the end point of inducing acute cardiac failure, the cellular events and the mechanisms involved may differ considerably.

Ionic and electrical effects of ouabain on isolated rabbit hearts

The effects of ouabain (0.5 × 10−6m) on the intracellular ionic concentrations and the transmembrane potential were studied. Isolated rabbit hearts were perfused at 33°C and constant coronary flow. Krebs solution containing [14C]inulin was used to measure the extracellular space (ECS). The cell Na and K content were determined after 10 and 60 min perfusion with ouabain. The membrane potential was measured throughout the experiment. Ouabain produced a decrease in ECS and cell potassium ([K]i) and an increase in intracellular sodium ([Na]i) and water. There was a time lag between the change in [Na]i and the change in [K]i. A positive inotropic effect occurred during the first 10 to 20 min of perfusion with ouabain. The resting potential (RP) and action potential amplitude (APA) decreased with a similar time course, reaching values of 63 and 55% of the control by the end of the 60 min period. The maximum rate of depolarization (Vmax) decreased after 10 min, levelling off at about 50% of the control at 30 min. An early lengthening of the plateau was followed by progressive shortening of the action potential. It is postulated that the depolarization is due to inhibition of an electrogenic ionic pump, that the changes in APA are due to ionic shifts and depolarization, and that the values of Vmax are largely determined by shifts in the h∞ curve.

Influence of varied [Ca2+]o and [Na+]o on electrical activity of clusters of cultured cardiac cells from neonatal rats

The effects of sodium and calcium concentration on the action potential of cell clusters derived from rat ventricle were studied. The maximum rate of rise (Vmax) decreased in low sodium (50 mm) medium, but the overshoot did not change. The Vmax did not vary with [Ca2+]o within the range of 0.4 to 5.0 mm, but the size of the overshoot increased with increasing [Ca2+]o. A change of 8.8 mV/decade of change of [Ca2+]o was observed. These results indicate that both calcium and sodium ions carry the inward current responsible for the upstroke. The insensitivity of Vmax to changes in [Ca2+]o can be explained if high calcium shifts the inactivation characteristic of the slow current (f∞) towards more negative potentials. The slope of the diastolic depolarization decreased in low sodium and in low calcium media, and increased in 5 mm [Ca2+]o. These findings, together with the different sensitivity of the upstroke and the diastolic depolarization to TTX [15] lead us to postulate that although calcium and sodium carry the current(s) involved in electrogenesis and automaticity, these phenomena may be mediated by two distinct types of ionic channels having different kinetics.

Partial contribution of the ATP-sensitive K+ current to the effects of mild metabolic depression in rabbit myocardium.

The object of the study was to compare the capability of glibenclamide to block the effects of K+-ATP channel activators on action potential duration and steady state whole cell current to its efficiency in counteracting the effects of hypoxia or metabolic poisons in the presence of glycolytic substrate. The modulation of action potential duration by 30 μM glibenclamide was tested in perfused hearts subjected to hypoxia or to the K+-ATP channel opener pinacidil. Similar protocols were used to study the modifications of the steady state whole cell current in isolated ventricular myocytes. It was found that glibenclamide did not prevent early action potential shortening induced by hypoxia but produced a partial recovery after 15 min of exposure. At the steady state the action potential duration had lengthened by 53±6% at plateau level and 42±3% at 95% repolarization. In contrast, action potential shortening induced by 100 μM pinacidil was fully reversed by glibenclamide within 2 min. Freshly dispersed ventricular myocytes were characterized in control conditions as for the properties of the steady state current. This current, measured at the end of 450 ms long pulses showed typical inward rectification that was abolished by 50 μM Ba2+. Cyanide (2 mM), carbonyl-cyanide m-chlorophenylhydrazone (CCCP, 200 nM) and BRL 38227 (30 μM) produced characteristic increases in time independent outward currents. Glibenclamide abolished the outward current induced by BRL 38227 and the concomitant action potential shortening. Addition of cyanide in the presence of glibenclamide and BRL 38227 produced a new increase in outward current accompanied by action potential shortening. In the absence of K+-ATP channel activators, glibenclamide partly inhibited the CCCP induced current. Our data suggested that the delayed onset of glibenclamide action in hypoxic hearts is not due to diffusion barriers. They rather support the view that mechanisms other than K+-ATP channel activation could determine the early action potential shortening in whole hearts. The partial recovery observed under glibenclamide may be due, in part, to channel desensitization but also reflect the contribution of more than one current system to the action potential shortening because the glibenclamide insensitive fraction of the CCCP induced current is partly blocked by low concentrations of Ba2+. Differences with other data in the literature are attributed to the degree to metabolic blockade, to species differences, and to the inherent heterogeneities of the whole heart model where non-muscle cells may modulate the response to hypoxia.

Effects of amphotericin B on isolated rabbit hearts

Abstract The effects of amphotericin B on contraction and electrical activity of isolated rabbit hearts were tested. The hearts were perfused with Krebs Henseleit solution at 33°C and constant coronary flow. Amphotericin B was used at concentrations of 1.25, 2.5, 5 and 10 μg/ml. Surface electrograms were displayed synchronously with atrial transmembrane potentials. In non-driven preparations, amphotericin caused progressive lengthening of PR interval and P wave. AV conduction block progressed towards complete atrioventricular dissociation. The effects were the same with all the concentrations used but the time course of the changes observed was related to the concentration. In driven preparations, the antibiotic produced a decrease in action potential amplitude. This was characterized by the progressive abolition of the slow phase of depolarization followed by a decrease in the amplitude of the fast phase of the upstroke. The maximum depolarization rate decreased. Progressive inactivation of atrial fibers occurred. Alternance of action potentials with electrotonic potentials was currently observed. Finally, the preparation became irreversibly unexcitable. It is proposed that amphotericin exerts its effects by progressive blocking of the ionic currents involved in excitation, and that it also interferes with the processes of activation and removal of inactivation.