Johan Vereecke

Adrenaline and the plateau phase of the cardiac action potential

SummaryConduction block in heart cells by K+ rich, or Na+ depleted solutions can be overcome by adrenaline. In order to explain this phenomenon, the effect of adrenaline on the membrane resting and action potentials of cow Purkinje fibers was measured at various extracellular concentrations of Na+, K+ and Ca++, in the presence of tetrodotoxin, Mn++ and beta-receptor antagonists.It was found that adrenaline specifically increases the amplitude and duration of the plateau phase of the cardiac action potential. Plateu-like action potentials, without preceding Na+-spike, can be generated and conducted in an all-or-nothing way. In K+ rich solutions and under the influence of adrenaline, the depolarization proceeds in two steps. The first step corresponds to the Na+-spike. The second step or secondary depolarization corresponds to the plateau; it was not modified by changes of the membrane potential between −85 and −55 mV, or by reduction of extracellular Na+ ions, but was specifically blocked by Mn++ ions and beta-receptor antagonists. Its amplitude increased by 17 mV for a tenfold change in extracellular Ca++. Tetrodotoxin preferentially blocked the Na+-spike, but also slowed the rate of potential change during the secondary depolarization.The simplest explanation for the observed phenomena can be found in an increase of Ca++ inward current under the influence of adrenaline. The existence of an inward Na++ current, different in characteristics from the Na+ conductance during the fast upstroke, cannot be ruled out. Some data are in accord with a decrease in K+ conductance.

Stereoselective effects of the enantiomers of bupivacaine on the electrophysiological properties of the guinea‐pig papillary muscle

1 Direct myocardial effects of the S(−)‐ and R(+)‐enantiomers of bupivacaine were compared in the guinea‐pig isolated papillary muscle by recording transmembrane action potentials with the standard microelectrode technique. 2 In 5.4 mm K+, at a stimulation rate of 1 Hz, the maximal rate of depolarization () was reduced to 59.9 ± 1.4% (n = 10) of control (mean ± s.e.mean) in the presence of 10 μm R(+)‐bupivacaine, and to 76.7 ± 1.2% (n = 14) in the presence of the same concentration of S(−)‐bupivacaine. This was mainly due to a difference in time constant at which block dissipated during the diastolic period. Recovery was slower in the presence of R(+)‐bupivacaine. The slower recovery in the presence of R(+)‐bupivacaine resulted also in a more pronounced frequency‐dependent block of . 3 Time constants for recovery from use‐dependent block became significantly faster for both enantiomers on hyperpolarization, while no significant change was observed at depolarization. At all membrane potentials recovery was slower in the presence of R(+)‐bupivacaine. 4 The action potential duration (APD) was shortened to a greater extent in the presence of R(+)‐bupivacaine over a large range of stimulation frequencies. 5 We conclude that S(−)‐bupivacaine affects and APD in the guinea‐pig papillary muscle less than the R(+)‐enantiomer at different rates of stimulation and resting membrane potentials.

Ionic currents during hypoxia in voltage-clamped cat ventricular muscle.

To explore the mechanisms underlying the shortening of the cardiac action potential in hypoxia, we studied the effect of hypoxia on the ionic currents in cat papillary and trabecular muscles using the single sucrose gap-voltage clamp technique. For potentials positive to −70 mV, hypoxia induces an increase in time-independent outward current. The changes in the tail current suggest that time-dependent outward current is not increased but, rather, reduced. Because the time course of IK remains unchanged, we concluded that the shortening of the action potential is not a result of a change in the time-dependent outward current. In the potential range of the plateau, the amplitude of the slow inward current is not affected by hypoxia. Its time constant of inactivation appears slightly decreased. The prolongation of the action potential by epinephrine during hypoxia is accompanied by an increase in the slow inward current. As a result of these studies, we conclude that the shortening of the cardiac action potential in the early stage of hypoxia results from an increase in K+outward background current. Circ Res 47: 501-508, 1980

The shortening of the action potential by DNP in guinea-pig ventricular myocytes is mediated by an increase of a time-independent K conductance

Abstract1.The mechanism of the well known shortening of the cardiac action potential by 2,4-dinitrophenol (DNP, a classical uncoupler of oxidative phosphorylation) was studied in single ventricular myocytes using a two microelectrode voltage clamp technique.2.Single ventricular cells were isolated from the heart of adult guinea-pigs. These cells were superfused with Tyrode solution containing 1.8 or 3.6 mM Ca.3.After application of 0.1 mM DNP initially a small depolarization and a prolongation of the action potential is observed. This effect is most likely related to an inhibition of the electrogenic sodium pump caused by ATP depletion.4.The marked shortening of the action potential which follows the initial prolongation is accompanied by a very pronounced increase of the outward current. This DNP-induced outward current component is time-independent. This current shows a reversal potential negative to the resting potential indicating that it is mainly carried by potassium ions.5.The DNP-induced current attenuates and abolishes the N-shape of the steady-state current-voltage relationship. When the inward-rectifying potassium current is blocked by pretreatment with 20 mM Cs or 1 mM Ba, large DNP-induced currents which show outward rectification can be seen. The increase in outward-rectifying potassium current by DNP is responsible for the shortening of the action potential and the loss of plateau.6.In addition, DNP also seems to cause an increase of inward-rectifying potassium current. This effect appears later than the increase in outward rectifier; it does not contribute to the shortening of the action potential but causes a hyperpolarization of the cell.7.In the latest phase of the DNP effect, which occurs only after the action potential has completely lost its plateau, changes of the amplitude and time constant of inactivation of the slow inward current can sometimes be observed.8.The effect of DNP on the potassium conductance is discussed in terms of increased cytosolic calcium activity.

Three different potassium channels in human atrium. Contribution to the basal potassium conductance.

We applied the cell-attached and inside-out patch-clamp technique under symmetrical isotonic potassium conditions on single human (and guinea pig) atrial cells. The human cells were isolated by a modified method to that described earlier. Our aim was twofold: 1) to study the single-channel characteristics of potassium channels in human atrial single cells, present under basal conditions (iK1 and iK(ATP] or when stimulated with 10(-5) M acetylcholine; and 2) to calculate the contribution of these three channel types to the total basal potassium conductance in human atrial cells, and to compare the results with data on guinea pig atrial cells under the same conditions. We found that in human cells 58% of the patches (n = 42/74) contained acetylcholine-sensitive potassium channels: their conductance was 42 +/- 1.2 pS and mean open time (tau o) was 1.7 +/- 0.5 msec. They showed sporadic openings in the absence of agonist, and activation by acetylcholine was G-protein dependent. In 16% of the patches (n = 7/44), adenosine (10(-4) M) activated the same channels, but the activity was lower than when stimulated by acetylcholine. In 18% of the patches (n = 9/51), an iK1 channel was present (conductance, 27 pS; tau o, 8.7 msec), whereas in the cell-attached mode, ATP-dependent channels were never seen. However, they were present in half of the inside-out patches on washout of ATPi (conductance, 73 pS; tau o, 1.4 msec). The basal potassium conductance (i.e., in the absence of any exogenous hormone or neurotransmitter) was mainly due to iK1 channels in both human and guinea pig cells, a finding that is in contrast with previous reports. However, the potassium current that is induced by acetylcholine is much higher in guinea pig than in human isolated cells; a fraction of it would suffice to fully determine the resting potassium conductance in guinea pig atrial cells, whereas it can play only a modulatory role in human cells. This difference could be important in species-specific autonomic modulation and antiarrhythmic drug action.

The mechanism of the inactivation of the inward-rectifying K current during hyperpolarizing steps in guinea-pig ventricular myocytes

The time course of the inward-rectifying K current during hyperpolarizing clamp steps was investigated in single myocytes isolated from guinea-pig ventricles. The experiments were done using a two-electrode voltage-clamp technique with two patch pipettes in the whole-cell configuration. Hyperpolarizations to potentials negative to −100 mV, induced large inward-rectifying K currents (iK1), which showed a marked decay. The current-voltage relation of the peak inward current was almost linear, but the steadystate current-voltage relation had a region of negative slope at potentials negative to −140 mV. These findings indicate that the channel inactivates during hyperpolarizing steps. When Na ions in the extracellular solution were replaced by choline, Tris, TMA or sucrose, the decay of the inward currents was largely reduced, and the negative slope in the steady-state current-voltage relation was absent. When divalent ions were removed from the Na-free bathing solution, a marked increase iniK1 was found, and the currents became time-independent. These experiments demonstrate that the inactivation during hyperpolarization is largely due to a block of the channel by external Na ions. The block by Na is most pronounced at very negative potentials, and is strongly voltage-dependent. External Ca and Mg ions also cause a marked block of the channel. The block by these divalent ions is however much less voltage-dependent than the one by Na, but is already present at the cells resting potential.

Magnesium‐inhibited, TRPM6/7‐like channel in cardiac myocytes: permeation of divalent cations and pH‐mediated regulation

Cardiac tissue expresses several TRP proteins as well as a Mg2+‐inhibited, non‐selective cation current (IMIC) that bears many characteristics of TRP channel currents. We used the whole‐cell voltage clamp technique in pig and rat ventricular myocytes to characterize the permeation, blockage properties and regulation of the cardiac IMIC channels in order to compare them with TRP channels, in particular with Mg2+‐sensitive TRPM6 and TRPM7. We show that removing extracellular divalent cations unmasks large inward and outward monovalent currents, which can be inhibited by intracellular Mg2+. Inward currents are suppressed upon replacing extracellular Na+ by NMDG+. Divalent cations block monovalent IMIC and, at 10–20 mm, carry measurable currents. Their efficacy sequence in decreasing outward IMIC (Ni2+= Mg2+ > Ca2+ > Ba2+) and in inducing inward IMIC (Ni2+≫ Mg2+= Ca2+≈ Ba2+), and their permeabilities calculated from reversal potentials are similar to those of TRPM6 and TRPM7 channels. The trivalent cations Gd3+ and Dy3+ also block IMIC in a voltage‐dependent manner (δ= 0.4–0.5). In addition they inhibit the inward current carried by divalent cations. IMIC is regulated by pH. Decreasing or increasing extracellular pH decreased and increased IMIC, respectively (pH0.5= 6.9, nH= 0.98). Qualitatively similar results were obtained on IMIC in rat basophilic leukaemia cells. These effects in cardiac myocytes were absent in the presence of high intracellular buffering by 40 mm Hepes. Our results suggest that IMIC in cardiac cells is due to TRPM channels, most probably to TRPM6 or TRPM7 channels or to their heteromultimeres.

Differential effects of verapamil and flunarizine on cardiac L-type and T-type Ca channels

SummaryWhole cell experiments were used to study whether the L-type and the T-type Ca channel in guinea-pig ventricular myocytes are blocked similarly by verapamil and flunarizine. The L-type current is blocked by 5 μol/l verapamil and 5 μol/l flunarizine in a use-dependent way, and block can be relieved by hyperpolarizing pulses in a potential-dependent way. The T-type current is not affected by 10 μmol/l verapamil while it is blocked by 10 μol/l flunarizine in a use-dependent way. Verapamil selectively blocks the L-type channel in contrast to flunarizine.

Existence of a calcium-dependent potassium channel in the membrane of cow cardiac Purkinje cells

Single-channel currents were recorded in the membrane of cow cardiac Purkinje cells using the patch-clamp technique. Recordings from cell-attached and cell-free patches demonstrated large outward single-channel currents associated with depolarizing voltage-clamp pulses. The time course of the reconstructed mean current showed a rapid activation phase followed by a slower inactivation following a single exponential time course with a time-constant in the range 30 ms to 100 ms. The current-voltage relation of the channel was linear in the voltage range between + 10 mV and + 110 mV with a slope conductance of 120 pS in 10.8 mM external K. The results indicated that the channel is selective for K ions. In inside-out patches, when the internal Ca activity was raised from 0.01 μM to 1 μM, the frequency of opening of the K channel during a depolarizing pulse was markedly increased, indicating Ca-dependence of these channels. The relation between this ion channel and the previously described transient outward current in cow Purkinje fibres is discussed. In sheep Purkinje cells a channel, carrying a transient outward current, with different properties was found.

A combined study of sodium current and T-type calcium current in isolated cardiac cells

Sodium currents (INa) and T-type calcium currents (ICa,T) of isolated guinea-pig ventricular myocytes were recorded using the whole-cell voltage-clamp technique. Separation of the two currents was obtained by using the difference current method in the presence and absence of 2 mM extracellular Na (Nao). Time to peak and the time constant of inactivation of. INa were about 5 times faster than that of ICa,T (test potential −30 mV). and ICa,T had an activation range positive to −50 mV, were inactivated at −50 mV, and their current/voltage relationships peaked at −22.3±1.8 mV (n=18) and −29.3±0.5 mV (n=18) respectively, with a reversal potential of +40.3±4 mV (n=18) and +30±10 mV (n=18), respectively [2 mM Nao; 5.4 mM extracellular Ca (Cao)]. INa was blocked by 30 μM tetrodotoxin (TTX), 500 μM lidocaine, partly inhibited by 1 mM amiloride, but not affected by 100 μM nickel (Ni). ICa,T was neither affected by 30 μM TTX nor 500 μM lidocaine, but blocked by 100 μM Ni, 1 mM amiloride, 10 μM R 56865 and use-dependently reduced by 5 μM flunarizine. Adenosine (500 μM) affected neither INa nor ICa,T, whereas 1 μM isoprenaline did not affect ICa,T, but slightly increased INa. Our results demonstrate that the characteristics of ICa,T are not affected by the concomitant activation of INa, and vice versa. We conclude that ICa,T are not Ca currents through Na channels.