R E Ten Eick

On the mechanism of lysophosphatidylcholine-induced depolarization of cat ventricular myocardium.

Lysophosphatidylcholine, a putative biochemical mediator of ischemia-induced arrhythmias, reduces the resting potential of ventricular muscle. To elucidate possible mechanisms of lysophosphatidylcholine-induced depolarization, we investigated the effects of lysophosphatidylcholine on the electrophysiological properties of cat ventricular muscle, using potassium ion-selective electrodes and conventional microelectrode, current-, and voltage-clamp techniques. Lysophosphatidylcholine (50 microM) decreased the sensitivity of the resting potential to changes in extracellular potassium concentration. Hyperpolarization of lysophosphatidylcholine-depolarized fibers by current-clamp methods failed to reveal two stable levels of resting potential. Depolarizing concentrations of lysophosphatidylcholine did not reduce the potassium equilibrium potential, as determined from the reversal potential of the time-dependent potassium current and measurements of intracellular potassium activity using potassium ion-selective electrodes. Lysophosphatidylcholine induced a depolarizing shift of the reversal potential for steady state current, and did not induce the formation of a negative slope region in the steady state current-voltage or background current-voltage relationships. Lysophosphatidylcholine induced an inward shift and linearization of the background current-voltage relationship negative to -30 mV, and the lysophosphatidylcholine-sensitive component of the background current was an inward rectifier with a reversal potential approximately equal to the potassium equilibrium potential. Lysophosphatidylcholine also reduced the amplitudes of the time-dependent potassium current, slow inward current, and the potassium accumulation and depletion currents. These results indicate that lysophosphatidylcholine-induced depolarization is due, in part, to reduced potassium conductance at voltages near the normal resting potential, and that lysophosphatidylcholine may act as a nonspecific depressant of membrane channels.

Electrophysiological properties of diseased human atrium. I. Low diastolic potential and altered cellular response to potassium.

Isolated specimens of right atrial appendage from 138 subjects with atrial disease or dysfunction and from seven subjects with clinically normal atria were examined electrophysiologically by conventional microelectrode techniques. The endocardial surfaces of diseased atrial appendage were found to be characterized primarily by cells with diastolic transmembrane potentials of − 56 ± 0.7 (SEM) mV, whereas potentials of normal atria were −74.4 ± 1.0 mV. The response of the hypopolarized cells of diseased atria to K* was radically different from that defined for normally polarized cells of normal atria. The diastolic potential of hypopolarized cells was insensitive to changes in K+ concentration in the Tyrodes perfusion solution of between 2 and approximately 12 mM. Between 20 and 50 miu, the cells depolarized 34 mV per 10-fold increase in K+ concentration. The K+ electrode properties of hypopolarized cells were unaffected by reducing Na+ concentration by as much as 50-fold or by varying Ca1+ concentration from 1 to 5 mM. Acetylcholine hyperpolarized cells of diseased atria to within 5 mV of the mean resting potential for cells of normal atria. Voltage and current clamp studies on a trabecula of diseased atrium indicated that the steady state current-voltage relationship may be different from that described for nonhuman mammalian atria. These data suggest that the K+ conductance is not the principal determinant of the diastolic potential of cells in diseased atria, that the K* conductance relative to other significant membrane conductances may be substantially reduced, and that the absolute level of the K+ conductance may be reduced in cells of diseased atrium. The functional importance of the hypopolarization to the electrical and mechanical activity of the diseased atrial appendage is explored. Circ Res 44: S4S-5S7, 1979

Evidence for two components of sodium channel block by lidocaine in isolated cardiac myocytes.

The effects of lidocaine on sodium current in cardiac myocytes isolated from cat and guinea pig were investigated using the whole-cell variation of the patch-damp technique. Lidocaine (43- 200 μM) reduced sodium current during repetitive depolarizing pulses in a use-dependent manner. To clarify the nature of the use-dependent block, we characterized the time course of block development using a two-pulse protocol. Two distinct phases of block development were found: a rapid phase ( ± = 1-6 msec) having a time course concurrent with the time course of channel activation, and a slower phase (±= 100-900 msec), which developed after channels inactivated. The amplitude of the block during the rapid phase of development was a steep function of transmembrane voltage over the range of - 70 to +20 mV. The voltage-dependence was similar to that for sodium channel activation (sodium conductance) but was too steep to be attributed solely to the passive movement of a singly charged molecule under the influence of the transmembrane voltage gradient. These results suggest that use-dependent block of sodium channels in cardiac tissue may result from an interaction of lidocaine with sodium channels in the activated as well as the inactivated channel states. Possible mechanisms underlying the fast component of block are discussed.

Sodium current kinetics in cat atrial myocytes.

1. Na+ current kinetics were studied in isolated atrial myocytes from the adult cat using the single suction‐pipette voltage‐clamp technique. 2. Current‐voltage and conductance‐voltage relationships were similar to those described in other cardiac myocyte preparations. 3. Analysis of Na+ current decay using single‐pulse, double‐pulse and tail current measurements were in agreement and demonstrate a second‐order process of current decay. 4. Voltage dependence of steady‐state inactivation curves was not symmetrical, having an inflexion at about ‐90 mV. These results suggest more than a single inactivation process for Na+ channel in the negative potential region. 5. Recovery of Na+ current from inactivation had a sigmoid time course: an initial slow component (delay) followed by a fast and then a second slow component. Increasing the pre‐pulse duration slowed the time course of recovery. 6. Taken together, the results were consistent with the presence of multiple inactivated states for the atrial myocyte Na+ channel.

Beta‐adrenergic and cholinergic modulation of inward rectifier K+ channel function and phosphorylation in guinea‐pig ventricle.

1. To clarify the nature of the inhibition of whole‐cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell‐attached and excised inside‐out patches obtained from guinea‐pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell‐attached patches. This was inhibited by propranolol. Bath‐applied forskolin or dibutyryl cAMP mimicked the effect of bath‐applied Iso. 3. Exposure of the cytosolic face of inside‐out patches to purified catalytic subunit of the cAMP‐dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath‐applied Iso on iK1 recorded from cell‐attached patches. 4. When applied directly to cell‐attached patches via the patch pipette solution, ACh antagonized Iso‐induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath‐applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell‐attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso‐induced suppression of iK1 channels. When inside‐out patches were used, bath‐applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath‐applied catalytic subunit of PKA (PKA‐CS), suggesting that a PTX‐sensitive G1 alpha mediates ACh‐induced antagonism of Iso‐induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath‐applied PKA‐CS in inside‐out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA‐CS. 7. Findings in guinea‐pig ventricular myocytes suggest that iK1 can be suppressed by a PKA‐mediated phosphorylation of the iK1 channel occurring in response to Iso‐induced beta‐adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane‐delimited pathways. The membrane‐delimited pathway appears to involve M2‐cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.

Protein kinase-dependent Cl- currents in feline ventricular myocytes.

A Cl- current (ICl) induced by isoproterenol (ISO) has been identified in isolated guinea pig ventricular myocytes. This ISO-induced ICl can be inhibited by propranolol and mimicked by forskolin (FSK), suggesting that beta-receptors, cAMP, and protein kinase A (PKA) are involved in regulating the involved Cl- channel. Because activation of protein kinase C (PKC) mediated via alpha-adrenergic receptor stimulation is also known to regulate several ion channels, the idea that activation of PKC also can induce ICl was investigated by using isolated feline ventricular myocytes and the whole-cell patch-clamp technique. We found that extracellularly applied phorbol 12-myristate 13-acetate (PMA) could activate ICl in feline ventricular cells. Control experiments indicated that in the absence of PMA or other interventions, the steady-state current-voltage relation of patches maintained for more than 40 minutes was unchanged over a voltage range from -100 to +80 mV. This suggests that the present findings are not complicated by the development over time after patching of a steady-state ICl, similar to the findings reported for canine atrial myocytes. When induced by PMA, ICl was noninactivating and outwardly rectifying; it reversed polarity at approximately the equilibrium potential for Cl- and was sensitive to the Cl- channel blocker 9-anthracene carboxylic acid. In contrast, PMA failed to induce ICl when either staurosporine or calphostin C was added to the patch pipette solution used to internally dialyze the myocytes. The kinetic properties of PMA- and FSK-induced ICl were similar. When supramaximal concentrations of both ISO (1 mumol/L) and PMA (6 mumol/L) were applied simultaneously, the size of the induced ICl was the same as that induced by the same concentrations of either agonist applied alone. In addition, maximal induction of ICl with PMA (6 mumol/L) prevented the effects of FSK (1 mumol/L, the concentration causing approximately 40% of the maximal response [approximately EC40]), yet the effects of simultaneously applied submaximal concentrations (eg, approximately EC25 to approximately EC40) of both 0.5 mumol/L PMA and 1 mumol/L FSK were roughly additive. The results suggest that (1) both PMA and ISO or FSK can induce ICl with approximately equal efficacy, (2) the PMA- and ISO- or FSK-induced ICls are similar, and (3) they all flow through the same set of Cl- channels, implying that channel phosphorylation via either PKA or PKC can activate this feline cardiac ICl.

beta‐adrenergic and cholinergic modulation of the inwardly rectifying K+ current in guinea‐pig ventricular myocytes.

1. Whole‐cell patch‐clamp technique was used to study the beta‐adrenergic and cholinergic regulation of the inwardly rectifying K+ conductance (gK1) in isolated guinea‐pig ventricular myocytes. 2. In Cl(‐)‐free solutions or in the presence of 9‐anthracenecarboxylic acid or Co2+, bath‐applied isoprenaline (Iso) partially inhibited the steady‐state whole‐cell conductance (gss) calculated from the steady‐state current (Iss)‐voltage (Iss‐V) curve at membrane voltages (Vm) negative to the equilibrium potential for potassium (EK). Iss was also inhibited at Vm positive to EK when the extracellular [K+] was 20 mM. The Iso‐sensitive component of gss exhibited the characteristics of the inwardly rectifying K+ conductance (gK1). 3. The Iso‐induced inhibition of gK1 was reversible, concentration dependent, blocked by propranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP‐dependent protein kinase (PKA) inhibitor in the pipette solution. These findings suggest that PKA mediates the Iso‐induced inhibition of gK1. 4. The apparent dissociation constant (KD) for the concentration dependence of Iso‐induced inhibition was 0.035 microM and the Hill coefficient was approximately 1.0. A maximal Iso concentration (1 microM) inhibited gK1 by 40 +/‐ 4.1% (mean +/‐ S.E.M.; n = 13). 5. Bath application of acetylcholine (ACh, 0.1 microM or more) antagonized the Iso‐induced (1 microM) inhibition of gK1; [ACh] > 1.0 microM antagonized 88 +/‐ 2.1% (n = 10) of the inhibition. ACh increased the KD for Iso to inhibit Iso‐sensitive gK1 and also reduced the maximal Iso‐induced inhibition. 6. ACh‐induced antagonism could be abolished by pre‐incubating myocytes with pertussis toxin (PTX), suggesting that a muscarinic receptor‐coupled, PTX‐sensitive G protein, Gi, is involved. 7. ACh (10 microM) also antagonized approximately 70% of the dibutyryl cyclic AMP (1 mM)‐induced inhibition of gK1 (n = 3), suggesting that the ACh‐induced antagonism involves more than simply inhibiting the Iso‐mediated activation of adenylyl cyclase via the activated Gi. 8. Intracellularly applied okadaic acid (OkA, 1 microM) did not alter gK1 (control = 134 +/‐ 5.1 nS vs. OkA = 136 +/‐ 6.1 nS), but the Iso‐induced decrease in gK1 was less (P < 0.001) with OkA present (42.1 +/‐ 2.4 nS, n = 5) than when absent (54.0 +/‐ 2.2 nS, n = 10). However, ACh (10 microM) failed to antagonize Iso‐induced inhibition with OkA present, suggesting involvement of a protein phosphatase.

Two stable levels of diastolic potential at physiological K+ concentrations in human ventricular myocardial cells.

Cells in many specimens of human ventricle can exhibit either of two stable levels of diastolic potential (DP) when exposed to 4 mM K+ in vitro (i.e., -78 +/- 4 mV or -45 +/- 5 mV, mean +/- SEM). In this report we show that the DP of some partially depolarized human ventricular cells developed a sustained 25-35 mV hyperpolarization (n = 28) when bath K+ concentration (K+b) was raised from 4 to 7 mM. On return of K+b to 4 mM, the DP of most, but not all, of these cells returned to the original depolarized levels. In other cells, the transition between the two levels of DP occurred at variable K+b ranging from 1 to 20 mM. We investigated the ionic mechanism(s) underlying the shifts between the two levels of potential by studying the K+ dependence of the DP in partially depolarized cells in 22 specimens of human ventricle. DP hyperpolarized an average of 25.6 mV (from -44.4 +/- 1.3 to -70.0 +/- 1.3 mV; n = 25) when K+b was increased from 4 to 7 mM. Intracellular K+ activity, determined by K+-selective microelectrodes, was within the range of normal reported for other mammalian species (106.7 +/- 4.4 mM in 4 mM K+; n = 22) and was unaffected by increasing K+b to 7 mM (111.7 +/- 6.6 mM; n = 6). Ba2+ (0.05 mM), a blocker of the inward rectifying K+ current, reversibly prevented the hyperpolarization, whereas acetylstrophanthidin (9 microM) failed to inhibit it. These results suggest that the hyperpolarization was due to a K+-dependent increase in K+ permeability and that electrogenic sodium pumping did not contribute significantly to the process. The ionic basis of the depolarization from a hyperpolarized level of DP also was investigated. Decreasing bath Na+ concentration and exposure to 30 microM tetrodotoxin did not prevent the depolarization. However, the depolarization could be inhibited by 2 mM Mn2+. These findings suggest that the depolarization may have been due to a Mn2+-sensitive inward current.

Ionic diffusion in voltage-clamped isolated cardiac myocytes. Implications for Na,K-pump studies

The whole-cell voltage-clamp technique employing electrolyte-filled micro-pipette suction electrodes is widely used to investigate questions requiring an electrophysiological approach. With this technique, the ionic composition of the cytosol is assumed to be strongly influenced (as result of diffusion) by the ionic composition of the solution contained in the electrode. If this assumption is valid for isolated cardiac myocytes, the technique would be particularly powerful for studying the dependence of their Na,K-pump on the intracellular [Na+]. However, the relationship between the concentrations of ions in the solution filling the electrode and those in the cytosol has not been established. The relationship was investigated to determine in particular whether the [Na+] at the intracellular cation ligand binding sites for the Na-pump ([ Na+]ps) can be set and clamped by [Na+] in the pipette electrode ([ Na+]pip). If [Na+]pip can set and clamp [Na+]ps, this would provide a means for defining the dependence of the Na,K-pump on intracellular [Na+]. The relationship between [Na+]pip and [Na+]ps was analyzed using two approaches. First, a mathematical model of three-dimensional ionic diffusion within a whole-cell patch-clamped myocyte was developed and the effects of experimental parameters on mean [Na+]ps were investigated. When typical experimental values were simulated, the time course to achieve steady state mean [Na+]ps was found to be most sensitive to variations in electrode pore size, cell length and the Na+ pumping rate, but at steady state, mean [Na+]ps varies from [Na+]pip by 5% or less depending on pump rate. Second, to provide experimental support for the validity of the simulations, isolated ventricular myocytes were voltage-clamped and the reversal potential for the Na current was determined in order to estimate steady state intracellular [Na+]. The results of the mathematical and experimental analyses suggest that steady state [Na+]ps can be regulated by the [Na+] in suction pipette electrodes. These findings, while also having a broader significance, indicate for isolated cardiac myocytes that whole-cell suction micro-electrodes can provide a means to assess the dependence of the Na,K-pump on [Na+]ps.