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I S Cohen

Publications and source records attributed to I S Cohen.

At least 19 recordsLinked to original sources
PubMed1995

Pacemaker current i(f) in adult canine cardiac ventricular myocytes.

1. Single cells enzymatically isolated from canine ventricle and canine Purkinje fibres were studied with the whole-cell patch clamp technique, and the properties of the pacemaker current i(f) compared. 2. Steady-state i(f) activation occurred in canine ventricular myocytes at more negative potentials (-120 to -170 mV) than in canine Purkinje cells (-80 to -130 mV). 3. Reversal potentials were obtained in various extracellular Na+ (140, 79 or 37 mM) and K+ concentrations (25, 9 or 5.4 mM) to determine the ionic selectivity of i(f) in the ventricle. The results suggest that this current was carried by both sodium and potassium ions. 4. The plots of the time constants of i(f) activation against voltage were 'bell shaped' in both canine ventricular and Purkinje myocytes. The curve for the ventricular myocytes was shifted about 30 mV in the negative direction. In both ventricular and Purkinje myocytes, the fully activated I-V relationship exhibited outward rectification in 5.4 mM extracellular K+. 5. Calyculin A (0.5 microM) increased i(f) by shifting its activation to more positive potentials in ventricular myocytes. Protein kinase inhibition by H-7 (200 microM) or H-8 (100 microM) reversed the positive voltage shift of i(f) activation. This effect of calyculin A also occurred when the permeabilized patch was used for whole-cell recording. 6. These results indicate i(f) is present in ventricular myocytes. If shifted to more positive potentials i(f) could play a role in ischaemia-induced ventricular arrhythmias. The negative shift of i(f) in the ventricle might play a role in differentiating non-pacing regions of the heart from those regions that pace.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Mechanism of acetylcholine action on pacemaker current (i(f)) in canine Purkinje fibers.

We have recently reported in canine Purkinje fibers that acetylcholine (ACh) can reverse the positive voltage shift of the pacemaker current (i(f)) induced by beta-adrenergic stimulation while having no direct action of its own. We have now investigated this effect of ACh on the cyclic adenosine monophosphate (cAMP) cascade in more detail. We find that addition of a membrane permeable analogue of cAMP (8-chlorophenylthio cAMP), 0.5-1 mM, increased the amplitude of i(f). This action was not reversed by 1 microM ACh, implying that ACh acts at a step prior to cAMP action. We then looked at the steps controlling intracellular concentration of cAMP. Inhibiting the phosphodiesterase with 100 microM isobutyl-1-methylxanthine (IBMX) increased i(f). This action, however, was reversed by ACh. Finally we investigated whether the action of forskolin, a direct activator of adenylyl cyclase, could be reversed by ACh. Forskolin (10-20 microM) increased i(f), and ACh at 1 microM partially reversed this action of forskolin. These results suggest that, in canine Purkinje fibers, ACh reverses the positive action of beta-adrenergic agents on i(f) via a decrease in cAMP production.

Acetylcholine↗
PubMed1992

Isoprenaline, Ca2+ and the Na(+)-K+ pump in guinea-pig ventricular myocytes.

1. The whole-cell patch clamp technique was employed to study the effects of the beta-agonist isoprenaline (ISO) on the Na(+)=K+ pump current, Ip, in acutely isolated ventricular myocytes from guinea-pig hearts. Propranolol, a beta-adrenergic antagonist, was used to demonstrate that all of the effects of ISO, stimulatory or inhibitory, are mediated by beta-receptors. 2. Below about 150 nM [Ca2+]i, we find that ISO reduces Ip, while above this [Ca2+]i ISO increases Ip. The stimulatory and inhibitory effects of ISO on Ip are independent of either intracellular sodium ([Na+]i) or extracellular potassium ([K+]o). These results suggest that the end-effect of ISO is directly on the maximum pump turnover rate (Vmax) rather than indirectly through changes in [Na+]i or [K+]o or modulatory effects on Na+ or K+ affinity. 3. The maximum effect of ISO increases Ip by 25% when [Ca2+] is buffered at 1.4 microM. A half-maximal effect is reached at roughly 10 nM-ISO and a near-maximal effect by 0.5 microM. 4. The permeabilized patch technique, using amphotericin B (Horn & Marty, 1988; Rae, Cooper, Gates & Watsky, 1991), was employed to minimize changes in the normal second messenger systems and calcium buffers. In these experiments, we used a high intracellular sodium solution (pipette sodium was 50 mM), thus sodium-calcium exchange was depressed and we expected [Ca2+]i to be above 150 nM. ISO increases Ip in these conditions as in the dialysed cells. 5. Our results suggest that beta-stimulation can increase Ip, but only if [Ca2+]i is above about 150 nM. In the beating heart [Ca2+]i rises well above this value during systole and the average [Ca2+]i, which depends on heart rate, is expected to normally be above this level. During beta-stimulation, the increase in Ip along with a concomitant increase in IK (Giles, Nakajima, Ono & Shibata, 1989; Duchatelle-Gourdon, Hartzell & Lagrutta, 1989) helps prevent action potential lengthening and allows an increase in heart rate even in the presence of increased calcium current. Further, beta-stimulation will compensate for the effects on Ip of either hypokalaemia or digitalis toxicity, and so reduce the expected rise in both [Na+]i and [Ca2+]i.

Animals↗
PubMed1992

Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes.

1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two-microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time-dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of -50.7 per tenfold change in [K+]o. Extracellular Cl- and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell-shaped and the peak value was at a potential 30-50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10-31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression-cloning of the inward rectifier channel.

Animals↗
PubMed1991

The effects of manganese and barium on the cardiac pacemaker current, if, in rabbit sino-atrial node myocytes.

The isolation of ionic fluxes contributing to electric currents through cell membranes often requires block of other undesired components which can be achieved, among others, by divalent cations. Mn2+ and Ba2+ are often used, for example, to block Ca and K currents. Here we have investigated the effects of these two cations on the properties of the hyperpolarization-activated pacemaker current if, in rabbit sino-atrial node myocytes, as obtained by voltage clamp analysis. We find that 2 mM Mn2+ shifts the if activation curve by 3.2 +/- 0.3 mV towards more positive values. However, when 1 mM Ba2+ is also added, the positive shift is more than halved (1.3 +/- 0.2 mV). We find, too, that in the absence of blocking cations the ACh-induced if inhibition is slightly higher than in their presence. These results indicate that the alteration of if kinetic properties by Ba2+ plus Mn(2+)-containing solutions is minimal.

Acetylcholine↗
PubMed1991

Block of the cardiac pacemaker current (If) in the rabbit sino-atrial node and in canine Purkinje fibres by 9-amino-1,2,3,4-tetrahydroacridine.

We have investigated the action of 9-amino-1, 2, 3, 4-tetrahydroacridine (THA) on the pacemaker current If in rabbit sino-atrial node myocytes and in canine Purkinje fibers. THA at concentrations in the range 3-300 microM blocked If in a voltage-independent manner, as revealed by measurements on the fully activated I/V relation for If. The dose/response relationship of the If maximal slope conductance (Gf) can be fitted by assuming a cooperative binding reaction where two THA molecules are required to block one If channel. Half-maximal block occurred at 18.2 microM in the sino-atrial node and 36.6 microM in Purkinje fibers. THA also affected the If kinetic properties. This was examined in the sino-atrial node where the current activation curve was shifted in the negative direction on the voltage axis (- 21 mV at 30 microM THA). The delayed rectifier current, IK, was also reduced by THA in sino-atrial node myocytes: at - 40 mV the IK fully activated value was decreased to 37% of its control value by 30 microM THA, with only a minor modification of the position of the activation curve at the same potential. Thus, although THA blocks If at a lower concentration than other known If-channel blockers [DiFrancesco (1982) J Physiol (Lond) 329:485-507], its action on the pacemaker current is not specific.

Animals↗
PubMed1991

Cooling/heating module for tissue chambers and solutions: theoretical considerations and practical design.

We provide a theoretical framework for the estimation of the performance of a modular cooling/heating device for tissue baths. The framework can be adapted to other designs using Peltier elements for cooling and heating. The design employs a Peltier as a heat pump and a flat heat pipe to transport heat to or from a 'remote' site. In the cooling mode heat from the hot side of the Peltier is removed by a heat sink cooled by a fan. The small cross section of the heat pipe permits cooling/heating of tissue chambers on microscope stages or in locations where it would be impractical to mount a Peltier element. The faces of the heat pipe can be used to pre-cool/heat solutions using a simple capillary heat exchanger.

Computers↗
PubMed1991

Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current i(f).

1. The effects of the protein kinase inhibitors H-7 and H-8 were investigated on diastolic depolarization of the action potential with microelectrodes and on the pacemaker current if with the two-microelectrode voltage clamp in canine cardiac Purkinje fibres. 2. Both 200 microM-H-7 and 100 microM-H-8 had no significant effect on the slope of diastolic depolarization but eliminated the actions of isoprenaline (1 microM). 3. We examined the actions of H-7 and H-8 on if in the presence and absence of isoprenaline. H-7 (200 microM) shifted the pacemaker current if in the negative direction on the voltage axis, whereas 100 microM-H-8 had no significant effect by itself. Both 200 microM-H-7 and 100 microM-H-8 can reverse or prevent the actions of isoprenaline (1-5 microM) on if. 4. We applied activators of the cyclic AMP cascade down-stream to the beta-receptor, to further evaluate where H-7 and H-8 might be exerting their effects. When exposing Purkinje fibres to an adenylyl cyclase activator (forskolin, 10-50 microM), a phosphodiesterase inhibitor (IBMX, 100 microM) and a permeable cyclic AMP analogue (8-chlorophenylthio-cyclic AMP, 200 microM-1 mM), the amplitude of if was increased. H-7 and H-8 at 100-200 microM eliminated each of these actions. 5. These results suggest that a phosphorylation process is involved in the modulation of the pacemaker current, if, in Purkinje fibres. The different actions of H-7 and H-8 on basal if suggest the hypothesis that other protein kinases, possibly protein kinase C, might also be involved in regulating basal phosphorylation of if in Purkinje fibres.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.

Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that binding of Ca(+)+ to the exchanger can be affected by the mobile buffer.

Animals↗
PubMed1990

A novel action of isoproterenol to inactivate a cardiac K+ current is not blocked by beta and alpha adrenergic blockers.

The K+ current iKl sets the resting potential in cardiac cells. Here we report that isoproterenol (ISO), a prototypical beta agonist, increases inactivation of iKl. This action of ISO on iKl is mimicked by permeant analogues of cAMP but is not blocked by the beta blockers propranolol and pindolol or the alpha blockers prazosin or yohimbine. We suggest that this novel action of ISO may contribute to pacemaker activity in the Purkinje strand and be mediated through a class of receptors different from classical beta's or alpha's.

Animals↗
PubMed1990

Interaction of intracellular ion buffering with transmembrane-coupled ion transport.

The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.

Biological Transport↗
PubMed1990

The mechanism of rectification of iK1 in canine Purkinje myocytes.

We have characterized the inward rectifying background potassium current, iK1, of canine cardiac Purkinje myocytes in terms of its reversal potential, voltage activation curve, and "steady-state" current-voltage relation. The latter parameter was defined from the difference current between holding currents in the presence and absence of 20 mM cesium. Our data suggest that iK1 rectification does not arise exclusively from voltage-dependent gating or exclusively from voltage-dependent blockade by internal magnesium ions. The voltage activation curve constructed from tail currents fit to a Boltzmann two-state model predicts less outward current than is actually observed. The magnesium-dependent rectification due to channel blockade is too fast to account for the time-dependent gating of iK1 that gives rise to the tail currents. We propose a new model of rectification that assumes that magnesium blockade of the channel occurs simultaneously with voltage-dependent gating. The new model incorporates the kinetic schema elaborated by Matsuda, H. (1988. J. Physiol. 397:237-258) to explain the appearance of subconducting states of the iK1 channel in the presence of blocking ions. That schema suggested that iK1 channels were composed of three parallel pores, each of which could be blocked independently. In our model we considered the consequences of partial blockade of the channel. If the channels are partially blocked at potentials where normally they are mostly gated closed, and if the partially blocked channels cannot close, then blockade will have the paradoxical result of enhancing the current carried by iK1.

Adenosine Triphosphate↗
PubMed1989

Internal and external K+ help gate the inward rectifier.

Recent investigations have demonstrated substantial reductions in internal [K+] in cardiac Purkinje fibers during myocardial ischemia (Dresdner, K.P., R.P. Kline, and A.L. Wit. 1987, Circ. Res. 60: 122-132). We investigated the possible role these changes in internal K+ might play in abnormal electrical activity by studying the effects of both internal and external [K+] on the gating of the inward rectifier iK1 in isolated Purkinje myocytes with the whole-cell patch-clamp technique. Increasing external [K+] had similar effects on the inward rectifier in the Purkinje myocyte as it does in other preparations: increasing peak conductance and shifting the activation curve in parallel with the potassium reversal potential. A reduction in pipette [K+] from 145 to 25 mM, however, had several dramatic previously unreported effects. It decreased the rate of activation of iK1 at a given voltage by several-fold, reversed the voltage dependence of recovery from deactivation, so that the deactivation rate decreased with depolarization, and caused a positive shift in the midpoint of the activation curve of iK1 that was severalfold smaller than the associated shift of reversal potential. These changes suggest an important role of internal K+ in gating iK1 and may contribute to changes in the electrical properties of the myocardium that occur during ischemia.

Animals↗
PubMed1988

Stimulation of cardiac alpha receptors increases Na/K pump current and decreases gK via a pertussis toxin-sensitive pathway.

Alpha-adrenergic amines exert concentration-dependent actions on the automaticity of cardiac Purkinje fibers (Posner, P., E. L. Farrar, and C. R. Lambert. 1976. Am. J. Physiol. 231:1415-1420; Rosen, M. R., A. J. Hordof, J. P. Ilvento, and P. Danilo, Jr. 1977. Circ. Res. 40:390-400; Rosen, M. R., R. M. Weiss, and P. Danilo, Jr. 1984. J. Pharmacol. Exp. Ther. 231:1415-1420). At high concentrations they induce a largely beta adrenergic increase in the spontaneous firing rate of adult canine Purkinje fibers, whereas at concentrations less than 10(-6) M, their effect is mediated through alpha-adrenergic receptors and is seen predominantly as a decrease in the fibers' spontaneous firing rate. The mechanism for this decrease in spontaneous firing rate remains unexplained. We report here that phenylephrine (10(-7) M) increases the activity of the Na/K pump and decreases background gK in Purkinje myocytes. Both effects appear to be alpha-1 adrenergic and, in addition, are abolished on pretreatment with pertussis toxin. These results suggest that like the atrial muscarinic receptor (Pffafinger, P. J., J. M. Martin, D. D. Hunter, N. M. Nathanson, and B. Hille. 1985. Nature [Lond.]. 317:536-538; Breitwieser, G. E., and G. Szabo. 1985. Nature [Lond.]. 317:538-540) the Purkinje fiber alpha-1 receptor is coupled to background gK via a GTP-regulatory protein. Further, they suggest that the phenylephrine-induced decrease in spontaneous firing rate is due to stimulation of the Na/K pump via a novel coupling of the Na/K pump to a pertussis toxin-sensitive GTP regulatory protein.

Animals↗
PubMed1988

Calculation of time constants for intracellular diffusion in whole cell patch clamp configuration.

We present a simplified model to identify and analyze the important variables governing the diffusion of substances from pipettes into canine cardiac Purkinje cells in the whole cell patch clamp configuration. We show that diffusion of substances through the pipette is the major barrier for equilibration of the pipette and cellular contents. We solve numerically the one-dimensional diffusion equation for different pipette geometries, and we derive a simple analytic equation which allows one to estimate the time necessary to reach the steady state of intracellular concentration. The time constant of the transient to steady state is given by a pipette geometric factor times the cell volume divided by the diffusion coefficient of the substance of interest. The geometric factor is shown to be given by the ratio of pipette resistance to the resistivity of the filling solution. Additionally from our modeling, we concluded that pipette perfusion at distances greater than 20 microns from the pipette tip would not substantially reduce the time necessary to achieve the steady state.

Animals↗