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.

Gating of IsK expressed in Xenopus oocytes depends on the amount of mRNA injected.

IsK is a K+ channel of the delayed rectifier type widely distributed throughout both excitable and nonexcitable cells. Its structure is different from other cloned K+ channels and molecular details of its gating remain obscure. Here we show that the activation kinetics of IsK expressed in Xenopus oocytes depend upon the amount of its mRNA injected, with larger amounts resulting in slower activation kinetics with a longer initial delay during activation. Similar changes in activation kinetics occur with time after a single injection of IsK mRNA. We present two kinetic schemes which illustrate how our experimental results could arise. Both imply an interaction among individual channel proteins during IsK activation. The dependence of channel gating on mRNA concentration provides a novel mechanism for long term regulation of ion current kinetics.

Rapid in vivo monitoring of chemotherapeutic response using weighted sodium magnetic resonance imaging.

A novel pulse sequence strategy uses sodium magnetic resonance imaging to monitor the response to chemotherapy of mouse xenograft tumors propagated from human prostate cancer cell lines. An inversion pulse suppresses sodium with long longitudinal relaxation times, weighting the image toward intracellular sodium nuclei. Comparing these weighted sodium images before and 24 h after administration of antineoplastics, we measured a 36 +/- 4% (P < 0.001; n = 16) increase in signal intensity. Experiments with these same drugs and cells, treated in culture, detected a significant intracellular sodium elevation (10-20 mM) using a ratiometric fluorescent dye. Flow cytometry studies showed that this elevation preceded cell death by apoptosis, as determined by fluorescent end-labeling of apoptotic nuclei or Annexin V binding. Histopathology on formalin-fixed sections of explanted tumors confirmed that drug administration reduces proliferation (2.2 versus 8.6 mitotic figures per high power field; P < 0.0001), an effect that inversely correlates with the sodium magnetic resonance image response on a tumor-to-tumor basis (P < 0.02; n = 10). Morphological features, such as central zones of nonviable cells, rims of active apoptosis, and areas of viable tumor, could be distinguished by comparing weighted and unweighted images. Advantages of this sodium imaging technique include rapid determination of drug efficacy, improved diagnosis of lesions, ease of coregistration with high resolution proton magnetic resonance imaging, and absence of costly or toxic reagents.

Light-induced potassium fluxes in the skate retina.

Changes in the extracellular concentration of potassium [K+]0 in response to photic stimulation were studied in the skate retina with the aid of ion-selective electrodes. The results confirm earlier studies in demonstrating that the light-evoked changes originate at three intraretinal sites; an efflux of K+ was recorded in the regions of the two plexiform (synaptic) layers, whereas a decrease in [K+]0 occurred in the extracellular space surrounding the photoreceptors. Prolonged illumination induced long-term alterations in the levels of [K+]0 which, depending upon the retinal depth of the recording electrode, contained contributions from the various sinks and sources of [K+]0. In addition, the marked undershoot of the baseline level of [K+]0 that followed termination of the stimulus suggested the activity of a metabolically driven process for the uptake of extracellular potassium.

Effects of phencyclidine on cardiac action potential: pH dependence and structure-activity relationships.

The effects of phencyclidine [1-(1-phenylcyclohexyl)-piperidine; PCP] on cardiac action potential duration (APD) were compared to those of some of its derivatives, in strips of isolated frog ventricular muscle perfused with normal Ringer solution. We studied compounds with PCP-like behavioral actions (N-ethyl-1-phenyl-cyclohexylamine: PCE; and m-amino-PCP) as well as behaviorally inactive analogs (m-nitro-PCP; the quaternary derivative PCP-methyl iodide; and various fragments of the PCP molecule). Exposure to PCP, 3 microM to 1 mM, produced reversible, dose- and pH-dependent prolongations, of the APD to over 100% above control. The observed effects of the drugs are compatible with a mechanism of blockade of potassium conductance. An intracellular site for this action is suggested by: (i) the inactivity of the quaternary analog; (ii) the marked increase in the potency of the compounds when the external pH is changed in the region of their respective pKa values to increase the concentration of the unionized species; and (iii) the pronounced acceleration of the termination of the PCP effect by washout with a series of buffer solutions with decreasing pH values. The rank order of potency of the compounds in lengthening APD (PCE greater than m-amino PCP greater than PCP much much greater than m-nitro-PCP) is the same as reported from other pharmacological studies of specific PCP actions, and matches the rank of behavioral activity of the drugs.

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.

Potassium efflux in heart muscle during activity: extracellular accumulation and its implications.

1. Extracellular K+ activity and transmembrane potential were simultaneously monitored with a K+-selective micro-electrode placed in the extracellular space and a standard KCl-filled micro-electrode in the intracellular space of the frog ventricular muscle. 2. K+ was found to accumulate during activity and had the approximate magnitude and time course to account for the measured membrane depolarization. 3. The magnitude of the K+ accumulation depended on the frequency of stimulation, diameter of the muscle and temperature of the bathing solution. 4. The time constants of accumulation and decay were dependent only on the diameter and the temperature of the strip. A Q10 of 2 was measured for the decay of accumulated K+. 5. Double barrelled K+-electrodes were used to monitor the change in K+ activity accompanying a single action potential, since the reference barrel allowed for rapid compensation of the electrical potential fluctuations encountered in the subendothelial space. 6. K+ accumulated continuously during the plateau to a level which increased external K concentration by about 1 mM. This increase in the subendothelial space corresponds to about 1-3 muA/cm2 or 10-30 pmole/cm2-sec-1 of net K+ efflux. These values are at least an order of magnitude larger than required to discharge the membrane capacitance. 7. There is no direct relation between action potential duration and rate of development or magnitude of K+ accumulation during that action potential. 8. Increase in the external K concentration, while shortening the action potential and depolarizing the membrane, does not lead to an increased rate of accumulation of K+. The presence of Ni2+, on the other hand, prolongs the action potential and decreases the rate of K+ accumulation. 9. The results suggest that there is a substantial and continuous efflux of K+ during the action potential, which sums during rapid beating, resulting in membrane depolarization and alteration of action potential duration. The change in action potential duration in response to rate may be caused by alteration of EK in the local micro-environments.

Effects of extracellular potassium accumulation and sodium pump activation on automatic canine Purkinje fibres.

1. Double barrel potassium-sensitive micro-electrodes were used to measure fluctuations in extracellular potassium ion concentration in large automatic canine Purkinje fibres.2. Slow accumulations of potassium were seen in the extracellular space during prolonged beating. Following cessation of prolonged beating, a depletion of extracellular potassium ions was noted.3. The time course of these slow changes in extracellular potassium concentration were shown to be a function of the diffusion properties of the preparation, and the rate and degree of activation of the sodium pump.4. Action potential duration and maximum diastolic potential were simultaneously monitored during and after these periods of rapid stimulation, using conventional intracellular micro-electrodes.5. Initial depolarization and later hyperpolarization of the maximum diastolic potential appeared to occur as a direct result of changes in extracellular potassium concentration and level of pump activation induced by the sudden and prolonged alteration in stimulus rate.6. Following prolonged stimulation, dramatic changes in the automatic beating rate were correlated with changes in extracellular potassium and pump rate, and their effects on the maximum diastolic potential and other parameters of the diastolic potential depolarization.7. For some locations of the potassium-sensitive electrode tip, large fluctuations in extracellular potassium were seen during single beats. Alterations in action potential duration during abrupt rate changes appear to derive in part from modulation by these extracellular potassium concentration fluctuations altering net membrane currents. Slower shifts in action potential duration appear correlated to the degree of sodium pump activation.

Effects of alpha-adrenergic stimulation on intracellular sodium activity and automaticity in canine Purkinje fibers.

Approximately 60% of adult canine Purkinje fibers respond to alpha 1-adrenergic stimulation with a decrease in automaticity. Recent studies of disaggregated Purkinje myocytes have suggested that this negative chronotropic effect results from alpha 1-adrenergic activation of the Na-K pump. In this study we evaluated 1) whether Na-K pump activation is associated with the negative chronotropic effect of alpha 1-adrenergic stimulation in adult canine Purkinje fibers and 2) if the effect of alpha-agonists on the pump is direct or mediated by an increase in intracellular sodium activity (aNai). We used sodium selective microelectrodes to determine the effects of 5 x 10(-9) and 5 x 10(-8) M phenylephrine on aNai. Phenylephrine decreased automaticity in five of eight Purkinje fibers while an increase occurred in the other three. The rate decrease was always accompanied by a decrease in aNai (-3.9 mM; p less than 0.05), whereas in fibers showing an increase in rate, aNai was unchanged. To evaluate the effect of phenylephrine in the absence of changes in automaticity, 10 Purkinje fibers were studied during pacing. A clear-cut reduction in aNai (-2.8 mM) was present in six fibers; no change was seen in the other four. The effect of phenylephrine was blocked by prazosin but not by propranolol. We conclude that the effect of alpha 1-adrenergic stimulation to reduce aNai is consistent with activation of the Na-K pump. Moreover, this action of alpha 1-adrenergic stimulation is closely linked to its negative chronotropic effect.

Statistical properties predicted by the ball and chain model of channel inactivation.

It has been proposed that part of a voltage gated channel is a tethered ball and that inactivation occurs when this wandering ball binds to a site in the channel. In order to be able to quantitatively test this model by comparison to experiments we developed analytical solutions and numerical simulations of the distribution of times it takes the ball to reach the binding site when the motion of the ball is random and when it is also influenced by a directed force. If the motion of the ball is one-dimensional, at long times this distribution is a single exponential with a rate constant that is inversely proportional to the square of the length of the chain and does not depend on the starting position of the ball. This dependence on the chain length is not significantly altered if there are short range electrical forces between the ball and its binding site. These predictions suggest that to confirm the validity of this model additional experiments should be done to more precisely determine the form of this distribution and its dependence on the length of the chain.

Zatebradine slows ectopic ventricular rhythms in canine heart 24 hours after coronary artery ligation.

Arrhythmias occur 24 h after occlusion of the left anterior descending (LAD) coronary artery in the canine heart and have been attributed to the abnormal spontaneous activity in subendocardial Purkinje fibers, which are markedly depolarized. The major current underlying normal automaticity in these fibers is i(f). Although the i(f) activation range is generally considered to be more negative than the diastolic membrane potential in these depolarized fibers in infarcts, this activation range has been shown to shift in a positive direction in response to hormonal influences. Thus i(f) could still mediate automaticity in these fibers in infarcts. Furthermore, recent reports indicate that a depolarizing diastolic current, probably i(f), also can be measured in ventricular muscle during abnormal experimental conditions, which may occur during ischemia. To test whether there is a role of i(f) currents in sustaining ventricular ectopy, we administered the selective i(f) channel blocker, zatebradine, 24 h after LAD ligation in canine hearts. We report that intravenous injections of zatebradine (0.25 or 1.0 mg/kg) significantly slow ventricular rhythms (with average reductions of 19 or 26%, respectively). Moreover, because zatebradine also slows sinus nodal rate, it can lead to an increased incidence of ectopic beats. However, during right atrial pacing, when sinus slowing has no effect on ventricular rhythms, capture of ventricular rhythms occurs at lower rates in the presence of zatebradine. The reduction of capture threshold is comparable to the reduction in the rate of the ectopic rhythm. Thus zatebradine eliminated the arrhythmia when the right atrium was paced at the original sinus rate.

Intracellular pH of canine subendocardial Purkinje cells surviving in 1-day-old myocardial infarcts.

A large reduction of intracellular potassium activity in depolarized subendocardial Purkinje fibers 24 hours after coronary artery ligation is accompanied by a much smaller increase in intracellular sodium activity. Similar intracellular ionic changes also occur during acute ischemia in ventricular muscle and are consistent with mechanisms based on intracellular acidification, which is known to occur in acutely ischemic muscle. To determine if canine subendocardial Purkinje cells 24 hours after myocardial infarction are also acidic, their intracellular pH, surface pH, and maximum diastolic potential (MDP) were measured with double-barrel pH-sensitive microelectrodes and compared with control fibers in noninfarcted hearts. In 12 mM bicarbonate Tyrode's solution (5% CO2-95% O2), the average intracellular pH was not significantly different (p greater than 0.25) for normal tissue (6.83 +/- 0.08, SD, MDP = -83.5 +/- 3.2 mV), for depolarized Purkinje fibers in infarct preparations during the first hour of superfusion (6.88 +/- 0.11, MDP = -47.8 +/- 11.8 mV), and for partially recovered Purkinje fibers in infarcts averaged over the third to sixth hours of superfusion (6.85 +/- 0.12, MDP = -74.5 +/- 9.6 mV). In 24 mM bicarbonate Tyrode's solution, infarct intracellular pH during both the first hour of superfusion (7.08 +/- 0.13, MDP = -57.6 +/- 15.7 mV) and during the third to sixth hours of superfusion (7.06 +/- 0.15, MDP = -76.5 +/- 9.6 mV) was significantly alkaline (p less than 0.0005) compared with average control pH (6.92 +/- 0.12, MDP = 82.1 +/- 3.7 mV). In 24 mM bicarbonate Tyrode's solution, the intracellular pH did vary with MDP (0.0032 pH units/mV). During superfusion of normal Purkinje fibers with hypoxic Tyrode's solution, intracellular pH acidified by 0.22 pH units as they depolarized. Therefore, intracellular acidification does not seem to be a cause of the depolarization of subendocardial Purkinje cells 24 hours after myocardial infarction.

Generation of b-wave currents in the skate retina.

Potassium kinetics within the skate retina were monitored extracellularly with K+-selective electrodes. Two sources of K+ efflux were detected in response to photic stimulation: one in the distal retina in the region of the outer plexiform layer, and the other at a more proximal location near the border between the inner nuclear and inner plexiform layers. The magnitude of the K+ efflux at these retinal depths was affected differently by spot and full-field illumination, suggesting that the two sources originate from different classes of neuron. There is evidence that both sources are associated with current sinks provided by the Müller cells, thereby establishing radial current paths along the lengths of these elements. We have proposed a model in which asymmetries in the magnitudes of these currents give rise to the b-wave of the electroretinogram. Extracellular field potentials recorded differentially at various retinal depths, and in response to changes in stimulus configuration, were consistent with predictions of the model.

Activity-dependent extracellular K+ fluctuations in canine Purkinje fibres.

Cardiac Purkinje fibres are important in cardiac electrophysiology and pathology due to their specialized role in the ventricular conducting system, and their properties as a second cardiac pacemaker. Changes in extracellular K+ activity (Ko) affect the conduction velocity and the natural automatic rate of the Purkinje fibre. Both anatomical and electrophysiological studies of the Purkinje fibres have demonstrated the possibility of Ko fluctuations in the narrow intercellular clefts. We investigated using K+ selective electrodes, the effect of the Ko fluctuations observed during activity in canine Purkinje fibres. Summation of Ko fluctuations during single action potentials leads to elevation of baseline Ko during rapid trains. However, unlike in ventricular muscle where depolarization is seen, membrane potential is non-automatic Purkinje fibres hyperpolarizes in response to rapid beating. Prolonged depletions of extracellular K+ following long periods of overdrive are associated with slowly changing membrane currents, which markedly influence automaticity, action potential duration, and membrane potential.

Voltage-clamp studies on the canine Purkinje strand.

Purkinje strands were excised from the left and right ventricles of adult mongrel dogs and cut to lengths of less than 2.0 mm in order to apply the two-microelectrodes voltage-clamp technique. A sizeable fraction of these preparations fully recover following dissection, with resting potentials more negative than--80 mV and upstroke velocities faster than 290 V s-1. Analysis of the voltage response to small current pulses shows that the short Purkinje strands can be treated as simple finite one-dimensional cables with ends of infinite resistance. The average length constant is 2.5 mm. In keeping with the relatively long length constant, insertion of a third microelectrode along the strand demonstrates a high degree of longitudinal homogeneity of the voltage clamp. Analysis of the capacity transient gives an estimate of the total capacity, normalized to cylindrical surface area, of 11.5 muF cm-2. The final decay of the capacity transient is a single exponential with an average time constant of 1 ms. A second slower component to the final decay of the capacity transient is absent in solutions of normal conductivity as well as in solutions of reduced (13%) conductivity. This suggests that the extracellular series resistance may be relatively small. The magnitude of the K+ depletion current was estimated by measuring the ratio of depletion current to instantaneous current. This ratio averaged 10%. These two results are consistent with the morphometric data described in the accompanying paper, which show that the canine preparation has wider extracellular clefts than the ungulate preparation. The existence of the full complement of inward and outward currents, including the pacemaker current, is demonstrated. The presence of wide clefts does not affect the potential at which the pacemaker current reverses (about--107 mV in 4 mM [K+] Tyrode solution), since the pacemaker current reverses at approximately the same potential in the canine Purkinje preparation as it does in the ungulate.

Intracellular K+ activity, intracellular Na+ activity and maximum diastolic potential of canine subendocardial Purkinje cells from one-day-old infarcts.

The basis for the reduced maximum diastolic potential of canine cardiac subendocardial Purkinje fibers surviving one day after extensive transmural infarction was investigated, using double-barrel potassium and sodium ion-sensitive microelectrodes. The maximum diastolic potential of Purkinje fibers in infarct preparations from the left ventricular apex measured during the first hour of superfusion in a tissue bath was -50.1 +/- 13.7 mV, a value markedly reduced from the value in control Purkinje fibers from noninfarcted preparations (-85.0 +/- 4.5 mV). The intracellular potassium ion activity was reduced by 50.4 mM during this time (intracellular potassium ion activity equals 61.6 +/- 16.1 mM, as compared to control intracellular potassium ion activity of 112 +/- 19.8 mM). The potassium equilibrium potential was reduced by 16.0 mV (from -97.2 +/- 4.7 mV in controls to -81.2 +/- 6.9 mV), thus accounting for about one half of the reduction in the maximum diastolic potential. After 6 hours of superfusion, the maximum diastolic potential increased to -78.9 +/- 8.7 mV (still significantly less than control). The potassium equilibrium potential had largely recovered (-93.8 +/- 5.9 mV). The intracellular sodium ion activity of Purkinje fibers in the infarcts (15.6 +/- 6.9 mM) was elevated during the first hour of superfusion by 6.2 mM compared to control (9.4 +/- 2.6 mM), and this was only 12% as much as the initial intracellular potassium ion activity decrease. Sodium ion activity after 3-6 hours of superfusion was not significantly different than normal (12.1 +/- 4.9 mM). In conclusion, only a portion of the maximum diastolic potential changes can be explained by a reduction of the potassium equilibrium potential. It is likely that change(s) in the cell membrane sodium-potassium pump's function and in the membrane conductance are also involved. Furthermore, the lack of a compensatory increase in intracellular sodium ion activity accompanying the large reduction of intracellular potassium ion activity may be a consequence of the cellular acidosis, which is known to occur during myocardial ischemia.

Triggered activity in atrial fibres of canine coronary sinus: role of extracellular potassium accumulation and depletion.

1. Bursts of triggered activity can be induced in atrial fibres of the canine coronary sinus exposed to catecholamines. During a triggered burst there is an initial acceleration of rate accompanied by depolarization of the maximum diastolic potential (m.d.p.) followed by slowing of the rate and termination accompanied by hyperpolarization. 2. We have used extracellular K+-sensitive micro-electrodes (potassium ISE) to monitor extracellular K+ concentration ([K+]o) during and following triggered activity, while simultaneously measuring membrane potential with conventional intracellular micro-electrodes. 3. We found that the initial increase in rate during triggered activity is accompanied by increased [K+]o and depolarization. Later rate slowing and m.d.p. hyperpolarization is accompanied by decline of extracellular K+ accumulation. Following termination of triggered activity, extracellular K+ depletion occurred. 4. The decline of [K+]o and slowing of rate are known responses to enhanced Na+-K+ pump activation, as is the post-triggering depletion of extracellular K+. 5. Strophanthidin, which blocks the Na+-K+ pump, also blocks the [K+]o decline, the slowing of rate seen towards the end of the triggered episode, and the post-triggering depletion of extracellular K+. 6. Separate experiments studying the effects of elevated bath K+ and depolarizing current on triggering rate and delayed after-depolarization amplitude support our hypothesis that the rate profile of the triggered episode is to a large extent controlled by variations in m.d.p. subsequent to extracellular K+ accumulation and Na+-K+ pump activation.

Time course of changes in intracellular K+, Na+, and pH of subendocardial Purkinje cells during the first 24 hours after coronary occlusion.

We investigated the basis for the alterations in the intracellular potassium and sodium activity occurring in subendocardial Purkinje fibers surviving in 24-hour infarcts by examining ion activities in these Purkinje fibers removed from infarcting hearts at earlier times. Specifically, we examined intracellular potassium activity, sodium activity, and pH at 1 and 3 hours after ligation of the left anterior descending coronary artery, and we correlated the changes in ion activity with changes in maximum diastolic potential. We tested various mechanistic hypotheses relating to how the ion activity changes develop and how they affect membrane potential. We found that intracellular sodium activity in tissue removed 1 hour after ligation was on average already maximally elevated by a factor of 2 over control (19.2 +/- 2.0 mM [mean +/- SEM] versus 9.4 +/- 0.4 mM). Potassium activity diminished progressively over the first 24 hours (from normal of 112.0 +/- 2.7 to 61.6 +/- 2.8 mM), although half of the decrease occurred during the first hour (to 86.8 +/- 4.1 mM). Intracellular pH did not change at either 1 or 3 hours. Whereas maximum diastolic potential depolarization exceeded the calculated depolarization of the potassium equilibrium potential by a factor of 2 in 24-hour infarcts, the depolarization at 1 and 3 hours could be more nearly attributed to the loss of potassium. The change in the dependence of maximum diastolic potential on potassium equilibrium potential may be due to changes in membrane conductance caused by ionic or biochemical factors. The changes in ion activity continuously develop during the first day after ligation and may be due to multiple factors and mechanisms.

Endothelin-dependent actions in cultured AT-1 cardiac myocytes. The role of the epsilon isoform of protein kinase C.

The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC)-epsilon immunoreactivity from the soluble to the particulate cell fraction. The subcellular distributions of PKCalpha and PKCzeta (also expressed by AT-1 cells) are not influenced by endothelin. Using quantitative fluorescence microscopy with fura 2, we examined the effects of endothelin on intracellular calcium. In electrically driven myocytes, endothelin induces a rapid and transient increase in the amplitude of the calcium transient. This is blocked by both phorbol 12-myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKCepsilon plays a critical role in endothelin receptor-dependent increases in intracellular calcium. Endothelin also stimulates mitogen-activated protein kinase (MAPK). MAPK activation is markedly attenuated by pretreatment with PMA or pertussis toxin (PTX, to activate susceptible G protein alpha subunits); it is completely prevented by combined pretreatment with PMA and PTX. In contrast, it is not attenuated by chelation of intracellular calcium with BAPTA. These findings indicate that the pathway for endothelin receptor stimulation of MAPK involves PKCepsilon and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKCepsilon as a mediator of endothelin receptor-dependent increases in cytosolic calcium and MAPK activity in AT-1 cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKCepsilon and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes.