Atilla B. Goknur

Inhibition of Sodium/Calcium Exchange and Calcium Channels of Heart Cells by Volatile Anesthetics

Background Volatile anesthetics exert profound effects on the heart, probably through their effect on Calcium2+ movements during the cardiac cycle. Calcium2+ movements across the sarcolemma are thought to involve mainly Calcium2+ channels and the Sodium sup +/Calcium2+ exchanger. We have therefore investigated the action of halothane, isoflurane, and enflurane on Sodium sup +/Calcium2+ exchange and Calcium2+ channel activity to assess the contribution of these pathways to the observed effect of the anesthetics on the myocardium. Methods Sarcolemmal ion fluxes were investigated using radioisotope uptake by isolated adult rat heart cells in suspension. Sodium sup +/Calcium2+ exchange activity was measured from45 Calcium2+ uptake by Sodium sup + ‐loaded cells. Calcium2+ channel activity was measured from verapamil‐sensitive trace54 Manganese2+ uptake during electric stimulation. Results Halothane, isoflurane, and enflurane inhibited Sodium sup +/Calcium2+ exchange completely, with similar potency when concentrations were expressed in millimolar units in aqueous medium but not when expressed as minimum alveolar concentration (MAC). The inhibition by enflurane was particularly strong, > 50%, at 2 MAC. In contrast, the three anesthetics inhibited Calcium2+ channels with similar potency when concentrations were expressed as MAC but not when expressed in millimolar units in aqueous medium. Hill plots of pooled data with all three anesthetics showed a slope of 3.87 plus/minus 0.50 for inhibition of Sodium sup +/Calcium2+ exchange and 1.73 plus/minus 0.19 for inhibition of Calcium2+ channels. Conclusions Halothane, isoflurane, and enflurane inhibit both Sodium sup +/Calcium2+ exchange and Calcium2+ channels at concentrations relevant to anesthesia, although they exhibit differences in potency and number of sites of action. At 1.5 MAC, halothane inhibits Calcium2+ channels more than Sodium sup +/Calcium2+ exchange, whereas enflurane inhibits Sodium sup +/Calcium2+ exchange more than Calcium2+ channels. Isoflurane inhibited both systems equally. The inhibition of Calcium2+ influx by these agents is likely to contribute to their negative inotropic effect in the heart. The inhibition of Sodium sup +/Calcium2+ exchange by enflurane may account for its observed action of delaying relaxation in species lacking sarcoplasmic reticulum.

Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion.

Using 45Ca, indo1, and quin2, calcium uptake was measured in isolated quiescent adult rat heart cells under different metabolic conditions. Exposure of cells in a medium containing 1 mM CaCl2 to rotenone and uncoupler resulted in adenosine triphosphate (ATP) depletion from 17.08 +/- 2.26 to 0.63 +/- 0.11 nmol/mg within 8 minutes, and the cells went into contracture. In this time, the cells lost 1.65 +/- 0.1 nmol Ca/mg of total rapidly exchangeable cellular calcium, and the level of free cytosolic calcium as measured by indo1 rose from 47.4 +/- 16.3 nM to 79.8 +/- 27.6 nM. The subsequent rate of rise of intracellular free calcium concentration was just 4 nM/min for at least 40 minutes. Therefore, we investigated the effect of ATP depletion on the rate of calcium entry. In cells loaded with sodium by ouabain treatment without calcium, the initial rate of calcium influx on calcium addition was inhibited by 82-84% when cellular ATP was depleted, as measured by 45Ca or indo1. Quin2 also showed a strong inhibition of calcium influx by ATP depletion, but itself also caused a strong inhibition of calcium influx. The rate of calcium influx declined even further in ATP-depleted cells after the initial influx: Between 1 and 12 minutes after calcium addition, the residual 45Ca uptake rate of the first minute was inhibited by an additional 90%. We conclude that ATP depletion per se does not quickly elevate cytoplasmic free calcium and that such an elevation is prevented by a very strong inhibition of the rate of calcium entry.

Inhibition of ATP-sensitive potassium channels of adult rat heart cells by antiarrhythmic drugs.

We have investigated the effect of antiarrhythmic drugs on the increased potassium conductance induced in isolated adult rat heart cells by ATP depletion. The rate of 86Rb uptake in the presence of ouabain was used as a measure of potassium conductance. Treatment of cells with rotenone plus p-trinuoromethoxyphenylhydrazone (FCCP) rapidly depleted ATP levels and strongly stimulated the rate of 86Rb uptake. The stimulated uptake and the ATP depletion were inhibited by oligomycin; thus, the uptake was not induced by roteuone plus FCCP directly. The stimulated uptake, but not the ATP depletion, was inhibited potently by glyburide (ICJO, 38.3 nM), quinidine (IC50, 2.7 μM), verapamil (IC50, 4.5 μM), and amiodarone (IC50, 19.1 μM). The stimulated uptake was also inhibited by tetraethylammonium ion and by 4-aminopyridine but not by tetrodotoxiii or manganese. We conclude that 1) the stimulated 86Rb uptake is measuring ATP-sensitive potassium channel activity, 2) the ATP-sensitive potassium channel is strongly inhibited by quinidine, verapamil, and amiodarone, and 3) this inhibition may contribute to the antiarrhythmic action of these drugs.

Inhibition of Na-Ca exchange by general anesthetics.

General anesthetics, typically octanol, were found to inhibit the influx of calcium in isolated sodium-loaded adult rat heart cells, using 45Ca, quin 2, or indo 1. Inhibition by octanol, like inhibition by sodium, was competitive with calcium. Octanol and sodium together inhibited calcium influx synergistically. At physiological levels of extracellular calcium and sodium, the EC50 was 177 ± 37 μM for octanol and 48 ± 5 μM for decanol. These values are threefold to fourfold larger than those reported to cause 50% loss of righting reflex in tadpoles, a measure of their anesthetic effectiveness. We conclude that general anesthetics inhibit Na-Ca exchange at the sarcokmma. We suggest that octanol inhibits like sodium, and the synergism stems from the cooperativity of sodium inhibition at the binding and regulatory sites of the exchanger. Insofar as Na-Ca exchange may regulate inotropy, the inhibition of Na-Ca exchange by general anesthetics could contribute to their negative inotropic effect.

Some determinants of quality and yield in the isolation of adult heart cells from rat.

We investigated the effect of changes in perfusate substrate and Ca content on the quality and yield of isolated adult rat heart cells. When 1 mM Ca was added to the recirculating perfusate 15 min after collagenase addition, the ATP level of cells in the heart 15 min later, and their morphology in histological section, was no different from when no Ca was added back. The cells subsequently isolated were of similar yield, but a greater percentage were rod-shaped, compared with cells isolated without Ca restoration to the perfusate. Increased yield could be obtained by including substrates in the perfusate in addition to glucose. Either fatty acids or amino acids were effective. We conclude that: (1) all cells in the heart are Ca tolerant at the end of enzyme perfusion; (2) the presence of substrates in addition to glucose can help cells survive the isolation process.

Synchronous depletion of ATP in isolated adult rat heart cells.

We tested the hypothesis that isolated adult rat heart cells could be depleted of most of their ATP without undergoing contracture. Two strategies for ATP depletions were employed. First, cells were exposed to a high level of rotenone plus FCCP. The cells lost 90% of their ATP within 3 min without change in sarcomere length before undergoing contracture. Even though ATP levels were so low, glycolysis from glycogen was maximally activated at this time. Second, cells exposed to repeated cycles of acidic anoxia were depleted of 77% of their ATP without change in sarcomere length and remained rod-shaped when restored to normoxia and neutral pH. The hypothesis was thus confirmed. The results support the previously developed concept that ATP decline in cells can be synchronous, with a similar decline in all cells, or asynchronous, with a sudden decline in different cells at different times. Whether the decline is synchronous or asynchronous depends on the conditions of metabolic impairment. This concept can explain the pattern of ATP decline observed in whole hearts during ischemia, and also the mechanism by which glycolytic ATP appears to protect against contracture.

ATP dependence of calcium uptake by the Na-Ca exchanger of adult heart cells

The ATP dependence of the Na-Ca exchanger was investigated in isolated adult rat heart cells to evaluate the extent to which ATP depletion after a period of ischemia plus reperfusion in whole hearts could limit calcium uptake by Na-Ca exchange. A standard state for measurement of Na-Ca exchange activity that could be used with cells depleted of ATP to different degrees was defined. This was a state of zero sarcolemmal gradient for sodium, potassium, and pH and was achieved by incubation of the cells for 5 minutes with EDTA, EGTA, ouabain, and nigericin. Heterogeneity of cell ATP levels was minimized by using a protocol of total ATP depletion by incubation under conditions similar to ischemia, followed by reoxygenation to give partial restoration of ATP levels. No ATP was regenerated when cells were reoxygenated in the presence of rotenone, and such cells showed a very low rate of calcium uptake. Without rotenone, cells showed an almost complete restoration of Na-Ca exchange activity, in spite of a restoration of ATP levels to only one third of control values. Thus, the dependence of calcium uptake on ATP was highly nonlinear under these conditions. The calculated Km for ATP was no more than 10% of normal ATP levels. We conclude that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of calcium uptake through Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. In addition, we measured the apparent ATP dependence of calcium uptake by Na-Ca exchange in cells under conditions in which we previously had concluded that cell ATP distributions were very heterogeneous: when cells undergo contracture during incubation with oligomycin and without glucose. A linear relation between calcium uptake rate and ATP was observed at all ATP levels. This can be understood if cells in contracture that are incubated with oligomycin cannot take up calcium because of low ATP, whereas rod-shaped cells are able to retain a full uptake capability. This result further supports our conclusion that the ATP level declines catastrophically to near zero in these oligomycin-incubated cells just before contracture.

Control of the Na-Ca exchanger in isolated heart cells. I. Induction of Na-Na exchange in sodium-loaded cells by intracellular calcium.

Isolated adult rat heart cells in suspension were loaded with sodium by incubation with ouabain in the absence of calcium for 30 minutes. Addition of low levels of calcium induced accelerated rates of sodium influx and efflux, as measured with 22Na. The magnitude of calcium-induced 22Na efflux was 50-fold greater than the net rate of calcium uptake and required extracellular sodium, but not extracellular calcium, once some calcium was taken up. Calcium did not induce 86Rb efflux. The accelerated rate of 22Na efflux was prevented by verapamil, but verapamil was ineffective when added after calcium. Addition of EGTA after calcium reversed the effect of calcium, but only after incubation. Dichlorobenzamil, unlike verapamil, both prevented and reversed the induction of sodium fluxes by calcium. We conclude 1) that intracellular calcium induces Na-Na exchange through the Na-Ca exchanger in sodium-loaded cells exposed to calcium; and 2) that Na-Na exchange can be activated by calcium that enters the cell through calcium channels. We propose that this Na-Na exchange reflects the intrinsic activity of the Na-Ca exchanger.

Control of the Na-Ca exchanger in isolated heart cells. II. Beat-dependent activation in normal cells by intracellular calcium.

Electrical stimulation of isolated adult rat heart cells in suspension at 4 Hz resulted in a fourfold increase in the rate of sodium influx and efflux across the sarcolemma, with no change in total cell sodium, as measured with 22Na. The magnitude of stimulation-dependent sodium fluxes under these conditions averaged 17 nmol/min/mg protein. The increased rate of efflux was inhibited by tetrodotoxin, verapamil, or dichlorobenzamil and required extracellular calcium. The inhibition by tetrodotoxin was overcome by Bay K 8644. The basal rate of 22Na efflux in cells at rest was inhibited only slightly by dichlorobenzamil. The stimulation-induced efflux was not inhibited by ouabain, but in the presence of ouabain, stimulation increased the rate of accumulation of total sodium by 4 nmol/min/mg. This increase was inhibited by tetrodotoxin or verapamil. A calcium-dependent increase in rate of 22Na influx and efflux could also be induced by KCl addition. This was inhibited by verapamil and dichlorobenzamil but not by tetrodotoxin and was reversed by EGTA, but only after a delay. We conclude the following. 1) The Na-Ca exchanger in cells at rest is no more than 10% activated. 2) The exchanger becomes activated directly or indirectly by calcium that enters the cell through calcium channels during excitation. 3) In this preparation the major part of excitation-induced sodium fluxes are mediated by the Na-Ca exchanger, with only a relatively small direct participation of sodium channels. These channels participate indirectly by promoting calcium channel activation. 4) If all the calcium-dependent sodium fluxes were Na-Ca exchange, then calcium flux through the exchanger per beat would be about sevenfold larger than that through the calcium channels. An undetermined part of the calcium-dependent sodium fluxes, however, could be a direct Na-Na exchange through the activated Na-Ca exchanger.

Regulation of Sodium‐Calcium Exchange in Intact Myocytes by ATP and Calcium

Regulation of Na-Ca exchange activity by ATP and by intracellular Ca (Cai) has been studied in suspensions of intact Na-loaded adult rat cardiac myocytes using 45Ca uptake and exchange of 22Na. ATP depletion of Na-loaded myocytes results in a strong inhibition of the Na-Ca exchanger, manifested as a strong inhibition of intracellular Na-dependent Ca uptake. Ca uptake by Na-loaded cells in the course of ATP depletion can be very heterogeneous because of the heterogeneity amongst cells of the extent of ATP depletion. This can result in a false measure of the dependence of exchanger activity on cell ATP content. Under conditions intended to maximize the uniformity of cell ATP content amongst cells we found a half maximal rate of Ca uptake with a cell ATP content of 1.96 nmol/mg, about 10% of the normal cell ATP level. The results suggest that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of Ca uptake by Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. Ca addition to myocytes loaded with Na in the absence of Ca results in a strong activation of the Na-Ca exchanger at an intracellular site, manifested as a large activation of Na-Na exchange activity. A similar activation of the exchanger is observed in cells with a normal level of intracellular Na, suspended in a medium containing physiological levels of Ca, when the cells are stimulated to beat by application of an electric field. This suggests that regulation of the exchanger by Cai is important physiologically, in the regulation of excitation-contraction coupling. Cells depleted of ATP show not only a strongly inhibited rate of Na-Ca exchange and Na-Na exchange, but also a strongly reduced degree of activation by Cai, even in ATP-depleted cells with no acidosis. This could result from the combined effect of ATP loss and an elevated intracellular Mg concentration on Ca binding affinity at the regulatory site.