Using45 Ca, indol, 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 CaCl 2 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 indol 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 45 Ca or indol. 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 4SCa 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. (Circulation Research 1987;60:586-594) C ellular calcium overload is thought to be a contributing and perhaps decisive factor in heart cell necrosis under a variety of pathologic conditions including hypoxia, ischemia, catecholamine-induced damage, and cardiomyopathy.1 " 3 Therefore, it is desirable to understand how calcium fluxes across the sarcolemma are controlled, and especially how changes in cellular high-energy phosphates affect these fluxes since in many of the above pathologic conditions cellular high-energy phosphates are depleted. Data on this question at present appear to conflict. ATP depletion had no effect on the initial rate of cellular calcium uptake. These results suggest that intracellular calcium levels could rise on ATP depletion, perhaps as a result of an inhibition of energy-dependent calcium efflux or sequestration. On the other hand, rabbit ventricular tissue exposed to hypoxia showed an inhibited rate of 47 Ca influx at the time when hypoxic contracture was induced.9 With aequorin, little or no increase in the level of intracellular free calcium has been found in whole tissue 10 and single cells" when contracture was induced by anoxia or metabolic inhibitors. Anoxic contracture is paralleled by an ATP loss in whole tissue 12 and in isolated adult heart cells' 3 consistent with contracture being caused by a calcium-independent process that is activated by extremely low levels (<100 (xM) of ATP. Thus, these results suggest that very low levels of calcium can be maintained in the face of...

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-trifluoromethoxyphenylhydrazone (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 rotenone plus FCCP directly. The stimulated uptake, but not the ATP depletion, was inhibited potently by glyburide (IC50, 38.3 nM), quinidine (IC50, 2.7 microM), verapamil (IC50, 4.5 microM), and amiodarone (IC50, 19.1 microM). The stimulated uptake was also inhibited by tetraethylammonium ion and by 4-aminopyridine but not by tetrodotoxin 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.

General anesthetics, typically octanol, were found to inhibit the influx of calcium in isolated sodium-loaded adult rat heart cells, using ^Ca, 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 EC M was 177±37 /ttM for octanol and 48±5 fiM 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. (Circulation Research 1989;65:1021-1023)

Halothane, isoflurane, and enflurane inhibit both Na+/Ca2+ exchange and Ca2+ channels at concentrations relevant to anesthesia, although they exhibit differences in potency and number of sites of action. At 1.5 MAC, halothane inhibits Ca2+ channels more than Na+/Ca2+ exchange, whereas enflurane inhibits Na+/Ca2+ exchange more than Ca2+ channels. Isoflurane inhibited both systems equally. The inhibition of Ca2+ influx by these agents is likely to contribute to their negative inotropic effect in the heart. The inhibition of Na+/Ca2+ exchange by enflurane may account for its observed action of delaying relaxation in species lacking sarcoplasmic reticulum.

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 8'Rb 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. (Circulation Research 1991;69:1506-1513 T he Na-Ca exchanger is an active component of the sarcolemma in heartl-5 and nerve.6-10 Although the participation of the exchanger in excitation-contraction coupling in heart has long been postulated, its role has remained controversial. There is mounting evidence that the exchanger does have a major role in calcium efflux11-14 and perhaps also in calcium influx during excitation.1516 To elucidate this role it is important that the properties of the exchanger in heart be well understood. To date, most of the detailed studies on Na-Ca exchange in intact tissue have been done with squid axon, where both internal and external solutes can be controlled. Two significant properties revealed by these studies are that the exchanger is controlled by ATP7 and by intracellular calcium.1017 The exchanger in heart is likewise controlled by ATP18 and by intracellular calcium.19-21 In squid axon, Na-Na exchange activated by intracellular calcium has also been demonstrated.10These authors showed that Nai-Na0 exchange and Nai-Ca0 exchange had a similar affinity for activation by intracellular calcium and concluded that the Na-Na exchange was a mode of operation of the Na-Ca exchanger. We show here that a similar Na-Na exchange mode exists for the Na-Ca exchanger in heart and that this activity can be induced by calcium that enters the cell through calcium channels. We propose that this Na-Na exchange activity is indicative of the extent of activation of the Na-Ca exchanger by intracellular calcium. The significance of this result goes beyond a comparison between heart and nerve. In the following article we demonstrate that in normal cells at rest there is a near absence of intracellular calcium-dependent Na-Na exchange activity through the exchanger, but such activity can be induced by electrical stimulation. On the basis of our conclusions here, this implies that, at rest, the...