Heterogeneous response of isolated adult rat heart cells to insulin

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...

Section: Resultssupporting

confidence: 82%

Section: Measurement Of Cell 45 Ca Contentmentioning

confidence: 99%

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

Haworth¹,

Goknur²,

Hunter³

et al. 1987

“…Cells as isolated have an extremely low basal rate of 3-Omethylglucose uptake. This basal rate is biphasic, reflecting a small population of cells with finite uptake rates and most cells with rates that are essentially zero (Haworth et al, 1984). This property allowed us to monitor 3-O-methylglucose uptake continually on the same time scale and in the same experiment as we monitored the onset of contracture.…”

Section: Chronology Of Contracture and Induction Of Glucose Transportmentioning

confidence: 98%

“…Consequently, 3-0-methylglucose equilibrates with the cell water at a rate governed by the glucose permeability of the cell membrane (Whitesell and Gliemann, 1979;Haworth et al, 1984). Low concentrations of 3-O-methylglucose ( « Km) were used in order to gain a measure of uptake rate uncomplicated by variable rates of back exchange (Whitesell and Gliemann, 1979).…”

Section: Ijmentioning

confidence: 99%

The control of sugar uptake by metabolic demand in isolated adult rat heart cells.

Haworth¹,

Berkoff²

1986

SUMMARY. To investigate the control of sugar uptake by metabolic demand, we used isolated quiescent adult rat heart cells in suspension, under conditions similar to those found during anoxia. Metabolic demand was varied by exposing cells to rotenone plus various levels of ptrifluoromethoxyphenylhydrazone. Without glucose, the time taken for half of the cells to undergo contracture was inversely proportional to the metabolic demand as measured by the rate of lactate production. For any metabolic demand, the onset of contracture was preceded by a sudden drop in adenosine triphosphate. The permeability of contracted cells to glucose was investigated using 3-0-methylglucose. The rate of 3-0-methylglucose uptake by such cells was strongly dependent on the time taken for half the cells to undergo contracture, with low rates at low times to half contracture, and insulin-like rates at high times to half contracture. This suggests that the full induction of glucose transport by metabolic demand can be prematurely curtailed by the loss of adenosine triphosphate. This phenomenon appeared to limit glucose utilization in cells with a high metabolic demand when glucose was present: such cells underwent contracture unless insulin was also present, the rate of glucose uptake as measured with 2-deoxyglucose was inhibited, and the rate of lactate production was inhibited. Isoproterenol depressed glucose transport by two mechanisms. First, by stimulating the basal metabolic demand of the cell it reduced the time taken for half the cells to undergo contracture and, hence, the level of induced sugar transport. Second, it significantly delayed the onset of sugar permeability with respect to the contracture event. Consequently, cells treated with isoproterenol were more prone to contracture than cells without isoproterenol. (Circ Res 58: 157-165, 1986) EXPOSURE of whole isolated rat hearts to periods of anoxia results in activation of glucose transport (Morgan et al., 1959). The complexity of the whole heart has made it difficult to investigate in detail the way in which glucose uptake is controlled under these conditions. The recent advent of preparations of isolated adult heart cells that resist physiological levels of calcium has made it possible to investigate this question. In this study, anoxia was simulated by the addition of rotenone to inhibit mitochondrial respiration. Various amounts of p-trifluoromethoxyphenylhydrazone (FCCP) were added to achieve increased levels of metabolic demand. By 'metabolic demand,' we mean the rate of adenosine triphosphate (ATP) utilization by cells sustaining normal levels of ATP. For such cells, the metabolic demand is equal to the rate of ATP production. Normally, most ATP resynthesis would occur by oxidative phosphorylarion. In the presence of rotenone, ATP production is restricted to that from glycolysis, plus a transient and limited contribution from creatine phosphate. Indeed, since there is an obligatory link between glycolysis and ATP production, glycolysis cannot proceed unless there...

“…The method is an extension of that previously used in our laboratory for measuring solute fluxes. [24][25][26] The new feature of the method is that concentrated cells are labeled with 22Na to a high specific activity and then diluted 10-fold into unlabeled medium before centrifugation. The dilution results in a 10-fold higher specific activity of 22Na inside the cell compared with 22Na outside and hence allows a 10-fold enhancement of the sensitivity of measurement of intracellular sodium compared with that of our basic method.…”

Section: Isotope Flux Measurementsmentioning

confidence: 99%

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

Haworth¹,

Goknur²,

Hunter³

1991

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...