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Effect of diuretics on ion transport of kidney cortex mitochondria. III. Species difference in calcium accumulation and in ethacrynic acid effect.

Effect of inorganic phosphate (4 X 10(-3) M) on Ca++-accumulation was examined in kidney cortex mitochondria. Ca++-accumulation of rat kidney cortex mitochondria was slightly influenced by inorganic phosphate. On the other hand, dog kidney cortex mitochondria did not accumulate calcium from the incubation medium until the inorganic phosphate had been added. Ca++-accumulation of rabbit kidney cortex mitochondria was markedly stimulated by inorganic phosphate. When ethacrynic acid was added to the reaction medium in the absence of inorganic phosphate, Ca++-accumulation of rat kidney cortex mitochondria was depressed and the decrease in calcium content of rabbit and dog kidney cortex mitochondria was enhanced. In the presence of inorganic phosphate, the inhibition of Ca++-accumulation by ethacrynic acid was observed only on dog kidney cortex mitochondria. Subsequently, the effect of inorganic phosphate (4 X 10(-4) M) and ethacrynic acid (1 X 10(-4) M on Ca++-ATPase was examined in kidney cortex mitochondria. The low concentration of inorganic phosphate (4 X 10(-4) M) activated Ca++-ATPase of kidney cortex mitochondria in all animal species. The greatest activation of Ca++-ATPase occurred in rabbits, but the activity of the enzyme was lower than that in rats and dogs. Inhibition of Ca++-ATPase by ethacrynic acid was depressed by the addition of inorganic phosphate in kidney cortex mitochondria of experimental animals. Ca++-accumulation may be regulated through the stimulating effect of inorganic phosphate and the inhibitory effect of ethacrynic acid on Ca++-ATPase in kidney cortex mitochondria. Species difference in ethacrynic acid effect on Ca++-accumulation in kidney cortex mitochondria of rats, rabbits and dogs is discussed.

Abstract-Effect of inorganic phosphate (4 x 10-3 M) on Ca++-accumulation was ex amined in kidney cortex mitochondria. Call-accumulation of rat kidney cortex mitochondria was slightly influenced by inorganic phosphate. On the other hand, dog kidney cortex mitochondria did not accumulate calcium from the incubation medium until the inorganic phosphate had been added. Call-accumulation of rabbit kidney cortex mitochondria was markedly stimulated by inorganic phosphate. When ethacrynic acid was added to the reaction medium in the absence of inorganic phosphate, Ca`-accumulation of rat kidney cortex mitochondria was depressed and the decrease in calcium content of rabbit and dog kidney cortex mitochondria was enhanced. In the presence of inorganic phosphate, the inhibition of Ca++-accumula tion by ethacrynic acid was observed only on dog kidney cortex mitochondria. Sub sequently, the effect of inorganic phosphate (4 x 10-' M) and ethacrynic acid (1 x 10-' M) on Call-ATPase was examined in kidney cortex mitochondria. The low con centration of inorganic phosphate (4 x 10-' M) activated Call-ATPase of kidney cor tex mitochondria in all animal species. The greatest activation of Ca"-ATPase occurred in rabbits, but the activity of the enzyme was lower than that in rats and dogs. Inhibition of Ca' -ATPase by ethacrynic acid was depressed by the addition of inorganic phosphate in kidney cortex mitochondria of experimental animals. Ca accumulation may be regulated through the stimulating effect of inorganic phos phate and the inhibitory effect of ethacrynic acid on Call-ATPase in kidney cortex mitochondria. Species difference in ethacrynic acid effect on Ca"-accumulation in kidney cortex mitochondria of rats, rabbits and dogs is discussed.
It is well known that renal tubule cells, especially proximal tubule cells, contain a great number of large sized mitochondria.
It is assumed that ion accumulation in mito chondria plays an important role in cell membrane transport mechanism through a regula tion of intracellular calcium concentration. Ethacrynic acid has different diuretic effects on different species of experimental ani mals, i.e., potent diuretic effect is observed in the dog, a mild effect in the rabbit, while that in the rat is slight (3,4). Whether or not ethacrynic acid reveals differences among the species regarding inhibitory effects on subcellular organella of experimental animals is a subject which holds great interest.
The present investigation was undertaken to compare these effects in rats, rabbits and dogs.
MATERIALS AND METHODS Animals were anesthetized with sodium pentobarbital, and the kidneys were removed from male Wistar rats, weighing 250 to 350 g, male rabbits, weighing 2 to 3 kg, and mongrel dogs of both sexes, weighing 10 to 20 kg. Kidneys were cooled immediately in ice cold sucrose (0.25 M) and the cortex was separated from medulla. Kidney cortex mitochondria were prepared with cold sucrose (0.25 M) by the method of Hogeboom (5).
Accumulation of calcium in mitochondria. The mitochondria (ca. 2.5 mg protein) were incubated in a reaction medium containing 10 mM Tris-HC1, pH 7.4, 2 mM MgCl2, 0.5 mM CaC12, 6 mM NaCI, 4 mM KCI, 1 mM ATP (tris salt), 7.8 mM succinate, 250 mM sucrose and other additions depending upon experimental conditions in a total volume of 5.0 ml at 30°C, while shaking gently. After a 10 min incubation, the reaction medium was filtered through a millipore filter (type DAWP, size 0.65 p) to rapidly separate the mitochondria.
Ca' '-accumulation was estimated by determining its appearance in the extract (99% of formic acid) of mitochondria as described previously (1). Calcium was determined by an atomic absorption spectrophotometer (Hitachi 207).
Assay of ATPase. ATP hydrolysis by kidney cortex mitochondria was measured by estimating inorganic phosphate released in the medium containing 12.5 mM Tris-HCI, pH 7.4, 2 mM ATP (tris salt), 2 mM MgC12, 250 mM sucrose and other additions depend ing upon experimental conditions in a total volume of 1.0 ml at 30'C for 10 min as de scribed in a previous paper (2). Ca"-ATPase activity was calculated as follows; ATP ase activity observed in the presence of MgCl2 (2 mM) was subtracted from total ATPase activity obtained in the reaction medium which contained both MgCl2 (2 mM) and CaC12 (0.5 mM). ATPase activity was expressed as ni zmoles of liberated inorganic phosphate/ mg protein/min. Protein concentration of mitochondria was estimated according to the procedure of Lowry et al. (6). Crystalline bovine serum albumin was used as a protein standard. ATP (ditris salt) was obtained from Sigma Chemical Co.. ATP and succinic acid were employed after the pH had been adjusted to 7.4 with tris (hydroxymethyl) aminomethane. Ethacrynic acid, 2,3-dichloro-4-(2 methylenbutyryl)-phenoxyacetic acid, was kindly provided by Japan Merck Banyu Co. and prepared in Tris-HCI, pH 7.4.

RESULTS
After incubation of kidney cortex mitochondria in a standard medium, a similar Ca"-accumulation was observed in rats and rabbits; 741.3 ±76.6 (SE) m,eequiv/mg protein/ The medium was incubated for 10 min at 30°C. The indicated final con centration of phosphate was added to the medium as KH2PO4-Na2HPO4 buffer, p14 7.4. Each bar denotes the mean±standard error (SE). n=5.
10 min for rats and 685.9+122.5 (SE) mpequiv/mg protein/10 min for rabbits. On the other hand, dog mitochondria did not accumulate calcium from the standard medium but rather extruded it as shown in Fig. 1. Chapell et al. reported that calcium uptake by rat liver mitochondria was stimulated by the addition of inorganic phosphate (7). We, there fore, examined the effect of inorganic phosphate on Cal '-accumulation to make a com parison among the three species. Generally, inorganic phosphate (4 x 10-3 M) stimulated Ca `-accumulation in the mitochondria of all these species, but the extent was different.
The greatest stimulation was observed in rabbits and the least in rats. In the dog, calcium movement turned from efflux to influx by the addition of inorganic phosphate (Fig. 1).
In our previous work, we reported that ethacrynic acid inhibited Ca "-accumulation of rat kidney cortex mitochondria (1). In the present paper, the inhibitory effect of the diuretic was examined in three different animal species and compared. Ethacrynic acid (1 x 10-4 M) decreased Ca "-accumulation of mitochondria in all species. In rats, Cal' accumulation was depressed by 82.1 %, and the value was almost the same as that re ported previously. In rabbits, Ca"-accumulation turned to extrusion by the addition of ethacrynic acid, and in dogs, calcium extrusion was enhanced by ethacrynic acid (Fig. 2). phosphate in the control experiment using all three species. The highest Ca"-accumula tion occurred in rabbits, and next in rats. Dog kidney cortex mitochondria had the lowest Ca++-accumulation rate among the three species.
In a second series of experiments, mitochondrial ATPase of each animal was determined with and without CaCl2. With the absence of CaCl2 in the reaction medium, the order of ATPase activity was rats, rabbits and dogs (Fig. 4). Although the extent of Ca++-ATP ase in rats and dogs was almost the same, the activity was less in the rabbit.  6 shows that the addition of inorganic phosphate resulted in a decrease in the percent in hibition of Ca"-ATPase by ethacrynic acid in all species. In rat kidney cortex mito chondria, the percent inhibition of Ca"-ATPase by ethacrynic acid was decreased from 91.0% in the control experiment to 64.6% following the addition of inorganic phosphate.

DISCUSSION
It has been reported that Ca"-ATPase activity is related to Ca `-accumulation in heart mitochondria (8). ATP and calcium appear to have an influence on each other in their transport across the mitochondrial inner membrane. Spencer and Bygrave have reported that calcium stimulated adenine nucleotide translocase activity in rat liver mitochondria (9). The increased ATP translocated in the presence of calcium ion would be available for hydrolysis by ATPase. The energy produced by ATP breakdown would then serve for Ca "-accumulation. Previous work in this series has shown that the main inhibitory effect of ethacrynic acid on Ca "-accumulation depends on the action on Ca"-ATPase in rat kidney cortex mitochondria (2). The present experiment showed that ethacrynic acid inhibited Ca"-ATPase of kidney cortex mitochondria not only in rats but also in rabbits and dogs (Fig. 4). In rabbit kidney cortex mitochondria, Ca"-ATPase was in hibited completely by ethacrynic acid, and the decrease of calcium content was observed by the addition of the diuretic (Fig. 2). The decrease of calcium content was also en hanced by the diuretic in dog kidney cortex mitochondria. On the basis of these data, it is reasonable to assume that inhibition of Ca++-ATPase by ethacrynic acid may relate not to the inhibition of calcium efflux but to the inhibition of calcium influx in kidney cortex mitochondria. Inorganic phosphate did not affect ATPase activity observed in the presence of MgCl2, but rather stimulated Ca"-ATPase in kidney cortex mitochondria (Fig. 5). It is more likely that inorganic phosphate may function to regulate the activity of Ca"-ATPase of kidney cortex mitochondria in the intact cells of these experimental animals. Stimulative action of phosphate and inhibitory action of ethacrynic acid on mitochondrial Ca"-ac cumulation may appear through the influence on Ca"-ATPase activity. Whether or not inorganic phosphate competes with ethacrynic acid concerning the action on mito chondrial Ca"-ATPase is a subject of further investigation.
Our present results show that dog kidney cortex mitochondria releases calcium in the absence of inorganic phosphate and that the ability to accumulate calcium was lower than in rabbits and rats even in the presence of inorganic phosphate. It would thus appear that the rate of calcium efflux is highest in dogs. In addition, the differences in the re sponse of mitochondrial Ca "-accumulation among rats, rabbits and dogs may be re lated to differences in the activity of Ca"-ATPase and the extent of sensitivity of inorganic phosphate or ethacrynic acid to the enzyme. It is of further interest that ethacrynic acid increased ATPase activity in the presence of MgCl2 in dog kidney cortex mitochondria unlike the results in rats and rabbits (Fig. 4), thus resembling the action of uncoupling agents on the ATPase of mitochondria. We also have data which show that 2,4-dinitro phenol (5 x 10-5 M), well known as an uncoupler of mitochondria, stimulates 92.0 % of ATPase observed in the presence of MgCI2 and depresses 53.8 % of Ca `-accumulation in rat kidney cortex mitochondria (Values are not shown in the figure). The action of ethacrynic acid resembling the action of uncoupler on the ATPase was not depressed by inorganic phosphate (unpublished), and therefore, may relate in part to the inhibition of Cal '-accumulation observed in the presence of inorganic phosphate in dog kidney cortex mitochondria (Fig. 3). Lehninger has suggested that the ion transport function of mitochondria might play an important role in physiological regulation of calcium and active transcellular trans port of reabsorptive substances in renal tubule cells (10). From the present results, it may be concluded that ethacrynic acid affects the intracellular calcium homeostasis of the kidney, to a different extent in each of the three species. Further investigation of the intracellular concentration of inorganic phosphate in the kidney cells of animal species employed in the present study is warranted.