Calcium Transport in Canine Renal Basolateral Membrane Vesicles

Introduction The effects of parathyroid hormone were studied on Ca" fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na' gradient, and this was inhibited by the Na' ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2" exchange activity. There was a Na' gradient independent Ca2+ flux, but membrane binding of Ca2" was excluded from contributing to the Na' gradient-dependent efflux. The Na' gradient-dependent flux of Ca2" was very rapid, and even 2and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2" and Na' with an apparent (5 s) K. for Na'-dependent Ca2" uptake of 10 ,uM, and an apparent (5 s) V.,.,, of 0.33 nmol/mg protein per 5 s. The Na' concentration that yielded half maximal Ca2" efflux (2 s) was 11 mM, and the Hill coefficient was two or greater. Both Na+ gradient dependent and independent Ca2" efflux were decreased in BLMV prepared from kidneys of thyroparathyroidectomized (TPTX) dogs, and both were stimulated by parathyroid hormone (PTH) infusion to TPTX dogs. BLMV from TPTX dogs exhibited significantly reduced maximal stimulation of Na+ gradient-dependent Ca2+ uptake with an apparent (5 s) V,,, of 0.23 nmol/mg protein per 5 s, but the apparent K. was 8 ,uM, which was unchanged from normal. The Na+ gradient independent Ca2+ uptake was also reduced in BLMV from TPTX dogs compared with normal. Thus, PTH stimulated both Na+/Ca2" exchange activity and Na+ independent Ca2" flux. In vivo, the latter could result in an elevation of cytosolic Ca2" by PTH, and this might contribute to the observed decrease in solute transport in the proximal tubule. Parts of these data were presented to the American Society of Nephrology, Washington, DC, 1983, and were published in abstract form in Kidney Int., 1984, 25:144. While this manuscript was under review for publication, Drs. A. Jayakumar, L. Cheng, C. T. Liang, and B. Sacktor have published work showing Na/Ca exchanger activity in BLMV J. Biol. Chem. 259:10827-10833, 1984. Their kinetics were based on longer incubation times but gave similar results to these. They reported an effect of PTH but no kinetic data for this. Dr. Hruska is an Established Investigator of the American Heart Association. Address reprint requests to Dr. Hruska. Received for publication 29 May 1984 and in revised form 26 December 1984. Approximately 60% of the filtered Ca2" is reabsorbed in the proximal convoluted tubule (1). The bulk of Ca2" reabsorption in this segment parallels the reabsorption of Na+, such that tubular fluid (TF) to plasma ultrafiltrate (UF) TF/UFNa+/TF/ UFca2+ concentration is nearly a unity under nondiuretic conditions (1, 2). In situ microperfusion experiments demonstrate high permeability to Ca2+ in this segment, such that the unidirectional effilux of Ca2+ is 2-3 times larger than the net flux (3-5). These observations have been interpreted to indicate that the bulk of Ca2' reabsorption in the proximal convoluted tubule is a passive process, and some investigators (6) have speculated that a paracellular Ca2+ flux through the tight junctions is the major path of Ca2' reabsorption in the proximal convoluted tubule. However, Ullrich et al. (4) found that more Ca2+ was transported than could be accounted for by the electrochemical gradient when equilibrium solutions were present in the lumen and the bath of isolated perfused proximal segments. This suggested that an element of Ca2+ reabsorption was active in this segment. These findings have been criticized on technical grounds (6). However, recent findings of Bomsztyk and Wright (7, 8) confirm the presence of net Ca2+ absorption in the absence of transepithelial electrochemical driving forces which indicate active Ca2+ transport. In these studies, there was also clear dissociation between Na+ and Ca2+ transport rates in certain experimental settings. Recent studies, as reviewed by Schaefer (9), have also demonstrated that transepithelial volume flow in the proximal tubule is established through transcellular solute transport (9). Thus, transcellular Ca2+ transport is an important mechanism of reabsorption in the proximal convoluted tubule. The fact that parathyroid hormone (PTH)' stimulates renal Ca2" reabsorption is well established (10-15). In the proximal convoluted tubule, where Ca2+ transport is isotonic and generally associated with fluid and Na+ fluxes (1-4), PTH has been shown to inhibit solute reabsorption isotonically (16-19), or alternatively to have no effect on Ca2+ reabsorption (20, 21). In the hamster, Harris et al. (13) have reported stimulatory effects of PTH on Ca2' transport in the proximal convoluted tubule, but this may be a species-specific effect. Only a preliminary report (22) supports PTH stimulation of proximal tubular Ca2+ transport in other rodents. In summary, the effect of PTH on Ca2+ reabsorption in the mammalian proximal 1. Abbreviations used in this paper: BLMV, basolateral membrane vesicles; BBMV, brush border membrane vesicles; DOC, deoxycholate; IOV, inside out vesicle; PTH, parathyroid hormone; ROV, right side out vesicle; TPP+, tetraphenylphosphonium; TPTX, thyroparathy-