Two vitamin D3-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. I. Effect of CaBP 28K.

Several clearance and micropuncture studies suggest that vitamin D deficiency decreases calcium (Ca2+) reabsorption by the nephron. In confirmation of these observations, we reported recently that in vitamin D depleted rabbits, Ca2+ transport by both the luminal and the basolateral membranes of the distal tubule was significantly diminished. It is also in the distal tubule that vitamin D stimulates the synthesis of two calcium binding proteins (CaBP) with molecular weights of 28K and 9K. The purpose of this study was to investigate the role of the CaBP 28K in the regulation of Ca2+ transport by the two polar membranes of the distal nephron of the rabbit. Results were compared to those obtained with the corresponding membranes of the proximal tubule. The basolateral and luminal membranes were purified from proximal and distal tubule suspensions separately. Ca2+ uptake was measured by the rapid filtration technique. Preloading the luminal membrane vesicles from proximal and distal tubules with 3 microM CaBP 28K strongly enhanced the initial Ca2+ uptake by the two membranes. When the vesicles, preloaded with CaBP 28K and incubated with 0.5 mM Ca2+, were abruptly diluted in a medium containing 10 microM A 23187 and 2 mM EGTA, the release of Ca2+ was slower than with control vesicles, suggesting that the excess of transported Ca2+ was bound inside the vesicles. The effect of CaBP 28K was dose-dependent with a half-maximal effect at 10(-6) M and a maximal effect at 5 x 10(-6) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Two vitamin D3-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. II. Effect of CaBP 9K.

The distal tubule is a major site of hormone regulated Ca2+ reabsorption. We have previously shown that in the rabbit, vitamin D depletion decreases both the Ca2+ uptake by the luminal membrane of nephron distal segments and the ATP-dependent Ca2+ transport by the basolateral membrane of these same segments. We also reported that in in vitro experiments, the vitamin D-dependent calcium binding protein (CaBP) 28K enhances Ca2+ uptake by the high-affinity Ca2+ transport system of the distal luminal membranes. The purpose of the present study was to investigate the role of CaBP 9K in Ca2+ transport by the two polar membranes of the proximal and distal nephron segments. Preloading the luminal membranes of the two series of tubules with 10 microM CaBP 9K did not modify 0.5 mM Ca2+ uptake by these membranes. In contrast, preincubation of distal tubule basolateral membranes with 10 microM CaBP 9K strongly enhanced ATP-dependent Ca2+ transport by these membranes. A similar although more modest effect was observed in proximal basolateral membranes. This effect was present only in calmodulin depleted membranes. Calmodulin also increased the ATP-dependent Ca2+ uptake by the depleted membranes. The two actions were not additive. A dose-response curve showed that the maximal CaBP 9K effect was obtained at 10(-5) M. CaBP 9K increased the Vmax of ATP-dependent uptake from 0.43 +/- 0.03 to 0.79 +/- 0.06 nmol/mg/10 sec (P < 0.005), leaving the Km unchanged. It is concluded that CaBP 9K activates the Ca2+ pump in the basolateral membrane of both the proximal and distal tubule.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone and hydrochlorothiazide increase calcium transport by the luminal membrane of rabbit distal nephron segments through different pathways.

The present study was designed to investigate the effect of PTH on calcium (Ca2+) transport through the luminal membrane of proximal and distal rabbit tubule segments. Proximal tubule and distal tubule segment suspensions were incubated with the hormone, and the luminal membranes were subsequently purified. Incubation with 10(-8) M human PTH(1-34) strongly increased initial Ca2+ uptake by the distal membranes. The effect of PTH was dose dependent, with an apparent ED50 of 8.2 +/- 1.0 nM. We recently reported the presence of two kinetics of Ca2+ uptake by the distal luminal membranes. PTH affected exclusively the high affinity component, increasing the maximum velocity from 0.31 +/- 0.02 to 0.76 +/- 0.07 pmol/micrograms.10 sec (P < 0.001), and leaving the Michaelis-Menten constant Ca2+ unchanged. The addition of 500 microM hydrochlorothiazide (HCTZ) to the luminal membranes of distal tubules incubated with PTH further enhanced Ca2+ uptake. The effect of HCTZ was on the low affinity system. HCTZ (100 microM) enhanced the maximum velocity from 2.5 +/- 0.3 to 3.7 +/- 0.6 pmol/micrograms protein.10 sec (P < 0.01) without affecting the Michaelis-Menten constant. Whereas 1 microM nitrendipine alone did not affect Ca2+ transport by the distal tubule luminal membranes, the Ca2+ channel inhibitor completely abolished the effect of PTH. Conversely, 1 microM Bay K 8644 increased Ca2+ uptake by membranes from PTH-treated distal tubules but was ineffective in membranes from control tubules. Neither PTH nor nitrendipine nor Bay K 8644 had any effect on the luminal membranes from proximal tubules. These results suggest that: 1) the high affinity, low velocity Ca2+ transport system in the luminal membrane from distal cortical segments is sensitive to PTH; 2) the effects of nitrendipine and Bay K 8644 on Ca2+ uptake were observed only in membranes from tubules incubated with PTH; 3) this uptake is distinct from the thiazide-sensitive Ca2+ transport system; and 4) PTH does not influence Ca2+ transport by the luminal membrane of proximal tubules.

Effect of vitamin D depletion on calcium transport by the luminal and basolateral membranes of the proximal and distal nephrons.

To study the effect of vitamin D on calcium (Ca2+) reabsorption by the kidney, we measured Ca2+ uptake by the basolateral and luminal membranes of proximal and distal tubules obtained from rabbits fed a vitamin D-deficient diet for 3 weeks. Results were compared to those obtained with a group of control animals fed a normal diet. Serum Ca2+ concentrations were comparable in both groups. In the control group, serum PTH, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 remained relatively stable. In the vitamin D-deficient animals, serum PTH levels slightly, but not significantly, increased, and the levels of vitamin D metabolites abruptly fell. Vitamin D depletion produced a 40% decrease in ATP-dependent Ca2+ uptake by the basolateral membrane of the distal tubule. There was no change in the activity of the Na+/Ca2+ exchanger. A very significant effect was also observed in the luminal membrane of the distal tubule, where a 50% decrease in Ca2+ uptake was observed after the third week of vitamin depletion. Administration of 0.1 microgram 1,25-dihydroxyvitamin D3 16 and 2 h before death partially reestablished normal uptake. In contrast, no change in Ca2+ uptake could be detected in the basolateral or luminal membranes of the proximal tubule. These observations provide the first evidence of an effect of vitamin D on Ca2+ transport at both the basolateral and luminal membranes of the distal segment of the nephron.

The mechanism of parathyroid hormone action on calcium reabsorption by the distal tubule.

PTH increases calcium reabsorption exclusively in the distal nephron. Two mechanisms of Ca++ transport through the basolateral membrane (BLM) have been described: the ATP-dependent and the sodium gradient-dependent transport. In the present study, we investigated the effect of PTH and (Bu)2cAMP on these two mechanisms. We recently reported, using 100 microM Ca++ as the substrate, that whereas the ATP-dependent system was present in the proximal and distal tubule (DCT), the Na+/Ca++ exchanger was located only in the DCT. Using 2 microM Ca++ as the substrate, the Na+/Ca++ exchanger was again found to be present only in the DCT. Incubation of DCT suspension with 10(-8) M bovine PTH (1-34) resulted in a significant increase in the Na(+)-dependent Ca++ uptake by the corresponding BLM vesicles. This effect was dose dependent. The half-maximal stimulation was obtained with 1 X 10(-8) M PTH. At this concentration, PTH increased the maximum velocity (Vmax) from 0.34 +/- 0.06 t 0.54 +/- 0.02 nmol/mg/10 s (P less than 0.05) without influencing the Michaelis-Menten constant (Kms). Incubation of DCT suspensions with (Bu)2cAMP mimicked this effect. The dose-response curve showed a peak action at 1 mM (Bu)2cAMP. In contrast, neither (Bu)2cAMP nor PTH influenced the ATP-dependent Ca++ transport through BLM from proximal tubule nor DCT. It is proposed that PTH influences Ca++ reabsorption by the DCT because the target molecule, the Na+/Ca++ exchanger, is located exclusively at this site.

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes.

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K+ acetate.