CELLULAR MECHANISM OF CA TRANSPORT IN THE NEPHRON SEGMENTS AND REGULATION BY HORMONES AND DRUGS

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Renal tubular reabsorption of Ca is important in the regulation of body Ca balance. Distal nephron segments plays important roles for Ca balance by responding to hormones or autacoids released for emergent Ca loss. The present project was designed to clarify the cellular mechanisms of Ca transport in the distal nephron segments by using the in vitro microperfusion technique of isolated renal tubules. Methods to explore Ca transport in the nephron segments included measurement of net Ca flux, intracellular Ca concentration (Ca^<2+>i) by microscopic fluorometory with fura2, electrophysiological studies and cell volume measurement. PTH and calcitonin were found to act on the connecting tubule (CNT) and distal convoluted tubule (DCT), respectively, to increase net Ca absorption. In both segments, intact Na/Ca antiporter in the basolateral membrane is essential for the effect of these hormones. We focused our attention on the mechanisms of action of PTH in the CNT. Based on the observation … More that PTH caused biphasic responses of transmural voltage (Vt), we speculated that PTH, via cAMP, increases Ca entry from the apical membrane by opening a non-selective cation channel. Na entry along with Ca through this channel may reduce transmembrane Na gradient which would reduce Ca extrusion by Na/Ca exchanger. This unfavorable effect for Ca is prevented by inhibiting apical Na entry through amiloride sensitive Na channel by increased Ca^<2+>i. Thus an inhibition of Ca entry from the apical membrane is expected to enhance net Ca transport. This hypothesis was supported by the observations that the maneuvers which reduce Na entry from the apical membrane enhanced net Ca transport across the CNT in the presence of PTH. Such maneuvers included elimination of Na from the luminal fluid and administration of amiloride or trichlormethiazide in the lumen. Thus, the interaction between Na and Ca in the CNT may account for the mechanism of anticalciuric effect of these diuretics.Interaction between Na and Ca was further explored in the other protocols in which we examined effect of PGE_2 on the ion transport in the CNT. We found for the first time that PGE_2 added to the bath markedly increases cell volume of the CNT in the absence of osmotic gradient. I contradictory to the generally believed assumption that PGE_2 inhibits Na, K-pump, we found that Na entry from the basolateral membrane via Na/Ca exchanger is the cause of cell swelling induced by PGE_2. This hypothesis was supported by the observations that the cell swelling response was prevented by an inhibitor of Na/Ca exchanger. Existence of dihydropyridine sensitive Ca channel in the basolateral membrane is also essential for the cell swelling. By cellular impalement of a microelectrode, we found that PGE_2 causes biphasic changes in the apical membrane voltage (Va). By careful analysis of this phenomenon, we concluded that PGE_2, in addition to the basolateral action, opens the non-selective cation channel in the apical membrane by a cAMP mediated process and then inhibits amiloride sensitive Na channel by a Ca medicated process. Less