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

肾小管对钙的重吸收在机体钙平衡的调节中起重要作用。远端肾单位段通过响应紧急钙丢失释放的激素或自育激素对钙平衡起重要作用。本研究旨在通过离体肾小管微灌注技术，探讨钙离子在远侧肾单位段转运的细胞机制。方法采用Fura 2荧光显微镜下测定肾单位内Ca ~（2+）浓度（Ca ~（2+）i）、Ca ~（2+）净流量、细胞电生理和细胞体积等方法，探讨肾单位内Ca ~（2+）的转运。发现PTH和降钙素分别作用于连接小管（CNT）和远曲小管（DCT），以增加净钙吸收。在这两个部分中，基底外侧膜中完整的Na/Ca反向转运蛋白对于这些激素的作用是必不可少的。我们将注意力集中在PTH在CNT中的作用机制上。基于以下观察 ...更多信息 PTH引起跨壁电压（Vt）的双相反应，我们推测PTH通过cAMP，通过开放非选择性阳离子通道增加Ca从顶膜的进入。Na与Ca通过该通道沿着进入可降低跨膜Na梯度，这将减少Na/Ca交换器的Ca挤出。通过增加Ca 2 + i来抑制顶端Na进入阿米洛利敏感性Na通道，可防止Ca的这种不利作用。因此，抑制钙从顶膜进入，预计将增强净钙转运。这一假设得到了以下观察结果的支持：在PTH存在下，减少Na从顶膜进入的操作增强了穿过CNT的净Ca转运。这些操作包括从腔液中消除Na和在腔中给予阿米洛利或三氯噻嗪。因此，CNT中Na和Ca的相互作用可能是这些利尿剂抗钙作用的机制，在我们研究PGE_2对CNT中离子转运的影响的其他方案中，我们进一步探讨了Na和Ca的相互作用。我们首次发现，在没有渗透压梯度的情况下，加入PGE_2可以显著增加CNT的细胞体积。与一般认为的PGE_2抑制Na，K泵的假设相反，我们发现，Na通过Na/Ca交换器从基底外侧膜进入细胞是PGE_2引起细胞肿胀的原因。这一假设得到了以下观察结果的支持：Na/Ca交换抑制剂可阻止细胞肿胀反应。基底外侧膜上二氢吡啶敏感性钙通道的存在也是细胞肿胀的必要条件。用微电极穿刺细胞，发现前列腺素E_2（PGE_2）引起细胞膜电压（Va）的双相变化。PGE_2除了对基底外侧膜有作用外，还通过cAMP介导的过程开放了顶膜非选择性阳离子通道，然后通过Ca介导的过程抑制了阿米洛利敏感性Na通道。少

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