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

肾单位段 CA 运输的细胞机制以及激素和药物的调节

• 批准号: 03454147
• 负责人: IMAI Masashi
• 金额: $ 4.03万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454147/
• 关键词: Connecting tubule; Calcium transport; Calcium channel; Sodium transport; Na; Ca exchanger; Prostaglandins; amiloride; カルシウム; PTH; カルシトニン; 遠位曲尿細管; Ca^<2+>; Na^+交換系; サイアザイド利尿薬

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

肾小管钙重吸收在调节体内钙平衡中起重要作用。远端肾单位段通过对激素或类金丝桃体的反应对钙的平衡起着重要作用。本研究旨在利用体外肾小管微灌流技术，阐明远端肾小管钙转运的细胞机制。研究肾单位段钙转运的方法包括用FURA2显微荧光法测定细胞内钙离子浓度(Ca^&lt；2+&gt；i)、电生理研究和细胞体积测量。甲状旁腺激素和降钙素分别作用于连接小管(CNT)和远端曲管(DCT)，增加钙的净吸收。在这两个节段，基底膜上完整的Na/Ca逆向转运体对这些激素的作用是必不可少的。我们将注意力集中在甲状旁腺素在CNT中的作用机制上。基于观察值的…除了PTH引起跨壁电压(Vt)的双相反应外，我们推测，PTH通过cAMP，通过开放非选择性阳离子通道，增加了从根尖膜的钙内流。Na与Ca一起进入该通道可能会降低跨膜Na梯度，从而减少Na/Ca交换器对Ca的排泄。这种对钙的不利影响可以通过增加钙离子浓度来抑制通过阿米洛利敏感钠通道的钠离子进入顶端。因此，抑制从顶膜的钙离子进入有望增强钙的净转运。这一假说得到了以下观察的支持：在甲状旁腺素存在的情况下，减少钠从顶膜进入的动作增加了通过CNT的净钙转运。这些操作包括从管腔液体中清除钠，以及在管腔内给药阿米洛利或三氯甲肼。因此，钠和钙在碳纳米管中的相互作用可能解释了这些利尿剂的抗钙尿作用的机制。在其他方案中，我们进一步探讨了钠和钙的相互作用，其中我们检测了PGE_2对碳纳米管中离子转运的影响。我们首次发现，在无渗透梯度的情况下，加入前列腺素E_2能显著增加CNT的细胞体积。与普遍认为的PGE_2抑制Na，K泵的假设相反，我们发现通过Na/Ca交换器从基底膜进入Na是PGE_2引起细胞肿胀的原因。这一假说得到了Na/Ca交换器抑制剂阻止细胞肿胀反应的观察的支持。基底膜上存在二氢吡啶敏感的钙通道也是细胞肿胀所必需的。通过细胞穿透微电极，我们发现PGE_2引起顶膜电压(Va)的双相变化。通过对这一现象的仔细分析，我们得出结论：PGE_2除了基侧作用外，还通过cAMP介导的过程开放根尖膜上的非选择性阳离子通道，然后通过钙离子调节过程抑制阿米洛利敏感的钠通道。较少

项目成果

Shimizu T, Nakamura, Yoshitomi K, Imai M: "Interaction of trichlormethiazide or amiloride with PTH in stimulating Ca^<2+> absorption in rabbit CNT." Am J Physiol. 261 (Renal Fluid Electrolyte Physiol 30). F36-F43 (1991)

Shimizu T、Nakamura、Yoshitomi K、Imai M：“三氯甲噻嗪或阿米洛利与 PTH 的相互作用刺激兔 CNT 中的 Ca^2 吸收。”

Shimizu T,: "Mechanism of PGE_2 induced cell swelling in distal nephron segments." Am J Physiol. 263. F824-F832 (1992)

Shimizu T，：“PGE_2 诱导远端肾单位段细胞肿胀的机制。”

Muto S, Yasoshima K, Yoshitomi K, Imai M, Asasno Y: "Electrophysiological identification of alpha- and beta-intercalated cells and their distribution along the rabbit distal nephron segments." J Clin Invest. 86. 1829-1839 (1990)

Muto S、Yasoshima K、Yoshitomi K、Imai M、Asasno Y：“α 和 β 嵌入细胞的电生理学鉴定及其沿兔远端肾单位段的分布。”

Imai M,Yoshitomi K,Taniguchi J:"Loop of Henle function In:New Insight in Vertebrate Kidney Function." Brown JA,Balment RJ(Editors) Cambridge University Press,

Imai M，Yoshitomi K，Taniguchi J：“Henle 功能环：脊椎动物肾功能的新见解。”

Shimizu T,Nakamura,Yoshitomi K,Imai M:"Interaction of trichlomethiazide or amilorride with PTH in stimulating Ca^<2+> absorption in rabbit CNT." Am J Physiol. 261. F36-F43 (1991)

Shimizu T，Nakamura，Yoshitomi K，Imai M：“三氯甲噻嗪或阿米洛利与 PTH 的相互作用刺激兔 CNT 中的 Ca^2 吸收。”