Mrkic B, Forgo J, Murer H, Helmle-Kolb C
Department of Physiology, University of Zurich, Switzerland.
J Membr Biol. 1992 Dec;130(3):205-17. doi: 10.1007/BF00240478.
Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10(-8) M; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10(-8) M); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10(-8) M) resulted in a rapid and transient increase in [Ca2+]i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/Pi cotransport.
甲状旁腺激素（PTH）可抑制肾近端小管的钠/氢交换。为了进一步分析这种调节所涉及的细胞机制，我们使用了在可渗透滤膜支架上生长的MCT细胞（一种SV - 40永生化小鼠皮质小管细胞培养物）。通过单细胞荧光显微镜（BCECF）测量钠/氢交换，并通过示踪技术测量磷酸盐转运（用于比较）。MCT细胞表达顶端和基底外侧的钠/氢交换体，它们对乙基异丙基氨氯吡咪和二甲基氨氯吡咪的抑制反应不同，基底外侧膜转运体更敏感。顶端膜钠/氢交换受到PTH（10^(-8) M；平均抑制25%）的抑制；当细胞暴露于福斯可林、8 - 溴 - cAMP或佛波酯时，观察到类似程度的抑制。基底外侧膜钠/氢交换通过与PTH孵育（升至对照水平以上129%）或添加佛波酯（升至对照水平以上120%）而受到刺激；在暴露于福斯可林或8 - 溴 - cAMP后受到抑制。PTH和佛波酯（顶端和基底外侧）的上述作用可通过用蛋白激酶C拮抗剂、星形孢菌素和钙磷蛋白C预孵育细胞来预防；这两种化合物均不影响福斯可林或8 - 溴 - cAMP诱导的作用。PTH还抑制顶端钠依赖性磷酸盐内流（10^(-8) M时抑制29%）；它对基底外侧磷酸盐通量（钠依赖性和非钠依赖性）没有影响。用PTH（10^(-8) M）孵育导致细胞内钙离子浓度（[Ca2+]i）迅速短暂升高（用荧光指示剂fura - 2测量），这是由于细胞内储存的钙离子释放受到刺激。将MCT细胞暴露于PTH不会提高细胞内cAMP水平。综上所述，这些结果表明PTH在MCT细胞中利用磷脂酶C/蛋白激酶C途径来不同地控制钠/氢交换体（顶端与基底外侧）并抑制顶端钠/磷酸盐共转运。
