Ando Y, Jacobson H R, Breyer M D
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Clin Invest. 1988 May;81(5):1578-84. doi: 10.1172/JCI113491.
Activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration ([Ca++]i) result from phosphatidylinositol biphosphate (PIP2) breakdown. We previously demonstrated that PKC activation inhibits arginine vasopressin (AVP)-induced osmotic water flow in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. To estimate the potential significance of PIP2 turnover as a modulator of water transport in this nephron segment, we examined the effect of Ca on AVP action and explored the mechanisms of action of PKC and increased [Ca++]i. In rabbit CCTs perfused at 37 degrees C, pretreatment with bath A23187 (2 x 10(-8) M, 2 x 10(-6) M), a Ca ionophore, almost totally suppressed AVP (10 microU/ml)-induced peak hydraulic conductivity (Lp). The suppression by 2 x 10(-8) M A23187 was as potent as that by 2 x 10(-6) M A23187, and significant even when it was administered 10 min after AVP. When phorbol myristate acetate (PMA, 10(-9) M), a PKC activator, and A23187 (2 x 10(-8) M) were placed in the bath simultaneously, the combined suppressive effect on peak Lp was greater than that of either inhibitor alone. However, the mechanisms of inhibition by PMA and A23187 were different. While both 10(-7) and 10(-9) M PMA suppression are primarily post-cAMP, A23187 predominantly suppressed a pre-cAMP step: 10(-4) M chlorophenylthio-cAMP-induced peak Lp was not affected by 2 x 10(-8) M A23187, and only partially inhibited by 2 x 10(-6) M A23187. The PMA (10(-7) M) suppression of AVP-induced peak Lp was totally reversed by bath staurosporine (10(-7) M), a PKC inhibitor, but not attenuated by either bath indomethacin (5 x 10(-6) M) or low Ca (1-2 x 10(-6) M) bath medium. In contrast, the A23187 (2 x 10(-8) M) suppression of the peak Lp was not affected by staurosporine, but was significantly reversed by indomethacin or low Ca bath medium. We conclude: (a) Elevation of [Ca++]i, as well as activation of PKC, suppresses the hydroosmotic effect of AVP on CCT at 37 degrees C. (b) When stimulated simultaneously these two intracellular mediators are additive in their antagonism of AVP action. These results suggest that stimulated PIP2 breakdown may be an important modulator of water transport in CCT. (c) Different mechanisms underlie PKC and Ca-mediated suppression of the AVP-induced water transport. The inhibition of AVP action by increased [Ca++]i is primarily pre-cAMP, and involves a cyclooxygenase metabolite(s) of arachidonic acid, while the inhibition by PKC is post-cAMP, and independent of cyclooxygenase products of arachidonic acid.
磷脂酰肌醇二磷酸（PIP2）分解导致蛋白激酶C（PKC）激活和细胞内钙离子浓度（[Ca++]i）升高。我们先前证明，在37℃体外灌注的兔皮质集合管（CCT）中，PKC激活会抑制精氨酸加压素（AVP）诱导的渗透水流。为了评估PIP2周转作为该肾单位节段水转运调节剂的潜在意义，我们研究了钙离子对AVP作用的影响，并探讨了PKC和升高的[Ca++]i的作用机制。在37℃灌注的兔CCT中，用钙离子载体A23187（2×10−8 M、2×10−6 M）预处理几乎完全抑制了AVP（10微单位/毫升）诱导的峰值水力传导率（Lp）。2×10−8 M A23187的抑制作用与2×10−6 M A23187一样有效，即使在AVP给药10分钟后给药也具有显著抑制作用。当蛋白激酶C激活剂佛波酯（PMA，10−9 M）和A23187（2×10−8 M）同时置于浴液中时，对峰值Lp的联合抑制作用大于单独使用任何一种抑制剂的作用。然而，PMA和A23187的抑制机制不同。虽然10−7 M和10−9 M PMA的抑制作用主要是在环磷酸腺苷（cAMP）之后，而A23187主要抑制cAMP之前的步骤：10−4 M氯苯硫基-cAMP诱导的峰值Lp不受2×10−8 M A23187影响，仅被2×10−6 M A23187部分抑制。浴液中PKC抑制剂星形孢菌素（10−7 M）可完全逆转PMA（10−7 M）对AVP诱导的峰值Lp的抑制作用，但浴液中吲哚美辛（5×10−6 M）或低钙（1 - 2×10−6 M）浴液介质均不能减弱该抑制作用。相反，A23187（2×10−8 M）对峰值Lp的抑制作用不受星形孢菌素影响，但可被吲哚美辛或低钙浴液介质显著逆转。我们得出结论：（a）[Ca++]i升高以及PKC激活会抑制37℃时AVP对CCT的水渗透作用。（b）当这两种细胞内介质同时受到刺激时，它们对AVP作用的拮抗作用具有相加性。这些结果表明，受刺激的PIP2分解可能是CCT中水转运的重要调节剂。（c）PKC和钙离子介导的对AVP诱导的水转运的抑制作用存在不同机制。[Ca++]i升高对AVP作用的抑制主要在cAMP之前，涉及花生四烯酸的环氧化酶代谢产物，而PKC的抑制作用在cAMP之后，且与花生四烯酸的环氧化酶产物无关。
