三氯化铝抑制激动剂刺激的肌醇磷酸积累的潜在机制。钙、G蛋白、磷脂酶C和蛋白激酶C的作用。

Mechanisms underlying AlCl3 inhibition of agonist-stimulated inositol phosphate accumulation. Role of calcium, G-proteins, phospholipase C and protein kinase C.

作者信息

Shafer T J, Nostrandt A C, Tilson H A, Mundy W R

机构信息

Cellular and Molecular Toxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.

出版信息

Biochem Pharmacol. 1994 Apr 20;47(8):1417-25. doi: 10.1016/0006-2952(94)90342-5.

DOI:10.1016/0006-2952(94)90342-5

PMID:8185649

Abstract

Possible mechanisms of AlCl3-induced inhibition of agonist-stimulated inositol phosphate (IP) accumulation were investigated using rat brain cortex slices, synaptosomes or homogenates. Under conditions in which AlCl3 inhibits carbachol (CARB)-stimulated IP accumulation (Gp-mediated), AlCl3 did not affect CARB (100 microM)-induced decreases (Gi-mediated) in 30 microM forskolin-stimulated cAMP accumulation, suggesting that AlCl3 may be specific for Gp-mediated signal transduction. To determine whether AlCl3 interfered with Gp function and/or phosphatidylinositol-specific phospholipase C (PiPLC) activity, effects of AlCl3 on CARB- and Ca(2+)-stimulated IP accumulation were examined in cortical synaptosomes. AlCl3 (500 microM) decreased CARB (1 mM)- and Ca2+ (20 microM ionomycin)-stimulated IP accumulation to 77 and 75% of control, respectively, suggesting that AlCl3 may not directly affect Gp activity, but does inhibit PiPLC activity. In cortical homogenates, AlCl3 (10-500 microM) inhibited hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by PiPLC in a concentration-dependent manner with an estimated IC50 of 100 microM. The effects of AlCl3 on modulation of IP accumulation by extracellular Ca2+ and PKC were also examined as potential mechanisms. Decreasing the extracellular Ca2+ concentration ([Ca2+]e) from 1.0 to 0.1 mM decreased CARB-stimulated IP accumulation in slices. AlCl3 (500 microM) decreased significantly 1 mM CARB-stimulated IP accumulation in 1.0 and 0.1 mM Ca2+ solutions; however, the effect of AlCl3 on IP accumulation did not depend on [Ca2+]e. In cortical slices, inhibition of 1 mM CARB-stimulated IP accumulation by 500 microM AlCl3 was not altered by the PKC activator phorbol 12,13-dibutyrate (PdBu, 1 microM), or the PKC inhibitor H-7 (10 microM), suggesting that AlCl3 does not interfere with IP accumulation by activation of PKC. Other studies found that AlCl3 (10-100 microM) inhibited PKC activity in a concentration-dependent manner in both cytosolic and membrane fractions of cortical homogenates with an estimated IC50 of 60 microM. These results support the hypothesis that AlCl3 inhibition of agonist-stimulated IP accumulation may be mediated by inhibition of PiPLC activity, rather than disruption of G-protein function or modulation of the IP signalling system by Ca2+ or PKC.

摘要

利用大鼠脑皮质切片、突触体或匀浆，研究了氯化铝诱导抑制激动剂刺激的肌醇磷酸（IP）积累的可能机制。在氯化铝抑制卡巴胆碱（CARB）刺激的IP积累（Gp介导）的条件下，氯化铝不影响CARB（100μM）诱导的30μM福斯可林刺激的环磷酸腺苷（cAMP）积累的减少（Gi介导），这表明氯化铝可能对Gp介导的信号转导具有特异性。为了确定氯化铝是否干扰Gp功能和/或磷脂酰肌醇特异性磷脂酶C（PiPLC）活性，在皮质突触体中检测了氯化铝对CARB和Ca²⁺刺激的IP积累的影响。氯化铝（500μM）分别将CARB（1 mM）和Ca²⁺（20μM离子霉素）刺激的IP积累降低至对照的77%和75%，这表明氯化铝可能不直接影响Gp活性，但确实抑制PiPLC活性。在皮质匀浆中，氯化铝（10 - 500μM）以浓度依赖性方式抑制PiPLC对[³H]磷脂酰肌醇4,5 - 二磷酸（PIP₂）的水解，估计半数抑制浓度（IC₅₀）为100μM。还研究了氯化铝对细胞外Ca²⁺和蛋白激酶C（PKC）调节IP积累作用的影响，作为潜在机制。将细胞外Ca²⁺浓度（[Ca²⁺]e）从1.0 mM降至0.1 mM可降低切片中CARB刺激的IP积累。氯化铝（500μM）在1.0 mM和0.1 mM Ca²⁺溶液中均显著降低1 mM CARB刺激的IP积累；然而，氯化铝对IP积累的影响不依赖于[Ca²⁺]e。在皮质切片中，500μM氯化铝对1 mM CARB刺激的IP积累的抑制作用不受PKC激活剂佛波醇12,13 - 二丁酸酯（PdBu，1μM）或PKC抑制剂H - 7（10μM）的影响，这表明氯化铝不会通过激活PKC来干扰IP积累。其他研究发现，氯化铝（10 - 100μM）在皮质匀浆的胞质和膜部分均以浓度依赖性方式抑制PKC活性，估计IC₅₀为60μM。这些结果支持以下假设：氯化铝对激动剂刺激的IP积累的抑制作用可能是通过抑制PiPLC活性介导的，而不是通过破坏G蛋白功能或由Ca²⁺或PKC调节IP信号系统。