Mechanisms underlying AlCl 3 inhibition of agonist-stimulated inositol phosphate accumulation : Role of calcium, G-proteins, phospholipase C and protein kinase C

Abstract

Possible mechanisms of AlCl 3-induced inhibition of agonist-stimulated inositol phosphate (IP) accumulation were investigated using rat brain cortex slices, synaptosomes or homogenates. Under conditions in which AlCl 3 inhibits carbachol (CARB)-stimulated IP accumulation (G p-mediated), AlCl 3 did not affect CARB (100 μM)-induced decreases (G i-mediated) in 30 μM forskolin-stimulated cAMP accumulation, suggesting that AlCl 3 may be specific for G p-mediated signal transduction. To determine whether AlCl 3 interfered with G p function and/or phosphatidylinositol-specific phospholipase C (PiPLC) activity, effects of AlCl 3 on CARB- and Ca 2+-stimulated IP accumulation were examined in cortical synaptosomes. AlCl 3 (500 μM) decreased CARB (1 mM)- and Ca 2+ (20 μM ionomycin)-stimulated IP accumulation to 77 and 75% of control, respectively, suggesting that AlCl 3 may not directly affect G p activity, but does inhibit PiPLC activity. In cortical homogenates, AlCl 3 (10–500 μM) inhibited hydrolysis of [ 3H]phosphatidylinositol 4,5-bisphosphate (PIP 2) by PiPLC in a concentration-dependent manner with an estimated IC 50 of 100 μM. The effects of AlCl 3 on modulation of IP accumulation by extracellular Ca 2+ and PKC were also examined as potential mechanisms. Decreasing the extracellular Ca 2+ concentration ([Ca 2+] e) from 1.0 to 0.1 mM decreased CARB-stimulated IP accumulation in slices. AlCl 3 (500 μM) decreased significantly 1 mM CARB-stimulated IP accumulation in 1.0 and 0.1 mM Ca 2+ solutions; however, the effect of AlCl 3 on IP accumulation did not depend on [Ca 2+] e. In cortical slices, inhibition of 1 mM CARB-stimulated IP accumulation by 500 μM AlCl 3 was not altered by the PKC activator phorbol 12,13-dibutyrate (PdBu, 1 μM), or the PKC inhibitor H-7 (10 μM), suggesting that AlCl 3 does not interfere with IP accumulation by activation of PKC. Other studies found that AlCl 3 (10–100 μM) inhibited PKC activity in a concentration-dependent manner in both cytosolic and membrane fractions of cortical homogenates with an estimated IC 50 of 60 μM. These results support the hypothesis that AlCl 3 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 Ca 2+ or PKC.