MECHANISMS UNDERLYING ALCL3 INHIBITION OF AGONIST-STIMULATED INOSITOLPHOSPHATE ACCUMULATION - ROLE OF CALCIUM, G-PROTEINS, PHOSPHOLIPASE-CAND PROTEIN-KINASE-C

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 (G(p)-medi ated), AlCl3 did not affect CARB (100 mu M)-induced decreases (G(i)-me diated) in 30 mu M forskolin-stimulated cAMP accumulation. suggesting that AlCl3 may be specific for G(p)-mediated signal transduction. To d etermine whether AlCl3 interfered with G(p) function and/or phosphatid ylinositol-specific phospholipase C (PiPLC) activity, effects of AlCl3 on CARB- and Ca2+-stimulated IP accumulation were examined in cortica l synaptosomes. AlCl3 (500 mu M) decreased CARB (1 mM)- and Ca2+ (20 m u M ionomycin)-stimulated IP accumulation to 77 and 75% of control, re spectively, suggesting that AlCl3 may not directly affect G(p) activit y, but does inhibit PiPLC activity. In cortical homogenates, AlCl3 (10 -500 mu M) inhibited hydrolysis of [H-3]phosphatidylinositol 4,5-bisph osphate (PIP2) by PiPLC in a concentration-dependent manner with an es timated IC50 of 100 mu M. The effects of AlCl3 on modulation of IP acc umulation by extracellular Ca2+ and PKC were also examined as potentia l mechanisms. Decreasing the extracellular Ca2+ concentration ([Ca2+]( e)) from 1.0 to 0.1 mM decreased CARB-stimulated IP accumulation in sl ices. AlCl3 (500 mu M) 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 sli ces, inhibition of 1 mM CARB-stimulated IP accumulation by 500 mu M Al Cl3 was not altered by the PKC activator phorbol 12,13-dibutyrate (PdB u, 1 mu M), or the PKC inhibitor H-7 (10 mu M), suggesting that AlCl3 does not interfere with IP accumulation by activation of PKC. Other st udies found that AlCl3 (10-100 mu M) inhibited PKC activity in a conce ntration-dependent manner in both cytosolic and membrane fractions of cortical homogenates with an estimated IC50 of 60 mu M. These results support the hypothesis that AlCl3 inhibition of agonist-stimulated IP accumulation may be mediated by inhibition of PiPLC activity, rather t han disruption of G-protein function or modulation of the IP signallin g system by Ca2+ or PKC.