Mechanisms underlying AlCl3 inhibition of agonist-stimulated inositol phosphate accumulation. Role of calcium, G-proteins, phospholipase C and 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 (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.
Shafer
T J
TJ
Cellular and Molecular Toxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
Nostrandt
A C
AC
Tilson
H A
HA
Mundy
W R
WR
D000077410:Aluminum Chloride; D017607:Aluminum Compounds / Q000494:pharmacology*; D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002217:Carbachol / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000096:biosynthesis*; D008297:Male; D010710:Phosphates / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D017607:Aluminum Compounds; D002712:Chlorides; D007295:Inositol Phosphates; D010710:Phosphates; D000077410:Aluminum Chloride; D002217:Carbachol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium