MECHANISMS UNDERLYING ALCL3 INHIBITION OF AGONIST-STIMULATED INOSITOLPHOSPHATE ACCUMULATION - ROLE OF CALCIUM, G-PROTEINS, PHOSPHOLIPASE-CAND PROTEIN-KINASE-C
Tj. Shafer et al., MECHANISMS UNDERLYING ALCL3 INHIBITION OF AGONIST-STIMULATED INOSITOLPHOSPHATE ACCUMULATION - ROLE OF CALCIUM, G-PROTEINS, PHOSPHOLIPASE-CAND PROTEIN-KINASE-C, Biochemical pharmacology, 47(8), 1994, pp. 1417-1425
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.