Ms. Nijjar et B. Grimmelt, DOMOIC ACID INHIBITS ADENYLATE-CYCLASE ACTIVITY IN RAT-BRAIN MEMBRANES, Molecular and cellular biochemistry, 136(2), 1994, pp. 105-111
Adenylate cyclase activity measured by the formation of cyclic AMP in
rat brain membranes was inhibited by a shellfish toxin, domoic acid (D
OM). The inhibition of enzyme was dependent on DOM concentration, but
about 50% of enzyme activity was resistant to DOM-induced inhibition.
Rat brain supernatant resulting from 105,000 x g centrifugation for 60
min, stimulated adenylate cyclase activity in membranes. Domoic acid
abolished the supernatant-stimulated adenylate cyclase activity. The b
rain supernatant contains factors which modulate adenylate cyclase act
ivity in membranes. The stimulatory factors include calcium, calmoduli
n, and GTP, In view of these findings, we examined the role of calcium
and calmodulin in DOM-induced inhibition of adenylate cyclase in brai
n membranes. Calcium stimulated adenylate cyclase activity in membrane
s, and further addition of calmodulin potentiated calcium-stimulated e
nzyme activity in a concentration dependent manner. Calmodulin also st
imulated adenylate cyclase activity, but further addition of calcium d
id not potentiate calmodulin-stimulated enzyme activity. These results
show that the rat brain membranes contain endogenous calcium and calm
odulin which stimulate adenylate cyclase activity. However, calmodulin
appears to be present in membranes in sub-optimal concentration for a
denylate cyclase activation, whereas calcium is present at saturating
concentration. Adenylate cyclase activity diminished as DOM concentrat
ion was increased, reaching a nadir at about 1 mM. Addition of calcium
restored DOM-inhibited adenylate cyclase activity to the control leve
l. Similarly, EGTA also inhibited adenylate cyclase activity in brain
membranes in a concentration dependent manner, and addition of calcium
restored EGTA-inhibited enzyme activity to above control level. The f
act that EGTA is a specific chelator of calcium, and that DOM mimicked
adenylate cyclase inhibition by EGTA, indicate that calcium mediates
DOM-induced inhibition of adenylate cyclase activity in brain membrane
s. While DOM completely abolished the supernatant-, and Gpp(NH)p-stimu
lated adenylate cyclase activity, it partly blocked calmodulin-, and f
orskolin-stimulated adenylate cyclase activity in brain membranes. The
se results indicate that DOM may interact with guanine nucleotide-bind
ing (G) protein and/or the catalytic subunit of adenylate cyclase to p
roduce inhibition of enzyme in rat brain membranes.