Disruption by lithium of phosphatidylinositol-4,5-bisphosphate supply and inositol-1,4,5-trisphosphate generation in Chinese hamster ovary cells expressing human recombinant m1 muscarinic receptors

Abstract

Inhibitory effects of the anti-manic agent lithium on carbachol-stimulated phosphoinositide signaling have been investigated in Chinese hamster ovary (CHO) cells transfected with human m1 muscarinic receptor cDNA (Bmax, 816 fmol/mg of protein). In the presence of Li+, a time-dependent inhibition of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass accumulation was observed within 10 min of agonist addition (IC50 for lithium inhibition at 20 min after carbachol addition, 0.5 mM). The Li(+)-induced decrease in agonist-stimulated Ins(1,4,5)P3 levels was preceded by a dramatic increase in CMP-phosphatidate accumulation. The idea that Li+ blockade of inositol monophosphatase caused a rapid depletion of the cellular myo-inositol pool in CHO-m1 cells was supported by the reversal of Li+ effects by exogenous myo-inositol. Carbachol (1 mM) alone caused a rapid and dramatic decrease in phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)-P2]in CHO-m1 cells labeled to equilibrium with [3H]-inositol. Carbachol-evoked decreases in PtdIns(4,5)P2 were time-dependently accentuated by Li+ (IC50 for Li+ inhibition at 20 min after carbachol addition, 1.2 mM). Measurements of changes in PtdIns(4,5)P2 mass demonstrated that the effect of Li+ was completely and concentration-dependently reversed by addition of myo-inositol. Sequential 30-min periods of carbachol stimulation resulted in similar time courses of Ins(1,4,5)P3 accumulation when an intervening 20-min recovery period was included in the protocol. Inclusion of Li+ throughout resulted in a more rapid and dramatic attenuation of Ins(1,4,5)P3 during the agonist rechallenge period, which could be correlated with accentuated changes in PtdIns(4,5)P2. These data demonstrate that, although mechanisms operate to efficiently resynthesize PtdIns(4,5)P2, the temporal correlation of carbachol-evoked decreases in PtdIns(4,5)P2 levels in the presence of Li+ strongly suggests that phosphoinositide-specific phospholipase C substrate depletion may be causal in the subsequent decrease in Ins(1,4,5)P3 levels.