Characterization of the effects of lithium on phosphatidylinositol (PI) cycle activity in human muscarinic m1 receptor-transfected CHO cells.

1. The effects of lithium on [3H]-inositol and [3H]-cytidine incorporation into [3H]-inositol monophosphates ([3H]-InsP1) and [3H]-cytidine monophosphorylphosphatidate ([3H]-CMP-PA), respectively, and inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) mass were studied in carbachol-stimulated human m1 muscarinic receptor-transfected Chinese hamster ovary cells (m1 CHO cells). 2. Lithium alone (10 mM) had no appreciable effects on any of the four parameters measured; it was only in carbachol-stimulated cells that the effects of lithium became apparent. 3. In the presence of carbachol (1 mM), lithium (10 mM) caused a relatively rapid (within 5 min) accumulation of [3H]-InsP1 and [3H]-CMP-PA which continued up to about 20-30 min, after which accumulation slowed down. On the other hand, the elevation in InsP3 and InsP4 levels produced by carbachol was not altered by lithium in the short-term and only at later times (> 20-30 min) was the response attenuated, with InsP3 and InsP4 levels approaching basal. 4. The effects of lithium on carbachol-stimulated [3H]-InsP1 and [3H]-CMP-PA accumulation and the attenuation of the carbachol-induced elevation of InsP3 and InsP4 were all dose-dependent, with EC50s in the region of 1 mM. 5. The lithium-induced effects on [3H]-CMP-PA and InsP3 and InsP4 in carbachol-stimulated cells could be reversed, in a dose-dependent manner, by preincubation with exogenous myo-inositol (EC50 = 2-3 mM) but not by the inactive analogue scyllo-inositol, indicating that these effects occur as a consequence of depletion of inositol. 6. The temporal effects of lithium are consistent with lithium inhibiting inositol monophosphatase,causing accumulation of InsP1, resulting in lower free inositol levels. This leads to accumulation of CMP-PA and reduced PI synthesis which, once agonist-linked membrane inositol phospholipids are depleted, produces attenuated InsP3 and InsP4 responses.7. These results in ml CHO cells support the hypothesis that lithium affects the PI cycle cell signalling pathway by depletion of inositol due to inhibition of inositol monophosphatase.