EFFECTS OF L-690,488, A PRODRUG OF THE BISPHOSPHONATE INOSITOL MONOPHOSPHATASE INHIBITOR L-690,330, ON PHOSPHATIDYLINOSITOL CYCLE MARKERS

In order to enhance the entry into cells of L-690,330, a bisphosphonat e inhibitor of inositol monophosphatase (IMPase; a key, enzyme in the phosphatidylinositol (PI) cell signaling pathway), the tetrapivaloylox ymethyl ester prodrug, L-690,488 [tetrapivaloyloxymethyl 1-(4-hydroxyp henoxy)ethane-1,1-bisphosphonate], was synthesized. The effects of L-6 90,488 were studied in cholinergically (carbachol)-stimulated rat cort ical slices and Chinese hamster ovary cells stably transfected with th e human muscarinic m1 receptor (m1 CHO cells). The accumulation of [H- 3]inositol monophosphates or [H-3]cytidine monophosphoryl-phosphatidat e ([H-3]CMP-PA) after [H-3]inositol or [H-3]cytidine prelabeling, resp ectively, and inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrak isphosphate mass were measured. In rat cortical slices and m1 CHO cell s, the maximum response and time course of accumulation of [H-3]inosit ol monophosphates for L-690,488 and lithium were similar. However, the concentrations of L-690,488 required to produce these effects (EC(50) values of 3.7 +/- 0.9 and 1.0 +/- 0.2 mu M in cortical slices and m1 CHO cells, respectively) were much lower than with lithium (0.3-1.5 mM ). Likewise, the time course and maximum accumulation of [H-3] CMP-PA in L-690,488-treated m1 CHO cells was similar to lithium but L-690,488 was again much more potent (EC(50) values = 3.5 +/- 0.3 mu M and 0.52 +/- 0.03 mM for L-690,488 and lithium, respectively). In addition, L- 690,488 attenuated the carbachol-induced elevation of inositol 1,4,5-t risphosphate and inositol 1,3,4,5-tetrakisphosphate in m1 CHO cells, a n effect reported previously with lithium. These results are all consi stent with L-690,488 and lithium both depleting intracellular inositol as a consequence of inhibition of IMPase. That these effects of L-690 ,488 on the PI cycle are indeed due to inositol depletion is shown by the observation that the effects of L-690,488 on CMP-PA accumulation c ould be overcome by addition of exogenous myo-inositol (EC(50) = 1.7 /- 0.5 mM). These data show that inhibition of IMPase produces effects on the PI cycle comparable to lithium. As a corollary, the effects of lithium on the PI cycle are therefore consistent with its major mecha nism of action being inhibition of IMPase.