Inhibition of agonist-stimulated inositol 1,4,5-trisphosphate production and calcium signaling by the myosin light chain kinase inhibitor, wortmannin.

The myosin light chain kinase inhibitor, wortmannin (WT), caused dose-dependent inhibition of the sustained increases but not the initial spikes of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and cytoplasmic Ca2+ concentration ([Ca2+]i) in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In contrast, WT did not affect the [Ca2+]i increases evoked by K(+)-induced depolarization or by depletion of intracellular Ca2+ stores with ionomycin or thapsigargin, suggesting that its inhibitory effects on Ins-1,4,5-P3 and [Ca2+]i responses are closely related. The inhibitory effect of WT on the Ins-1,4,5-P3 response was not secondary to its reduction of Ca2+ entry, since ionomycin-induced elevation of [Ca2+]i did not restore Ins-1,4,5-P3 formation. Also, WT inhibited agonist- and GTP gamma S-stimulated Ins-1,4,5-P3 formation in permeabilized cells maintained at fixed Ca2+ concentrations. Thus, inhibition of Ca2+ influx by WT is a consequence of reduced Ins-1,4,5-P3 formation and could reflect the resultant refilling of the agonist-sensitive Ca2+ pool. Indeed, AII-depleted Ca2+ pools were found to refill by a thapsigargin-sensitive mechanism in WT-treated cells. Other structurally unrelated inhibitors of myosin light chain kinase (MS-347a, KT5926, and ML-9) also inhibited AII-induced inositol phosphate formation, albeit with lower potencies than WT. MS-347a, like WT, inhibited only the second phase of the inositol phosphate and [Ca2+]i responses to AII. These findings demonstrate that WT is a potent inhibitor of the Ins-1,4,5-P3-Ca2+ signaling pathway and acts primarily on a mechanism that promotes Ins-1,4,5-P3 formation and is sensitive to inhibitors of myosin light chain kinase activity.