Modulation of nucleocytosolic [Ca2+] gradient in smooth muscle by protein phosphorylation

Abstract

In resting DDT1MF-2 smooth muscle cells, the cytosolic free Ca2+ concentration ([Ca2+]c) was higher than the free Ca2+ concentration in the nucleus ([Ca2+]n). However, this nucleocytosolic [Ca2+] gradient was reversed by Ca2+ agonists like ATP or, as is shown here, by the epidermal growth factor (EGF). The ATP-induced reversal of the nucleocytosolic [Ca2+] gradient was blocked by stimulation of protein kinase C with phorbol 12-myristate 13-acetate or with the diacylglycerol kinase inhibitor R59949, or by inhibition of the Ser/Thr-specific protein phosphatases-1 and -2A with okadaic acid or calyculin A. Moreover, the magnitude of the ATP-induced reversal of the [Ca2+] gradient diminished during prolonged culture of the cells. The EGF-induced [Ca2+] rise in the cytosol and nucleus was blocked by okadaic acid and by the tyrosine kinase inhibitors herbimycin A and psi-tectorigenin. Our data suggest that the nucleocytosolic [Ca2+] gradient is modulated by (de)phosphorylation processes catalyzed by tyrosine protein kinases, by protein kinase C, and by Ser/Thr protein phosphatases-1 and -2A.