In situ Ca2+ dependence for activation of Ca2+/calmodulin-independent protein kinase II in vascular smooth muscle cells

Activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) and development of the Ca2+/CaM-independent (autonomous) form of the kinase was investigated in cultured vascular smooth muscle (VSM) cells, Within 15 s of ionomycin (1 mu M) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintained for at least 10 min, This correlated with P-32 phosphorylation of CaM kinase II delta-subunits in situ and was abolished by pretreatment with the CaM kinase II inhibitor KN-93, The in situ Ca2+ dependence for generating autonomous CaM kinase II was determined in cells selectively permeabilized to Ca2+ and depleted of sarcoplasmic reticulum Ca2+ by pretreatment with thapsigargin. Analysis of the resulting curve revealed an EC(50) (concentration producing 50% of maximal response) of 692 +/- 28 nM [Ca2+](i), a maximum of 68 +/- 2% of the total activity becoming autonomous reflecting nearly complete activation of CaM kinase II and a Hill slope of 3, indicating a highly cooperative process, Based on this dependence and measured [Ca2+](i) responses in intact cells, increases in autonomous activity stimulated by angiotensin II, vasopressin and platelet-derived growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were unexpectedly high, In intact cells stimulated by ionomycin, the correlation between autonomous activity and [Ca2+](i) resulted in a parallel curve with an EC(50) of 304 +/- 23 nM [Ca2+](i), This apparent increase in Ca2+ sensitivity for generating autonomous activity in intact VSM cells was eliminated by thapsigargin pretreatment. We conclude that alteration of [Ca2+](i) over a physiological range activates CaM kinase II in VSM and that this process is facilitated by release of Ca2+ from intracellular pools which initiates cooperative autophosphorylation and consequent generation of autonomous CaM kinase II activity.