Activation of Ca/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) and development of the Ca/CaM-independent (autonomous) form of the kinase was investigated in cultured vascular smooth muscle (VSM) cells. Within 15 s of ionomycin (1 µM) 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 phosphorylation of CaM kinase II -subunits in situ and was abolished by pretreatment with the CaM kinase II inhibitor KN-93. The in situ Ca dependence for generating autonomous CaM kinase II was determined in cells selectively permeabilized to Ca and depleted of sarcoplasmic reticulum Ca by pretreatment with thapsigargin. Analysis of the resulting curve revealed an EC (concentration producing 50% of maximal response) of 692 ± 28 nM [Ca], 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 [Ca] 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 [Ca] resulted in a parallel curve with an EC of 304 ± 23 nM [Ca]. This apparent increase in Ca sensitivity for generating autonomous activity in intact VSM cells was eliminated by thapsigargin pretreatment. We conclude that alteration of [Ca] over a physiological range activates CaM kinase II in VSM and that this process is facilitated by release of Ca from intracellular pools which initiates cooperative autophosphorylation and consequent generation of autonomous CaM kinase II activity.

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