IN-SITU CA2+ DEPENDENCE FOR ACTIVATION OF CA2+ CALMODULIN-INDEPENDENTPROTEIN-KINASE-II IN VASCULAR SMOOTH-MUSCLE CELLS/

Activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM k inase II) and development of the Ca2+/CaM-independent (autonomous) for m of the kinase was investigated in cultured vascular smooth muscle (V SM) cells, Within 15 s of ionomycin (1 mu M) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintaine d for at least 10 min, This correlated with P-32 phosphorylation of Ca M kinase II delta-subunits in situ and was abolished by pretreatment w ith the CaM kinase II inhibitor KN-93, The in situ Ca2+ dependence for generating autonomous CaM kinase II was determined in cells selective ly permeabilized to Ca2+ and depleted of sarcoplasmic reticulum Ca2+ b y pretreatment with thapsigargin. Analysis of the resulting curve reve aled an EC(50) (concentration producing 50% of maximal response) of 69 2 +/- 28 nM [Ca2+](i), a maximum of 68 +/- 2% of the total activity be coming autonomous reflecting nearly complete activation of CaM kinase II and a Hill slope of 3, indicating a highly cooperative process, Bas ed on this dependence and measured [Ca2+](i) responses in intact cells , increases in autonomous activity stimulated by angiotensin II, vasop ressin and platelet-derived growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were unexpectedly high, In intact cells stimulated by io nomycin, the correlation between autonomous activity and [Ca2+](i) res ulted in a parallel curve with an EC(50) of 304 +/- 23 nM [Ca2+](i), T his apparent increase in Ca2+ sensitivity for generating autonomous ac tivity 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.