In situ Ca2+ dependence for activation of Ca2+/calmodulin-dependent 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 microM) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintained for at least 10 min. This correlated with 32P 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 EC50 (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 EC50 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.
Abraham
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Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
Benscoter
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Schworer
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CM
Singer
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HA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium