The effect of Ca 2+ on the translocation of protein kinase C in bovine tracheal smooth muscle

Abstract

The role of calcium in protein kinase C redistribution was studied in bovine tracheal smooth muscle preparations contracted by methacholine. Previous results have shown that, in the presence of normal extracellular Ca 2+, 10 μM methacholine produced a sustained contraction and a sustained translocation of protein kinase C from the cytosol to the membrane. In the present study, when tissues were preincubated in Ca 2+-free buffer containing 1 mM EGTA, methacholine produced a rapid but transient elevation in membrane-associated protein kinase C activity which was detected at 30 s and had returned to basal within 20 min. The redistribution of protein kinase C from the cytosol to the membrane induced by 1 μM methacholine in normal Ca 2+ was reversed by removal of the extracellular Ca 2+ and addition of 2 mM EGTA during agonist stimulation. Removal of the Ca 2+ caused approximately 50% relaxation after 10 min. Verapamil (30 μM) partially reversed the methacholine-induced protein kinase C redistribution and caused approximately 40% relaxation after 15 min. Sodium nitroprusside (10 μM) caused a rapid relaxation and complete reversal of the protein kinase C redistribution induced by methacholine. High K + (60 mM) also induced a sustained contraction and redistribution of protein kinase C from the cytosol to the membrane. Suitable antagonists were added to the bathing medium to block the effects of endogenous mediators which could be released by KCl-induced depolarization. Thus, translocation of protein kinase C is obtained in the absence of receptor activation. These observations indicate that alterations in [Ca 2+] i may be responsible for the changes in protein kinase C redistribution observed during agonist-induced contractions of bovine tracheal smooth muscle.