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Articles in PresS, published online ahead of print July 24, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00105.2002
Submitted on March 7, 2002
Accepted on July 17, 2002
and PKC
on basolateral membrane dynamics in intestinal epithelia
1 Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA
2 Medicine, McMaster University, Hamilton, ON, Canada
* To whom correspondence should be addressed. E-mail: jeffrey.matthews{at}uc.edu.
Protein kinase C (PKC) is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC is poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKC followed by late activation of the conventional isoform PKC in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKC was shown to be responsible for stimulation of basolateral endocytosis but the role of PKC was not defined. In the present study, we used detailed time course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKC on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor Go976 (5 µM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKC-selective agonist carbachol (CCh, 100 µM) induced prolonged stimulation of endocytosis devoid of inhibitory phase. Actin disassembly caused by PMA was completely blocked by Go850 but not by Go976, implicating PKC as the key isoform responsible for actin disruption. The Ca+2 agonist thapsigargin (Tg, 5 µM) induced early activation of PKC when added simultaneously with PMA. This early activation of PKC blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. In conclusion, activation of PKC stabilizes f-actin and thereby opposes the effect of PKC on membrane remodeling in T84 cells.
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