Calyculin A-induced endothelial cell shape changes are independent of [Ca(2+)](i) elevation and may involve actin polymerization.
Changes in endothelial cell (EC) shape result in inter-EC gap formation and subsequently regulate transendothelial passage. In this work, we investigated the effects of protein phosphorylation (induced by inhibition of protein phosphatases) on EC shape changes. Treatment of bovine pulmonary artery endothelial cells (BPAEC) with calyculin A (100 nM, an inhibitor of protein Ser/Thr phosphatases 1 and 2A) resulted in cell retraction, surface bleb formation and cell rounding. Trypan blue and electrophysiological experiments suggested that the plasma membrane of these rounded cells maintained functional integrity. Calyculin A-induced morphological changes were strongly inhibited by staurosporine, but not affected by specific inhibitors of the myosin light chain (MLC) kinase, protein kinases A, C and G, and tyrosine kinases. The calyculin A effects were not mimicked by phorbol myristate acetate, dibutyryl cAMP, 8-bromo-cGMP or ionomycin. Cytochalasin B (an inhibitor of actin polymerization) almost completely abolished such shape changes while colchicine (an inhibitor of microtubule polymerization) had no inhibitory effect at all. Ca(2+) imaging experiments showed that the morphological changes were not associated with any global or local cytosolic Ca(2+) concentration ([Ca(2+)](i)) elevation. The results suggest that calyculin A unmasked the basal activities of some protein Ser/Thr kinases other than MLC kinase and protein kinases A, C and G; these unknown kinases might cause BPAEC shape changes by a mechanism involving actin polymerization but not [Ca(2+)](i) elevation.
Leung
Yuk Man
YM
Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada. yukman.leung@utoronto.ca
Kwan
Tony K
TK
Kwan
Chiu Yin
CY
Daniel
Edwin E
EE
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002413:Cations, Divalent; D002460:Cell Line / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D003078:Colchicine / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology*; D011108:Polymers / Q000737:chemistry; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013997:Time Factors
D000199:Actins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D011108:Polymers; D003571:Cytochalasin B; C059041:calyculin A; D011493:Protein Kinase C; D003078:Colchicine; D002118:Calcium