Thrombin-stimulated phosphorylation of myosin light chain and its possible involvement in endothelin-1 secretion from porcine aortic endothelial cells.
Thrombin-stimulated secretion of endothelin-1 (ET-1) from porcine aortic endothelial cells was inhibited in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) also prevented the thrombin-stimulated secretion of ET-1 but it enhanced the accumulation of ET-1 in the endothelial cells. When the endothelial cells were treated with thrombin, the phosphorylation of a 20-kDa protein which was identified as myosin light chain (MLC) was detected. Phosphorylation was augmented in a time-dependent manner. As in the case of ET-1 secretion, MLC phosphorylation was prevented by TMB-8, trifluoperazine, W-7 and ML-9 at the same concentrations which were effective in inhibiting the ET-1 secretion. The site of phosphorylation of MLC was identified as a serine residue. Parallel to the phosphorylation of MLC, thrombin increased the amounts of the 43- and 200-kDa proteins in the Triton-insoluble fraction; these proteins were identified as actin and myosin heavy chain, respectively. These results suggest that the MLC phosphorylation elicited by MLC kinase may facilitate the formation of filamentous myosin and actin which are probably involved in ET-1 secretion, possibly in the transport of ET-1-containing vesicles in thrombin-stimulated endothelial cells.
Kitazumi
K
K
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Tasaka
K
K
D000818:Animals; D001011:Aorta; D001381:Azepines / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D014268:Trifluoperazine / Q000494:pharmacology
D001381:Azepines; D016232:Endothelins; D013449:Sulfonamides; C056218:ML 9; D014268:Trifluoperazine; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; C017967:W 7; D011494:Protein Kinases; D013917:Thrombin; D009218:Myosins