Thrombin-stimulated events in cultured vascular smooth-muscle cells.
Thrombin is present in high concentrations at sites of clots and may have important post-clotting effects on adjacent vascular tissue. This may be particularly important for vascular smooth-muscle cells (VSMC), whose growth and contractility are altered following atherosclerotic-associated thromboses. To study the cellular signal events by which thrombin exerts its actions, the effects of purified human alpha-thrombin were examined in cultured rat aortic VSMC. alpha-Thrombin stimulated a biphasic change in intracellular pH (pHi), causing an early rapid acidification, followed by a sustained alkalinization. The increase in pHi was dependent on extracellular Na+ and inhibited by 5'-(NN-dimethyl)amiloride, consistent with mediation by Na+/H+ exchange. alpha-Thrombin rapidly increased free intracellular [Ca2+] ([Ca2+]i). The increase in [Ca2+]i was secondary to activation of phospholipase C, as demonstrated by increases in InsP3 (226%) and InsP2 (387%) and decreases in polyphosphoinositides at 15 s. Expression of the mRNA for the proto-oncogene c-fos was induced by alpha-thrombin. Stimulation of c-fos mRNA was not dependent on alterations in pHi, but required a rise in [Ca2+]i. Despite many growth-related signals shared by alpha-thrombin with platelet-derived growth factor, alpha-thrombin failed to stimulate [3H]thymidine incorporation or cell division, although there was a maximal increase of 52% in protein synthesis. The data suggest that there are cellular signal events not activated by alpha-thrombin which are required for proliferation of these aortic VSMC.
Berk
B C
BC
Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.
Taubman
M B
MB
Griendling
K K
KK
Cragoe
E J
EJ
Fenton
J W
JW
Brock
T A
TA
D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction; D024510:Muscle Development; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000254:growth & development / Q000503:physiopathology*; D009693:Nucleic Acid Hybridization; D010743:Phospholipids / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000502:physiology*; D013927:Thrombosis / Q000378:metabolism / Q000503:physiopathology; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; C000715470:MAS1 protein, human; D010743:Phospholipids; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D006859:Hydrogen; D012964:Sodium; D010738:Type C Phospholipases; D013917:Thrombin; D002118:Calcium