Endothelin-1 activates phospholipase C and mobilizes Ca2+ from extra- and intracellular pools in osteoblastic cells.
The effect of endothelin-1 (ET), a novel vasoactive peptide derived from endothelial cells, on osteoblastic MC3T3-E1 cells was studied. ET specifically binds to a single class of high-affinity receptors in MC3T3-E1 cells and induces phospholipase C activation with the production of two second messengers, inositol trisphosphate and 1,2-diacylglycerol, and a biphasic increase in intracellular free Ca2+ concentration ([Ca2+]i), which consists of an initial transient increase and an ensuing sustained plateau, as measured with a fluorescent indicator, fura-2. The second plateau phase but not the initial transient increase in [Ca2+]i induced by ET is abolished by removal of extracellular Ca2+ but not by either nicardipine, verapamil, or diltiazem. The ET-stimulated production of inositol trisphosphate is not abolished by removal of extracellular Ca2+, indicating that ET-stimulated phospholipase C activation is not a consequence of an increase in Ca2+ influx across the plasma membrane. ET causes stimulation of DNA synthesis and reduction of alkaline phosphatase activity in MC3T3-E1 cells. A protein kinase C activator phorbol 12,13-dibutyrate mimics these effects of ET. The results demonstrate that ET activates the inositol lipid signaling pathway and induces mobilization of Ca2+ from both extra- and intracellular pools and activation of protein kinase C in osteoblastic MC3T3-E1 cells.
Takuwa
Y
Y
Department of Internal Medicine, University of Tsukuba, Ibaraki, Japan.
Ohue
Y
Y
Takuwa
N
N
Yamashita
K
K
D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D003470:Culture Media; D004261:DNA Replication / Q000187:drug effects; D016232:Endothelins; D004730:Endothelium, Vascular / Q000502:physiology; D004789:Enzyme Activation; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism; D017466:Receptors, Endothelin; D010738:Type C Phospholipases / Q000378:metabolism*
D002121:Calcium Channel Blockers; D003470:Culture Media; D016232:Endothelins; D007295:Inositol Phosphates; D010455:Peptides; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D015240:Phorbol 12,13-Dibutyrate; D010738:Type C Phospholipases; D002118:Calcium