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NR Danthuluri and TA Brock
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
Endothelin (ET), a peptide that is released from cultured endothelial cells, is a potent vasoconstrictor that induces characteristically long- lasting contractions. We used the A10 vascular smooth muscle cell (VSMC) line to probe mechanisms underlying ET-induced contractions. Intracellular Ca2+ ([Ca2+]i) and pH were monitored in A10 monolayers using the fluorescent dyes Fura-2 and 2,7-bis-carboxyethyl-5,6- carboxyfluorescein, respectively. Synthetic porcine ET induced rapid and transient increases in [Ca2+]i (EC50 value, 0.75 nM; maximum, approximately 6-fold above basal). External Ca2+ removal did not block the ability of ET (0.5 or 50 nM) to increase initial [Ca2+]i, although [Ca2+]i returned to prestimulus levels faster as compared with that seen in the presence of external Ca2+. Total cell 45Ca2+ content decreased within 30 sec and remained below prestimulus values for at least 20 min (34 +/- 2% decrease after 5 min, n = 3) in ET-stimulated VSMC. ET stimulated a transient rise in inositol trisphosphate formation in [3H]myo-inositol labeled VSMC, peaking in 30 sec (62 +/- 20% increase, n = 3). In contrast, ET-stimulated diacylglycerol formation in [3H]arachidonic acid-labeled VSMC was sustained and biphasic, exhibiting two peaks at 15 sec (41 +/- 16% increase) and at 5 min (75 +/- 7% increase, n = 3). ET (50 nM) also induced an intracellular alkalinization of 0.17 +/- 0.02 (n = 10) pH units above basal.(ABSTRACT TRUNCATED AT 250 WORDS)
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