Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts.
Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC(50)) and maximal, saturable binding (B(max)) were estimated from Scatchard analyses and were found to be 24.2 +/- 3.3 pM and 56500 +/- 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of ? 100 nM to a peak level of 600-800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (?250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC(50) of ? 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of (45)Ca from cells loaded to isotopic equilibrium (3 h) with (45)Ca. The intracellular second messenger, IP(3), also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with (45)Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 +/- 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP(3)-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.
Johns
A
A
Berlex Laboratories, Inc., Cedar Knolls, NJ 07927, U.S.A.
Rewers-Felkins
K
K
Devesly
P
P
Rubanyi
G M
GM
Parker-Botelho
L H
LH