ENDOTHELIN-1 AND ENDOTHELIN-3 REGULATE DIFFERENTLY VASOCONSTRICTOR RESPONSES OF SMOOTH-MUSCLE OF THE PORCINE CORONARY-ARTERY

1 Using front-surface fluorometry of fura-2 and medial strips of the p orcine coronary artery, we investigated mechanisms by which endothelin -1 (ET-1) and ET-3 function as vasoconstrictors. 2 In the presence of extracellular Ca2+(1.25 mM), ET-1 (10(-10)-10(-7) M) increased cytosol ic Ca2+ concentrations ([Ca2+](i)) and tension, in a concentration-dep endent manner. ET-1, at concentrations greater than 10(-8) M, induced an abrupt elevation of [Ca2+](i) which reached a transient peak (the f irst component, [Ca2+](i)-rising phase) and subsequently declined ([Ca 2+](i)-declining phase) to reach a lower sustained phase (the second c omponent, steady-state phase), while the tension rose monotonically to reach a peak and then slightly and gradually declined. ET-1, at conce ntrations lower than 10(-8) M, induced slowly developing and sustained increases in [Ca2+](i) and tension ([Ca2+](i)-rising phase followed b y steady-state phase). All concentrations of ET-1 increased tension mo re slowly than [Ca2+](i). 3 In the presence of extraceliular Ca2+, ET- 3 (10(-8)-10(-5) M) induced concentration-dependent increases in [Ca2](i) and tension. However, the maximal elevations of [Ca2+](i) and ten sion induced by ET-3 were substantially smaller than those induced by ET-1, indicating the involvement of an ETA receptor subtype. ET-3, at concentrations greater than 6 x 10(-7) M, caused biphasic slowly devel oping increases in [Ca2+](i) and tension. At concentrations lower than 10(-6) M, ET-3 caused monophasic increases in [Ca2+](i) and tension. At all concentrations of ET-3, the time courses of increases in [Ca2+] (i) and tension were similar. 4 The biphasic increases in [Ca2+](i) an d tension induced by 10(-5) M ET-3 and by 10(-7) M ET-1 were significa ntly inhibited by pretreatment with 10(-5) M of the Ca2+ entry blocker , diltiazem, although the inhibition of the first component of ET-1-in duced [Ca2+](i) increase was partial. 5 In the absence of extracellula r Ca2+, ET-1 induced a concentration-dependent transient increase in [ Ca2+](i), possibly due to release of Ca2+ from intracellular stores, a nd a sustained contraction. In contrast, ET-3 (greater than or equal t o 10(-6) M) caused little, if any, transient increase in [Ca2+](i) and a small sustained contraction. 6 Temporal changes in the relationship s between [Ca2+](i) and tension ([Ca2+](i)-tension relationship) durin g contractions induced by ET-1 and ET-3 were compared with the [Ca2+]( i)-tension relationship of Ca2+-induced contractions (Ca2+-contraction s) obtained by cumulative applications of extracellular Ca2+ (0-7.5 mM ) to tissues depolarized in the presence of 118 mM K+. In the [Ca2+](i )-rising phase, ET-1 increased tension more slowly than [Ca2+](i), the reby shifting the [Ca2+](i)-tension relation to the right from that fo r Ca2+-contractions. In the [Ca2+](i)-declining and the steady-state p hases, ET-1, at concentrations higher than 10(-9) M, produced greater tension development than that expected from a given change in [Ca2+](i ), resulting in a leftward shift of the [Ca2+](i)-tension relation. Du ring ET-3-induced contractions, ([Ca2+](i)-rising, [Ca2+](i)-declining and steady-state phases), fhe [Ca2+](i)-tension relation was similar to that of Ca2+-contractions. 7 BQ-123, a selective ETA receptor antag onist, completely inhibited the increases in [Ca2+](i) and tension ind uced by ET-1 and ET-3. 8 These results suggest: (1) That ET-1 elicits vasoconstriction by increasing [Ca2+](i) through the activation of Ca2 + influx from the extracellular space and Ca2+ release from intracellu lar storage sites, and by increasing the Ca2+ sensitivity of the contr actile apparatus, whereas ET-3 induces vasoconstriction by increasing [Ca2+](i) mainly through Ca2+ influx from the extracellular space. (2) Distinct mechanisms of time-dependent modulation of the Ca2+ sensitiv ity function in the vasoconstrictor responses to ET-1 and ET-3. (3) Th at both ET-1- and ET-3-induced contractions seem to be mediated via ET A-receptors in porcine coronary artery, and that the ETA-receptor-medi ated effects of ET-1 and ET-3 can be dissociated at the sub-receptor l evels of the signal transduction pathway.