MECHANISMS OF VASOCONSTRICTION INDUCED BY ENDOTHELIN-1 IN SMOOTH-MUSCLE OF RABBIT MESENTERIC-ARTERY

1. The mechanism underlying the vasoconstriction induced by endothelin-1 (ET-1) was investigated by measuring the intracellular concentration of Ca2+ ([Ca2+](i)), isometric force and phosphorylation of the myosin light chain (MLC) in endothelium-denuded unskinned and beta-escin-treated skinned smooth muscle from resistance vessels of the rabbit mesentery. The role of protein kinase C (PKC) in the action of ET-1 was studied in skinned smooth muscle using a synthetic peptide, PKC19-36, which corresponds to the autoinhibitory domain of PKC. 2. ET-1 (> 0.1 nM) induced slowly developing, maintained increases in [Ca2+](i) and force. Nicardipine completely blocked the ET-1-induced increase in [Ca2+](i). BQ-123 (an inhibitor of the ET(A) receptor) blocked the ET-1-induced contraction but IRL 1620 (Suc-[Glu(9), Ala(11,15)]-ET-1(8-21), an agonist of the ET(B) receptor) failed to induce contraction. 3. In ionomycin- and 70 mM K+-treated strips, ET-1 shifted the [Ca2+](i)-force relationship to the left and enhanced the maximum amplitude of contraction induced by 2.6 mM Ca2+. In skinned smooth muscle treated with ionomycin, Ca2+ (0.1-3 mu M) increased both force and MLC phosphorylation, in a concentration-dependent manner. ET-1 with GTP shifted both the Ca2+-force and Ca2+-MLC phosphorylation relationships to the left without significant changes in the maximum responses. ET-1 with GTP did not change the relationship between force and MLC phosphorylation. Similar effects were observed with phorbol 12,13-dibutyrate (PDBu, an activator of PKC). These results indicate that the sensitivity of MLC phosphorylation to Ca2+ is enhanced both by ET-1 with GTP and by PDBu. 4. PKC19,36 (an inhibitor of PKC) modified neither the contraction nor MLC phosphorylation induced by 0.3 mu M Ca2+ but blocked the PDBu-induced enhancement of both these Ca2+-induced responses. However, PKC19-36 only partly inhibited the enhancement produced by ET-1 with GTP on the Ca2+-induced responses. PKC19-36 did not modify the relationship between force and MLC phosphorylation in the presence either of ET-1. with GTP or of PDBu. By contrast, BQ-123, neomycin and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) each abolished the ET-1-induced enhancement of the contraction induced by 0.3 mu M Ca2+. 5. These results suggest that ET-1 acts on the ET(A) receptor and increases Ca2+ influxes through an activation of the dihydropyridine-sensitive Ca2+ channel, causing a longlasting and maintained contraction in resistance vessels of the rabbit mesentery. ET-1 increases Ca2+-induced MLC phosphorylation through an activation of both PKC-dependent and -independent mechanisms and thus causes an increase in the sensitivity of contractile proteins to Ca2+.