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1 We have recently shown that endothelin-1 (ET-1) activates two types of Ca 2+ -permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca 2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca 2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca 2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) of single smooth muscle cells using these blockers. 2 LOE 908 or a blocker of voltage-operated Ca 2+ channel nifedipine had no e ect on the contractions and increases in [Ca 2+ ] i induced by thapsigargin or ionomycin, whereas SK&F 96365 abolished them. 3 The contractions and increases in [Ca 2+ ] i induced by ET-1 depended on extracellular Ca 2+ but were resistant to nifedipine. The responses to lower concentrations (40.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (51 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. 4 SK&F 96365 inhibited the LOE 908-resistant contractions induced by higher concentrations of ET-1 with IC 50 values similar to those for contractions induced by thapsigargin or ionomycin. 5 These results show that the contractions and increases in [Ca 2+ ] i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca 2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca 2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.

We previously showed a role for a nonselective cation channel (NSCC) in the ETA-dependent action of endothelin-1 in mouse fibroblast and rabbit aortic smooth-muscle cell. To clarify the physiological significance of NSCCs in endothelin-1 (ET-1)-induced vasocontraction, we examined the effects of NSCC blockers such as mefenamic acid and SK&F 96365 on the contractions of deendothelialized rabbit aortic rings induced by a low (10[-10] M) or high (10[-8] M) concentration of ET-1. Mefenamic acid (< or =10[-3] M) had little effect on the contraction induced by 45 x 10(-3) M K+ or 1 x 10(-6) M Bay K-8644 in combination with 15 x 10(-3) M K+, indicating that it does not affect voltage-operated calcium channels (VOCs) and contractile mechanisms. The contraction by a low concentration of ET-1 was abolished after removal of extracellular Ca2+, but it was reduced only to 50% by a maximally effective concentration (10[-5] M) of nifedipine, an inhibitor of L-type VOCs (L-VOC). Mefenamic acid and SK&F 96365 inhibited the ET-1-induced contraction with 50% inhibitory concentration (IC50) values of 10(-4) M and 2 x 10(-5) M, respectively, and abolished it at 10(-3) M and 10(-4) M. By contrast, nifedipine, mefenamic acid, or SK&F 96365 had little effect on the contraction by a high concentration of ET-1. The contraction induced by a low or high concentration of ET-1 was abolished by an ETA antagonist, BQ-123, but not by an ETB antagonist, BQ-788. These results demonstrate that the contraction induced by ET-1 is totally mediated exclusively by ETA, but that Ca2+ entry through NSCCs in addition to L-VOCs plays an important role in contractions induced by low concentrations of ET-1, whereas it plays only a minor role in contractions induced by high concentrations of ET-1.

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.

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