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.

These results show that overexpression of HO-1 inhibits arterial contractions induced by hemoglobin and can reduce vasospasm after experimental SAH.

1 Recombinant human ETA receptors were expressed in a mouse fibroblast cell line (Ltk-cell) and functional coupling of the receptors with Ca24 permeable channels at low concentrations of endothelin-l (ET-1) was investigated using whole-cell recordings and monitoring the changes in intracellular free Ca2" concentrations ([Ca24] evoked an initial transient peak and a subsequent sustained elevation in [Ca24]i whereas a lower concentration of ET-1 (10-10 M) evoked only a sustained elevation of [Ca2"i. After removal of extracellular Ca2", ET-l evoked only an initial peak without a sustained elevation of [Ca24]i. The sustained elevation induced by 101 M ET-1 was blocked by 300 pM mefenamic acid (a cation channel blocker) but not by 10 JIM nifedipine (a blocker of voltage-operated Ca24 channel). 3 In whole-cell recordings with Ltk-cells, a brief (3-5 min) application of ET-l (1010 M) induced a sustained inward current at a holding potential of -60 mV. The current-voltage relationship revealed that the reversal potential of the ET-l-induced current was close to 0 mV (1.9 mV) and was not altered by reducing the concentration of Cl-in the bath solution, indicating that the current is carried by cations. The current was reversibly blocked by 300 JiM mefenamic acid, and it persisted after all cations in the bath solution had been replaced by Ca2" (5 or 30 mM) and nonpermeant cation N-methyl-Dglucamine, indicating that the ET-1-activated channel is permeable to Ca24. Activation of the current was independent of membrane potential and the current was induced even after addition of a high concentration (10 mM) of a Ca2" chelator, EGTA, to the pipette solution.4 In whole-cell recordings from rabbit aortic VSMCs, ET-l (101-M) induced a sustained inward current at a holding potential of -60 mV. The reversal potential was -12 mV and was not altered when the concentration of Cl-in the pipette solution was decreased, indicating that the current is carried by cations

1 In single vascular smooth muscle cells (VSMCs) isolated from the aortae of male Wistar rats, we examined the e ects of nitric oxide (NO) donors such as sodium nitroprusside (SNP) and S-nitroso-Nacetyl-DL-penicillamine (SNAP), and 8-bromo-guanosine-3':5'-cyclic monophosphate (8-bromo-cyclic GMP) on endothelin-1 (ET-1)-activated Ca 2+ -permeable nonselective cation channel by use of whole-cell recordings of patch-clamp technique and monitoring of intracellular free Ca 2+ concentration ([Ca 2+ ] i ) with fura-2 real-time digital micro¯uorometry. 2 ET-1 evoked an initial transient peak and a subsequent sustained elevation in [Ca 2+ ] i . After removal of extracellular Ca 2+ , ET-1 evoked only an initial transient peak without a sustained phase. Nifedipine (1 mM), a speci®c blocker of the L-type voltage-operated Ca 2+ channel (VOC), reduced the sustained phase to about 40% of the control level. The remaining part of the sustained phase was abolished by 30 mM SK&F 96365, a blocker of nonselective cation channels. 3 The nifedipine-resistant sustained elevation in [Ca 2+ ] i was abolished by 100 mM SNP, 10 mM SNAP and 300 mM 8-bromo-cyclic GMP. Neither SNP, SNAP nor 8-bromo-cyclic GMP signi®cantly a ected the basal level of [Ca 2+ ] i . 4 In a VSMC clamped at a holding potential of 760 mV with K + in the pipette solution replaced by Cs + , application of 10 78 M ET-1 induced an inward current with an increase in baseline¯uctuation. With¯uctuation analysis, unit conductance of the ET-1-induced current was calculated to be about 21 pS. The ET-1-induced current was linearly related to the membrane potentials with its reversal potential of 75.5 mV. 5 The ET-1-induced current was reversibly and completely inhibited by 30 mM SK&F 96365 or 500 mM Cd 2+ . The current inhibited by SK&F 96365 or Cd 2+ was linearly related to membrane potential with a reversal potential of about 75 mV. 6 The ET-1-induced current was reversibly and completely inhibited by 100 mM SNP, 10 mM SNAP and 300 mM 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about 75 mV. 7 In a bath solution in which all cations were replaced by 30 mM Ca 2+ and 100 mM nonpermeant cation N-methyl-D-glucamine (NMDG), ET-1 evoked a current with a reversal potential of 711 mV, from which P Ca 2+ /P Cs + was calculated to be 2.1. This Ca 2+ current was also abolished by 100 mM SNP, 10 mM SNAP and 300 mM 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about 711 mV. 8 These results taken together indicate that NO through a cyclic GMP signalling pathway inhibits ET-1-activated Ca 2+ -permeable nonselective cation channels, thereby suppressing the sustained increase in [Ca 2+ ] i . Thus, the present study indicates that this Ca 2+ -permeable nonselective cation channel is an important target for nitrovasodilators.

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.