No abstract

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.

The coupling between the dopamine1 (DA1) receptor and the G protein/adenylyl cyclase (AC) enzyme complex is defective in the proximal convoluted tubule (PCT) of 20-wk-old spontaneously hypertensive rats (SHRs). Because this coupling defect could have been due to desensitization secondary to elevated renal dopamine levels in the adult animal, we studied the interaction between DA1 receptors and AC in PCT of rats as early as 3 wk of age, a time when renal dopamine levels are similar in SHRs and their normotensive controls (Wistar-Kyoto rats, WKYs). Maximum receptor density did not change with age and was similar in WKYs and SHRs in all the age groups studied (3, 8, and 20 wk). Basal-, forskolin-, and guanyl nucleotide-stimulated AC activities were also similar in WKYs and SHRs and did not change with age. However, the DA1 agonist-stimulated AC activity was greater in WKYs than in SHRs and increased with age in WKYs but not in SHRs. Moreover, the ability of a nonhydrolyzable analogue of GTP, Gpp(NH)p, to enhance DA1 agonist (SND-919-C12, 1 microM)-stimulated AC activity increased with age in WKY but not in SHRs. To determine if the defect noted in the PCT of SHRs is due to a defective D1A receptor gene, parallel studies were performed in the striatum, since this receptor is expressed predominantly in the latter tissue. In contrast to the results in PCT, radioligand binding and AC studies in striatum revealed no differences between WKYs and SHRs.(ABSTRACT TRUNCATED AT 250 WORDS)

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 A7r5 cells loaded with the Ca 2+ indicator fura-2, we examined the e ect of a Ca 2+ channel blocker SK&F 96365 on increases in intracellular free Ca 2+ concentrations ([Ca 2+ ]i) and Mn 2+ quenching of fura-2¯uorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed.