Purpose:
Endothelin (Et-1) is an endothelial derived constrictor, stimulating rises in [Ca2+] and contraction in vascular myocytes. We tested the hypothesis that activation of Ca2+-sensitive Cl- currents contributes to Et-1 signaling using high-speed confocal Ca2+-imaging.
Methods:
Adult male Sprague-Dawley rats were anaesthetized with CO2 and killed by cervical dislocation. Retinas were removed and intact arteriole segments isolated. The smooth muscle was loaded with the Ca2+-indicator fluo-4 and normalized fluorescence (F/F0) used as a measure of [Ca2+]. Responses to Et-1 (10nM) were recorded before and during treatment with the Cl- channel blocker anthracene 9-carboxylate (9AC; 10mM). Three representative cells were analyzed for each of 4 repeat experiments.
Results:
Et-1 increased the frequency and amplitude of oscillations (Fig.1). Oscillation frequency rose from 0.17±0.03 s-1 (mean±SEM) under control conditions, to 0.34±0.04 s-1 after 10s in the presence of Et-1 (P<0.01, non-parametric ANOVA, Dunn’s multiple comparison test). Oscillation amplitude increased from 0.12±0.01 to 0.29±0.04 for control and Et-1 treatment, respectively (ΔF/F0, P<0.001). Baseline [Ca2+] was also elevated, rising from 1.01±0.03 during the control period to 1.38±0.12 after 10s of Et-1 superfusion. Addition of 9AC abolished the effects of Et-1 on oscillations (Fig. 1).Oscillation frequency was decreased from 0.34±0.04 s-1 with Et-1, to 0.14±0.04 s-1 when 9AC was also present (P<0.001). Similarly, oscillation amplitude was reduced from 0.29±0.04 in the presence of Et-1 to 0.13±0.02 when 9-AC was added (P<0.001). There were no significant differences in oscillation frequency or amplitude between the control period and the period when both Et-1 and 9AC were present (P>0.05). Baseline [Ca2+], however, remained elevated after 9AC was added, at 1.15±0.05 (P<0.05 v. control; NS v. Et-1 alone).
Conclusions:
Activation of 9AC-sensitive Cl- currents plays an important role in the stimulation of Ca2+-oscillations by Et-1.
Keywords: vascular cells • calcium • blood supply