Positive and negative coupling of the endothelin ET_A receptor to Ca²⁺‐permeable channels in rabbit cerebral cortex arterioles

Abstract

1. Arteriolar segments were isolated from pial membrane and studied within 10 h. Current-clamp and voltage-clamp measurements were made by patch-clamp recording from smooth muscle cells within arterioles. [Ca2+]i was measured from the smooth muscle cell layer by digital imaging of emission from fura-PE3 which was loaded into arterioles by pre-incubation with the acetoxymethyl ester derivative. The external diameter of arterioles was measured using a video-dimension analyser. 2. Endothelin-1 (ET1) was a potent constrictor of isolated arterioles and induced a sustained depolarization up to -27 mV and reduced membrane resistance (EC50 140-170 pm). At a constant holding potential of -60 mV ET-1 induced a transient followed by a sustained inward current. ET1 inhibited L-type voltage-dependent Ca2+ current. 3. ET1 induced a transient followed by sustained elevation of [Ca2+]i. The sustained effect was dependent on extracellular Ca2+. It occurred at a constant holding potential of -60 mV and was not inhibited by the Ca2+ antagonists nicardipine (1 microM) or D600 (10 microM). Thapsigargin (1 microM) completely depleted Ca2+ from caffeine- and ET1-sensitive sarcoplasmic reticulum but did not inhibit the ET1-induced sustained elevation of [Ca2+]i. ET1 effects on [Ca2+]i were prevented by the ETA receptor antagonist BQ123 (cyclo-D-Asp-Pro-D-Val-Leu-D-Trp). 4. The data suggest that ETA receptors are negatively coupled to L-type Ca2+ channels and positively coupled to receptor-operated Ca2+-permeable channels. Inhibition of L-type Ca2+ channel activity may suppress autoregulation, and Ca2+ influx through receptor-operated channels may have a major functional role in the potent long-lasting constrictor effect of endothelin-1 in the cerebral microcirculation.