Endothelin 1 stimulates Ca2+-sparks and oscillations in retinal arteriolar myocytes via IP3R and RyR-dependent Ca2+ release

Abstract

Purpose: To investigate endothelin 1 (Et1)-dependent Ca(2+)-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.

Methods: Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.

Results: Basal [Ca(2+)](i), subcellular Ca(2+)-sparks, and cellular Ca(2+)-oscillations were all increased during exposure to Et1 (10 nM). Ca(2+)-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (Et(A)R) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca(2+)-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 μM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), xestospongin C (10 μM), 2-aminoethoxydiphenyl borate (100 μM), and tetracaine (100 μM), a blocker of ryanodine receptors (RyRs).

Conclusions: Et1 stimulates Ca(2+)-sparks and oscillations through Et(A)Rs. The underlying mechanism involves the activation of phospholipase C and both IP(3)Rs and RyRs, suggesting crosstalk between these Ca(2+)-release channels. These findings suggest that phasic Ca(2+)-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca(2+)-sparks in these vessels.

Figure 1.

Et1 effects on Ca²⁺-oscillations. (A) Confocal image of retinal arteriolar myocytes loaded with Fluo-4 (left). Adjacent myocytes were scanned along the marked line to generate a line scan image (right). (B) Time series data for three cells in (A) (c2, c4, c5) showing Et1-induced Ca²⁺-oscillations. (C) Summary data (mean ± SEM) for oscillation frequency and amplitude in the presence of Et1 at 1 nM (314 cells, 18 arterioles, 5 animals) or 10 nM (57 cells, 8 arterioles, 4 animals). Results have been normalized using oscillation data recorded under control conditions (362 cells in 26 arterioles from 9 animals).

Figure 2.

Effects of Et1 on Ca²⁺-sparks. (A) 2D scan (upper) and high-speed line scan image showing a single Ca²⁺-spark (lower and graph). (B) Line scan images (upper) and time-series data (lower) during control and Et1 treatments. Asterisks: sparks sites at x. (C) Spark frequency summary data (59 spark sites, 7 arterioles, 4 animals).

Figure 3.

Effects of EtR antagonists on Et1 actions. Summary data show the effects of Et1 (10 nM) on Ca²⁺-oscillations in the presence of (A) BQ123 (100 nM; 61 cells, 7 arterioles, 3 animals) and (B) BQ788 (100 nM; 200 cells, 18 arterioles, 10 animals). All data have been normalized using control data recorded before the addition of the receptor antagonists.

Figure 4.

Effects of inhibitors of PLC inhibition and SR Ca2⁺-release on Et1-induced Ca²⁺-oscillations. Summary data showing reductions in (A) oscillation frequency and (B) oscillation amplitude when inhibitors were added to arteriole segments prestimulated with Et1 (10 nM). The drugs used were U73122 (10 μM; 89 cells, 18 arterioles, 7 animals), xestospongin C (10 μM; 26 cells, 4 arterioles, 3 animals), 2APB (100 μM; 26 cells, 4 arterioles, 2 animals), and tetracaine (100 μM, 30 cells, 5 arterioles, 3 animals). Data have been normalized to observations recorded in Et1 before the addition of the relevant inhibitor. P values indicate statistically significant reductions from recordings made in the presence of Et1 alone.