Distinguishing receptor‐ versus store‐operated calcium entry in arteriolar endothelium

Abstract

The rise in [Ca+2]i for native endothelial cells responding to acetylcholine (ACh) consists of an initial rapid peak (internal release) succeeded by a sustained plateau (influx). Whereas the plateau reflects receptor‐activated Ca+2 entry (RACE) plus store‐operated Ca+2 entry (SOCE), respective contributions are not well defined in the microcirculation. The goal of this study was to distinguish the contributions of RACE and SOCE in arteriolar endothelium. Abdominal muscle feed arterioles were dissected from anesthetized C57BL/6 mice (n=6). Intact endothelial tubes were isolated enzymatically with gentle trituration, loaded with Fura‐2 dye and superfused (physiological saline, PSS; 2.5 ml/min; pH 7.4, 32 °C). With zero [Ca+2] in PSS, cells were emptied of Ca+2 using ACh (1 μM) or cyclopiazonic acid (CPA; 5 μM). Total Ca+2 influx (RACE + SOCE) was recorded by adding back 2 mM Ca+2 PSS with ACh; SOCE was distinguished by adding back Ca+2 with CPA. Peak Fura‐2 ratios were greater with ACh (2.73±0.18) vs. CPA (1.92±0.04, p<0.05; n=3). Integrated responses (arbitrary ratio units) were greater when restoring Ca+2 with ACh (240±27) than with CPA (89±9, p<0.05; n=3). By subtraction, SOCE accounted for 37% while RACE accounted for 63% of total Ca+2 influx. We suggest that ACh‐stimulated Ca+2 influx in mouse arteriolar endothelium occurs predominantly via receptor‐activated signaling events. (NIH HL41026 & T32‐AR048523)