Pregnancy-enhanced store-operated Ca2+ channel function in uterine artery endothelial cells is associated with enhanced agonist-specific transient receptor potential channel 3-inositol 1,4,5-trisphosphate receptor 2 interaction

Abstract

We have previously shown that endothelial cells (EC) derived from the uterine artery (UA) of both pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes show a biphasic intracellular free Ca2+ ([Ca2+]i) response after ATP stimulation. In each case, the initial transient peak, caused by the release of Ca2+ from the intracellular Ca2+ stores, is mediated by purinergic receptor-Y2 and is very similar in both cell types. However, the sustained phase in particular, caused by the influx of extracellular Ca2+, is heightened in the P-UAEC, and associates with an increased ability of the cells to demonstrate enhanced capacitative Ca2+ entry (CCE) via store-operated channels (SOCs). Herein we demonstrated that the difference in the sustained [Ca2+]i response is maintained for at least 30 min. When 2-aminoethoxydiphenyl borate (2APB) (an inhibitor of the inosital 1,4,5-trisphosphate receptor (IP3R) and possibly SOC) was used in conjunction with ATP, it was capable of completely inhibiting CCE. Since 2APB can inhibit SOC in some cell types and 2APB was capable of inhibiting CCE in the UAEC model, the role of SOC in CCE was first evaluated using the classical inhibitor La3+. The ATP-induced sustained phase was inhibited by 10 μM La3+, implying a role for SOC in the [Ca2+]i response. Since canonical transient receptor potential channels (TRPCs) have recently been identified as putative SOCs in many cell types, including EC, the expression levels of several isoforms were evaluated in UAEC. Expression of TRPC3 and TRPC6 channels in particular was detected, but no significant difference in expression level was found between NP- and P-UAEC. Nonetheless, we were able to show that IP3R2 interacts with TRPC3 in UAEC, forming a protein complex, and that this interaction is considerably enhanced in an agonist sensitive manner by pregnancy. Thus, while IP3R and TRPC isoforms are not altered in their expression by pregnancy, enhanced functional interaction of TRPC3 with IP3R2 may underlie pregnancy-enhanced CCE in the UAEC model and so explain the prolonged [Ca2+]i sustained phase seen in response to ATP.