Smooth Muscle α1‐Adrenergic Receptor Activation Initiates a Negative Feedback Mechanism That Involves Ca2+ Influx Through Endothelial TRPV4 Channels
Authors: Hong Kwangseok , Cope Eric L , Marziano Corina , Sonkusare Swapnil K 
Published: November 2023

Endothelial cell (EC) Ca2+ is a key regulator of endothelial vasodilations. ECs negatively regulate the vasoconstrictions caused by sympathetic nerve stimulation or activation of α1‐adrenergic receptors (α1ARs) on smooth muscle cells (SMCs). Previous studies have indicated that activation of Gq‐protein coupled α1ARs generates inositol trisphosphate (IP3) and Ca2+ in the SMCs, which can be transported to the ECs through myoendothelial gap junctions. This results in an increase in EC Ca2+ thereby generating a dilatory signal that opposes α1AR‐induced SMC contraction. We recently showed that elementary Ca2+ influx through EC TRPV4 (transient receptor potential vanilloid 4) channels, called Ca2+ sparklets, regulates endothelial vasodilations in resistance‐sized arteries (Sonkusare et al., Science, 2012). Although TRPV4 channel activity is potentiated by both IP3 and Ca2+, the role of EC TRPV4 channels in the negative feedback regulation of α1AR‐induced constrictions remains unknown. Therefore, we hypothesized that activation of α1ARs in the SMCs induces TRPV4 Ca2+ sparklets in the ECs, which limits α1AR‐induced vasoconstrictions in resistance‐sized arteries. Using high‐speed spinning disk confocal microscopy, local Ca2+ signals in the ECs were recorded in the en face 3rd order mesenteric arteries (MAs) from the mice expressing genetically encoded Ca2+ biosensor (GCaMP2) or fluo‐4 loaded MAs from C57BL6 mice. The differences in fluorescence kinetics were used to discern TRPV4 Ca2+ sparklets (long, square events) and IP3 receptor (IP3R)‐Ca2+ release events (short spikes) (Sonkusare et al., Science, 2012). Phenylephrine (PE, 10 μM, α1AR agonist) markedly activated both TRPV4 Ca2+ sparklets and IP3R‐Ca2+ release in the ECs. In the presence of cyclopiazonic acid (CPA, 20 μM, an inhibitor of sarco/endoplasmic reticulum Ca2+‐ATPase) used to eliminate IP3R‐Ca2+ release, PE induced a 3‐fold increase in TRPV4 sparklet activity, which was completely abolished by TRPV4 inhibitor GSK2193874 (GSK219, 100 nM). Moreover, phospholipase C inhibitor U73122 (3 μM) and gap junction uncoupling agent 18β‐glycyrrhetinic acid (30 μM) inhibited the effect of PE on TRPV4 sparklets. SMC L‐type Ca2+ channel agonist BayK8644 (1 μM) did not alter EC TRPV4 sparklet activity, ruling out a role for SMC Ca2+ in the activation of EC TRPV4 sparklets. Taken together, these results suggested that SMC‐derived IP3 may be involved in the activation of EC TRPV4 channels by PE. Uncaging IP3 in the presence of CPA induced a 3‐fold increase in TRPV4 sparklet activity, confirming the activation of TRPV4 sparklets by IP3. PE (0.1–10 μM)‐induced constrictions in cannulated and pressurized MAs were significantly higher in the presence of GSK219 and in the MAs from TRPV4−/− mice. These results reveal an important role for EC TRPV4 channels in regulating α1AR‐induced vasoconstrictions and support the novel paradigm that SMC‐derived IP3 can activate EC TRPV4 channels.

Support or Funding Information

This study was supported by NIH grant (R00 HL121484 to S.K.S.).