Abstract
Background and PurposeCoordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β‐glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers.Experimental ApproachWe investigated the effects of CBX and 18βGA on intercellular Ca2+ waves, evoked by inositol 1,4,5‐trisphosphate (IP3) in the endothelium of intact mesenteric resistance arteries.Key ResultsAcetycholine‐evoked IP3‐mediated Ca2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca2+ release. Localised photolysis of caged IP3 (cIP3) was used to provide precise spatiotemporal control of site of cell activation. Local cIP3 photolysis generated reproducible Ca2+ increases and Ca2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca2+ waves in a time‐dependent manner by inhibiting the initiating IP3‐evoked Ca2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K+‐channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential.Conclusion and ImplicationsCBX and 18βGA inhibit IP3‐mediated Ca2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell–cell communication by acting at sites that are unrelated to gap junctions.
Highlights
Cell–cell communication is a central component of endothelial function that is required for propagated vasodilation, transfer of signals from activated cells and emergent signalling (Bagher & Segal, 2011; Lee et al, 2018; Longden et al, 2017; McCarron et al, 2019; Socha, Domeier, Behringer, & Segal, 2012; Tallini et al, 2007)
CBX and 18β-glycyrrhetinic acid (18βGA) each rapidly and reversibly collapsed the mitochondrial membrane potential. These results suggest that CBX and 18βGA act at sites outwith gap junctions by inhibiting inositol 1 (IP3)-mediated Ca2+ release and depolarising mitochondrial membrane potential (ΔΨM)
To explore the role of gap junctions in the intercellular propagation of Ca2+ waves, we examined the effects of the two widely used putative gap junction blockers, CBX and 18βGA, on ACh-evoked endothelial cell Ca2+ signalling (Figure 2)
Summary
Cell–cell communication is a central component of endothelial function that is required for propagated vasodilation, transfer of signals from activated cells and emergent signalling (Bagher & Segal, 2011; Lee et al, 2018; Longden et al, 2017; McCarron et al, 2019; Socha, Domeier, Behringer, & Segal, 2012; Tallini et al, 2007). Ca2+ increases begin as highly localised subcellular events caused by the opening of a single or multiple IP3 receptors in the internal store (Bagher et al, 2012; Ledoux et al, 2008; Sonkusare et al, 2012; Wilson et al, 2019) These local signals rapidly grow and propagate among cells to transmit information. CBX and 18βGA each blocked Ca2+ waves in a time-dependent manner by inhibiting the initiating IP3-evoked Ca2+ release event rather than block of gap junctions This effect was reversed on drug washout and was unaffected by small or intermediate K+-channel blockers. Conclusion and Implications: CBX and 18βGA inhibit IP3-mediated Ca2+ release and depolarise the mitochondrial membrane potential These results suggest that CBX and 18βGA may block cell–cell communication by acting at sites that are unrelated to gap junctions
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