Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function

Abstract

Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca²⁺ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca²⁺ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca²⁺ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca²⁺-induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca²⁺ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca²⁺ entry through the ubiquitous store-operated Ca²⁺ entry channel Orai1, global Ca²⁺ entry across the plasma membrane, and Ca²⁺ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca²⁺ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca²⁺ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca²⁺-induced endothelial contraction is a cum hoc fallacy that should be abandoned.

Keywords: G protein-coupled receptor (GPCR); calcium release-activated calcium channel protein 1 (ORAI1); calcium signaling; endothelial cell; endothelial dysfunction; stromal interaction molecule 1 (STIM1).