The sigma‐1 agonist PRE‐084 protects against energy depletion induced barrier disruption and also enhances glycolysis in HUVECs

Abstract

The sigma‐1 receptor (σ1) is a well‐known endoplasmic reticulum (ER) chaperone that resides at the mitochondria‐associated ER membrane, having an important role in calcium signaling between ER and mitochondria. Expression of σ1 is present in all human tissues with specific predominance in arteries, liver, fibroblasts and brain. Our laboratory has shown that σ1 activation in lymphatic vessels elicits nitric oxide‐dependent lymphatic relaxation, a finding that suggested the potential important role of σ1 in endothelium. Other recent work has highlighted that σ1 may contribute to healthy endothelial barrier function, although little is known about the mechanisms involved. We hypothesized that σ1 helps protect the endothelial barrier disruption by stabilizing cellular bioenergetics. Cultured human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIS) was used to study the contribution of σ1 to endothelial barrier function. Western blotting was used to quantify σ1 protein expression. Endothelial bioenergetics were determined with extracellular flux analysis using glycolytic rate and ATP rate assays. The endothelial‐specific contribution of σ1 was assessed using with the σ1‐selective agonist PRE‐084 and by specific siRNA knockdown of σ1. The mitochondrial un‐coupler Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) was used to model mitochondrial dysfunction in HUVEC. Our results show that activation of σ1 with PRE‐084 50 μM, 100 μM, 150 μM, 200 μM significantly enhances barrier function of HUVEC monolayers in a concentration‐dependent manner. Knockdown of σ1 with siRNA reduces baseline barrier function and diminishes the ability of PRE‐084 to enhance TER. The decrease in TER elicited by CCCP (10 μM) is significantly amplified in cells with siRNA‐mediated knockdown of σ1 expression, and is reversed by PRE‐084 (100 μM) in cells with normal σ1 expression. HUVEC treated with PRE‐084 display higher levels of glycolysis and glycolytic ATP production than control cells. This response to PRE‐084 was not evident in the presence of σ1 knockdown. Collectively, our results suggest that σ1 plays an important role in maintenance of baseline endothelial barrier function, and that activation of σ1 can enhance barrier function. Moreover, σ1 activation also can rescue endothelial barrier dysfunction caused by disruption of mitochondrial ATP production, likely due to its ability enhance glycolysis. Thus, σ1 may represent a future therapeutic target to rescue endothelial barrier dysfunction.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.