Abstract
Purposes: Since the role of store-operated calcium entry (SOCE) in endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation of mesenteric arteries in health and colitis is not fully understood, cyclopiazonic acid (CPA), a specific inhibitor of the sarco(endo) plasmic reticulum calcium-ATPases (SERCA), was used as a SOCE activator to investigate its role in normal mice and its alteration in colitis mice.Methods: The changes in Ca2+ signaling in vascular endothelial cells (VEC) were examined by single cell Ca2+ imaging and tension of mesenteric arteries in response to CPA were examined using Danish DMT520A microvascular measuring system.Results: CPA activated the SOCE through depletion of the endoplasmic reticulum (ER) Ca2+ in endothelial cells. CPA had a concentration-dependent vasorelaxing effect in endothelium-intact mesenteric arteries, which was lost after endothelial removal. Both nitric oxide (NO) and prostacyclin (PGI2) inhibitors did not affect CPA-induced vasorelaxation; however, after both NO and PGI2 were inhibited, KCa channel blocker [10 mM tetraethylammonium chloride (TEA)] inhibited CPA-induced vasorelaxation while KCa channel activator (0.3 μM SKA-31) promoted it. Two SOCE blockers [30 μM SKF96365 and 100 μM flufenamic acid (FFA)], and an Orai channel blocker (30 μM GSK-7975A) inhibited this vasorelaxation. The inhibition of both Na+/K+-ATPase (NKA) and Na+/Ca2+-exchange (NCX) also inhibited CPA-induced vasorelaxation. Finally, the CPA involved in EDH-induced vasorelaxation by the depletion of ER Ca2+ of mesenteric arteries was impaired in colitis mice.Conclusion: Depletion of ER Ca2+ by CPA induces a vasorelaxation of mesenteric arteries that is mediated through EDH mechanism and invokes the activation of SOCE. The CPA-induced endothelium-dependent dilation is impaired in colitis which may limit blood perfusion to the intestinal mucosa.
Highlights
Ca2+ as an important second messenger plays a critical role in the regulation of cell function and participates in various human physiological processes
We examined the CPA-induced store-operated calcium entry (SOCE) in Human umbilical vein endothelial cells (HUVECs)
After basal [Ca2+]i was stable in normal PSS containing 2 mM extracellular Ca2+, application of CPA (10 μM) induced a marked increase in [Ca2+]i in HUVEC (Figure 1A)
Summary
Ca2+ as an important second messenger plays a critical role in the regulation of cell function and participates in various human physiological processes. The rapid influx of extracellular Ca2+ increases the concentration of [Ca2+]i, which is an important signal that triggers many physiological activities in the cells (Garland et al, 2017). Putney first proposed the concept of the store-operated calcium entry (SOCE), a physiological phenomenon that the depletion of Ca2+ store in the endoplasmic reticulum (ER) activates the influx of extracellular Ca2+ (Putney, 1990). The molecular mechanism of the SOCE is comprised of the STIM protein of the endoplasmic reticulum membrane and the Orai protein family of the cell membrane. The STIM protein senses a decrease in the ER Ca2+, and activates the Orai protein located on the cell membrane through protein-protein interactions, thereby causing the influx of extracellular Ca2+ (Liou et al, 2005; Zhang et al, 2006). The SOCE can be activated by GPCR/PLC/IP3-mediated ER Ca2+ release (Taylor, 2006)
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