Regulation of Endothelin-1 Stimulated-Ca²⁺-Activated K⁺ Channel Activity by Protein Disulfide Isomerase Gene Knockdown in Endothelial Cells

Abstract 1080

Endothelial cell activation is as an important contributor to vaso-occlusion in the pathophysiology of Sickle Cell Disease. We have reported a role for dual endothelin-1 (ET-1) receptor antagonists in improving sickle cell pathophysiology in vivo via modulation of the Ca²⁺-activated K⁺ channel, KCNN4 (Rivera A., 2008, Amer J Physiol). Protein disulfide isomerase (PDI) catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. Thus KCNN4 maybe regulated by PDI in endothelial cells. We now report on the detection of KCNN4, PDI and ET-1 receptor B by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We studied the effects ET-1 on cytosolic Ca²⁺ levels by spectrofluorimetry of FURA-2AM loaded endothelial cells and observed that 10⁻⁹−10⁻⁷ M ET-1 led to dose-dependent increases in cytosolic Ca²⁺ levels that were blocked by pre-incubation with the ET-1 receptor B antagonist, BQ-788. We then studied the effects of 100 nM ET-1 on Ca²⁺-activated K⁺ channels and observed increased activity (153.7 vs. 187.0 mmol/L × h × mg protein) that was sensitive to clotrimazole (95.8 mmol/L × h × mg protein), a well-known inhibitor of KCNN4. We then studied the effects of two well-described PDI inhibitors, monoclonal antibodies to PDI and bacitracin, on channel activity and showed that these inhibitors significantly decreased channel activity in EA cells. Consistent with these observations, we show that siRNA against PDI likewise led to a reduction in channel activity (153.7 vs 29.5 mmol/L × h × mg protein, P<0.01, n=6) that was associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with TaqMan detection probes and GAPDH and beta-2 microglobulin as endogenous controls. We then studied the effects of ET-1 on EA cells as well as in early cultures of mouse aortic endothelial cells (MAEC) using quantitative RT-PCR with TaqMan probes and GAPDH and beta-actin as endogenous controls and observed that stimulation of EA cells with 100 nM ET-1 for 4 hr was associated with increased mRNA expression of PDI levels that was 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Similar results were observed on PDI mRNA expression in MAEC incubated with 100 nM ET-1 for 4 hr. Furthermore, incubation of EA cells for 12 hr with 10 nM ET-1 led to increases in cell-associated PDI levels by western blot analyses. We then tested the in vivo effects of ET receptor antagonist in sickle transgenic BERK mice and observed a reduction of plasma PDI activity when compared to vehicle treatment (67.7±3 to 34.3±6, Relative Fluorescence Units, P<0.03, n=3). In these mice, we also observed a positive correlation in erythrocyte Gardos channel activity and PDI activity. Thus, our results implicate PDI as a novel regulator of KCNN4. We posit that regulation of PDI activity represents a novel target aimed at ameliorating complications associated with the pathophysiology of Sickle Cell Disease. Supported by NIH R01HL090632 to AR.