Biomedical Sciences ETDs

Publication Date

12-1-2011

Abstract

Background and Specific Aims: Chronic hypoxia (CH) induced pulmonary hypertension is mediated in part by endothelial dysfunction, involving reduced intracellular Ca2+ levels and decreased production of endothelium-derived vasodilators and anti-mitogenic compounds. Agonist-induced endothelial Ca2+ entry is decreased following CH due to a derangement in T-type voltage-gated calcium channels (T-channels) and caveolin-1 containing membrane lipid domains that regulate calcium influx in these cells. Considering the importance of store-operated Ca2+ entry (SOCE) to agonist-induced Ca2+ entry, we hypothesized that CH impairs pulmonary endothelial SOCE through altered caveolin-1 regulation of T-channels. To test this hypothesis, we addressed the following specific aims: Specific Aim 1: Determine the role of T-channels in agonist-induced calcium entry and SOCE in pulmonary arterial endothelial cells (PAEC) from control and CH rats. Specific Aim 2: Determine the contribution of caveolin-1 to SOCE in PAEC from control and CH rats. Experimental Approach: Fura-2 fluorescence microscopy was used to assess either ATP-induced Ca2+ influx or SOCE resulting from depletion of intracellular Ca2+ stores in freshly isolated PAEC from control and CH (4 wk at 0.5 atm) rats. Experiments were conducted in the presence or absence of the T-channel inhibitor mibefradil. In separate protocols, SOCE was assessed following pretreatment with either a peptide containing the scaffolding domain of caveolin-1 (AP-CAV) or a scrambled control peptide. Immunofluorescence microscopy was used to evaluate the distribution of endothelial T-channels and caveolin-1, while the density of caveolae was determined from electron micrographs of PAEC from each group. Results: Both ATP-induced Ca2+ influx and SOCE were attenuated in PAEC from CH compared to control rats. Although T-channel inhibition selectively attenuated Ca2+ responses to ATP in cells from control rats and normalized responses between groups, mibefradil was without effect on SOCE in control cells. Furthermore, AP-CAV augmented SOCE in cells from CH rats while having no effect in controls. We observed similar immunofluorescent staining for T-channels and caveolin-1 between groups, and the incidence of endothelial caveolae was unchanged by CH. Conclusions: CH impairs caveolin-1 regulation of SOCE in PAEC without affecting intracellular caveolin-1 distribution or caveolar density. However, decreased SOCE following CH appears to be independent of Ca2+ influx through T-channels.

Keywords

Hypoxia, Calcium, Arterial, Vascular, Pulmonary, Endothelial

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Masters

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Walker, Ben

Second Committee Member

Jernigan, Nikki

Third Committee Member

Valenzuela, Fernando

Fourth Committee Member

Gonzalez Bosc, Laura

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