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Pulmonary endothelial calcium entry following chronic hypoxia


Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/17441

Pulmonary endothelial calcium entry following chronic hypoxia

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Title: Pulmonary endothelial calcium entry following chronic hypoxia
Author: Menicucci, Steven
Advisor(s): Resta, Thomas
Committee Member(s): Walker, Ben
Jernigan, Nikki
Valenzuela, Fernando
Gonzalez Bosc, Laura
Department: University of New Mexico. Biomedical Sciences Graduate Program
Subject(s): Hypoxia
LC Subject(s): TRP channels.
Membrane proteins
Pulmonary endothelium.
Degree Level: Masters
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.
Graduation Date: December 2011
URI: http://hdl.handle.net/1928/17441

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