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Assessment of Endoplasmic Reticulum Stress by Graded Sarcoplasmic Reticulum Ca 2+ Store Depletion in Coronary Smooth Muscle
Author(s) -
Dineen Stacey,
Sturek Michael
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.638.2
Subject(s) - endoplasmic reticulum , caffeine , chemistry , medicine , cytosol , coronary arteries , calcium , endocrinology , artery , biochemistry , enzyme
This study examined the threshold of sarcoplasmic reticulum (SR) Ca 2+ store depletion necessary for induction of endoplasmic reticulum (ER) stress in coronary smooth muscle cells (CSM). Coronary arteries from Ossabaw swine with and without coronary artery disease (CAD) were sectioned into arterial rings and denuded of endothelium for assessment of ER stress. Phosphorylation of eukaryotic initiation factor 2α was examined by immunoblot as an index of ER stress and was elevated in CAD arteries, compared to healthy. CSM were enzymatically isolated and loaded with the fluorescent intracellular Ca 2+ indicator, fura‐2. In arteries with overt CAD, CSM from atherosclerotic plaques were isolated separately from the medial layer. The SR Ca 2+ store was released with caffeine in Ca 2+ ‐free physiologic salt solution (0Ca‐PSS) and the resultant transient rise in cytosolic Ca 2+ was examined as an index of SR Ca 2+ store. The SR Ca 2+ store was increased ~1.5‐fold in CSM from arteries with mild CAD compared to healthy, but was decreased in overt CAD. Sarco/endoplasmic reticulum Ca 2+ ATPase activity as assessed by recovery of cytosolic Ca 2+ below baseline followed the same pattern. Following release with 1, 5, or 10 mM caffeine, the SR Ca 2+ store was clamped in 0Ca‐PSS for 30 minutes, after which the remainder of the SR Ca 2+ was released. This SR Ca 2+ store release was assessed as a percentage of maximum. The SR Ca 2+ store was depleted in a dose‐dependent manner, indicating the feasibility of clamping SR Ca 2+ independent of cytosolic Ca 2+ . Together, these results indicate that CSM display ER stress in CAD and that SR Ca 2+ store is decreased in overt CAD, providing clues to the mechanisms behind CSM changes underlying CAD progression. (Support: NIH HL062552)