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Am J Physiol Cell Physiol (December 11, 2002). doi:10.1152/ajpcell.00158.2002
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Articles in PresS, published online ahead of print December 11, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00158.2002
Submitted on April 9, 2002
Accepted on December 5, 2002

Effects of Organ Culture on Arterial Gene Expression and Hypoxic Relaxation: Role of the Ryanodine Receptor

George D Thorne1 and Richard J Paul2*

1 Pharmacolgy/Biochemistry, USAMRICD, APG, MD, USA; Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, USA
2 Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, USA

* To whom correspondence should be addressed. E-mail: Richard.Paul{at}uc.edu.

Organ culture specifically inhibits vasorelaxation to acute hypoxia (25) and preferentially decreases specific voltage-dependent K+ channel expression over other K+ and Ca2+ channel subtypes (24). To isolate further potential oxygen sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and cultured coronary arterial rings. We hypothesize that organ culture results in altered gene expression important for vascular smooth muscle (VSM) contractility important to the mechanism of hypoxia-induced relaxation. Our results indicate a milieu of changes suggesting both up and downregulation of several genes. The altered expression pattern of two positive clones was verified by Northern analysis. Subsequent screening of a porcine cDNA library indicated homology to the ryanodine receptor (RYR). RT-PCR using specific primers to the three subtypes of RYR shows an upregulation of RYR2 and RYR3 after organ culture. Additionally, the caffeine/ryanodine sensitive intracellular Ca2+ store was significantly more responsive to caffeine activation after organ culture. Our data indicate that organ culture increases expression of specific RYR subtypes and inhibits hypoxic vasorelaxation. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RYR might play a novel role in altered intracellular Ca2+ handling during hypoxia.




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