Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor

Abstract

. Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor. Am J Physiol Cell Physiol 284: C999-C1005, 2003. First published December 11, 2002 10.1152/ajpcell.00158.2002.-Organ culture specifically inhibits vasorelaxation to acute hypoxia and preferentially decreases specific voltage-dependent K ϩ channel expression over other K ϩ and Ca 2ϩ channel subtypes. 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 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-and/or ryanodine-sensitive intracellular Ca 2ϩ 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 Ca 2ϩ handling during hypoxia. ryanodine receptor; vascular smooth muscle; hypoxia A DECREASE IN OXYGEN TENSION (PO 2 ), or hypoxia, modulates coronary, and most systemic, arterial tone, causing a vasodilation to maintain delivery of adequate oxygen to the heart and other organs. The mechanism(s) by which systemic vascular smooth muscle (VSM) senses these changes in PO 2 remains unclear. Common theories include the modulation of ion channel function, an acute decrease in available ATP necessary for force maintenance, direct regulation of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), and modulation of Ca 2ϩ sensitivity (6,8,9,19). We have shown that these theories cannot completely account for the hypoxiainduced relaxation of porcine coronary arteries (20).Additionally, considerable attention has been given to small heme-containing proteins as the oxygen sensor where an PO 2 -dependent change in redox status may transduce the appropriate response (12,14,15).In a recent investigation, we demonstrate the specific inhibition of relaxation to acute hypoxia after organ culture of porcine coronary arteries (25). Organ culture of VSM is reported to cause changes in [Ca 2ϩ ] i handling (4, 7) and has been associated with altered gene expression (21). Chronic hypoxia leads to the regulation of several hypoxia-inducible genes that modulate long-term responses to decreases in PO 2 (1,13,22). It is conceiva...

Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

“…8A) is consistent with findings in other rat arterial smooth muscle preparations, including aorta, superior and inferior mesenteric (59), coronary (70), and main pulmonary arteries (80), but not basilar arteries (65). Our real-time PCR data, however, show that RyR2 is the most abundant transcript in rat cerebral artery myocytes, with RyR3 less highly expressed and RyR1 barely detected (Fig.…”

Stimulation of the adenosine A3 receptor reverses vascular hyporeactivity after hemorrhagic shock in rats

“…Consistent with others' reports, our results showed that RyR-mediated Ca 2+ release is over-activated in hypoxic VSMCs, which might be owing to the increased expression of RyR and the increased sensitivity of RyR to Ca 2+ [25,26] . Furthermore, the over-activation of RyRmediated Ca 2+ release is closely associated with the occurrence of vascular hyporeactivity to NE in hemorrhagic shock rats.…”

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+stores in pulmonary arterial smooth muscle cells

“…However, expression of multiple RyR subtypes is common in many other tissues. In rat aorta, mesenteric arteries, cerebral arteries, portal veins, and porcine coronary arteries, coexpression of all three RyR subtypes has been reported (6,32,35,49). Evidence suggests that RyR1 and RyR2 are required for Ca 2ϩ spark generation in these vascular myocytes, whereas RyR3 participates in global Ca 2ϩ responses induced by agonists and/or in the modulation of Ca 2ϩ spark frequency (6,32,33).…”