Effects of chronic exposure of cultured atrial myocytes to R-N6-(2-phenylisopropyl)-adenosine (R-PIA) on the A1 adenosine receptor-mediated inhibition of adenylate cyclase activity and myocyte contractility were examined. Chronic exposure of atrial myocytes cultured from 14-day-old chick embryos to R-PIA desensitized the myocyte to the inhibitory effects of R-PIA on contractility and adenylate cyclase activity in a time- and dose-dependent manner. Desensitization of the negative inotropic response was only partial, whereas the adenosine receptor-mediated inhibition of adenylate cyclase activity was almost completely absent after 24 hours of R-PIA (1 microM) exposure. Furthermore, the contractile response to R-PIA desensitized more slowly than the desensitization of A1 adenosine receptor-mediated inhibition of adenylate cyclase (t1/2 = 11.4 +/- 0.7 hours versus 7.5 +/- 1 hours, mean +/- SEM, n = 12 and 6, respectively). Thus, the two A1 adenosine receptor-linked functional responses desensitized differently in response to chronic exposure of the myocyte to R-PIA. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine [( 3H]CPX) in membranes from myocytes preexposed to R-PIA demonstrated a time-dependent decrease in receptor density without any change in the affinity for the antagonist radioligand. Computer analyses of agonist competition with [3H]CPX binding in membranes from control and R-PIA-treated myocytes revealed a conversion of the high-affinity A1 adenosine receptor to a low-affinity form such that after 24 hours of 1 microM R-PIA exposure, all of the receptors were in a low-affinity form.(ABSTRACT TRUNCATED AT 250 WORDS)

Aims Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis. Methods and results Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad−/− mice were crossed to an ApoE−/− background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad−/−ApoE−/− mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad−/− mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt. Conclusion The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway.

Background: Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries.Methods: Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), Cu2FL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or en face imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1 µM), and NO donor (SNAP, 10 µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR0.Results: In cell-free chambers using either Cu2FL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both Cu2FL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with Cu2FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR0 following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30 min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP.Conclusion: These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.