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The etiology of the atherosclerosis that occurs in diabetes mellitus is unclear. Adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM D-glucose, which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT-1) and ENT-2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT-1 and inhibited by nitrobenzylmercaptopurine riboside (NBMPR, IC50 = 0.69 ± 0.05 nM). Adenosine transport in HASMCs was increased by >30% after treatment for 48 h with 25 mM D-glucose, but not with equimolar D-mannitol and L-glucose. Kinetic studies showed that D-glucose increased Vmax of adenosine transport without affecting Kin. Similarly, D-glucose increased Bmax of high-affinity [³H]NBMPR binding, while the dissociation constant (Kδ) was not changed. Consistent with these observations, 25 mM D-glucose increased mRNA and protein expression of ENT-1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD-98059 (40 μM) and U-0126 (10 μM), abolished the effect of D-glucose on ENT-1. We conclude that D-glucose upregulates the protein and message expression and functional activity of ENT-1 in HASMCs, possibly via MAPK/ERK-dependent pathways. Pathologically, the increase in ENT-1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and, thus, alter vascular functions in diabetes. [ABSTRACT FROM AUTHOR]

Although acute adenosine preconditioning (PC) is well established, the signaling pathways mediating this cardioprotection remain unclear. Because adenosine receptor agonists activate p38 MAPK and this kinase has been implicated in ischemic and pharmacological PC, the purpose of this study was to determine the role of p38 MAPK in acute adenosine receptor PC. The role of p38 MAPK activation in discrete subcellular compartments during ischemia-reperfusion was also determined. The following groups were used in an in vivo rat ischemia-reperfusion model: 1) control (10% DMSO iv), 2) the A1/A2a adenosine receptor AMP-579 (50 μg/kg iv), 3) AMP-579 + the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 μg/kg iv), 4) AMP-579 + the p38 MAPK inhibitor SB-203580 (1 mg/kg iv), and 5) SB-203580 alone. p38 MAPK activation was measured by Western blot analysis in cytosolic, mitochondrial, membrane, and nuclear/myofilament fractions obtained from hearts at preischemic, ischemic, and reperfusion time points. A significant reduction in infarct size was observed with AMP-579 PC, an effect blocked by DPCPX or SB-203580 pretreatment. AMP-579 treatment was associated with a significant increase in p38 MAPK activation in the nuclear/myofilament fraction before ischemia, whereas no activation of this kinase occurred during ischemia or reperfusion. In contrast, p38 MAPK was activated in the mitochondrial fraction by ischemia and in the cytosolic, mitochondrial, and membrane fractions by reperfusion in the control group. SB-203580 blocked the AMP-579-induced increase in phosphorylation of the downstream p38 substrate activating transcription factor-2. These results suggest a role for p38 MAPK activation in discrete subcellular compartments in acute adenosine A1 receptor PC. [ABSTRACT FROM AUTHOR]