Regulation of the A3 adenosine receptor gene in vascular smooth muscle cells: role of a cAMP and GATA element.

In previous studies, we reported that the level of expression of the adenylyl cyclase inhibitory A3 adenosine receptor (AR) impacts vascular tone and that rat vascular smooth muscle cells (VSMCs) coexpress the A3 AR and the adenylyl cyclase stimulatory A2a- and A2b-type ARs. In the current study, we investigated the regulation of expression of the A3 AR gene, focusing on sequences conserved in the mouse and human promoters. Transient transfection of primary cultures of rat VSMCs, using the mouse A3 AR promoter, shows that mutation of a conserved cAMP response element (CRE) significantly up-regulates promoter activity in first passage cells, whereas mutation of a conserved GATA site reduces promoter activity. This suggests that an inhibitory protein binds the CRE, whereas an enhancing factor binds the GATA sequence. Electrophoretic mobility shift assays (EMSAs) indicate that the putative CRE and GATA sites indeed bind cAMP response element modulator 1/c-Jun and the GATA6 protein, respectively. A3 AR promoter activity is significantly up-regulated in the presence of forskolin, the nonselective agonist 5'-(N-ethylcarboxamido)adenosine, or the A2a AR agonist 4-[2-[[6-amino-9(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepro- panoic acid (CGS21680), reaching levels similar to those of the A3 AR promoter bearing a mutated CRE. EMSA indicates that in the presence of forskolin the binding to the CRE is inhibited, suggesting that cAMP elevation disturbs the formation of an inhibitory complex on the CRE. Finally, semiquantitative reverse transcription-polymerase chain reaction analysis reveals that endogenous A3 AR mRNA is elevated in response to forskolin. Our findings suggest the presence of a mechanism by which cAMP might control its own level in cells via regulation of genes involved in modulation of adenylyl cyclase activity.

A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice.

Adenosine administration has been reported to lower blood pressure by activating specific membrane receptors. The rat and human heart and aorta have been previously found to express both A2-type adenosine receptors, which activate adenylyl cyclase, and A3 adenosine receptors (A3AR), which inhibit adenylyl cyclase. In the current study, we used A3 adenosine receptor (A3AR) knock-out mice to examine the hypothesis that the relative levels of the A2-type adenosine receptors and A3AR determine the steady-state levels of cAMP in the cells and may affect blood pressure. We found that the A3AR knock-out mice express normal levels of the A1- and A2-type adenosine receptors. In situ hybridization demonstrated that the level of A3AR is high in the vascular smooth muscle layer of aortas derived from wild-type mice, but is not detectable in the knock-out mice. The steady-state level of cAMP is elevated in the aorta and heart of knock-out mice, as compared to wild-type mice, but is not altered in platelets, where A3AR is not expressed naturally. A3AR knock-out mice possess a blood pressure comparable to this in wild-type mice. However, when challenged with adenosine, the knock-out mice display a further increase in cAMP levels in the heart and vascular smooth muscle and a significant decrease in blood pressure, as compared to wild-type mice. In contrast, the effect of adenosine on ADP-induced platelet aggregation is similar in both types of mice. These studies indicate that the A3AR affects the steady-state level of cAMP in the tissues where it is naturally expressed, and that it influences the blood pressure in response to adenosine.

Reduced glutathione prevents nitric oxide-induced apoptosis in vascular smooth muscle cells.

The control of medial and neointimal growth, in which vascular smooth muscle (VSM) plays a central role, is most important to the development of hypertension and atherosclerosis, respectively. Growth of vascular smooth muscle cells is regulated by a number of factors, including the vasodilator nitric oxide (NO). In addition, NO modulates intracellular thiol redox states and the thiol redox state of the cell influences NO production. We, therefore, examined the nature of the effect of NO on growth of VSM cells and its modulation by cellular glutathione content. Here, we report that NO, either generated by NO donors or synthesized by iNOS in VSM cells, inhibited DNA synthesis and induced apoptosis in this cell type. NO-induced apoptosis was associated with a significant decrease in the intracellular concentration of reduced glutathione and with an increase in the level of the tumor suppressor gene p53 mRNA. Moreover, addition of glutathione monoethylester to the culture restored the level of reduced glutathione in VSM cells, and prevented the NO-induced increase in p53 expression and programmed cell death. Our findings suggest a role for reduced glutathione in protecting VSM cells exposed to NO from apoptosis.

An A3-subtype adenosine receptor is highly expressed in rat vascular smooth muscle cells: its role in attenuating adenosine-induced increase in cAMP.

Adenosine analogs are known to induce changes in the steady-state concentration of cAMP via binding to adenylyl cyclase-inhibitory or -stimulatory adenosine receptors. Although adenosine has been found to increase cAMP in vascular smooth muscle cells (VSMC), we found by the polymerase chain reaction of reverse-transcribed RNA and subsequently by Northern blot analysis that rat VSMC express high levels of an A3-subtype adenosine receptor cDNA which encodes an adenylyl cyclase-inhibitory adenosine receptor. The A3-specific agonist, N6-(3-iodobenzyl) adenosine-5'-N-mehylcarboxamide (IB-MECA) indeed decreases cAMP levels in VSMC cultured in the presence of forskolin. Antisense oligomers to the A3 adenosine receptor significantly reduce the level of this receptor in VSMC and potentiate endogenous adenosine- or 5'-N-ethylcarboxamido adenosine-induced increases in cAMP and of the proto-oncogene c-fos. Abrogating the expression of the A3 adenosine receptor also largely abolishes IB-MECA-induced inhibition of adenylyl cyclase. The level of A3 adenosine receptor mRNA and the extent of changes in cAMP in response to IB-MECA were lower in cultures of VSMC derived from adult rats, compared to VSMC from neonatal rats. The expression of a functional A3 adenosine receptor was also confirmed in preparations of isolated aortas. Our findings thus indicate that: (a) the A3-type receptor is a functional inhibitory adenosine receptor in VSMC; and (b) the regulation of expression of the A3 receptor is critical in determining effects of adenosine on the steady-state concentration of cAMP.