Chronic dietary L-arginine down-regulates adenosine receptor and nitric oxide synthase expression in rat heart.

L-Arginine increases myocardial nitric oxide production. Nitric oxide mediates many of the cardiovascular actions of adenosine and modulates adenosine metabolism. In this study, we examined the effect of chronic L-arginine (5%) intake on cardiac nitric oxide synthase (NOS) and adenosine receptor expression and cardiac function in rat Langendorff-isolated perfused hearts. Our results show that 4-week chronic l-arginine ingestion increases the weight of rat hearts by 17.6% (P < 0.05). L-Arginine treatment decreased the expression of all the cardiac adenosine receptors, with reductions in adenosine A(1) (20-fold), A(2A) (7.7-fold), A(2B) (76-fold) and A(3) (25.6-fold) mRNA (P < 0.05). NOS expression was variably affected with no change in the expression of NOS(1) and 4.2-fold down-regulation of NOS(3) expression with chronic L-arginine treatment (P < 0.05). NOS(2) was expressed in control tissues; however, in L-arginine-treated hearts the amount of NOS(2) mRNA was reduced to non-detectable levels. Following chronic L-arginine treatment, an increase in coronary perfusion pressure was observed (P < 0.05). Purine efflux was used as an indicator of metabolic efficiency. L-Arginine did not alter catecholamine-induced purine efflux (P > 0.05); however, noradrenaline-mediated increases in contractility and myocardial oxygen consumption were reduced. Vasodilator responses to 5'-N-ethylcarboxamidoadenosine (NECA) were reduced in hearts from l-arginine-treated rats and the NOS inhibitor N omega-nitro-L-arginine methyl ester (3 microM) did not inhibit responses to NECA. In conclusion, 4-week dietary supplementation of L-arginine reduced the expression of cardiac adenosine receptors and NOSs with a subsequent decrease in noradrenaline-stimulated cardiac function and adenosine receptor-mediated coronary vasodilation.

Loss of vascular adenosine A1 receptors with age in the rat heart.

OBJECTIVE: To investigate the effects of age on adenosine A1 receptor (ADORA1) mediated vascular, inotropic and chronotropic functional responses in isolated rat hearts. METHODS: NECA (5'-(N-ethylcarboxamido)adenosine) and R-PIA (R-N6-(1-methyl-2-phenylethyl)adenosine) concentration-response curves were produced in Langendorff prepared hearts isolated from immature (6 weeks), young (16 weeks) and mature (52 weeks) male Wistar rats and the effects of DPCPX (ADORA1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine, 30 nM) and pertussis toxin pre-treatment (PTX, 48 h, 10 microg/kg i.p., inhibits G(i/o)-protein) were observed. RESULTS: NECA mediated coronary vasodilation and induced biphasic concentration-response curves in hearts from immature rats (pEC50 8.5 (8.1-8.9) and 11.3 (10.3-12.3)). At the low sensitivity site, the potency of NECA increased in young but not mature rats and remained unchanged at the high sensitivity site. Both DPCPX and PTX each blocked NECA at the high sensitivity site in immature rats, producing monophasic concentration-response curves (pEC50 8.6 (8.5-9.9) for DPCPX and pEC50 8.7 (8.3-9.0) for PTX), but not in young and mature rats. A vasoconstrictor response was observed at low NECA concentrations in hearts from PTX pre-treated immature rats, but not in hearts from young and mature rats, and the response was inhibited by DPCPX. No age related changes were observed in R-PIA mediated negative inotropic and chronotropic responses (P>0.05). CONCLUSION: ADORA1 mediates a vasodilator response as well as a vasoconstrictor response in the coronary resistance vessels; the latter occurs via a PTX-insensitive pathway and declines with age.

Adenosine A(3) receptor mediated coronary vasodilation in the rat heart: changes that occur with maturation.

Adenosine A(2B) and A(3) receptors (ADOR) have been reported to induce coronary vasodilation in the rat. This study investigated the effect of age on ADORA(3) mediated coronary responses using hearts from rats aged 6-8 weeks (immature), 16-18 weeks (young) and 52-54 weeks (mature) perfused in Langendorff mode. APNEA (ADORA(3)>ADORA(1) agonist) was observed to activate at least two receptor subtypes to mediate a biphasic vasodilator response in hearts from immature rats. The potency of APNEA at the high affinity site was enhanced by alloxazine (ADORA(2B) antagonist) and reduced when combined with MRS1191 (ADORA(3) antagonist). This indicates that the high affinity phase is the ADORA(3), and ADORA(2B) signalling is likely to play a negative regulatory role towards the ADORA(3) mediated response. The activity at this site was also reduced with maturation. The low affinity site was inhibited by alloxazine but not MRS1191, indicating that this response is mediated by the ADORA(2B) or another receptor subtype. The response at this site did not alter with age. Cl-IB-MECA (ADORA(3) agonist) produced monophasic responses that were inhibited by alloxazine but remained unaffected by MRS1191 in all age groups. In addition the potency of Cl-IB-MECA does not change in hearts from PTX-treated rats. However, the maximal responses increased, indicating G(i) protein independent and dependent signalling. Q-PCR analysis of rat hearts indicated the presence of an ADORA(3) splice variant (ADORA(3i)), which increased in mRNA expression with age. Cl-IB-MECA responses may be mediated by this ADORA(3i). In conclusion, APNEA mediates coronary vasodilation in the rat heart via at least two receptor sites, the ADORA(3) and ADORA(2B). ADORA(3) responses are reduced while ADORA(2B) remain unchanged with maturation. In addition, the splice variant ADORA(3i) may contribute to coronary responses in the rat heart.

Age-related changes in cardiac adenosine receptor expression.

Adenosine is an important cardioprotective agent that works via several adenosine receptor (ADOR) subtypes to regulate cardiovascular activity. It is well established that functional responses to adenosine decline with age. What is unclear, though, is whether these changes occur at the receptor, second messenger or translational level. In this study we determined the effect of age on cardiac adenosine receptor expression using the housekeeping gene 18S rRNA versus the adenosine A(2B) receptor gene as internal controls. Absolute quantification showed that no age-related changes occurred in the expression of 18S rRNA or adenosine A(2B) receptor internal control genes. Subsequently, relative analysis of the adenosine receptor subtypes using 18S rRNA found a significant age-related reduction in the expression of the adenosine A(1) receptor (5.5-fold), with no changes in the expression of the adenosine A(2A), A(2B) and A(3) receptors. When using the expression of the adenosine A(2B) receptor as the internal control gene, a significant down regulation of both the adenosine A(1) (5.4-fold) and A(2A) (2.2-fold) receptors with no change in the expression of adenosine A(3) receptor was found. Therefore, the high level of expression of the 18S rRNA housekeeping gene was found to mask a significant change in expression of the adenosine A(2A) receptor with age. Ultimately, these findings show an age-related reduction in adenosine A(1) and A(2A) receptor expression in rat heart.