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.

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.

Glycolytic buffering affects cardiac bioenergetic signaling and contractile reserve similar to creatine kinase.

Creatine kinase (CK) and glycolysis represent important energy-buffering processes in the cardiac myocyte. Although the role of compartmentalized CK in energy transfer has been investigated intensely, similar duties for intracellular glycolysis have not been demonstrated. By measuring the response time of mitochondrial oxygen consumption to dynamic workload jumps (tmito) in isolated rabbit hearts, we studied the effect of inhibiting energetic systems (CK and/or glycolysis) on transcytosolic signal transduction that couples cytosolic ATP hydrolysis to activation of oxidative phosphorylation. Tyrode-perfused hearts were exposed to 15 min of the following: 1) 0.4 mM iodoacetamide (IA; n = 6) to block CK (CK activity <3% vs. control), 2) 0.3 mM iodoacetic acid (IAA; n = 5) to inhibit glycolysis (GAPDH activity <3% vs. control), or 3) vehicle (control, n = 7) at 37 degrees C. Pretreatment tmito was similar across groups at 4.3 +/- 0.3 s (means +/- SE). No change in tmito was observed in control hearts; however, in IAA- and IA-treated hearts, tmito decreased by 15 +/- 3% and 40 +/- 5%, respectively (P < 0.05 vs. control), indicating quicker energy supply-demand signaling in the absence of ADP/ATP buffering by CK or glycolysis. The faster response times in IAA and IA groups were independent of the size of the workload jump, and the increase in myocardial oxygen consumption during workload steps was unaffected by CK or glycolysis blockade. Contractile function was compromised by IAA and IA treatment versus control, with contractile reserve (defined as increase in rate-pressure product during a standard heart rate jump) reduced to 80 +/- 8% and 80 +/- 10% of baseline, respectively (P < 0.05 vs. control), and significant elevations in end-diastolic pressure, suggesting raised ADP concentration. These results demonstrate that buffering of phosphate metabolites by glycolysis in the cytosol contributes appreciably to slower mitochondrial activation and may enhance contractile efficiency during increased cardiac workloads. Glycolysis may therefore play a role similar to CK in heart muscle.

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis.

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A(1), A(2A), A(2B) and A(3), and estrogen receptors (ER) alpha and beta. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA(2A) (>4-fold down) and consistent (>2-fold) down-regulation of ADORA(1), ADORA(3), and ER-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.

Enhanced adenosine A(2B) mediated coronary response in reserpinised rat heart.

In this study, we investigated the effect of noradrenaline depletion on contractile recovery in rat isolated heart following myocardial ischaemia. Groups tested included control tissues and hearts from reserpinised rats. Reserpine 1 mg/kg s.c. was injected into rats 18 to 24 h prior to experiments. Hearts underwent 15 min global normothermic ischaemia followed by 30 min reperfusion. Functional data (end diastolic pressure (EDP), heart rate (HR), left ventricular developed pressure (LVDP), dP/dt(max), dP/dt(min)) showed that contractile function following ischaemia-reperfusion is unaffected by reserpinisation. However, pre- and post-ischaemic coronary flow rates (CFR) were increased by 16 to 38% in hearts from reserpinised rats versus control hearts. Pre-ischaemic CFRs in control hearts (11.17+/-0.67 ml/in(-1) x g tissue(-1), n=9) were significantly lower then CFRs derived from reserpinised rat hearts (14.57+/-0.72 ml/min(-1)/g tissue(-1), n=10). Post-ischaemic reactive hyperaemia was evident in all groups. CFRs in reserpinised hearts remained elevated when compared to pre-ischaemic values through reperfusion (P<0.05). Reserpine treatment did not significantly alter pre- or post-ischaemic adenosine efflux. The A(2B) adenosine receptor antagonist alloxazine (10 microM) attenuated pre- and post-ischaemic CFRs in both control and reserpinised hearts (P<0.05) without altering the hyperaemic response while the A(2A) adenosine receptor antagonist 8-(3-chlorostyryl) caffeine (1 microM) did not alter CFRs in both groups. The A(3) adenosine receptor antagonist MRS1191 (0.1 microM) increased CFR in control and reserpinised hearts (P<0.05). Catecholamine depletion with reserpinisation enhances the responsiveness of the coronary resistance vessels to endogenous adenosine through activation of the A(2B) adenosine receptor.

Study of the novel non-xanthine heterocyclic compound GU285 as a potent non-selective adenosine receptor antagonist in the rat.

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.

Effects of A(3) adenosine receptor activation and gene knock-out in ischemic-reperfused mouse heart.

OBJECTIVES: To characterize effects of A(3) adenosine receptor (A(3)AR) activation and gene knock-out on responses to ischemia-reperfusion in mouse heart. METHODS: Perfused hearts from wild-type and A(3)AR gene knock-out (A(3)AR KO) mice were subjected to 20 min ischemia and 30 min reperfusion. Functional responses were assessed and changes in energy metabolism and cytosolic pH monitored via 31P-NMR spectroscopy. RESULTS: Selective A(3)AR agonism with 100 nM 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (chloro-IB-MECA) enhanced post-ischemic contractile recovery without altering contracture development in wild-type hearts, an effect unrelated to non-selective activation of A(1) or A(2) adenosine receptors. Chloro-IB-MECA also improved recovery in hearts overexpressing A(1)ARs. Paradoxically, post-ischemic recovery was enhanced by A(3)AR KO. Developed pressure, +dP/dt, and -dP/dt all recovered to higher levels in A(3)AR KO (70-80% of pre-ischemia) vs. wild-type hearts (45-50% of pre-ischemia) (P<0.05). Enhanced recovery was unrelated to recoveries of ATP, phosphocreatine (PCr), inorganic phosphate (P(i)), energy state ([ATP]/[ADP] x [P(i)], DeltaG(ATP)) or cytosolic pH. CONCLUSIONS: Selective A(3)AR activation is cardioprotective in wild-type hearts and hearts overexpressing A(1)ARs, yet A(3)AR gene deletion generates an ischemia-tolerant phenotype without altering energy metabolism or pH. This may be due to compensatory changes or undefined genotypic differences in A(3)AR KO vs. wild-type hearts.