Roselyn Barbara Rose'Meyer

Cardiovascular adenosine receptors: expression, actions and interactions.

Intra- and extracellular adenosine levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenge and tissue injury. Extracellular adenosine engages members of the G-protein coupled adenosine receptor (AR) family to mediate generally beneficial acute and adaptive responses within all constituent cells of the heart. In this way the four AR sub-types-A1, A2A, A2B, and A3Rs-regulate myocardial contraction, heart rate and conduction, adrenergic control, coronary vascular tone, cardiac and vascular growth, inflammatory-vascular cell interactions, and cellular stress-resistance, injury and death. The AR sub-types exert both distinct and overlapping effects, and may interact in mediating these cardiovascular responses. The roles of the ARs in beneficial modulation of cardiac and vascular function, growth and stress-resistance render them attractive therapeutic targets. However, interactions between ARs and with other receptors, and their ubiquitous distribution throughout the body, can pose a challenge to the implementation of site- and target-specific AR based pharmacotherapy. This review outlines cardiovascular control by adenosine and the AR family in health and disease, including interactions between AR sub-types within the heart and vessels.

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.

Adenosine receptor subtypes mediating coronary vasodilation in rat hearts.

Adenosine receptor-mediated coronary vasodilation was studied in isolated hearts from young (1–2 months) and mature (12–18 months) Wistar rats. The nonselective agonist 5´-N-ethylcarboxamidoadenosine (NECA) induced biphasic concentration-dependant dilation with similar potencies in both age groups (p < 0.05). Despite similar potencies, responses to NECA were significantly depressed by 50% with age. NECA-mediated dilation was unaltered by selective A1 adenosine receptor (A1AR) antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 n M) or A2A adenosine receptor (A2AAR) antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261, 100 n M). However, the A2B adenosine receptor (A2B AR) selective antagonist alloxazine (10 &mgr;M) significantly reduced response magnitude to NECA in both age groups. Concentration–response curves to N6-2-(4-aminophenyl) ethyladenosine (APNEA) induced biphasic concentration-dependent dilation in hearts from young animals. In the presence of the three combined antagonists, 1 &mgr;M DPCPX, 100 n M SCH 58261, and 1 &mgr;M alloxazine, the response magnitude was significantly attenuated (p < 0.05). The addition of the A3 adenosine receptor (A3AR) antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191, 100 n M) to the combined antagonists further attenuated vasodilator responses to APNEA. The results suggest that multiple adenosine receptor subtypes mediate dilation in the rat coronary circulation. NECA mediates vasodilation via the A2BAR subtype, while dilator responses to APNEA in the presence and absence of A1, A2, and A3 AR antagonists provide evidence for a vasodilator role for A3 ARs in rat coronary circulation. The magnitude of the coronary dilator response is reduced with age and does not involve A2A or A1 ARs.

Cardiac adaptation to endurance exercise in rats

AbstractEndurance exercise is widely assumed to improve cardiac function in humans. This project has determined cardiac function following endurance exercise for 6 (n = 30) or 12 (n = 25) weeks in male Wistar rats (8 weeks old). The exercise protocol was 30 min/day at 0.8 km/h for 5 days/week with an endurance test on the 6th day by running at 1.2 km/h until exhaustion. Exercise endurance increased by 318% after 6 weeks and 609% after 12 weeks. Heart weight/kg body weight increased by 10.2% after 6 weeks and 24.1% after 12 weeks. Echocardiography after 12 weeks showed increases in left ventricular internal diameter in diastole (6.39 ± 0.32 to 7.90 ± 0.17 mm), systolic volume (49 ± 7 to 83 ± 11 μl) and cardiac output (75 ± 3 to 107 ± 8 ml/min) but not left wall thickness in diastole (1.74 ± 0.07 to 1.80 ± 0.06 mm). Isolated Langendorff hearts from trained rats displayed decreased left ventricular myocardial stiffness (22 ± 1.1 to 19.1 ± 0.3) and reduced purine efflux during pacing-induced workload increases. 31P-NMR spectroscopy in isolated hearts from trained rats showed decreased PCr and PCr/ATP ratios with increased creatine, AMP and ADP concentrations. Thus, this endurance exercise protocol resulted in physiological hypertrophy while maintaining or improving cardiac function. (Mol Cell Biochem 251: 51–59, 2003)

Cardioprotection induced by adenosine A1 receptor agonists in a cardiac cell ischemia model involves cooperative activation of adenosine A2A and A2B receptors by endogenous adenosine.

Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 μM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10−7M) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA; 10−7M) reduced the proportion of nonviable cells to 30.87 ± 2.49% and 35.18 ± 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 ± 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 ± 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 ± 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.

Adenosine A3 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.

Vascular adenosine receptors; potential clinical applications.

Adenosine is an endogenous purine nucleoside that is an important metabolic sensing molecule. It is released during conditions of low oxygen delivery to tissues and organs to activate a range of effects in vascular tissues. Adenosine has a role in the vasculature by mediating vasodilation, vessel remodelling, cell proliferation as well as antiplatelet and inflammatory responses. Also, adenosine stimulates vasculogenesis and angiogenesis during wound healing and tumour growth. Currently, the clinical uses of adenosine are limited to treatment of supraventricular tachycardia or as a coronary vasodilator during radionuclide myocardial perfusion imaging. Due to the involvement of adenosine in various pathological conditions, the targeting of specific adenosine receptor (ADOR) subtypes in the vasculature using selective ADOR agonists or antagonists could have potential therapeutic benefit. However, the distribution of the receptors differs between species. Therefore, cross-species testing is essential to validate drug function.

Adenosine receptor interactions alter cardiac contractility in rat heart

1. The effect of the adenosine A2 receptor (AdoA2R) agonist N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)‐ethyl]adenosine (DPMA) on adenosine A1 receptor (AdoA1R)‐mediated negative inotropic responses was investigated in rat heart.

Acute But Not Chronic Caffeine Impairs Functional Responses To Ischaemia–Reperfusion In Rat Isolated Perfused Heart

1. The effect of acute (50 μmol/L) and chronic (0.06% in drinking water for 14 days) caffeine on the response to ischaemia–reperfusion was studied in Wistar rat isolated perfused hearts.