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Age-related changes in adenosine-mediated relaxation of coronary and aortic smooth muscle. Am J Physiol Heart Circ Physiol 280: H2380-H2389, 2001.--We tested whether adenosine mediates nitric oxide (NO)-dependent and NO-independent dilation in coronary and aortic smooth muscle and whether age selectively impairs NO-dependent adenosine relaxation. Responses to adenosine and the relatively nonselective analog 5'-N-ethylcarboxamidoadenosine (NECA) were studied in coronary vessels and aortas from immature (1-2 mo), mature (3-4 mo), and moderately aged (12-18 mo) Wistar and Sprague-Dawley rats. Adenosine and NECA induced biphasic concentration-dependent coronary vasodilation, with data supporting high-sensitivity ([pEC.sub.50] = 5.2-5.8) and low-sensitivity ([pEC.sub.50] = 2.3-2.4) adenosine sites. Although sensitivity to adenosine and NECA was unaltered by age, response magnitude declined significantly. Treatment with 50 [micro]M [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) markedly inhibited the high-sensitivity site, although response magnitude still declined with age. Aortic sensitivity to adenosine declined with age ([pEC.sub.50] = 4.7 [+ or -] 0.2, 3.5 [+ or -] 0.2, and 2.9 [+ or -] 0.1 in immature, mature, and moderately aged aortas, respectively), and the adenosine receptor transduction maximum also decreased (16.1 [+ or -] 0.8, 12.9 [+ or -] 0.7, and 9.6 [+ or -] 0.7 mN/[mm.sup.2] in immature, mature, and moderately aged aortas, respectively). L-NAME decreased aortic sensitivity to adenosine in immature and mature tissues but was ineffective in the moderately aged aorta. Data collectively indicate that 1) adenosine mediates NO-dependent and NO-independent coronary and aortic relaxation, 2) maturation and aging reduce NO-independent and NO-dependent adenosine responses, and 3) the age-related decline in aortic response also involves a reduction in the adenosine receptor transduction maximum. adenosine receptors; aging; coronary vasculature; endothelium; maturation; nitric oxide; rat heart

Hinschen, Andrea K., Roselyn B. Rose'Meyer, and John P. Headrick. Age-related changes in [A.sub.1]-adenosine receptor-mediated bradycardia. Am. J. Physiol. Heart Circ. Physiol. 278: H789-H795, 2000.--The impact of age on functional sensitivity to [A.sub.1]-adenosine receptor activation was studied in Langendorff-perfused hearts from young (1-2 mo) and old (12-18 mo) male Wistar rats. Adenosine mediated bradycardia in young and old hearts, with sensitivity enhanced ~10-fold in old [negative logarithm of [EC.sub.50] (p[EC.sub.50]) = 4.56 [+ or -] 0.11] versus young hearts (p[EC.sub.50] = 3.70 [+ or -] 0.09). Alternatively, the nonmetabolized [A.sub.1] agonists [N.sup.6] -cyclohexyladenosine and (R)-[N.sup.6]-phenylisopropyladenosine were equipotent in young (p[EC.sub.50] = 7.43 [+ or -] 0.12 and 6.61 [+ or -] 0.19, respectively) and old hearts (p[EC.sub.50] = 7.07 [+ or -] 0.10 and 6.80 [+ or -] 0.11, respectively), suggesting a role for uptake and/or catabolism in age-related changes in adenosine sensitivity. In support of this suggestion, [[sup.3]H]-adenosine uptake was approximately twofold greater in young than in old hearts (from 3-100 [micro]M adenosine). However, although inhibition of adenosine deaminase and adenosine transport with 10 [micro]M erythro-9 -(2-hydroxy-3-nonyl)adenine hydrochloride and 10 [micro]M S-(4-nitrobenzyl)-6-thioinosine increased adenosine sensitivity three- to fourfold, it failed to abolish the sensitivity difference in old (p[EC.sub.50] = 4.95 [+ or -] 0.08) versus young (p[EC.sub.50] = 4.29 = [+ or -] 0.13) hearts. Data indicate that 1) age increases functional [A.sub.1] receptor sensitivity to adenosine without altering the sensitivity of the [A.sub.1] receptor itself, and 2) age impairs adenosine transport and/or catabolism, but this does not explain differing functional sensitivity to adenosine. This increased functional sensitivity to adenosine may have physiological significance in the older heart. metabolism; transport

Background: Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A3 receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A3 receptor agonists in coronary blood vessels using the isolated perfused heart preparation. Methods: mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A3 receptor mediated coronary vasodilation in the rat heart. Results: Adenosine A3 receptor agonists induced coronary vasodilation. The expression of adenosine A3 receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A3 receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A3 receptor agonists. Conclusions: This study demonstrated alterations in the expression of adenosine A3 receptors occurred in a tissue specific mode, and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A3 receptor in hypertensive hearts suggest that adenosine A3 receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A3 receptor agonists. [ABSTRACT FROM AUTHOR]

The effect of an allosteric modulator of the adenosine A1 receptors was investigated using an ischaemia-reperfusion protocol in murine isolated hearts. Isolated hearts were perfused with Kreb-Henseleit solution gassed with carbogen gas (95% O2 and 5% CO2) in Langendorff mode and electrically paced at 480 bpm. Following 20 min equilibration and 20 min global normothermic ischaemia, the allosteric modulator VCP333 (1 μM) or the adenosine A1 receptor partial agonist VCP102 (10 μM) were infused after 5 min of reperfusion for 15 min. Upon termination of the drug treatment, reperfusion continued for a further 40 min. At the end of 60 min reperfusion, treatment with VCP333 or VCP102 improved the recovery of the left ventricular developed pressure when compared to control group responses (p < 0.05). Neither compound affected end diastolic pressure, coronary flow rates or dP/dtmax values when compared to control tissues during reperfusion (p > 0.05). The infusion of VCP102 or VCP333 during reperfusion reduced cardiac troponin I efflux to 6.7% and 25% respectively of control heart efflux (p < 0.05). This data indicates that the allosteric modulator of the adenosine A1 receptor (VCP333) has similar characteristics to the adenosine receptor partial agonist VCP102 as it improves cardiac function and reduces myocardial cell death following an ischaemic episode. [ABSTRACT FROM AUTHOR]

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 chronicl-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 chronicl-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 A1 (20-fold), A2A (7.7-fold), A2B (76-fold) and A3 (25.6-fold) mRNA (P < 0.05). NOS expression was variably affected with no change in the expression of NOS1 and 4.2-fold down-regulation of NOS3 expression with chronicl-arginine treatment (P < 0.05). NOS2 was expressed in control tissues; however, inl-arginine-treated hearts the amount of NOS2 mRNA was reduced to non-detectable levels. Following chronicl-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 froml-arginine-treated rats and the NOS inhibitor Nϖ-nitro-l-arginine methyl ester (3 µM) did not inhibit responses to NECA. In conclusion, 4-week dietary supplementation ofl-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. [ABSTRACT FROM AUTHOR]

Abstract: 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-N 6-(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 μg/kg IP, inhibits Gi/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. [Copyright &y& Elsevier]

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/dtmax, dP/dtmin) 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.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 A2B adenosine receptor antagonist alloxazine (10 μM) attenuated pre- and post-ischaemic CFRs in both control and reserpinised hearts (P<0.05) without altering the hyperaemic response while the A2A adenosine receptor antagonist 8-(3-chlorostyryl) caffeine (1 μM) did not alter CFRs in both groups. The A3 adenosine receptor antagonist MRS1191 (0.1 μM) 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 A2B adenosine receptor. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) control essential metabolic processes in virtually every cell in the body and play a vital role in the development of fetal tissues and organ systems. The biological actions of GCs are mediated via glucocorticoid receptors (GRs), the cytoplasmic transcription factors that regulate the transcription of genes involved in placental and fetal growth and development. Several experimental studies have demonstrated that fetal exposure to high maternal GC levels early in gestation is associated with adverse fetal outcomes, including low birthweight, intrauterine growth restriction and anatomical and structural abnormalities that may increase the risk of cardiovascular, metabolic and neuroendocrine disorders in adulthood. The response of the fetus to GCs is dependent on gender, with female fetuses becoming hypersensitive to changes in GC levels whereas male fetuses develop GC resistance in the environment of high maternal GCs. In this paper we review GR function and the physiological and pathological effects of GCs on fetal development. We propose that GC-induced changes in the placental structure and function, including alterations in the expression of GR mRNA and protein levels, may play role in inhibiting in utero fetal growth.

The effect of an allosteric modulator of the adenosine A1 receptors was investigated using an ischaemia-reperfusion protocol in murine isolated hearts. Isolated hearts were perfused with Kreb-Henseleit solution gassed with carbogen gas (95% O2 and 5% CO2) in Langendorff mode and electrically paced at 480 bpm. Following 20 min equilibration and 20 min global normothermic ischaemia, the allosteric modulator VCP333 (1 μM) or the adenosine A1 receptor partial agonist VCP102 (10 μM) were infused after 5 min of reperfusion for 15 min. Upon termination of the drug treatment, reperfusion continued for a further 40 min. At the end of 60 min reperfusion, treatment with VCP333 or VCP102 improved the recovery of the left ventricular developed pressure when compared to control group responses (p < 0.05). Neither compound affected end diastolic pressure, coronary flow rates or dP/dtmax values when compared to control tissues during reperfusion (p > 0.05). The infusion of VCP102 or VCP333 during reperfusion reduced cardiac troponin I efflux to 6.7% and 25% respectively of control heart efflux (p < 0.05). This data indicates that the allosteric modulator of the adenosine A1 receptor (VCP333) has similar characteristics to the adenosine receptor partial agonist VCP102 as it improves cardiac function and reduces myocardial cell death following an ischaemic episode.

1. The effect of the adenosine A(2) receptor (AdoA(2)R) agonist N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) on adenosine A(1) receptor (AdoA(1)R)-mediated negative inotropic responses was investigated in rat heart. 2. Hearts from male Wistar rats (250-350 g) were perfused with Krebs'-Henseleit solution at constant flow in non-recirculating Langendorff mode. Hearts were paced at 5 Hz (5 ms duration, supramaximal voltage) via ventricular electrodes. After 30 min equilibration, (R)-N(6)-phenylisopropyl adenosine (R-PIA) concentration-response curves were constructed in the absence or presence of DPMA. 3. In paced hearts, R-PIA induced concentration-dependent decreases in triple product (heart rate x peak systolic developed pressure x dP / dt(max)), which were significantly attenuated by 1 nmol / L DPMA with a shift in pEC(50) from 8.0 +/- 0.5 (n = 9) in control hearts to 6.63 +/- 1.03 (n = 5) in treated tissues (P < 0.05). The AdoA(2A)R antagonist 8-(3-chlorostyryl)caffeine (1 micromol / L) and the adenylyl cyclase inhibitor cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL12330A; 100 nmol / L) reversed the effects of DPMA on AdoA(1)R-mediated negative inotropic actions, whereas the AdoA(2B)R antagonist alloxazine (3 micromol / L) had no effect on DPMA activity. 4. The results of the present study show that stimulation of the AdoA(2)R attenuates AdoA(1)R-dependent reductions in inotropic state. The receptor involved appears to be the AdoA(2A)R and its action involves stimulation of adenylyl cyclase activity.

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.

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.