|dc.description.abstract||Adenosine and adenosine receptors are important cardioprotective mediators. Age-related functional changes have previously been observed but factors that are likely to contribute remain unclear. This thesis examined the effect of age on (1) the mRNA expression of the ADOR and signalling molecules in whole heart and thoracic aorta preparations (chapter 3), and (2) the functional responses of the ADORA1, ADORA2B and ADORA3 in the rat heart (chapters 4-6). Quantitative real time PCR was employed to examine the effect of age on mRNA expression of the adenosine receptors (ADOR) and signalling messengers in hearts and thoracic aorta isolated immature (6-8 weeks), young (16-18 weeks), mature (52-54 weeks) and aged (104-106 weeks) rats, while protein expression of G?s-protein and Ca-L was examined using western blot. Conscious systolic blood pressure (sBP) was also measured in normotensive rats to demonstrate physiological variations that occur with maturation and ageing. Q-PCR analysis showed reduced mRNA expression of the ADORA1 with maturation but a 2.8-fold increase with ageing. In contrast, there was no detectable expression of the ADORA1 in isolated thoracic aorta. The ADORA2A, ADORA2B and ADORA3 were found to be expressed in hearts and thoracic aorta. In hearts they remained unchanged with maturation but were up-regulated (311, 317 and 309-fold, respectively) in aged rats. In thoracic aorta, the ADORA2A remained unaffected by age while the ADORA2B and ADORA3 were up-regulated in aged rats. In addition, up-regulation of the ADOR, NOS, Gi- and Go-proteins correlated with down-regulation of RyR and Ca-L and AC6. G7alpha;s-protein and Ca-L protein expression increased in young hearts, then decreased with maturation and ageing. Conscious systolic blood pressure increased from 98±1mmHg at 6 weeks to 134±5mmHg at 16 weeks (P less than 0.05), decreased to 85±4mmHg until 54 weeks and gradually increased again to 90mmHg by 104 weeks. The results indicate that cellular signalling systems in the rat heart change with maturation and ageing; while changes in the cardiac expression of the ADORA1, Gi2-, Gi3- and Gq-proteins, AC6, and NOS3 potentially play a role in age-related physiological changes in systolic blood pressure. The effect of age on ADORA1 mediated vascular, inotropic and chronotropic functional responses in rat isolated heart was examined in chapter 4. NECA (5`-(N-ethylcarboxamido)adenosine) and R-PIA (R-N6-(1-methyl-2-phenylethyl)adenosine) concentration-response curve experiments were conducted in Langendorff prepared hearts isolated from immature, young and mature male Wistar rats, and the effects of DPCPX (ADORA1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine, 30nM) and PTX pre-treatment (48h, 10µg/kg IP, inhibits Gi/o-protein) were observed. NECA mediated coronary vasodilation and induced a biphasic concentration-response curve in hearts from immature rats (pEC50 8.5 (8.1-8.9) and 11.3 (10.3-12.3)). At the low-sensitivity site, NECA potency increased in young but not mature rats and remained unchanged at the high-sensitivity site. DPCPX blocked NECA at the high sensitivity site in immature rats, producing a monophasic concentration-response curve (pEC50 8.6 (8.5-9.9)), but not in young and mature rats. A monophasic response to NECA was produced in PTX pre-treated immature hearts (pEC50 8.7 (8.3-9.0)) with a vasoconstrictor response at lower agonist concentrations, which disappeared with age and was inhibited by DPCPX. No age-related changes were observed in R-PIA mediated negative inotropic and chronotropic responses (Pless than 0.05). According to the results, ADORA1 causes vasoconstriction of coronary resistance vessels via a PTX-insensitive pathway and induces vasodilation in hearts from immature rats; responses that decline with age. This study also investigated ADOR subtype activity and the role of Gi-protein and NO signalling in NECA mediated responses, and determined the effects of maturation on these responses (chapter 5). NECA concentration-response curves were determined in hearts from each age group. The effect of selective antagonists, including MRS1191 (ADORA3), alloxazine (ADORA2B), pertussis toxin (Gi-protein) and L-NAME (NOS) were determined in each group. NECA produced a biphasic response in hearts from immature rats with pEC50 values of 11.4 (10.4-12.4) and 8.5 (8.1-8.9), respectively, with no age-related changes detected (P up to 0.05). MRS1191 (200nM) decreased the potency of NECA at the high sensitivity site in immature but not young, mature and aged rats (P up to 0.05), while alloxazine (3µM) shifted the low sensitivity phase to the right 8 fold, 83 fold, 35 fold and 12 fold in hearts from each age group (P less than 0.05). Pertussis toxin pre-treatment inhibited the first phase of the concentration-response curve in immature rats, instead a vasoconstrictor response was observed. The vasoconstrictor response was reduced with age and a vasodilator response maintained in young and mature rats. L-NAME (3µM) induced a monophasic concentration-response curve to NECA with a vasoconstrictor response at the lowest doses, while the low sensitivity site remained unchanged. The results show that NECA mediates a heterogenous coronary vascular response. Vasodilator responses at the low sensitivity site are mediated by the ADORA2B and increase with maturation. This site remains unaffected by nitric oxide synthase inhibition therefore is likely to be localized to the vascular smooth muscle. It is also not affected by PTX, indicating no role for Gi-protein signalling, as expected.
The effect of maturation and ageing on ADORA3 mediated coronary responses was investigated using isolated hearts perfused in Langendorff mode in chapter 6. APNEA (ADORA3 up to ADORA1 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 sensitivity site was enhanced by alloxazine (ADORA2B antagonist) and reduced when combined with MRS1191 (ADORA3 antagonist). This indicates that the high sensitivity phase is the ADORA3, and ADORA2B signalling is likely to play a negative regulatory role towards the ADORA3 mediated response. The activity at this site was also reduced with maturation. The low sensitivity site was inhibited by alloxazine but not MRS1191, indicating that this response is mediated by the ADORA2B or another receptor subtype. The response at this site did not alter with age. Cl-IB-MECA, (ADORA3 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 Gi-protein independent and dependent signalling. Q-PCR analysis of rat hearts indicated the presence of an ADORA3 splice variant (ADORA3i), which increased in mRNA expression with age. Cl-IB-MECA responses may be mediated by this ADORA3i. In summary, APNEA mediates coronary vasodilation in the rat heart via at least two receptor sites, the ADORA3 and ADORA2B. ADORA3 responses are reduced while ADORA2B remain unchanged with maturation. In addition, the splice variant ADORA3i may contribute to coronary responses in the rat heart. To summarize, this project investigated the gene expression and functional responses of the ADOR's in the cardiovascular system of the rat. All four ADOR subtypes are expressed in cardiac tissue, while only ADORA2A, ADORA2B and ADORA3 mRNA are expressed in isolated thoracic aorta. Pharmacological studies revealed that cardiac ADORA1 mediated responses do not change with age. In addition, the ADORA1, ADORA2B and ADORA3 mediate coronary vasodilator responses in hearts from immature rats, However, with advancing age, there is a change in the receptor population that mediates the vascular response, which involves the ADORA2B, ADORA3 and another unidentified receptor. Finally, the ADORA1 mediates a vasoconstrictor response, which is lost with age.||en_US