Browsing by Author "Alsalhin, Aisha Khlani Hassan"

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    The role of the beta3-adrenergic receptor (β3-AR) in cardioprotection
    (Stellenbosch : Stellenbosch University, 2015-12) Alsalhin, Aisha Khlani Hassan; Salie, Ruduwaan; Lochner, Amanda; Marais, Erna; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: It is well-established that transient activation of the β-adrenergic signalling pathway with ligands such as isoproterenol, formoterol and dobutamine, elicits cardioprotection against subsequent long periods of ischaemia. Initially the focus was on the β1- and β2-adrenergic receptors (β1-AR, β2-AR), but recently the β3-AR also emerged as a potential target in the treatment of heart disease. In heart failure, β1- and β2-AR are typically known to be down-regulated while β3-ARs, on the other hand, are up-regulated (Moniotte et al., 2001). Thus, it has become important to examine the significance of the β3-AR and its downstream signalling under similar states of stress. It has been shown that β3-AR stimulation is resistant to short term agonist-promoted desensitization in vitro and in vivo (Liggett et al., 1993) and after being activated, this receptor is able to convey continual intracellular signals (Lafontan et al., 1994). Thus, it could be an ideal target for therapeutic intervention, also in ischaemic heart disease. We hypothesized that selective β3-AR stimulation during ischaemia / reperfusion may be cardioprotective, whereas selective inhibition of this receptor may prove useful in the end stages of sustained ischaemia and early reperfusion. Methods: The isolated working rat heart, subjected to 35 min of regional ischaemia (RI) and 60 min reperfusion was used as model. The β3-AR agonist (BRL37344) (1 μM) or antagonist (SR59230A) (0.1 μM) were applied as follows: (i) before 35 min RI (PT), (ii) during the last 10 min of RI (PerT) and /or (iii) at the onset of reperfusion (PostT) and (iv) administration of BRL37344 during the last 10 min of RI BRL37344 (PerT) was followed by SR59230A during first 10 min of reperfusion SR59230A (Post). The contribution of nitric oxide synthase (NOS) in β3-AR was assessed, using the non-specific NOS inhibitor, L-NAME (50 μM). Endpoints were functional recovery and infarct size. In another set of experiments BRL37344 and SR59230A were applied according to the same protocols, but the left ventricle was dissected from the heart and freeze clamped at 10 min reperfusion for Western blot analysis of extracellular signal-regulated kinase (ERK p44/p42), protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS). Data were analyzed with one or two-way analysis of variance (ANOVA). Results: Administration of the selective β3-AR agonist (BRL37344) (1μM) before 35 min RI (BRL37344 (PT), significantly reduced infarct size when compared to the non-pretreatment group (NPT) (21.43±2.52 vs 43.17±1.20, p < 0.001). BRL37344 had similar effects on infarct size when applied during the last 10 min of regional ischaemia BRL37344 (PerT) (14.94±2.34, vs NPT, p < 0.001) or at the onset of reperfusion BRL37344 (PostT) (19.06±1.81, vs NPT, p < 0.001). When BRL37344 was applied as a (PerT+PostT) strategy, infarct size was once again significantly reduced (20.55±2.01 vs 43.17±1.20, p <0.001). In contrast, administration of the β3-antagonist SR59230A according to the same protocol did not reduce infarct size and values similar to those of untreated hearts (NPT) were obtained. Surprisingly, when BRL37344 was applied during the last 10 min of regional ischaemia followed by the administration of the β3-AR antagonist (SR59230A) at the onset of reperfusion, [BRL37344 (PerT) & SR59230A (PostT)], infarct size was significantly reduced to 20.78±3.02 (p <0.001 vs NPT and SR59230A (PerT + PostT). Involvement of nitric oxide (NO) was shown since the reduction in infarct size elicited by BRL37344 was totally abolished by, L-NAME, when administered in combination with BRL37344 for 10 minutes prior to RI or at the onset of reperfusion for 10 minutes (% infarct size: 41.48±3.18 and 35.75±3.54, p <0.001 vs BRL37344 (PT) and BRL37344 (PostT), respectively. Western blot results show that PKB/Akt is activated by BRL37344 regardless of the time of administration. The intervention BRL37344 (PerT+PostT), exhibited the most significant phosphorylation of PKB/Akt (fold increase: 14.2±3.71, p<0.01 vs NPT and p<0.05 vs BRL37344 (PostT). In addition, BRL37344 (PT), (PerT), (PostT) and [BRL37344 (PerT) +SR59230A (PostT)] showed significant activation of this kinase (2.92±0.22, 5.54±0.43, 4.73±0.47, and 6.60±0.78, respectively). ERKp44/p42 however, was not significantly activated by any of the treatments. Phosphorylation of eNOS and GSK-3β was significant only in the BRL37344 (PerT+PostT) and [BRL37344 (PerT) + SR59230A (PostT)] groups. The activation of eNOS-S-1177 in the BRL37344 (PerT+PostT) group was (2.82±0.46, p<0.01 and 0.05 vs NPT and BRL37344 (PostT), respectively) and in the [BRL37344 (PerT) + SR59230A (PostT)] group was (2.26±0.48, p<0.05 vs NPT). A very significant increased phosphorylation of GSK-3β was seen in the same two groups (68.8±7.73, p<0.001 vs NPT and 25.5±5.42 vs NPT, p<0.05, respectively). Conclusion: β3-AR has potent cardioprotective effects when administered either before, during and after ischaemia during early reperfusion as indicated by the reduction in infarct size as well as activation of PKB, GSK-3β and eNOS. These beneficial effects can be linked to NO production through activation of eNOS.