| Summary: | Orexin A (ORA) and orexin B (ORB) mediate diverse physiological processes including feeding behaviour, circardian pathway and cortisol secretion. Their effects are mediated through two G protein coupled receptors (GPCRs) termed orexin receptor l(OXIR) and orexin receptor 2 (OX2R). Additionally orexins have been shown to playa role in regulating mean arterial pressure, heart rate and also activate MAPK. Therefore study was conducted to (a) determine direct effects of orexins on cardiac function (heart rate), (b) investigate the effects of orexins on MAPK signalling and steroidogenesis in human adrenocortical (H295R) cells. The results from cardiac study clearly demonstrate the presence of orexin receptors in the atrium and ventricles. Furthermore I was able to show that the recovery time following bradycardia induced by perfusion with carbachol is significantly shorter with hearts treated with ORB, suggesting that orexins may well exert direct effects within the heart itself, possibly through inhibition of G protein activated inward rectifying potassium (GIRK) channels. Given these findings, further studies were conducted to investigate effects of orexins on GIRK channels in heterogeneous CHO cells over expre~sing cardiac GIRK channels. The second part of this project dissects the different MAPK pathways activated in H295R cell-line· upon stimulation by orexins. Both ERK1I2 and p38 were phosphorylated rapidly, with a subsequent decline, in a dose-dependent manner, in response to both orexins, whilst there was little or no direct activation of the ERK5 or JNK pathways. A broad analysis using the over-expression of dominantnegative G-proteins, MAPK inhibitors and receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1I2 and p38 activation were predominantly Gq- and to a lesser extent Gsmediated and p38 activation even had a small Gj-component. Effects were broadly comparable for both orexin peptides. Although most of the effects were transmitted through the OXIR sub-type, OX2R-mediated activation of both ERKl/2 and p38 was also observed. Furthermore, studies were conduced to investigate the effects of orexins on steroidogenesis and the precise signalling mechanism involved showed that lOOnM both ORA and ORB for 4 or 24 hours significantly up-regulate the mRNA of several key genes involved in steroid biosynthesis, including steroidogenic acute regulatory (StAR) protein a key rate-limiting enzyme in steroidogenesis. This study further assessed the dose-dependent and time-dependent characteristics of StAR up-regulation at the protein level, showing significant expression increases after 4 hours at a relatively low agonist concentration (lnM). Moreover, this study provides a broad analysis of the precise G-protein coupled signalling pathways required for the up-regulation of StAR protein in response to ORA and ORB. This study has demonstrated that a clear requirement for multiple G-protein coupling and MAPK-mediated signalling pathways leading to StAR expression, the exact nature of which is dependent on the orexin involved. Antagonist analysis also showed that both orexins effects on StAR were primarily acting through the OXIR, however, some OX2R involvement was also apparent. Finally involvement of T-type Ca2 + channel in orexin induced steroidogenesis was investigated using patch clamp technique. The results reveal that T-type Ca2+ channels are not involved in orexin induced steroidogenesis. Altogether the result obtained from this study suggests multiple roles for orexinmediated MAPK kinase activation and steroidogenesis in an adrenal cell-line and has wide-ranging consequences for our understanding of the mechanisms initiated by these complex steroidogenic molecules.
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