Regulation of beta-adrenoceptor function: A study of the three subtypes

The beta-adrenoceptors are located in a number of different tissues, from vital organs such as the heart (primarily a beta1-adrenoceptor population) and lung (mainly beta2-adrenoceptors) to adipose tissue (largely beta3-adrenoceptors), and are involved in a variety of regulatory processes. They are stimulated by the endogenous agonists adrenaline and noradrenaline, resulting in a range of effects on the body via the sympathetic nervous system; these include smooth muscle relaxation (beta2-adrenoceptors) and alterations in heart rate (beta1- adrenoceptors). The beta3-adrenoceptor effects thermogenesis and lipolysis, thus offering potential opportunities for beta3-agonists to be developed as anti-obesity and anti-diabetic drugs. In this study, my first aim was to investigate agonist-induced regulation of the beta-adrenoceptors, especially at the level of G-protein down-regulation. This included investigations into a number of the desensitization processes, including sequestration, G-protein down-regulation, and G-protein mRNA level changes (Chapter 3). Some areas of receptor desensitization have been researched a great deal, while other areas, such as at the level of the G-protein, have been less thoroughly investigated. In these studies I found that G-protein down- regulation occurred upon agonist occupancy of all three beta-adrenoceptor subtypes (after expression in CHO cells). This was surprising in the case of the beta3-adrenoceptor, as the receptor itself has previously been shown to be refractory to down-regulation (Chambers et d, 1994, Thomas et al, 1992). This, therefore, showed that co-down-regulation of the receptor and associated G-protein, previously thought to occur extensively, is not inextricably linked. There has been a growing awareness in recent years of the vital importance to account for the effect of receptor density in tissues or cells under investigation on the potency and/or efficacy of agonists at receptors. In Chapter 4,1 assessed the effects of receptor density of the beta2-adrenoceptor expressed in NCB20 cells, on the potency and efficacy of agonists such as isoprenaline, salbutamol, ephedrine and adrenaline. It was clear from my data that as receptor levels increased in the three cell lines assessed, the efficacy of the individual agonist increased also. It was also possible to observe that on increasing receptor levels, the EC50 value for each agonist at that cell line decreased, or that the potency was increased. In my final results Chapter, I studied the molecular basis of pharmacological differences previously observed between the rat and human beta3-adrenoceptor. Chimeric constructs of the human beta3-adrenoceptor were produced enabling evaluation of each transmembrane region of the receptor separately. Assays employed were two different functional assays, a cAMP accumulation assay and the microphysiometer, which detected minute pH changes in cells attached to a silicon sensor in response to receptor activation. Through the two assays, I was able to produce a thorough assessment of the role of each transmembrane region in the overall pharmacological response to agonist stimulation. The region which appeared to shift the pharmacology of the receptor from that of the human beta3-adrenoceptor to that of the rat beta3-adrenoceptor was transmembrane region 2. Further investigations employing computer modelling of the beta3- adrenoceptor indicated that this region was not involved in agonist binding, but agonist stimulation must somehow activate TM2 to cause alterations in a number of other transmembrane regions, thus producing an overall conformational change in the receptor