Characterisation of [Beta]-adrenoceptor subtypes in rat skeletal muscle: A study of the binding and functional properties of [beta]2 and putative [Beta] 3-adrenoceptors
thesisposted on 06.12.2017, 00:00 by Nicole Moore
Project aims to characterise the [Beta]-adrenoceptors of rat skeletal muscle.. The aim of the present study was to characterise the ß-adrenoceptor sUbtypes present in skeletal muscle and to determine the relative importance of each subtype in the control of protein accretion. The radioligand [¹²⁵I]iodocyanopindolol (I¹²⁵I]ICYP) labelled several binding sites in skeletal muscle. A low-density, high-affinity receptor population had binding characteristics of the ß2-adrenoceptor, including classical order of agonist affinity, high affinity for ICI118551, and stereoselectivity for isoprenaline and propranolol. I-Isoprenaline induced high-affinity, guanine nucleotide-sensitive binding to ß2-adrenoceptors resulting in the appearance of a second site. Unexpectedly, the high-affinity of d-[¹²⁵I]ICYP for binding to ß2-adrenoceptors caused an enantiomeric artefact suggestive of two high-affinity binding sites when d,l-[¹²⁵I]ICYP was used. This interference was avoided by using l-[¹²⁵I]ICYp exclusively in later studies. Finally, a low-affinity [¹²⁵I]ICYP binding site which was propranolol-resistant was also observed. This site was characterised in soleus muscle, revealing its high-density. The affinities of ß-adrenoceptor antagonists for the high-density site compared well with those determined at ß3-adrenoceptors (Emorine et al., 1989; Muzzin et al., 1991; Nahmias et al., 1991). The affinity of these ligands was not directly related to their lipophilicity. The putative ß3-adrenoceptor showed poor stereoselectivity for alprenolol and pindolol, but was highly stereoselective for cyanopindolol, favouring binding to the d-isomer. ß-Adrenoceptor agonists had unexpectedly low-affinity for this site. The nonselective agonist l-isoprenaline, stimulated adenylyl cyclase activity in rat BAT membranes at two sites, with different potency. Competitive inhibition studies using selective antagonists identified the two sites as ß1- and ß3-adrenoceptors. In contrast to isoprenaline, the ß3- adrenoceptor-selective agonist BRL28410 activated a single component of adenylyl cyclase. In soleus muscle membranes, l-isoprenaline stimulated a single component of adenylyl cyclase which was identified in inhibition studies as being ß2-adrenoceptor-mediated. BRL28410 also activated a single site, though only in membranes from reserpinised rats. Although basal cAMP production was not lowered by 1 M propranolol, high concentrations of both ICI118551 and CGP20712A inhibited cAMP production below basal levels, suggesting that these antagonists may also interact with cAMP-linked sites other than ß-adrenoceptors. The ß2-agonists, salmeterol and clenbuterol were tested in growth studies. The two compounds were equally anabolic when infused for ten days, though salmeterol was markedly less effective when given orally. Salmeterol and clenbuterol elicited a characteristic profile of muscle growth, apparently related to the muscle fibre composition. Increases in carcass weight were reflected well in the mixed-fibre muscles, but not in slow-twitch muscles. The ß3-adrenoceptor agonist BRL26830A, also promoted muscle growth at high doses (10 and 55 mg/day) when administered orally to rats for ten days. As with ß2-agonist treatment, growth was observed in the carcass and mixed-fibre muscles. However, at anabolic doses, and at some sub-anabolic doses, BRL26830A down-regulated ß2-adrenoceptors in gastrocnemius/plantaris muscle. This indicated that the growth response to this agonist was possibly ß2-adrenoceptor-mediated, and that ß2- adrenoceptor down-regulation is a more sensitive index of ß2-adrenoceptor activation than muscle growth. The ß3-adrenoceptor agonist ICID7114, which has less activity at ß2-adrenoceptors than BRL2841O, was also tested. The lack of a growth response to this compound confirmed that ß3- adrenoceptors are not linked to protein accretion in skeletal muscle. In conclusion, ß2- and putative ß3-adrenoceptors were identified in skeletal muscle using radioligand binding techniques. However, in vitro and in vivo functional studies showed that the ß2-adrenoceptor was predominantly responsible for the control of adenylyl cyclase activation and protein accretion in this tissue.