In Vitro and In Vivo Characterization of Intrinsic Sympathomimetic Activity in Normal and Heart Failure Rats

ABSTRACT Clinical studies conducted with carvedilol suggest that ␤-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many ␤-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective ␤ 1 -adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED 50 ϭ 5 and 40 g/kg, respectively) and SHHF rats (ED 50 ϭ 6 and 30 g/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (␤ 1 -adrenoceptor antagonist) but not by ICI 118551 (␤ 2 -adrenoceptor antagonist) in neonatal rat. When the ␤-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting ␤-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct ␤ 1 -adrenoceptor-mediated ISA in normal and heart failure rats

Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines

1. In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [(125)I]hU-II binding sites. 2. UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3. [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (K(d) 67.0±11.8 pM, B(max) 9687±843 sites cell(−1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (⩽15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667±165 sites cell(−1), K(d) 74±8 pM). 4. Consistent with the profile recorded in human recombinant UT-HEK293 cells, [(125)I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (K(i)s 0.5±0.1–1.2±0.3 nM) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; K(i)s 0.4±0.1 and 864±193 nM, respectively). 5. U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca(2+)](i) mobilization (EC(50) 6.9±2.2 nM) in SJRH30 cells. 6. The present study is the first to identify the presence of ‘endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and ‘native' UT receptors (K(i) 4.7±1.5 to 20.7±3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4±0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 μM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1±0.4 vs 5.8±0.9 nM and B(max) 3.1±1.0 vs 2.8±0.8 pmol mg(−1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6±0.4 nM, B(max) 0.86±0.12 pmol mg(−1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6±1.4 to 17.6±5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29–8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app)∼20 nM). 5. SB-706375 was a selective U-II antagonist with ⩾100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 μM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 μM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals