The Contribution of Classical (b1/2-) and Atypical b- Adrenoceptors to the Stimulation of Human White Adipocyte Lipolysis and Right Atrial Appendage Contraction by Novel b3-Adrenoceptor Agonists of Differing Selectivities

The role of b3and other putative atypical b-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine b3-adrenoceptor (b3AR) agonists with varying intrinsic activities and selectivities for human cloned bAR subtypes. The ability to demonstrate b1/2AR antagonist-insensitive (b3 or other atypical bAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective b3AR agonists (SB-226552, SB229432, SB-236923, SB-246982) consistently elicited b1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full b3AR agonist elicited full lipolytic and inotropic responses that were sensitive to b1/2AR antagonism, despite it having very low efficacies at cloned b1and b2ARs. A component of the response to another phenylethanolamine selective b3AR agonist (SB-215691) was insensitive to b1/2AR antagonism in some experiments. Because novel aryloxypropanolamine had a b1/2AR antagonistinsensitive inotropic effect, these results establish more firmly that b3ARs mediate lipolysis in human white adipocytes, and suggest that putative ‘b4ARs‘ mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned bARs which bARs will mediate responses to agonists in tissues that have a high number of b1and b2ARs or a low number of b3ARs. b3ARs were first identified by functional studies in animal gastrointestinal and adipose tissues (for review see Arch and Kaumann, 1993). Subsequently, the b3AR gene was cloned from human genomic DNA, and expression of b3AR mRNA has now been detected in various human tissues, including adipose, gastrointestinal and myocardial tissues (Krief et al., 1993; Berkowitz et al., 1995; Gauthier et al., 1996). However, although there are numerous reports describing functional responses mediated by b3ARs in animal tissues, evidence for such responses in human tissues is more limited, occasionReceived for publication October 13, 1997. 1 Current address: University of Buckingham, Hunter Street, Buckingham, MK18 1EG, UK. 2 Supported by the British Heart Foundation. ABBREVIATIONS: bAR, b-adrenoceptor; CHO, Chinese hamster ovary; MEM, minimal essential medium; CGP12177, (6)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one; ICI 118551, D-(6)-1-(7-methylylindan-4-yloxy)-3-isopropylaminobutan-2-ol; CGP 20712A, (6)-[2-(3-aminocarbamoyl-4-hydroxyphenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol hydrochloride; CL 316243, disodium (RR) -5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxazole-2,2-dicarboxylate; BRL-37344, (RR1SS)-(6)-4-[2-(2-(3-chlorophenyl)-2hydroxyethyl)amino)propyl]phenoxyacetate; SB-215691, (RS1RR) 3-hydroxypropyl 4-[2-[2-(3,4-dihydroxyphenyl)-2-hydroxyethylamino]propyl]phenoxymethylphosphonate ethyl ester, dihydrate; SB-220646, (R, R)-5-{2-[2-(3,4-dihydroxyphenyl)-2-hydroxyethylamino]propyl}-1,3-benzodioxole-2,2dicarboxylate; SB-226552, (S)-4-{2-[2-hydroxy-3-(4-hydroxyphenoxy)propylamino]ethyl}phenoxymethylcyclohexylphosphinic acid lithium salt; SB-229432, (SR)-4-{2-[2-hydroxy-3-(4-hydroxy-3-hydroxymethylphenoxy)propylamino]propyl}phenoxymethylphenylphosphinic acid, lithium salt; SB232602, (S)-N-{2-hydroxy-5-[2-hydroxy-3-(4-methoxyphenyl)ethylamino]propoxy}methanesulfonamide hydrochloride; SB-236923, (SR) 4-{2-[2-(2-hydroxy-3-(4-hydroxy-3-methanesulfonylaminophenoxy)-propyl)amino]propyl}phenoxymethylphenylphosphinic acid, hydrobromide; SB-246982, (S)-2-(4(2-(2-(3-(3-N-phenylsulfonylamino-4-hydroxyphenoxy)-hydroxypropyl)amino)ethyl)benzyloxy)benzoic acid, trifluoroacetate; SB-248320, (S)-diphenyl-4{2-[2-hydroxy-3-(4-hydroxy-3-iso-propylsulfonyl-aminophenoxy)propylamino]ethyl}phenoxymethyl phosphine oxide. 0022-3565/98/2853-1084$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 285, No. 3 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 285:1084–1095, 1998 1084 at A PE T Jornals on A ril 0, 2017 jpet.asjournals.org D ow nladed from ally contradictory and sometimes open to alternative interpretation. Much of the published evidence for b3AR-mediated responses in human tissues derives from studies using CGP 12177. In most tissues this aryloxypropanolamine is a potent antagonist of b1and b2ARs, but at concentrations about 1000-fold higher it is an agonist of b3ARs. It is able to stimulate human b1ARs when they are expressed in high density (Pak and Fishman, 1996) but its agonist activity in tissues is usually insensitive to standard b1and b2AR antagonists, indicating that responses are not mediated by b1or b2ARs. Thus both human colon (De Ponti et al., 1996) and taenia coli (Kelly et al., 1997) are relaxed by CGP 12177, and the taenia coli response has been shown to be resistant to nadolol (pA2 # 6). Similarly, in human white adipocytes Arner, Lonnqvist and their coworkers have consistently demonstrated lipolytic responses to CGP 12177 that are poorly blocked by standard b1and b2AR antagonists (Lonnqvist et al., 1993; Hoffstedt et al., 1996). Some reports support these findings (Sennitt et al., 1995; Portillo et al., 1995; Tavernier et al., 1996) although there are earlier reports that do not (Langin et al., 1991; Van Liefde et al., 1994). Again in human right atrial appendage, CGP 12177 elicits a small inotropic response that is resistant to blockade by propranolol. The response is antagonized by bupranolol but only at higher concentrations than those that block b1and b2ARs (Kaumann, 1996). In contrast to these results for CGP 12177, demonstration of b3AR-mediated responses in human tissues using phenylethanolamines that are agonists of b1and b2ARs as well as b3ARs has proved difficult. Isoproterenol, norepinephrine and the b3AR-selective agonist BRL-37344 each stimulate cyclic AMP accumulation in cells transfected with high numbers of human b3ARs, and they stimulate adenylyl cyclase in membranes from these cells (Emorine et al., 1994). However, responses to BRL-37344 (if they occur at all), and to isoproterenol and norepinephrine in human gut, adipose tissue and atrium are mediated primarily by b1or b2rather than b3ARs (MacLaughlin and MacDonald, 1991; Langin et al., 1991; Lonnqvist et al., 1993; Rosenbaum et al., 1993; Sennitt et al., 1995; Wang et al., 1996; Kaumann AJ and Sanders L, quoted in Arch and Kaumann, 1993), although responses to isoproterenol and norepinephrine in gut do involve a significant b3AR-mediated component (MacLaughlin and MacDonald, 1991; De Ponti et al., 1996; Kelly et al., 1997). Indeed, other than CGP 12177, only CL 316243, a phenylethanolamine with very low efficacy at b1and b2ARs, has been shown to elicit a lipolytic response in human white adipocytes that is totally resistant to antagonism by 10 M propranolol (Hoffstedt et al., 1996); and only in human ventricle, for which a negative inotropic effect has been reported (Gauthier et al., 1996; but see Molenaar et al., 1997), and possibly platelets (Gill et al., 1991) is there evidence for a strong b3AR component in the response to BRL-37344 in a human tissue. One possible explanation for the failure of BRL-37344 to elicit responses via b3ARs in human tissues is that its low efficacy and selectivity for the human (as compared with the rat or mouse) b3AR precludes it acting via these receptors in human tissues that express them in low numbers compared to b1and b2ARs (Arch and Wilson, 1996; Wilson et al., 1996). Another possibility is that CGP 12177 in fact elicits its effects not via b3ARs but via a related, putative ‘b4AR’ (Kaumann, 1997). Such an explanation has been used to interpret the pharmacology of cardiac responses in the rat, mouse, guinea pig, cat and ferret and has recently been extended to human atrium and ventricle(Kaumann, 1996; Kaumann, 1997; Kaumann and Molenaar, 1997; Molenaar et al., 1997). Thus cardiac pharmacology differs from colonic pharmacology not only by the failure of BRL-37344 and other b3AR-selective agonists to elicit positive inotropic responses via b3ARs, but also in a number of other respects (Kaumann and Molenaar, 1996; Kaumann, 1997). Finally, it is possible that in human tissues b3ARs adopt a conformation or associate with G proteins differently from in transfected cells, so that they are stimulated by CGP 12177 but not BRL-37344 (Kenakin, 1995; Arch, 1997). Further insight into the role of atypical bARs in mediating functional responses in human tissues may come from studies on novel b3AR agonists with differing efficacies and relative potencies at b1-, b2and b3ARs. We characterize a number of novel bAR agonists of both the phenylethanolamine and aryloxypropanolamine type in terms of their pharmacology at human cloned b1-, b2and b3ARs. We describe the lipolytic activities of these compounds in human white adipocytes, and in some cases their inotropic activities in human right atrial appendage, in the absence and presence of antagonists of b1and b2ARs. The results not only support a role for b3ARs in human adipocytes, but also illustrate the difficulties of predicting the tissue pharmacology of agonists, especially phenylethanolamines, from their profiles in cells expressing cloned bARs. The compounds studied may be broadly classified from their pharmacology and chemistry as: 1) the phenylethanolamines SB-215691 and SB-220646, which are similar to BRL-37344 in being agonists at b1and b2as well as b3ARs but have higher efficacy than BRL-37344 at human b3ARs; 2) the aryloxypropanolamines SB-226552, SB-229432 and SB232602, which are similar to CGP 12177 in being primarily antagonists at b1and b2ARs, but have far lower affinity than CGP 12177 at these receptors; 3) the aryloxypropanolamines SB-236923, SB-246982 and SB-248320 which are similar to the compounds in category 2) except that partial agonist activity at b1ARs was clearly demonstrated. Materials and Methods Cloned bARs and membrane preparation. Binding studies were conducted using membranes from CHO cells expressing 7100, 2300 and 3000 fmol/mg protein of human b1-, b2 and b3ARs, respectively. Adenylyl cyclase measurements were conducted using membranes from CHO cells expressing 210, 560 and 390 fmol/mg protein of human b1-, b2and b3ARs, respectively. The CHO cells expressing the higher numbers of b1and b2ARs were obtained from A D Strosberg (Tate et al., 1991) and those expressing the lower numbers from S B Liggett (Green et al., 1992). The CHO cells expressing b3ARs were derived in-house by transfecting cells with the short splice variant of the b3AR (Liggett, 1992) as described elsewhere (Wilson et al., 1996). We have found similar EC50 values and intrinsic activities (relative to isoproterenol) for isoproterenol, CGP 12177 and BRL-37344 for the short and long forms of the b3AR expressed at similar densities in CHO cells (Wilson S and Chambers JK, unpub-