Abstract
This study examines signaling pathways activated by the mouse β3-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHOβ3H) or low (CHOβ3L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (–)-Isoproterenol and the β3-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHOβ3H and CHOβ3L cells. For cAMP accumulation, the β3-AR ligand SR59230A was a partial agonist in CHOβ3H and an antagonist in CHOβ3L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (–)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHOβ3H cells, whereas p38 MAPK is a major contributor to ECAR in CHOβ3L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHOβ3H and CHOβ3L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHOβ3H and CHOβ3L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.
Footnotes
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This work was supported by the National Health and Medical Research Council (NHMRC) of Australia Project grant 236884 (to R.J.S.). D.S.H. is an NHMRC CJ Martin Fellow, M.S. is a Monash University Research Scholar.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.035337.
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ABBREVIATIONS: AR adrenoceptor; CHO Chinese hamster ovary; ECAR, extracellular acidification rate; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated protein kinase 1/2; CYP, cyanopindolol; SR59230A, 3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate; CL316243, (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl] 1,3-benzodioxole-2,2-decarboxylate; RWJ67657, 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; PD98059, 2′-amino-3′-methoxyflavone; DDA, 2′, 3′-dideoxyadenosine; H-89, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride; PKA, protein kinase A; IBMX, 3-isobutyl-1-methylxanthine; BSA, bovine serum albumin; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; MEK, mitogen-activated protein kinase kinase; 8-Br-cAMP, 8-bromoadenosine 3′, 5′-cAMP; BRL37344, (4-(2-((2-(3-chlorophenyl)-2-hydroxyethyl) amino) propyl) phenoxy) acetic acid; CGP12177A, 4-(3-tert-butylamino-2-hydroxypropoxy) benzimidazol-2-one; SR58611A, N-(7-hydroxy-1,2,3,4-tetrahydronaphth-2-yl)-2-hydroxy-2-(3-chlorophenyl) ethanol; ICI118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl) oxy]-3-[(1-methylethyl) amino]-2-butanol; CHOβ3H, β3 adrenoceptor expressed in Chinese hamster ovary cells at high levels; CHOβ3L, β3 adrenoceptor expressed in Chinese hamster ovary cells at low levels.
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↵1 Current affiliation: Department of Cellular and Molecular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, Japan.
- Received February 24, 2007.
- Accepted August 22, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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