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- Klotz, K N, et al.
(författare)
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Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells
- 1998
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Ingår i: Naunyn-Schmiedeberg's Archives of Pharmacology. - 0028-1298. ; 357:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.
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| 2. |
- Kull, B, et al.
(författare)
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Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors
- 1999
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Ingår i: Biochemical Pharmacology. - 0006-2952. ; 57:1, s. 65-75
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Tidskriftsartikel (refereegranskat)abstract
- To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.
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| 3. |
- Kull, B, et al.
(författare)
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Reciprocal interactions between adenosine A2A and dopamine D2 receptors in Chinese hamster ovary cells co-transfected with the two receptors
- 1999
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Ingår i: Biochemical Pharmacology. - 0006-2952. ; 58:6, s. 1035-1045
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Tidskriftsartikel (refereegranskat)abstract
- Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal gamma-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPgammaS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose-response curve for quinpirole on adenosine 3',5'-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPgammaS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in EC50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.
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