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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The cytoplasmic C terminus of the ␤ 2 -adrenergic receptor and many other G protein-coupled receptors contains a dileucine sequence that has been implicated in endosome͞lysosome targeting of diverse proteins. In the present study, we provide evidence for an essential role of this motif in the agonist-induced internalization of the ␤ 2 -adrenergic receptor. Mutation of Leu-339 and͞or Leu-340 to Ala caused little changes in surface expression, ligand binding, G protein coupling, and signaling to adenylyl cyclase, when these receptors were transiently or stably expressed in CHO or HEK-293 cells. However, agonist-induced receptor internalization was markedly impaired in the L 339,340 A double mutant and reduced in the two single mutants. This impairment in receptor internalization was seen by using various approaches to determine internalization: binding of hydrophobic vs. hydrophilic ligands, loss of surface ␤ 2 -adrenergic receptor immunoreactivity, and immunof luorescence microscopy. The selective effects of these mutations suggest that the C-terminal dileucine motif is involved in agonist-induced internalization of the ␤ 2 -adrenergic receptor.Many types of membrane-bound receptors undergo a dynamic trafficking between the cell surface and intracellular compartments. Such trafficking may be involved both in the transmission of receptor signals and in the termination of such signaling (1). For the ␤ 2 -adrenergic receptor, a prototypical G proteincoupled receptor, the most remarkable trafficking process is agonist-induced receptor internalization. This internalization is part of a whole spectrum of adaptive processes triggered by agonist stimulation (2, 3).Agonist-induced translocation of these receptors to an intracellular compartment has been established by several approaches: (i) After agonist stimulation, a certain (and variable) proportion of the receptors becomes inaccessible for hydrophilic ligands but remains accessible for hydrophobic ligands; this process is usually termed sequestration (4). (ii) Upon cell fractionation and sucrose density centrifugation, these receptors can be recovered in a fraction lighter than the plasma membrane fraction (5). (iii) Immunofluorescence studies show that agonist treatment results in the appearance of receptors in intracellular aggregates that might represent endosomes; these data thus provide evidence for true internalization (6-8).Whereas earlier studies assumed that internalization of ␤ 2 -adrenergic receptors was essentially a mechanism of receptor desensitization (9, 10), more recent studies have assigned it a recycling and sorting function. According to these models, receptor internalization may occur subsequent to receptor phosphorylation and desensitization and may serve to either dephosphorylate the receptors and recycle them back to the cell surface or-particularly in the case of prolonged receptor stimulation-to direct the receptors to lysosomes for degradation (8, 11-13).The mechanism of ␤ 2 -adrenergic receptor internalization is ill-defined at the...

The specific recognition of various DNA modifications by repair enzymes is exploited for the analysis of DNA damage induced by visible light in the presence of methylene blue in Salmonella typhimurium. The relative frequencies of various endonuclease-sensitive sites and strand breaks are determined in the plasmid pAQ1 of the treated bacteria and are compared with those observed after exposure of isolated DNA to various conditions. This comparison of damage profiles indicates that the cellular DNA damage by illumination in the presence of methylene blue is caused predominantly by the direct action of singlet oxygen. Indirect mechanisms, e.g. involving a generation of superoxide and hydroxyl radicals or the activation of cellular nucleases, do not contribute very much. The damage is dominated by base modifications. These are subject to an efficient repair that is not mediated by uvrABC proteins and therefore most probably involves recognition by specific glycosylases. Revertant frequencies observed under these conditions in the strains TA1535, TA100, TA2638 and TA104 indicate a pronounced mutagenicity of the lesions induced. On the other hand, the DNA damage does not contribute significantly to the cytotoxicity caused by the treatment as an excision repair deficiency (uvrB) has no influence on cell killing.