Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A1 and A2B receptors) or HEK-293 cells (A2A and A3 receptors). In binding studies using [3H]SCH 58261 as a radioligand, the three compounds were equally potent at A2A receptors, their K(i) value being less than 1 nM. Affinity for A1 and A3 receptors was measured using [3H]DPCPX and [125I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A1 receptors (K(i)3.5 nM) as at A2A receptors, showed moderate affinity for A3 receptors (K(i) 95 nM) and also interacted with A2B receptors (K(i) 44 nM; pA2 7.5). ZM 241385 showed little affinity for A1 receptors (K(i) 255 nM), and did not interact with A3 receptors (K(i)>10 microM); however, it displayed moderate affinity for A2B receptors (K(i) 50 nM; pA2 7.3). SCH 58261 had weak affinity for A1 receptors (K(i) 287 nM), no interaction with A3 receptors (K(i)>10 microM), and showed negligible interaction with A2B receptors (K(i) 5 microM; pA2 6.0). These data indicate that SCH 58261 is the most selective A2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies.

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It has been shown that the binding characteristics of one type of G protein-coupled receptor can be altered by the stimulation of another type of G protein-coupled receptor in crude membrane preparations (1). Such intramembrane interactions have been postulated to represent direct interactions between the receptor molecules and/or to involve G proteins or other mobile molecules associated with the membrane (1). There is increasing evidence suggesting that antagonistic intramembrane interactions between specific subtypes of adenosine and dopamine receptors constitute an important integrative mechanism in the basal ganglia (2, 3). Adenosine A 1 and A 2A receptors antagonistically and specifically modulate the binding characteristics of dopamine D 1 and D 2 receptors, respectively (2, 3). In membrane preparations from rat striatum, the stimulation of A 2A receptors decreases the affinity of D 2 receptors for agonists (4). On the other hand, the stimulation of A 1 receptors was shown to decrease the proportion of D 1 receptors in the high affinity state, without modifying the dissociation constants of high and low affinity D 1 agonist-binding sites (5).Thus, the A 1 receptor agonist had the same effect as that induced by the GTP analogue Gpp(NH)p.1 It was hypothesized that A 1 receptor stimulation might uncouple the striatal D 1 receptor from the G protein (5). There is evidence that the antagonistic A 2A -D 2 and A 1 -D 1 intramembrane interactions are involved in the motor depressant effects of adenosine receptor agonists and the motor stimulant effects of adenosine receptor antagonists, such as caffeine (2-5).The same changes in the binding characteristics of striatal D 2 receptors after A 2A receptor stimulation have been obtained in membrane preparations from a mouse fibroblast cell line (Ltk Ϫ ) stably cotransfected with the dog A 2A receptor and human D 2 (long-form) receptor cDNAs (6). In these transfection studies, it was also found that activation of adenylyl cyclase was not involved in the intramembrane A 2A -D 2 interaction (6). Altogether, these results showed that stably cotransfected cell lines constitute a valuable model to study the mechanistic aspects involved in the intramembrane receptor-receptor interactions. In the present work, this methodology has been applied to study the antagonistic interaction between A 1 and D 1 receptors. The first aim of the study was to demonstrate the existence of an antagonistic A 1 -D 1 intramembrane interaction in mammalian cells stably cotransfected with A 1 receptor and D 1 receptor cDNAs. The second aim was to demonstrate the existence of a functional antagonistic interaction between A 1 and D 1 receptors in the cotransfected cells by means of cAMP accumulation experiments. Finally, the third aim of the study was to find a functional significance of the antagonistic A 1 -D 1 intramembrane interaction. EXPERIMENTAL PROCEDURES Transfection and Maintenance of Fibroblast LtkϪ Cells-Cells from the mouse fibroblast Ltk Ϫ cell line previously transfected with the huma...

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Endothelin-1 (ET-1) is known to act via G-protein coupled receptors. It has therefore been suggested that any mitogenic activity it may possess, is due to activation of phospholipase C and protein kinase C (PKC). We have therefore examined both the ability of ET-1 to act as a mitogen and its ability to activate PKC. We found that ET-1 significantly increased thymidine incorporation and enhanced platelet-derived growth factor-induced DNA synthesis, as well as causing a prolonged translocation of PKC to the cell membrane. ET-1 significantly increased PKC dependent phosphorylation of two specific substrates. The phosphorylation of MBP4-14 (from myelin basic protein) was partially dependent on extracellular Ca2+, implicating activation of PKC-alpha, whereas phosphorylation of the so called epsilon-peptide was Ca(2+)-independent and prolonged. This could be due either to the delta or zeta isoform of PKC, known to be present in these cells. However, ET-1 induced little proliferation of PKC activity in a transformed smooth muscle cell line, DDT1 MF-2, which lacks expression of the PKC-alpha isoform, but expresses the zeta-isoform. Thus, it would appear the ET-1-induced mitogenicity in smooth muscle cells may be related to the sustained, Ca(2+)-independent activation of PKC-delta.