Kinetics of ternary complex formation with fusion proteins composed of the A(1)-adenosine receptor and G protein alpha-subunits.

High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.

Tricyclic theophylline derivatives with high water-solubility: structure-activity relationships at adenosine receptors, phosphodiesterases, and benzodiazepine binding sites.

A series of tricyclic, highly water-soluble theophylline derivatives (pyrimido[2,1-f]-theophyllines) containing a basic side chain was investigated in rat brain A1- and A2 adenosine receptor binding assays, phosphodiesterase assays, and benzodiazepine binding studies. Among the new compounds adenosine receptor antagonists with affinities in the same range as the parent compound theophylline were identified. In addition, some compounds were selective for the A1 adenosine receptor subtype. The compounds generally exhibited lower inhibitory activity at brain phosphodiesterases than the parent theophylline. Two compounds were found to show an about 10-fold affinity for benzodiazepine binding sites compared with caffeine and theophylline.

Pharmacokinetic-pharmacodynamic modelling of the cardiovascular effects of R- and S-N6-phenylisopropyladenosine in conscious normotensive rats.

Recently, the commercially available adenosine receptor agonist S-N6-phenylisopropyladenosine (S-PIA) has been demonstrated to be contaminated with the more potent R-diastereomer. The potency of S-PIA may therefore have been overestimated in previously published in vivo studies. Our objective was to determine the potency of both diastereomers in conscious normotensive rats by using an integrated pharmacokinetic-pharmacodynamic model. In a cross-over designed study, the animals received i.v. infusions of 177 micrograms/kg (0.51 mumol/kg) R-N6-phenylisopropyladenosine (R-PIA) and 4000 micrograms/kg (11.6 mumol/kg) S-PIA. The infusion of S-PIA corresponded to a simultaneous administration of 3823 micrograms/kg (11.1 mumol/kg) and 177 micrograms/kg (0.51 mumol/kg) of the S- and R-diastereomer, respectively. During the experiment, time courses of heart rate and blood pressure were recorded continuously. Serial arterial blood samples were collected and concentrations were determined by using a stereoselective high-performance liquid chromatography assay. After administration of R-PIA, the individual concentration-effect relationships could be quantified by the sigmoidal Emax model, yielding an EC50 value of 24 +/- 3 ng/ml for the reduction in heart rate (mean +/- S.E., n = 12). After administration of S-PIA, a similar EC50 value was obtained when heart rate was correlated to concentrations of R-PIA. Modelling of the concentration-effect data according to a competitive interaction model did not yield pharmacodynamic parameter estimates for S-PIA. In conclusion, the cardiovascular effects observed after infusion of S-PIA may be attributed entirely to the presence of R-PIA.

Correlation of (-)N6-phenylisopropyladenosine blood levels with cardiac responses in the anesthetized guinea pig.

This study was designed to develop a high-performance liquid chromatography-based analytical method to measure the blood concentration of (-)N6-phenylisopropyladenosine (R-PIA) in order to correlate such levels with cardiac responses, and to determine the pharmacokinetics of R-PIA. Experiments were carried out in anesthetized adult guinea pigs instrumented for measurement of the surface ECG. After i.v. (50 micrograms/kg; n = 9) or i.p. (3.5 mg/kg; n = 5) administration of R-PIA, the atrial rate and P-R interval were determined, and arterial blood samples (0.5 ml) were collected. The R-PIA content of plasma ultrafiltrates was determined by reversed-phase high-performance liquid chromatography. The ratio of R-PIA concentrations in whole blood and in ultrafiltrated plasma (free or unbound) at 35-37 degrees C was 2.51 +/- 0.10 (n = 7) and was relatively independent of R-PIA concentration. The concentration of unbound R-PIA in whole blood correlated well with cardiac responses. Two distinct patterns of cardiac response to varying R-PIA levels were observed. In 4 of 14 animals, R-PIA caused a prolongation of the P-R interval at a relatively constant atrial rate, whereas in 10 animals, R-PIA caused both a slowing of atrial rate and a prolongation of the P-R interval. An increase in the unbound concentration of R-PIA caused a decrease in atrial rate and an increase in P-R interval. Unbound R-PIA was rapidly cleared from blood (CL = 40 +/- 7 ml/kg/min) and had a large volume of distribution (VSS = 1.45 +/- 0.21 L/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Central adenosinergic system involvement in ethanol-induced motor incoordination in mice.

To clarify if the behavioral interaction between ethanol and adenosine reported previously occur centrally or due to a peripheral hemodynamic change, the effect of i.c.v. adenosine agonists, N6-(R-phenylisopropyl)adenosine (R-PIA), N6-(S-phenylisopropyl)adenosine, 5'-(N-cyclopropyl)-carboxamidoadenosine, antagonists, theophylline and 8-p-(sulfophenyl)theophylline as well as enprofylline on ethanol-(i.p.)-induced motor incoordination was evaluated by rotorod. Adenosine agonists and antagonists dose dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, thereby suggesting a central mechanism of adenosine modulation of this effect of ethanol and confirmed our previous reports in which adenosine agonists and antagonists were given i.p. Enprofylline, a weak adenosine antagonist but potent inhibitor of cyclic AMP phosphodiesterase, did not alter ethanol's motor incoordination, further supporting involvement of brain adenosine receptor mechanism(s) in ethanol-adenosine interactions. Results from R-PIA and N6-(S-phenylisopropyl)adenosine experiments showed nearly a 40-fold greater potency of R-vs. S-diastereoisomer, suggesting predominance of adenosine A1 subtype. However, 5'-(N-cyclopropyl)-carboxamidoadenosine data indicate complexity of the mechanism(s) and point toward an additional involvement of a yet unknown subtype of adenosine A2. No effect of ethanol on blood or brain levels of [3H]R-PIA was noted and sufficient amount of the latter entered the brain to suggest adenosine receptor activation adequate to produce behavioral interaction with ethanol. There was no escape of i.c.v.-administered [3H]R-PIA from brain to the peripheral circulation ruling out a peripheral and supporting a central mechanism of ethanol-adenosine interaction.(ABSTRACT TRUNCATED AT 250 WORDS)