Chronic treatment of rats with R-PIA 'in vivo' desensitized adenosine A1 receptor-mediated inhibition of adenylyl cyclase in brain plasma membranes and increased basal and forskolin-stimulated adenylyl cyclase. The adenosine A1 receptor agonist CHA (cyclohexyl adenosine) inhibited forskolin-stimulated adenylyl cyclase in synaptic plasma membranes from control rats but failed to do so in membranes isolated from rats treated with R-PIA. This loss of response was accompanied with a significant decrease in both, total number of adenosine A1 receptors and steady-state level of alpha-Gi in synaptic plasma membranes. An increase in the steady-state level of alpha-Gs in synaptic plasma membranes was also observed by R-PIA treatment. Concurrently, a significant increase of adenosine A1 receptors was observed in microsomes and coated vesicles. These results demonstrate adenosine A1 receptor desensitization in rat brain by 'in vivo' treatment with R-PIA and suggest a role for coated vesicles in the internalization of G-protein coupled receptors.

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.

Bovine brain coated vesicles display free calcium‐dependent phospholipase C activity. Gpp(NH)p and GTPγS inhibited phospholipase C at nanomolar concentrations. Increasing concentrations of Gpp(NH)p and GTPγS reversed the inhibitory effects and stimulated phospholipase C activity. Preincubation of coated vesicles with pertussis toxin blocked the poorly‐hydrolyzable GTP‐analogs' inhibitory effects on phospholipase C. These data indicate that guanine nucleotides exert a dual regulatory control of phospholipase C in coated vesicles and that the inhibitory pathway is mediated by a pertussis toxin‐sensitive G‐protein.

Glutamate metabotropic receptors (mGluRs) in bovine brain coated vesicles have been characterized by pharmacological and kinetic binding experiments. Saturation experiments revealed a single binding site with a Kd = 607.9 f 78.5 nM and a B,,,,, = 6.45 f 0.88 pmol/mg protein. The specific binding of Lj3H]glutamate to mGluRs is regulated by guanine nucleotides. Guanosine-5'-triphosphate (GTP; 100 PM) shifts the agonist competition curves to the right, increasing the XC, values. Pertussis toxin treatment produces a pharmacological binding profile for quisqualate similar to that obtained in the presence of 1OOpM GTP. These results indicate the presence of metabotropic glutamate receptors in coated vesicles and its coupling to a pertussis toxin sensitive G-protein.

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