Adenosine A(1) receptor in cultured neurons from rat cerebral cortex: Colocalization with adenosine deaminase

Adenosine A(1) receptors (A(1)Rs) have been characterized in primary cultur es of neurons from cerebral cortex. The specific adenosine A(1) antagonist 8-cyclopentyl-1,3-[H-3]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a K-D value of 0.76 n/M and a B-max of 57 fmol/mg of protein were obtained. Competitio n assays revealed a pharmacological profile characteristic of A(1)Rs. The p resence of this receptor was further confirmed by RT-PCR analysis. The expr ession of the receptor showed no significant changes during the period of c ulture studied, up to 12 days in vitro. A(1)R agonist inhibited forskolin-s timulated adenylyl cyclase, showing the functional coupling of these recept ors with the effector. alpha G(i1,2) protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlat ed with an increase in the mRNA level of alpha G(i1) but not alpha G(i2). B y immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A (1)Rs with microtubule-associated protein 2 and cell surface adenosine deam inase was shown by confocal microscopy. The high degree of colocalization o bserved between A(1)Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine-mediated neuromodulation.