Potentiation of ATP calcium responses by A(2B) receptor stimulation and other signals coupled to G(s) proteins in type-1 cerebellar astrocytes

We have studied the interaction between P1 and P2 purinoceptors in purified type-1 astrocyte cultures from postnatal days 7-8 rat cerebella using sing le cell microfluorimetry with fura-2. The stimulation of astrocytes with AT P elicits rapid [Ca2+](i) transients showing an EC50 value of 7.9 +/- 0.3 m u M. Costimulation of type-1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 mu M) evoked [Ca2+](i) transients that correspond to 60% of the maximal ATP response. NECA (5'-N-ethylcarboxamidoadenosine) w as the only agonist that mimicked the adenosine effect and showed an EC50 v alue of 0.17 +/- 0.01 mu M. This value was identical to that obtained for t he cAMP production stimulation, indicating that A(2B) receptors coupled to adenylate cyclase activation were involved. The presence of A(2B) adenosine receptors was also confirmed by immocytochemistry experiments. When astroc ytes were costimulated with isoproterenol and ineffective ATP concentration s similar [Ca2+](i) transients were observed. The treatment of astrocytes w ith cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 +/- 0.2 mu M. However, the pretreatment of cells with forsko lin or a permeable cAMP analogue had no effect on ATP calcium responses. Th ese results indicated that the potentiation mechanism was elicited before t he adenylate cyclase activation. We could conclude that in type-1 astrocyte s, the activation of A(2B) adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic c alcium responses to ATP. The potentiation was parallel but independent on c AMP accumulation suggesting the involvement of beta gamma subunits released after G(s) stimulation. GLIA26:119-128, 1999. (C) 1999 Wiley-Liss, Inc.