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

Abstract

We have studied the interaction between P1 and P2 purinoceptors in purified type-1 astrocyte cultures from postnatal days 7-8 rat cerebella using single cell microfluorimetry with fura-2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 +/- 0.3 microM. Costimulation of type-1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 microM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5'-N-ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 +/- 0.01 microM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 +/- 0.2 microM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type-1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of beta gamma subunits released after Gs stimulation.