A NOVEL P-2-PURINOCEPTOR EXPRESSED BY A SUBPOPULATION OF ASTROCYTES FROM THE DORSAL SPINAL-CORD OF THE RAT

1 Astrocytes from the dorsal spinal cord express P-2-purinoceptors whi ch, when stimulated, produce a rise in the intracellular level of free Ca2+ ([Ca2+](i)). Previously we have found that the P-2Y class of rec eptor is expressed by nearly all astrocytes from the dorsal horn. To d etermine whether other metabotropic P-2-purinoceptor classes are also present, in this study we investigated the effects of UTP. 2 Applicati on of UTP (1-500 mu M, 5-20 s) produced a transient rise in [Ca2+](i) in a subpopulation of astrocytes. The magnitude of the peak increase i n [Ca2+](i) was dependent upon UTP concentration and the EC(50) was fo und to be 5.2+/-0.2 mu M. Ca2+ responses were maximum at 100 mu M UTP. 3 The rise in [Ca2+](i) in response to UTP was not affected by remova l of extracellular Ca2+. On the other hand, application of the sarcopl asmic-endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, aboli shed responses to UTP. These findings indicate that UTP stimulates the release of Ca2+ from a thapsigargin-sensitive intracellular pool. 4 T he Ca2+ response to UTP was unaffected by treatment with pertussis tox in, suggesting that UTP responses may be mediated via a pertussis toxi n-insensitive G protein. 5 While all cells tested (n=52) responded to the P-2Y-purinoceptor agonist, 2-methylthio-ATP, only a subpopulation of astrocytes (n=67/93) was responsive to UTP. The presence of UTP-sen sitive and UTP-insensitive cells requires the existence of two discret e types of receptor. One receptor, expressed by UTP-insensitive cells, appears to be activated selectively by 2-methylthio-ATP. 6 To investi gate whether UTP and 2-methylthio-ATP activate a common type of recept or in UTP-responsive cells, a cross-desensitization strategy was used. Desensitization with prolonged exposure to a high concentration of 2- methylthio-ATP failed to affect responses to UTP and vice versa, indic ating that receptors activated by UTP are distinct from those activate d by 2-methylthio-ATP. 7 The P-2-purinoceptor antagonist, suramin (100 mu M), blocked Ca2+ responses to UTP and to 2-methylthio-ATP. 8 Pyrid oxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), has been rep orted to block responses mediated by P-2X- and P-2Y-purinoceptors in o ther systems and therefore we investigated its effects on responses to 2-methylthio-ATP and to UTP. PPADS was found to block Ca2+ responses to 2-methylthio-ATP in a concentration-dependent manner with an IC50 o f 0.92+/-0.1 mu M. PPADS also blocked UTP-evoked responses and the IC5 0 was 7.2+/-1.9 mu M. At a concentration of 10 mu M, PPADS produced a rightward shift in the dose-response curve for UTP and did not affect the maximum response. 9 Calcium responses evoked by the muscarinic ago nist, carbachol, were unaffected either by suramin (100 mu M) or by PP ADS (50 mu M). 10 The present results indicate the presence of a novel class of metabotropic P-2U-purinoceptor in dorsal spinal astrocytes. In contrast to P-2Y-purinoceptors, the P-2U-purinoceptor is expressed only by a subpopulation of astrocytes and its sensitivity to suramin a nd PPADS distinguish this receptor from P-2U-purinoceptors found in ot her tissues.