Fund. M.W.S. is an MRC Scholar. We thank Drs. J. F. MacDonald and Y. De Koninck for critical reading of the manuscript, and Ms. Kathleen Downie for assistance preparing the manuscript. We gratefully acknowledge Dr. J. Cobby at Bayer Canada Inc. for providing suramin. Ms. Lise Aasheim participated in some of the experiments.

Calcium signaling within astrocytes in the CNS may play a role comparable to that of electrical signaling within neurons. ATP is a molecule known to produce Ca2+ responses in astrocytes, and has been implicated as a mediator of intercellular Ca2+ signaling in other types of nonexcitable cells. We characterized the signal transduction pathway for ATP-evoked Ca2+ responses in cultured astrocytes from the dorsal spinal cord. Nearly 100% of these astrocytes respond to extracellularly applied ATP, which causes release of Ca2+ from an intracellular pool that is sensitive to thapsigargin and insensitive to caffeine. We found that intracellular administration of IP3 also caused release of Ca2+ from a thapsigargin-sensitive intracellular pool, and that IP3 abolished the response to ATP. The ATP-evoked Ca2+ response was blocked by the IP3 receptor antagonist heparin, applied intracellularly, but not by N-desulfated heparin, which is not an antagonist at these receptors. The Ca2+ response caused by ATP was also blocked by a phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. Increases in [Ca2+]i were elicited by intracellular application of activators of heterotrimeric G-proteins, GTP gamma S and AIF4-. On the other hand, [Ca2+], was unaffected by a G-protein inhibitor, GDP beta S, but it did abolish the Ca2+ response to ATP. Pretreating the cultures with pertussis toxin did not affect responses to ATP. Our results indicate that in astrocytes ATP-evoked release of intracellular Ca2+ is mediated by IP3 produced as a result of activating phospholipase C coupled to ATP receptors via a G-protein that is insensitive to pertussis toxin. ATP is known to be released under physiological and pathological circumstances, and therefore signaling via the PLC-IP3 pathway in astrocytes is a potentially important mechanism by which ATP may play a role in CNS function.

1. Astrocytes from the dorsal spinal cord express P2-purinoceptors which, when stimulated, produce a rise in the intracellular level of free Ca2+ ([Ca2+]i). Previously we have found that the P2Y class of receptor is expressed by nearly all astrocytes from the dorsal horn. To determine whether other metabotropic P2-purinoceptor classes are also present, in this study we investigated the effects of UTP. 2. Application of UTP (1-500 microM, 5-20 s) produced a transient rise in [Ca2+]i in a subpopulation of astrocytes. The magnitude of the peak increase in [Ca2+]i was dependent upon UTP concentration and the EC50 was found to be 5.2 +/- 0.2 microM. Ca2+ responses were maximum at 100 microM UTP. 3. The rise in [Ca2+]i in response to UTP was not affected by removal of extracellular Ca2+. On the other hand, application of the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin, abolished responses to UTP. These findings indicate that UTP stimulates the release of Ca2+ from a thapsigargin-sensitive intracellular pool. 4. The Ca2+ response to UTP was unaffected by treatment with pertussis toxin, suggesting that UTP responses may be mediated via a pertussis toxin-insensitive G protein. 5. While all cells tested (n = 52) responded to the P2Y-purinoceptor agonist, 2-methylthio-ATP, only a subpopulation of astrocytes (n = 67/93) was responsive to UTP. The presence of UTP-sensitive and UTP-insensitive cells requires the existence of two discrete types of receptor. One receptor, expressed by UTP-insensitive cells, appears to be activated selectively by 2-methylthio-ATP. 6. To investigate whether UTP and 2-methylthio-ATP activate a common type of receptor 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, indicating that receptors activated by UTP are distinct from those activated by 2-methylthio-ATP. 7. The P2-purinoceptor antagonist, suramin (100 microM), blocked Ca2+ responses to UTP and to 2-methylthio-ATP. 8. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), has been reported to block responses mediated by P2X- and P2Y-purinoceptors in other 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 of 0.92 +/- 0.1 microM. PPADS also blocked UTP-evoked responses and the IC50 was 7.2 +/- 1.9 microM. At a concentration of 10 microM, 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 agonist, carbachol, were unaffected either by suramin (100 microM) or by PPADS (50 microM). 10. The present results indicate the presence of a novel class of metabotropic P2U-purinoceptor in dorsal spinal astrocytes. In contrast to P2Y-purinoceptors, the P2U-purinoceptor is expressed only by a subpopulation of astrocytes ...