A novel P2‐purinoceptor expressed by a subpopulation of astrocytes from the dorsal spinal cord of the rat

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Abstract
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 μm, 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 μm. Ca2+ responses were maximum at 100 μm 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 Ca2+-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 μm), 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 μm. PPADS also blocked UTP-evoked responses and the IC50 was 7.2±1.9 μm. At a concentration of 10 μm, 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 μm) or by PPADS (50 μm). 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 and its sensitivity to suramin and PPADS distinguish this receptor from P2U-purinoceptors found in other tissues.