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

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 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 ...

The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization

“…In astrocytes from the dorsal spinal cord of the rat, PPADS inhibited UTP-induced changes in [Ca2+]i (Ho et al, 1995). This type of observation has led to the suggestion that there may be PPADS-sensitive and PPADS-insensitive P2U-purinoceptors (Ho et al, 1995). This possibility was not substantiated by molecular data.…”

“…In bovine aortic endothelial cells, PPADS inhibits the action of selective agonists of P2Y-purinoceptors (adenosine 5'-diphosphate (ADP) and 2-methylthio ATP) on phospholipase C activity; it did not inhibit the actions of agonists of P2U-purinoceptors (Brown et al, 1995). However, different results have been obtained in astrocytes from the dorsal spinal cord of the rat (Ho et al, 1995). In these cells, PPADS inhibited uridine 5'-triphosphate (UTP) as well as 2-methylthio ATP induced intracellular Ca2+ mobilization.…”

“…In aortic endothelial cells, PPADS did not inhibit UTP-induced activation of phospholipase C and it was suggested to be a selective antagonist of P2YI-purinoceptor responses (Brown et al, 1995). In astrocytes from the dorsal spinal cord of the rat, PPADS inhibited UTP-induced changes in [Ca2+]i (Ho et al, 1995). This type of observation has led to the suggestion that there may be PPADS-sensitive and PPADS-insensitive P2U-purinoceptors (Ho et al, 1995).…”

“…In these cells, PPADS inhibited uridine 5'-triphosphate (UTP) as well as 2-methylthio ATP induced intracellular Ca2+ mobilization. This led to the suggestion that astrocytes expressed a UTP receptor distinct from the P2U-purinoceptor of aortic endothelial cells (Ho et al, 1995).…”

Pharmacological characterization of uracil nucleotide-preferring P2Y receptors modulating intestinal motility: a study on mouse ileum

“…Altogether, these results may allow us to exclude even the involvement of P2Y 4 receptors in the contractile effects induced by UTP. Therefore, UTP contractile responses appear to be mediated by atypical UTP-sensitive receptors, resembling those described in the dorsal spinal astrocytes of rats [35] and in the superior cervical ganglion neurons of mice [36]. However, the pharmacological profile of UTP contractile responses is the same as UTPγS, which, as already underlined, is nominally a selective P2Y 2 / 4 receptors agonist.…”