P2Y and P2U receptors differentially release intracellular Ca2+ via the phospholipase c/inositol 1,4,5-triphosphate pathway in astrocytes from the dorsal spinal cord.
In astrocytes, raising intracellular Ca2+ concentration is a principal mechanism for transducing extracellular signals following activation of cell-surface receptors. Receptors that may be activated by purine nucleotides, P2 receptors, are known to be expressed by astrocytes from dorsal spinal cord; these astrocytes express two distinct subtypes of P2 receptor, P2Y and P2U. A main goal of the present study was to determine the intracellular signalling pathways mediating the Ca2+ responses produced by stimulating these receptors. Experiments were done using cultured astrocytes from rat dorsal spinal cord. Ca2+ responses were evoked by 2-methylthio-ATP or UTP, nucleotides previously shown to selectively activate P2Y and P2U receptors, respectively, in these cells. P2Y- and P2U-evoked Ca2+ responses were found not to depend upon extracellular Ca2+ and were blocked by thapsigargin, a Ca2+-ATPase inhibitor known to deplete inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Intracellular application of the inositol 1,4,5-triphosphate-sensitive receptor antagonist, heparin, or of the G-protein inhibitor guanosine 5'-O-(2-thiodiphosphate), blocked the P2Y- and P2U-evoked Ca2+ responses. Moreover, the responses were prevented by the phospholipase C inhibitor, U-73122, but were unaffected by the inactive analogue, U-73343. These results indicate that P2Y and P2U receptors on dorsal spinal astrocytes are linked via G-protein coupling to release of intracellular Ca2+ via the phospholipase C/inositol 1,4,5-triphosphate pathway. When we assessed the releasable pools of intracellular Ca2+, by repeated agonist applications in zero extracellular Ca2+, we found that the pool accessed by activating P2U receptors was only a subpool of that accessed by activating P2Y receptors. This implies that there are separable inositol 1,4,5-triphosphate-releasable pools of Ca2+ in dorsal spinal astrocytes and that these may be differentially released by activating distinct metabotropic P2 receptors. This differential release of Ca2+ may be important for physiological as well as pathophysiological events occurring within the spinal cord.
Hospital for Sick Children, Department of Physiology, University of Toronto, Ontario, Canada.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D015220:Calcium Channels; D004963:Estrenes; D053496:Inositol 1,4,5-Trisphosphate Receptors; C546387:P2ry2 protein, rat; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D018160:Receptors, Cytoplasmic and Nuclear; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013873:Thionucleotides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium; D014544:Uridine Triphosphate; C008501:2-methylthio-ATP