Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia.
Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.
Wang
X
X
Department of Pharmacology, University of British Columbia, Vancouver, Canada.
Kim
S U
SU
van Breemen
C
C
McLarnon
J G
JG
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D003470:Culture Media; D005453:Fluorescence; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
D003470:Culture Media; D007473:Ion Channels; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C030812:adenosine 5'-O-(2-thiodiphosphate); D008345:Manganese; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium