P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca(2+) rise and change in mitochondrial potential.
With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca(2+) level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y(1/2/4) receptor agonist in rat, caused a large cytosolic Ca(2+) increase in astrocytes and a considerably smaller Ca(2+) response in neighboring neurons. The P2Y(1) receptor antagonist MRS2179 completely blocked the ATP-induced Ca(2+) response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca(2+) level. Stimulation with UDP, a P2Y(6) receptor agonist; UTP, a P2Y(2/4) receptor agonist; 2MeSATP, a P2Y(1) receptor agonist; or 2MeSADP, a P2Y(1/12/13) receptor agonist, evoked significant Ca(2+) responses in astrocytes but small Ca(2+) responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca(2+) peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca(2+) level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain.
Kahlert
Stefan
S
Institut für Neurobiochemie, Medizinische Fakultät der Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany.
Blaser
Till
T
Tulapurkar
Mohan
M
Reiser
Georg
G
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*
C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; D009711:Nucleotides; D017382:Reactive Oxygen Species; D018048:Receptors, Purinergic P2; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium