Activation of P2Y1Nucleotide Receptors Induces Inhibition of the M-Type K+Current in Rat Hippocampal Pyramidal Neurons

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Abstract
We have shown previously that stimulation of heterologously expressed P2Y1nucleotide receptors inhibits M-type K+currents in sympathetic neurons. We now report that activation of endogenous P2Y1receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1agonist adenosine 5′-[β-thio]diphosphate trilithium salt (ADPβS) inhibited M-current by up to 52% with an IC50of 84 nm. The hydrolyzable agonist ADP (10 μm) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 μm) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 μm; neither ADPβS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPβS was prevented by a specific P2Y1antagonist, MRS 2179 (2′-deoxy-N′-methyladenosine-3′,5′-bisphosphate tetra-ammonium salt) (30 μm). Inhibition of the M-current by ADPβS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPβS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1and neurogranin, whereas 16% were only P2Y1positive. These results show the presence of functional M-current-inhibitory P2Y1receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium