ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca(2+)-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca(2+)](i) response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X(7) receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X(7) receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca(2+) influx that contributed a small component to the total [Ca(2+)](i) response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.
The expression of purinoceptor (P2)Y-subtypes on astrocytes in vivo under physiological conditions and after stab wound injury was investigated. Reverse transcriptase-polymerase chain reaction with specific primers for the receptor-subtypes P2Y1,2,4,6,12 in tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all P2Y receptor mRNAs investigated. Double immunofluorescence visualized with laser scanning microscopy indicated the expression of the P2Y1,4 receptors on glial fibrillary acidic protein (GFAP)-labeled astrocytes under physiological conditions. After stab wound injury the additional expression of the P2Y2 and P2Y6 receptors, and an up-regulation of the P2Y1,4 receptor-labeling on astrocytic cell bodies and/or processes was observed. Astrocytes of cortical, in contrast to accumbal areas exhibited P2Y1,2,4,6 receptor-immunoreactivity (IR) under control conditions, which was up-regulated after stab would injury. Labeling for the P2Y12 receptor was not observed on GFAP-positive cortical and accumbal astrocytes under any of the conditions used. For the first time, the co-localization of different P2 receptor-subtypes (e.g. P2Y1 and P2X3) on the same astrocyte was shown immunocytochemically. The up-regulation of P2Y1 receptor-IR on astrocytes and non-glial cells after mechanical injury could be facilitated by microinfusion of the P2Y1,12,13 receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS). Proliferative changes after ADPbetaS-microinjection were characterized by means of double-staining with antibodies against GFAP and 5-bromo-2'-deoxyuridine. The non-selective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, the P2Y1 receptor antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate and the P2Y1 receptor-antibody itself inhibited the agonist-induced effects. The data indicate the region-specific presence of P2Y receptors on astrocytes in vivo and their up-regulation after injury as well as the co-localization of P2X and P2Y receptor-subtypes on the same astrocyte. The dominant role of P2Y1 receptors in proliferation and the additional stimulation of non-P2Y1 receptors has been demonstrated in vivo suggesting the involvement of this receptor-type in the gliotic response under physiological and pathological conditions.
Membrane currents and changes in the intracellular Ca ] i was observed after the slow superfusion of ATP, ADPb-S and UTP; a,b-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to a,b-meATP is due to P2X 3 receptor activation, while the ATP-induced rise in [Ca 2+ ] i is evoked by P2Y 1 and P2Y 4 receptor activation. TCE depressed the a,b-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca 2+ ] i was much less sensitive to the inhibitory effect of TCE than the current response to a,b-meATP. The present study indicates that in HEK293-hP2X 3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X 3 receptors and, in addition, moderately interferes with G protein-coupled P2Y 1 and P2Y 4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.
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