Involvement Of P2 Purinoceptors Research Articles

Central CO2 chemoreception: a mechanism involving P2 purinoceptors localized in the ventrolateral medulla of the anaesthetized rat.

1. The involvement of P2 purinoceptors in chemosensory function in the ventrolateral regions of the medulla oblongata was investigated in the anaesthetized rat. We have investigated the effect of antagonizing, or desensitizing, P2 receptors in the retrofacial area of the ventrolateral medulla on factors modifying respiratory activity. 2. Bilateral microinjection of suramin (50 nl, 0.02 M), a P2 purinoceptor antagonist, into the retrofacial area in the artificially ventilated rat reduced resting phrenic nerve discharge. It also markedly affected the response of the phrenic nerve to increases in arterial CO2. Under conditions of hyperoxic, hypocapnic apnoea, the mean threshold for inducing phrenic nerve activity was raised significantly (from an end-tidal CO2 of 2.5 % to 4.5 %, n = 9). 3. In addition, the slope of the respiratory response curve to increases in CO2 was reduced after suramin. A similar effect was observed after desensitization of certain P2X receptors with alphabeta-methyleneATP. As arterial levels of O2 were greater than 100 mmHg, and an equivalent pattern of response was observed in sino-aortically denervated and vagotomized animals, we believe any contribution of the peripheral chemoreceptors to be minimal. 4. Our data suggest that respiratory neurones within the retrofacial area (Botzinger complex) represent part of the central site of action of CO2 on respiration. Moreover, our observations lead us to suggest that CO2-evoked changes in respiration are mediated at least in part by P2X purinoceptors.

Involvement of P2 purinoceptors in the regulation ofdna synthesis in the neural retina of chick embryo

The activation of P2 purinoceptors induces Ca2 mobilization in the earlyembryonic chick neural retina. This purinergic Ca2 response declines parallel with thedecrease in mitotic activity during retinal development. To investigate the role of P2purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2fn2fn2triphosphate ; [Ca2]i, intracellular Ca2 concentration ;DMSO, dimethyl sulfoxide ; E, embryonic day ; EC50, concentration for half maximalexcitation ; F340, fluorescence excited at 340 nm ; F380, fluorescenceexcited at 380 nm ; F340/F380, the ratio of F340/F380 ;FCS, fetal calf serum ; fura-2 AM, fura-2 acetoxymethyl ester ; IC50, concentrationfor half maximal inhibition ; PPADS, pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid ;UTP, uridine 5-triphosphate.,4-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organcultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidineincorporation in a dose-dependent manner (IC50 : ∼70 μM). PPADS alsoreduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 μM. Exogenous ATP enhanced [3H]-thymidine incorporation in adose-dependent manner to maximally 200% of control (EC50 : ∼70 μM). Indissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with theseobservations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chickamniotic fluid, the ATP concentration was nearly 3 μM at E3 and declined to 0.15 μM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrinerelease of ATP are involved in the regulation of DNA synthesis in the neural retina at earlyembryonic stages.

Selected P2 purinoceptor modulators prevent glutamate-evoked cytotoxicity in cultured cerebellar granule neurons.

Primary cultures of granule neurons derived from cerebella of postnatal rats are endowed with Glu receptors. Glu receptor agonists exert a trophic influence on differentiating granule cells but, with maturation, the cells become vulnerable to excitatory amino acids. Here we show that the P2 purinoceptor antagonist basilen blue abolishes in rat cerebellar granule neurons the cytotoxic action of glutamate with an IC50 in the 10-20 microM range. Within the same concentrations, basilen blue inhibits binding of [3H] ATP to cerebellar granule cells, glutamate-evoked release (but not uptake) of [3H] D-aspartate and Ca2+ uptake. Furthermore, the extracellular phosphorylation of a major 45-kDa endogenous ecto-protein substrate of cerebellar granule neurons is inhibited with an IC50 of about 1 microM. Similar effects are elicited by 5-adenylylimidodiphosphate, a P2 purinoceptor agonist, when supplied to the neurons for 8 days previously to the addition of glutamate. Our data point to the use of P2 purinoceptor modulators as novel elements for understanding and controlling glutamate-mediated excitatory neurotoxicity and neurotransmission. We suggest a possible involvement of P2 purinoceptors in these actions.

Selected P2 purinoceptor modulators prevent glutamate‐evoked cytotoxicity in cultured cerebellar granule neurons

Primary cultures of granule neurons derived from cerebella of postnatal rats are endowed with Glu receptors. Glu receptor agonists exert a trophic influence on differentiating granule cells but, with maturation, the cells become vulnerable to excitatory amino acids. Here we show that the P2 purinoceptor antagonist basilen blue abolishes in rat cerebellar granule neurons the cytotoxic action of glutamate with an IC50 in the 10–20 μM range. Within the same concentrations, basilen blue inhibits binding of [3H]ATP to cerebellar granule cells, glutamate-evoked release (but not uptake) of [3H] D-aspartate and Ca2+ uptake. Furthermore, the extracellular phosphorylation of a major 45-kDa endogenous ecto-protein substrate of cerebellar granule neurons is inhibited with an IC50 of about 1 μM. Similar effects are elicited by 5-adenylylimidodiphosphate, a P2 purinoceptor agonist, when supplied to the neurons for 8 days previously to the addition of glutamate. Our data point to the use of P2 purinoceptor modulators as novel elements for understanding and controlling glutamate-mediated excitatory neurotoxicity and neurotransmission. We suggest a possible involvement of P2 purinoceptors in these actions. © 1996 Wiley-Liss, Inc.

ATP-activated cationic and anionic conductances in cultured rat hippocampal neurons

The effects of adenosine 5′-triphosphate (ATP) on voltage-clamped and dissociated rat hippocampal neurons was investigated. Over 30% of neurons possessed ATP-activated inward currents at a holding potential of −70 mV. The ED 50 for these currents was 150 μM. At this concentration they were blocked by suramin (1 mM), indicating the involvement of P2-purinoceptors. The P2 purinoceptor agonist potency was 2-methylthio ATP > ATP > ADP > α,β-methylene ATP, thus identifying these purinoceptors as belonging to the P2x subclass. The reversal potential for the ATP-activated currents was −45 ± 8 mV. Ion substitution experiments showed that the permeability ratio for K +/Na +/Cs +/Cl −, was 18:3:2:1, according to the Goldman-Hodgkin-Katz equation, so that ATP activates cationic and anionic conductances in hippocampal neurons.