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P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

ABSTRACT: Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.

SUBMITTER: Soto F

PROVIDER: S-EPMC39672 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Soto F F Garcia-Guzman M M Gomez-Hernandez J M JM Hollmann M M Karschin C C Stühmer W W

Proceedings of the National Academy of Sciences of the United States of America 19960401 8

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional m ...[more]

PMID: 8622997

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Project description:Background: Mounting evidence indicate that reducing the sinoatrial node (SAN) activity may be a useful therapeutic strategy to control of heart failure. Purines, like ATP and its metabolite adenosine, consistently reduce the SAN spontaneous activity leading to negative cardiac chronotropy, with variable effects on the force of myocardial contraction (inotropy). Apart from adenosine A1 receptors, the human SAN expresses high levels of ATP-sensitive ionotropic P2X4 receptors (P2X4R), yet their cardiac role is unexplored. Methods: Here, we investigated the activity of P2 purinoceptors on isolated spontaneously beating atria (chronotropy) and on 2 Hz-paced right ventricular (RV, inotropy) strips from Wistar rats. Results: ATP (pEC 50 = 4.05) and its stable analogue ATPγS (pEC 50 = 4.69) concentration-dependently reduced atrial chronotropy. Inhibition of ATP breakdown into adenosine by NTPDases with POM-1 failed to modify ATP-induced negative chronotropy. The effect of ATP on atrial rate was attenuated by a broad-spectrum P2 antagonist, PPADS, as well as by 5-BDBD, which selectively blocks the P2X4R subtype; however, no effect was observed upon blocking the A1 receptor with DPCPX. The P2X4R positive allosteric modulator, ivermectin, increased the negative chronotropic response of ATP. Likewise, CTP, a P2X agonist that does not generate adenosine, replicated the P2X4R-mediated negative chronotropism of ATP. Inhibition of the Na+/Ca2+ exchanger (NCX) with KB-R7943 and ORM-10103, but not blockage of the HCN channel with ZD7288, mimicked the effect of the P2X4R blocker, 5-BDBD. In paced RV strips, ATP caused a mild negative inotropic effect, which magnitude was 2 to 3-fold increased by 5-BDBD and KB-R7943. Immunofluorescence confocal microscopy studies confirm that cardiomyocytes of the rat SAN and RV co-express P2X4R and NCX1 proteins. Conclusions: Data suggest that activation of ATP-sensitive P2X4R slows down heart rate by reducing the SAN activity while increasing the magnitude of ventricular contractions. The mechanism underlying the dual effect of ATP in the heart may involve inhibition of intracellular Ca2+-extrusion by bolstering NCX function in the reverse mode. Thus, targeting the P2X4R activation may create novel well-tolerated heart-rate lowering drugs with potential benefits in patients with deteriorated ventricular function.

Project description:Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E2 (PGE2) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE2 release and was attenuated by inhibition of either phospholipase A2 or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.

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P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Publications

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

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