A.M. Pugliese¹, C. Traini¹, G. Maraula¹, T. Mello², M.G. Giovannini¹, E. Coppi¹, R. Volpini³, G. Cristalli³ and F. Pedata^1
 
1Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy. ²Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Florence, Italy.
³School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy.
 
 

Extracellular adenosine-5’-triphosphate (ATP) is presently being given more and more importance as a messenger in the CNS during different physiological and pathological events exerting its effects via activation of P2 purinergic receptors: the ligand-gated ion channels P2X_(1-7) and metabotropic (P2Y_{1,2,4,6,11–14}). In the nervous system, including the hippocampus, P2X and P2Y receptors are broadly expressed on both neurons and glial cells. Evidences indicate that after brain ischemia ATP is released [1,2] and exerts a cytotoxic/deleterious role probably acting on one or more of the above mentioned receptors. Mitogen-activated protein kinases (MAP kinases) are important signaling factors in many cellular processes including cell proliferation, survival during development, synaptic plasticity and cerebral ischemia [3]. In this work the role of purinergic P2 receptors on CA1 synaptic transmission and MAP kinase activation under oxygen and glucose deprivation (OGD) was investigated. We conducted extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) evoked by a bipolar nichel-chromo stimulation electrode placed in the CA1 stratumradiatum. Electrophysiological recordings were followed by histochemical analysis (propidium iodide, PI) coupled to Western blot. Seven-min OGD (n=36) elicited an irreversible loss of fEPSP and the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage [4].
The effect of the following P2 antagonists, PPADS (unselective, 30 µM), MRS2179 (selective for P2Y₁ receptor, 10 µM), BBG (selective for P2X₇ receptor, 1 µM), A-317491 (selective for P2X₃ receptor, 10 µM) and of the new synthesized P2X₃ receptor antagonists PX21 (1 µM) and PX24 (1 µM) were then evaluated on the depression of fEPSPs and AD elicited by 7-min OGD. All antagonists significantly prevented these effects.
The extent of CA1 cell injury was assessed 3 hours after the end of 7 min OGD by PI staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 µM), MRS2179 (10 µM) and BBG (1 µM). Western blot analysis showed that 10 min after the end of the 7-min OGD, ERK1/2 MAP kinase activation was significantly increased. MRS2179, BBG, PPADS and A-317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors U0126 (10 µM) and SL327 (10 µM) showed a significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in CA1 neuronal damage induced by OGD.
In conclusion, our results demonstrate that P2Y₁, P2X₇ and P2X₃ receptors play a detrimental role in neurotransmission in CA1 hippocampus during OGD, bringing about a downstream activation of ERK1/2 that is involved in cell excitability and cell death.
 
[1] Melani, A.,et al., (2005) ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia. Neurochem.Int., 47, 442-448.
[2] Frenguelli, et al., (2007) Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus. J .Neurochem.,101, 1400-1413.
[3] Irving, E.A. and  Bamford, M. (2002) Role of mitogen- and stress-activated kinases in ischemic injury. J.Cereb.Blood Flow Metab, 22, 631-647.
[4] Somjen, G.G. (2001) Mechanisms of spreading depression and hypoxic spreading depression-like depolarization. Physiol Rev., 81, 1065-1096.