Increases in plasma osmolality activates the paraventricular nucleus of the hypothalamus (PVN) which in turn mounts a physiological response by increasing the release of arginine vasopressin and sympathetic nerve activity to end organs such as the kidney. The PVN expresses an abundance of purinergic receptors including P2X2 receptors. In the present study, we sought to determine (1) whether P2X2-expressing PVN neurons are activated by hypertonic saline or hypertonic mannitol and (2) what effects P2X receptor blockade has on sympathetic nerve activation mediated by a hyperosmotic stimulus. Male Wistar rats were randomly assigned to three groups and intravenously infused with either isotonic saline (0.154M, 0.5mL), hypertonic saline (3M, 0.5mL) or hypertonic mannitol (10% w/v, 0.5mL). Significantly greater numbers of Fos-positive cells were observed in the hypertonic saline (393±29)- and hypertonic mannitol (141±11)-infused rats compared with control, saline-treated, rats (47±2 neurons/PVN section). Furthermore, there was a significant increase in the number of activated (Fos-positive) P2X2 expressing PVN neurons in the hypertonic saline (65±7) and hypertonic mannitol (37±7)-treated rats compared with controls (16±2). Microinjection of a P2X receptor antagonist, PPADS, within the PVN significantly attenuated sympathetic nerve activation driven by a hyperosmotic stimulus. The hyperosmotically induced increase in lumbar sympathetic nerve activity was significantly blunted after PPADS pre-treatment. Collectively, our findings indicate that hyperosmotic stimulation activates a subset of P2X2 expressing PVN neurons that might facilitate increased sympathetic drive.

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

In the present work, we used a unique cortical/striatal/subventricular zone organotypic model in order to analyze the role of resident microglia in oxygen/glucose deprivation and to check the presence and modulation of several P2 receptors in the cortex. Immunofluorescence with the microglial marker OX42 and pharmacological experiments with indomethacin indicate that activation and recruitment of microglia after the insult is linked to cellular loss, mainly in the cortex. The confocal analysis with OX42 shows that, among the P2 receptors tested, P2X4, and P2X7 are expressed on microglia, while P2X1 and P2Y(1-2-12), although present in the slices, did not co-localize, whereas P2X6 is not detected. The upregulation of P2X4 and P2X7 on microglia and the toxic effect that different P2 agonists exert on cortical slices during oxygen/glucose deprivation indicate that a purinergic mechanism is related to the microglia activity; the protective effect of the P2 antagonist TNP-ATP is also described. In order to better understand the relationship between P2 receptors and OGD-activated microglia, we induced oxygen/glucose deprivation in co-cultures of organotypic slices and N9 microglia cell line. The presence of the N9 (which expresses P2X4 and P2X7 protein) in the cultures increases the damage in the cortex by 40% and the use of P2 antagonist PPADS reduced the cell damage due to the N9 activation. Our results show that microglia recruitment after a metabolic impairment is associated with cellular loss and that P2X4 and P2X7, are involved in microglia activity. The neuroprotective action exerted by TNP-ATP and PPADS and the possible use of purinergic antagonist in the pharmacological treatment of oxygen/glucose deprivation is also addressed.

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.