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Zornia latifolia is a plant suspected to possess psychoactive properties and marketed as a marijuana substitute under the name 'maconha brava'. In this study, the effects of fractions obtained from a 2-propanol extract of aerial portions of the plant were determined by multielectrode array (MEA) analyses on cultured networks of rat cortical neurons. Lipophilic (ZL_lipo, mainly containing flavonoid aglycones), and hydrophilic (ZL_hydro, mainly containing flavonoid glycosides) fractions were initially obtained from the raw extract. ZL_lipo significantly inhibited mean firing rate (MFR) and mean bursting rate (MBR) of MEA recordings, while ZL_hydro induced no inhibition. Column chromatography separation of ZL_lipo yielded five fractions (ZL1-ZL5), among which ZL1 induced the strongest MFR and MBR inhibition. NMR and HPLC-MS analyses of ZL1 revealed the prevalence of the common flavonoids genistein (1) and apigenin (2) (in about a 1:1 ratio), and the presence of the rare flavone syzalterin (6,8-dimethylapigenin) (3) as a minor compound. Exposures of MEA to apigenin and genistein standards did not induce the MFR and MBR inhibition observed with ZL1, whereas exposure to syzalterin standard or to a 1:9 mixture syzalterin-genistein induced effects similar to ZL1. These inhibitory effects were comparable to that observed with high-THC hashish, possibly accounting for the plant psychoactive properties. Data indicate that Z. latifolia, currently marketed as a free herbal product, should be subjected to measures of control. In addition, syzalterin showed distinctive pharmacological properties, opening the way to its possible exploitation as a neuroactive drug. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 113, 796–806. The P2X7 receptor (P2X7R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent cations and organic messengers such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg2+) and AA on P2X7R by measuring whole-cell currents and intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) dynamics in HEK293 cells stably expressing full-length P2X7R and in cells endowed with the P2X7R variant lacking the entire C-terminus tail (trP2X7R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X7R- and trP2X7R-mediated [Ca2+]i rise but did not affect the ionic currents in both conditions. Extracellular Mg2+ reduced the P2X7R- and trP2X7R-mediated [Ca2+]i rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X7R-mediated ionic current and [Na+]i rise were strongly dependent on Mg2+ concentration but occurred in a non-competitive manner. In contrast, in cells expressing the trP2X7R, the small ionic currents and [Na+]i signals were totally insensitive to Mg2+. Collectively, these results support the tenet of a functional structure of P2X7R possessing at least two distinct conductive pathways one for Ca2+ and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus. [ABSTRACT FROM AUTHOR]

Abstract: The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+-dependent vesicular glutamate release and significant Ca2+-independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R-mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals. [Copyright &y& Elsevier]

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X7 receptors, it is still unclear whether neuronal functions can be attributed to P2X7 receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X7 receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of ‘nude’ release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X7 receptors. Together, our findings suggest that (i) P2X7 receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X7 receptors are localized on glutamatergic nerve terminals; (iii) P2X7 receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca2+-dependent vesicular release; and (iv) the P2X7 receptor itself constitutes a significant Ca2+-independent mode of exit for glutamate. [ABSTRACT FROM AUTHOR]

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 μmol/L) increased endogenous glutamate or [3H]d-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode. [ABSTRACT FROM AUTHOR]