ATP (1 mM) induced a biphasic increase in intracellular Ca 2~concentration ([Ca2~]), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[17~-3methoxyestra-1,3, 5(10)-trien -1 7-yl]amino]hexyl]-1Hpyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca2~.We examined the mechanism of the AlP-elicited sustained [Ca2~], increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC 50 of ATP was -~0.75mM. The permeability ratio of various cations carrying this current was Na(defined as 1)> Li(0.92 ± 0.01; n = 5)> K(0.89 ± 0.03; n = 6) > Rb(0.55 ± 0.02; n = 6) > Cs~(0.51 ± 0.01; n = 5) > Ca~(0.22 ± 0.03; n = 3) > N-methyl-n-glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg Abbreviations used: ADP~3S, adenosine 5 '-O-(2-thiodiphosphate); AMPCPP, a~8-methylene-ATP;AMPPCP,~3,y-methylene-ATP; ATPyS, adenosine 5 '-O-(3-thiotriphosphate); BzATP, benzoylbenzoic-ATP; [Ca 2~]~, intracellular Ca2~concentration; I-V, current-voltage; 1P3, inositol 1 ,4,5-trisphosphate; 2MeSATP, 2methylthio-ATP; MS -, methanesulfonic acid; NMG N-methyl-Dglucamine; PLC, phosphoinositide-specific phospholipase C; U73122, 1-[6-[17~3-3-methoxyestra-l,3,5(10)-trien-l7-yl]amino]hexyl ] -lH-pyrrole-2,5-dione.

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1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.

Abstract. Ecto-5′-nucleotidase (NT5E), a predominant enzyme that produces extracellular adenosine from AMP, plays an important role in a variety of physiological and pathophysiological processes. This study was performed to identify agents that affect NT5E activity using C6 glioma cells. When cells were incubated with sodium nitroprusside (SNP), phorbol 12-myristate 13-acetate, forskolin, lipopolysaccharide, or interferon-γ, only SNP inhibited NT5E activity in a time-and concentration-dependent manner (IC 50 = 1.2 μM). The inhibitory effect of SNP was long-lasting even after SNP washout; and its action was not mimicked by nitric oxide generating agents, 8-bromo cyclic GMP, ferricyanide, ferrocyanide, or sodium cyanide. SNP did not change NT5E mRNA level or membrane surface protein expression. Similar to SNP, Fe 2+ inhibited NT5E activity, but to a lesser extent. Although Fe 2+ is known to increase oxidative stress, Fe 2+-mediated oxidative stress was not involved in SNP inhibition of NT5E because the inhibition of NT5E by SNP was not affected by superoxide dismutase and catalase. In contrast, addition of Zn 2+, an essential metal co-factor of NT5E activity, prevented SNP from inhibiting NT5E. These results suggest that SNP disrupts a critical Zn 2+ -dependent enzyme activity and might be useful as a pharmacological tool for inhibiting NT5E.

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