Satoko Ohkubo

Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response.

We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A2A receptors in NG108‐15 cells. Furthermore, the adenosine formation was found to be mediated by an ecto‐enzyme distinct from the ecto‐5′‐nucleotidase (CD73). In this study, we investigated the properties of the ecto‐AMP phosphohydrolase activity in NG108‐15 cells. NG108‐15 cells hydrolyzed AMP to adenosine with the KM value of 18.8±2.2 μM and Vmax of 5.3±1.6 nmol min−1 106 cells−1. This activity was suppressed at pH 6.5, but markedly increased at pH 8.5. The AMP hydrolysis was blocked by levamisole, an alkaline phosphatase (ALP) inhibitor. NG108‐15 cells released orthophosphate from 2′‐ and 3′‐AMP as well as from ribose‐5‐phosphate and β‐glycerophosphate, indicating that NG108‐15 cells express ecto‐ALP. The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p‐nitrophenylphosphate and β‐glycerophosphate, with a parallel decrease in the extracellular adenosine formation. Reverse transcriptase polymerase chain reaction analysis revealed that NG108‐15 cells express mRNA for the tissue‐nonspecific isozyme of ALP. These results demonstrate that AMP phosphohydrolase activity in NG108‐15 cells is due to ecto‐ALP, and suggest that this enzyme plays an essential role for the P1 antagonist‐sensitive ATP‐induced cyclic AMP accumulation in NG108‐15 cells.

Inhibitory effect of 2,3‐butanedione monoxime (BDM) on Na+/Ca2+ exchange current in guinea‐pig cardiac ventricular myocytes

The effect of 2,3‐butanedione monoxime (BDM), a ‘chemical phosphatase’, on Na+/Ca2+ exchange current (INCX) was investigated using the whole‐cell voltage‐clamp technique in single guinea‐pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. INCX was identified as a current sensitive to KB‐R7943, a relatively selective NCX inhibitor, at 140 mM Na+ and 2 mM Ca2+ in the external solution and 20 mM Na+ and 433 nM free Ca2+ in the pipette solution. In guinea‐pig ventricular cells, BDM inhibited INCX in a concentration‐dependent manner. The IC50 value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124±31 s (n=5). The effect of BDM was not affected by 1 μM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid‐sensitive protein phosphatases. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. PAM (pralidoxime), another oxime compound, also inhibited INCX in a manner similar to BDM. Isoprenaline at 50 μM and phorbol 12‐myristate 13‐acetate (PMA) at 8 μM did not reverse the inhibition of INCX by BDM. BDM inhibited INCX in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. We conclude that BDM inhibits INCX but the mechanism of inhibition is not by dephosphorylation of the Na+/Ca2+ exchanger as a ‘chemical phosphatase’.

Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1 Thromboxane A2 (TXA2) receptor‐mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2 The TXA2 agonist, U46619 (1 nM to 10 μ) caused shape change and aggregation in a concentration‐dependent manner. A forty‐times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 μ) than shape change (EC50 of 0.013 μ). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3 SQ29548 at 30 nM and GR32191B at 0.3 μ (TXA2 receptor antagonists) competitively inhibited U46619‐induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor‐mediated events. However, ONO NT‐126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619‐induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4 ONO NT‐126 (2 nM to 3 μ) by itself caused a shape change without aggregation in a concentration‐dependent manner, independent of external Ca2+. Therefore, ONO NT‐126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5 U46619 (10 nM to 10 μ) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration‐dependent manner with a similar concentration‐dependency. 6 U46619 (3 nM to 10 μ) also activated GTPase concentration‐dependently in the membranes derived from platelets. U46619‐induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7 The EC50 values of U46619 in Ca2+ mobilization (0.15 μ), PI hydrolysis (0.20 μ) and increase in GTPase activity (0.12 μ) were similar, but different from the EC50 value in shape change (0.013 μ), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8 U46619‐induced shape change was unaffected by W‐7 (30 μ), a calmodulin antagonist or ML‐7 (30 μ), a myosin light‐chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9 [3H]‐SQ29548 and [3H]‐U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/108 platelets and Kd value of 129.8 nM and Bmax of 170.4 fmol/108 platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H‐ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P<0.001 by Students t test) from the effect on shape change. 10 Neither U46619 nor ONO NT‐126 affected the adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11 The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq‐phospholipase C‐Ca2+ pathway.

Thromboxane A2 stimulates mitogen-activated protein kinase and arachidonic acid liberation in rabbit platelets.

U46619, a thromboxane A2 mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of 32P to a specific substrate for MAPK. U46619 also liberated [3H] arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [3H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca2+ ions. This is a first demonstration that TXA2 activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.

β,γ‐Methylene ATP‐induced cAMP formation in C6Bu‐1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor

The mechanism underlying β,γ‐methylene ATP (β,γ‐MeATP)‐induced cAMP elevation was investigated in rat glioma C6Bu‐1 cells. β,γ‐MeATP increased forskolin‐stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine‐induced response, did not eliminate the β,γ‐MeATP‐induced response. However, combination of ADA with α,β‐methylene ADP (α,β‐MeADP), an ecto‐5′‐nucleotidase inhibitor, blocked the β,γ‐MeATP‐induced response. AMP, the substrate for ecto‐5′‐nucleotidase, also induced cAMP formation in a manner sensitive to XAC and α,β‐MeADP inhibition. However, the AMP‐induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from β,γ‐MeATP and AMP. In addition, α,β‐MeADP inhibited the conversion of β,γ‐MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from β,γ‐MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto‐phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto‐5′‐nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT‐PCR. These results suggest that C6Bu‐1 cells possess ecto‐enzymes converting β,γ‐MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.

Baicalein inhibits Raf-1-mediated phosphorylation of MEK-1 in C6 rat glioma cells.

Baicalein is a flavonoid derived from the Scutellaria root. In investigations of the inhibitors of prostaglandin synthesis in C6 rat glioma cells, we found that baicalein had a potent inhibitory activity on prostaglandin synthesis induced by either histamine or A23187, a Ca(2+) ionophore. Baicalein inhibited histamine- or A23187-induced phosphorylation of p42/p44 extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), which causes the phosphorylation of cytosolic phospholipase A(2) (PLA(2)). Baicalein also inhibited the phosphorylation of MAPK kinase-1 (MEK-1) induced by histamine or A23187 in the cells. To examine the site of action of baicalein, MEK-1 and Raf-1 were prepared by immunoprecipitation with anti-MEK-1 and anti-Raf-1 antibodies, respectively. Baicalein inhibited the phosphorylation of exogenous MEK-1 by Raf-1 under cell-free conditions, while it did not change the phosphorylation of exogenous p42 MAPK by MEK-1. These results imply that baicalein inhibits the ERK/MAPK cascade, acting on the phosphorylation of MEK-1 by Raf-1.

P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y2 receptors are known to couple with Gq family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca2+ ([Ca2+]i) levels. In the present study, we investigated the involvement of lipid rafts in P2Y2 receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Gαq/11 and a part of P2Y2 receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-β-cyclodextrin (CD) disrupted not only lipid raft markers but also Gαq/11 and P2Y2 receptors in this fraction. In the presence of CD, P2Y2 receptor-mediated phosphoinositide hydrolysis and [Ca2+]i elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the Gq/11-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,–20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115–regulator of G protein signaling, which inhibits the G12/13 signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the Gq/11 signaling pathway. These results suggest that lipid rafts are critical for P2Y2 receptor-mediated Gq/11–PLC–Ca2+ signaling and this cascade is important for cell migration in NG 108-15 cells.

Effects of P1 and P2 receptor antagonists on β,γ‐methyleneATP‐ and CGS21680‐induced cyclic AMP formation in NG108‐15 cells

We have previously shown that ATP increased cyclic AMP in NG108‐15 cells, which was inhibited by P1 receptor antagonist methylxanthines. In the present study, we examined the effects of P1 and P2 receptor antagonists on cyclic AMP formation induced by β,γ‐methyleneATP (β,γ‐MeATP) and CGS21680, an A2A adenosine receptor agonist, in NG108‐15 cells. β,γ‐MeATP and CGS21680 increased intracellular cyclic AMP with EC50 values of 8.0±0.98 μM (n=4) and 42±7.5 nM (n=4), respectively. Several P1 receptor antagonists inhibited both β,γ‐MeATP‐ and CGS21680‐induced cyclic AMP increase with a similar rank order of potency; ZM241385>CGS15943>XAC>DPCPX. However, the pKi values of these antagonists for β,γ‐MeATP were larger than those for CGS21680. Alloxazine, a P1 receptor antagonist, and several P2 receptor antagonists (PPADS, iPPADS, reactive blue‐2) inhibited β,γ‐MeATP‐induced response, while these antagonists little affected CGS21680‐induced one. Suramin was effective only for β,γ‐MeATP‐induced response at 1 mM. 2‐chloroadenosine (2CADO) and 2‐chloroATP (2ClATP) increased cyclic AMP with similar potencies. The effects of these agonists were both inhibited by ZM241385, but only 2ClATP‐induced response was inhibited by PPADS. ATP‐ and β,γ‐MeATP‐induced responses were little affected by α,β‐methyleneADP, a 5′‐nucleotidase inhibitor. These results clearly demonstrate that ATP‐stimulated cyclic AMP formation can be distinguished from the A2A receptor agonist‐induced one by using the several P1 and P2 receptor antagonists.

Inhibition of ATP-induced cAMP formation by 5’-p-fluorosulfonylbenzoyladenosine in NG108-15 cells

ATP is known to increase intracellular cAMP levels in NG108-15 cells via a novel purinoceptor and this response is inhibited by the P1 purinoceptor antagonist methylxanthine. In the present study, we examined the effects of 5’-p-fluorosulfonylbenzoyladenosine (FSBA), an affinity ligand for ATP-binding proteins, on cAMP formation mediated by activation of adenylate cyclase (AC)-linked purinoceptors in NG108-15 cells. cAMP levels were determined by RIA using an anti-succinyl-cAMP antiserum. FSBA (100 µM) increased intracellular cAMP about 2.6-fold. However, FSBA-induced cAMP formation was abolished by pretreatment with adenosine deaminase, suggesting that adenosine, a breakdown product of FSBA, is involved in FSBA-induced cAMP formation. In contrast, pretreatment of cells with FSBA in the presence of adenosine deaminase inhibited cAMP formation induced by ATP and β,γ-methylene-ATP (β,γ-MeATP), without affecting the prostaglandin E1 (PGE1)-induced response. The inhibitory effect of FSBA on ATP-induced cAMP formation was concentration-dependent with a concentration required for half-maximal inhibition (IC50) of around 3 µM. The inhibitory effect of FSBA was not affected by pertussis toxin (PTX)-treatment. Pretreatment with FSBA (10 µM) depressed the maximal response to β,γ-MeATP by 60%, but did not affect the response to 5’-N-ethylcarboxamidoadenosine. The inhibitory effect of FSBA (100 µM) increased time-dependently during pretreatment and partly resisted wash-out. The inhibition by FSBA was protected by simultaneous addition of β,γ-MeATP during the FSBA pretreatment, indicating that both FSBA and the ATP analogue interacted with the same receptor site. The pretreatment with FSBA did not affect the increase in [Ca2+]i induced by ATP, UTP or benzoylbenzoic ATP. These results suggest that FSBA inhibits cAMP accumulation induced in NG108-15 cells by ATP or related agonists by selective modification of an AC-linked purinoceptor.

Effects of AMP derivatives on cyclic AMP levels in NG108-15 cells

In NG108‐15 neuroblastoma×glioma hybrid cells, ATP stimulates intracellular cyclic AMP formation, which is inhibited by both adenosine (P1) and P2 receptor antagonists. In the present study, we examined the effects of several AMP derivatives in NG108‐15 cells and mouse neuroblastoma N18TG‐2 cells. Adenosine 2′‐monophosphate (A2P), adenosine 3′‐monophosphate (A3P) and adenosine 5′‐phosphosulphate (A5PS) increased cyclic AMP levels with similar concentration‐dependencies in NG108‐15 cells. Increases in cyclic AMP by AMP derivatives were inhibited by the P2 receptor antagonist PPADS, but not by suramin. Effects of AMP derivatives were also inhibited by P1 receptor antagonists ZM241385, XAC, DPCPX and partially by alloxazine. The ecto‐nucleotidase inhibitor α,β‐methyleneADP was without effect. In contrast, AMP derivatives did not change cyclic AMP levels in N18TG‐2 cells. Accumulation of cyclic AMP in N18TG‐2 cells was stimulated by adenosine A2 receptor agonists CGS21680 and NECA, but not by ATP or β,γ‐methyleneATP, agonists for cyclic AMP production in NG108‐15 cells. Reverse transcription‐coupled polymerase chain reaction (RT–PCR) analyses revealed that N18TG‐2 cells express both A2A and A2B receptors, while NG108‐15 cells express mainly A2A receptors. AMP derivatives did not affect the P2X and P2Y receptors expressed in NG108‐15 cells. These results suggest that A2P, A3P and A5PS act as agonists for cyclic AMP production and that these compounds are valuable tools for determinating the mechanism of ATP‐stimulated cyclic AMP response in NG108‐15 cells.