No abstract

1 We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A 2A 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. 2 NG108-15 cells hydrolyzed AMP to adenosine with the K M value of 18.8+2.2 mM and V max of 5.3+1.6 nmol min 71 10 6 cells 71 . This activity was suppressed at pH 6.5, but markedly increased at pH 8.5. 3 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 b-glycerophosphate, indicating that NG108-15 cells express ecto-ALP. 4 The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p-nitrophenylphosphate and b-glycerophosphate, with a parallel decrease in the extracellular adenosine formation. 5 Reverse transcriptase polymerase chain reaction analysis revealed that NG108-15 cells express mRNA for the tissue-nonspeci®c isozyme of ALP. 6 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 ATPinduced cyclic AMP accumulation in NG108-15 cells.

1 The eect of 2,3-butanedione monoxime (BDM), a`chemical phosphatase', on Na + /Ca 2+ exchange current (I NCX ) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 ®broblast cells expressing canine NCX1. 2 I NCX was identi®ed as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na + and 2 mM Ca 2+ in the external solution and 20 mM Na + and 433 nM free Ca 2+ in the pipette solution.3 In guinea-pig ventricular cells, BDM inhibited I NCX in a concentration-dependent manner. The IC 50 value was 2.4 mM with a Hill coecients of 1. The average time for 50% inhibition by 10 mM BDM was 124+31 s (n=5). 4 The eect of BDM was not aected by 1 mM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5 Intracellular trypsin treatment via the pipette solution signi®cantly suppressed the inhibitory eect of BDM, implicating an intracellular site of action of BDM. 6 PAM (pralidoxime), another oxime compound, also inhibited I NCX in a manner similar to BDM. 7 Isoprenaline at 50 mM and phorbol 12-myristate 13-acetate (PMA) at 8 mM did not reverse the inhibition of I NCX by BDM. 8 BDM inhibited I NCX in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9 We conclude that BDM inhibits I NCX but the mechanism of inhibition is not by dephosphorylation of the Na + /Ca 2+ exchanger as a`chemical phosphatase'.

Abstract. Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP. Ecto-5'-nucleotidase (E-5'-NT) has been considered to play a principal role in conversion of AMP to adenosine. In addition to E-5'-NT, we have recently demonstrated that ecto-alkaline phosphatase is also involved in ATP-induced P1-receptor activation through a rapid and localized adenosine production on the membrane surface. In this minireview, we describe the pharmacological profile of ecto-nucleotidase-dependent P1-receptor activation by ATP and molecular bases of preferential delivery of metabolically generated adenosine to P1 receptors. Several lines of evidence suggest that the close association between ecto-nucleotidases and P1 receptors may constitute a functional receptor for extracellular ATP, and some physiological responses to ATP would occur through this mechanism.

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 gM) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (ECm of 0.58 gM) than shape change (ECm of 0.013 FM). 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 gM (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 gM) by itself caused a shape change without aggregation in a concentrationdependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA2 receptor in rabbit platelets.