Department of Bio- and Geoscience, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan1
Author for correspondence: Toshio Tanaka. Tel: +81 6 6605 3163. Fax: +81 6 6605 3164. e-mail: tanakato{at}sci.osaka-cu.ac.jp
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ABSTRACT |
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Keywords: Saccharomyces cerevisiae, plasma membrane H+-ATPase, signal transduction, nucleotide analogue
Abbreviations: AMPC12, adenosine 5'-dodecylphosphate; AMPC16, adenosine 5'-hexadecylphosphate; AMPC20, adenosine 5'-eicosylphosphate; DAG, diacylglycerol; DES, diethylstilboestrol; PKC, protein kinase C; UMPC12, uridine 5'-dodecylphosphate; UMPC16, uridine 5'-hexadecylphosphate; UMPC20, uridine 5'-eicosylphosphate
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INTRODUCTION |
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METHODS |
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Synthesis of uridine and adenosine 5'-alkylphosphates.
The following nucleotide analogues were synthesized as described previously (Machida et al., 1997 ): uridine 5'-dodecylphopshate (UMPC12), UMPC16, adenosine 5'-dodecylphosphate (AMPC12) and AMPC16. In the present study, uridine 5'-eicosylphosphate (UMPC20) was synthesized by exactly following the procedure for UMPC16 synthesis except that eicosyl alcohol was used as the alkyl donor in the reaction. Shiraishi et al. (1994)
have reported the synthesis of adenosine 5'-eicosylphosphate (AMPC20) by a condensation reaction between AMP and eicosyl alcohol, but we synthesized it by following our previously described method using AMP and the corresponding alkylbromide. 1H- and 13C-NMR spectral data of UMPC20 and AMPC20 were obtained using a JEOL JNM-400GX spectrometer in DMSO-d6 and showed good correlation to those obtained with UMPC16 (Machida et al., 1997
) and AMPC20 (Shiraishi et al., 1994
), respectively. The absolute chemical structures of UMPC20 and AMPC20 were ultimately confirmed by their mass spectra, with m/z 603 for C29H52N2O9P (M+-1) and m/z 626 for C30H53N5O7P (M+-1), respectively. Fig. 1
shows the chemical structures of these AMP analogues together with the structure of UMPC16.
HPLC analysis of AMPC16.
Cells of Sac. cerevisiae IFO 0203 were grown in YPD medium overnight at 30 °C, harvested and washed with 50 mM succinate buffer (pH 6·5). Washed cells were then suspended at the cell density of 107 ml-1 in the same buffer containing AMPC16 at 12·5 µg ml-1 and incubated with shaking at 30 °C. At various times, portions of the cell suspension were withdrawn and subjected to centrifugal filtration in a membrane-equipped tube (ULTRAFREE-MC, Millipore). Each filtrate was then assayed for its content of AMPC16 by HPLC on an ODS reverse phase column (6x150 mm) at the wavelength of 260 nm. Isocratic elution was done at room temperature with 5 mM (NH4)H2PO4/methanol (20:80, v/v) and a flow rate of 1·0 ml min-1.
Assay of glucose-induced proton extrusion.
Plasma membrane H+-ATPase activity was assayed in vivo by measuring the extracellular acidification due to glucose-induced proton extrusion by the intact cells, as described by Brandão et al. (1994) . Cells were grown overnight in YPG medium at 30 °C, collected by centrifugation and washed several times with distilled water. Washed cells were then suspended in distilled water at the cell density of 108 ml-1 and the pH of the cell suspension was adjusted to 7·0 with dilute sodium hydroxide. After preincubating the cell suspension at 30 °C for 10 min, glucose was added at the final concentration of 100 mM together with each of the other chemicals at the indicated concentrations. The pH of the cell suspension was measured using a Horiba pH meter model D-23 at 30 °C.
Preparation of plasma membrane fraction.
Cells were grown in YPG medium at 30 °C to mid-exponential phase, washed and suspended in 100 mM N-[tris(hydroxymethyl)methyl]glycine buffer containing 5 mM EDTA and 2 mM DTT at the cell density of 108 ml-1. The cell suspension was incubated at 30 °C for 10 min with supplementation with 100 mM glucose to fully activate the plasma membrane H+-ATPase (Brandão et al., 1994 ). Cells were then collected, washed and resuspended in the above buffer at the cell density of 1·52·0x108 ml-1, and then immediately frozen in liquid nitrogen and stored until use. The plasma membrane fraction was purified by ultracentrifugation according to the procedure described by Becher dos Passos et al. (1992)
. To measure the plasma membrane H+-ATPase activity in the cells with or without AMPC16 treatment, the plasma membrane fractions were prepared from the cells that had been incubated in distilled water containing both 100 mM glucose and 10 mM MgSO4 (adjusted to pH 7·0) at 30 °C for 10 or 30 min as well as from cells that had been further supplemented with 25 µg AMPC16 ml-1.
Assay of plasma membrane H+-ATPase.
The enzyme activity was assayed on the basis of ATP hydrolysis by the plasma membrane preparation according to the method of Rosa & Sá-Correia (1991) with a slight modification as follows. The standard reaction mixture contained 10 µmol ATP, 10 µmol MgSO4, 50 µmol KCl and plasma membrane preparation equivalent to 0·30·4 mg protein in 1·0 ml 50 mM Tris/Mes buffer (pH 6·0). KNO3, NaN3 or ammonium molybdate was added at 200 µM to inhibit vacuole-type ATPase, mitochondrial ATPase and acid phosphatase, respectively. After incubating the mixture at 30 °C for 10 min, inorganic phosphate liberated in the reaction was measured with a colorimetric phosphate assay kit (Wako Pure Chemicals). One unit of ATPase activity was defined as the amount of enzyme that liberated 1·0 nmol inorganic phosphate min-1 (mg protein)-1. Protein was measured according to the method of Bradford (1976)
using BSA as a standard.
Measurement of intracellular DAG level.
Cells were grown in YPG medium at 30 °C to mid-exponential phase, washed and suspended in distilled water containing both 100 mM glucose and 10 mM MgSO4 (adjusted to pH 7·0) at the cell density of 107 cells ml-1 and incubated at 30 °C with or without further supplementation with AMPC16 at 25 µg ml-1. At various times, 30 ml portions were withdrawn and the cells were collected by centrifugation. Lipids were extracted from the cell pellets by following our previously described method (Machida et al., 1999 ) for measurement of the DAG content using a radioenzymic assay (Preiss et al., 1986
).
Chemicals.
The following chemicals were purchased from Sigma: AMP, UMP, cerulenin, diethylstilboestrol (DES), amphotericin B, staurosporine and 1-oleoyl-2-acetyl-sn-glycerol (a membrane-permeable DAG analogue). The other chemicals were of analytical reagent grade.
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RESULTS AND DISCUSSION |
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Lapathitis & Kotyk (1998) reported that the glucose-induced extracellular acidification depends on both proton extrusion resulting from the action of the plasma membrane H+-ATPase and the cellular excretion of glucose-derived carbon dioxide and organic acids. In that study, the extent of contribution of the latter process was examined by using strain Y55 and its isogenic mutant, pmal-105, with a genetic lack of the enzyme. This process was estimated to contribute only 6·9% of the overall glucose-induced acidification on the basis of the rate of increase in the extracellular proton concentration predicted from the pH value. As shown in Fig. 4(a)
, the glucose-induced extracellular acidification initially proceeded without apparent inhibition in AMPC16-treated cells, but was later drastically inhibited in a dose-dependent manner. As can be deduced from the pH values (predicted proton concentrations) at the 30 min incubation, the extracellular acidification was inhibited by 25 µg ml-1 AMPC16 much more than expected from an inhibition of the glucose-induced process with no relation to plasma membrane H+-ATPase. The extracellular pH change was not provoked by incubating the cells only with AMPC16 at 25 µg ml-1 and 10 mM MgSO4, but was initiated with a similar inhibition pattern after addition of glucose. AMPC16 could cause complete cell death at 25 µg ml-1 in such an experiment using an increased cell density (108 cells ml-1), suggesting a correlation between its inhibitory effect on the action of plasma membrane H+-ATPase and the Mg2+-dependent cytotoxicity. The glucose-induced extracellular acidification was also markedly inhibited by 200 µM DES (Fig. 4b
), which can directly interfere with the action of plasma membrane H+-ATPase (Wach et al., 1990
). The extent of extracellular acidification, possibly due to excretion of carbon dioxide and organic acids, was likewise estimated to be less than 10% after a 30 min incubation. AMPC16 was not likely to exhibit a protonophoric activity such as promoting passive influx of extracellular protons since the extracellular pH remained unchanged in the cell suspension with no glucose when the initial pH was adjusted to 5·0. These findings indicate that the inhibition of glucose-induced extracellular acidification is due to the inhibitory effect of AMPC16 on the proton pumping function of the plasma membrane H+-ATPase. As already suggested, based on its mode of action, cerulenin did not cause any inhibition of the glucose-induced process during 30 min incubation (Fig. 4c
). Amphotericin B exerts an antifungal effect by causing damage of the plasma membrane phospholipid bilayers as a result of its complex formation with ergosterol (Herve et al., 1989
). Its inhibitory effect on the cellular proton extrusion thus could be a secondary effect resulting from the plasma membrane disruption (Fig. 4d
).
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Relationship between AMPC16-induced events and DAG
The plasma membrane H+-ATPase from Sac. cerevisiae is phosphorylated on multiple serine and threonine residues during its movement into the cell surface from the endoplasmic reticulum (Chang & Slayman, 1991 ). Phosphorylation of the enzyme is associated with an increased ATPase activity during growth on glucose. Upon glucose starvation, dephosphorylation occurs, together with a decrease in enzymic activity, and both are rapidly reversed upon re-addition of glucose. Site-specific phosphorylation is likely to regulate the ATPase activity in response to nutritional signals. It is known that the cellular cAMP level shows a rapid increase under the above conditions in which H+-ATPase-dependent proton extrusion occurs at the fully stimulated rate (Morishita et al., 1995
), suggesting a cAMP-dependent mechanism for the site-specific phosphorylation of the enzyme. However, yeast strains that are specifically deficient in the glucose-induced cAMP increase still show normal activation of plasma membrane H+-ATPase (Becher dos Passos et al., 1992
). Moreover, yeast mutants with widely divergent levels of cAMP-dependent protein kinase activity display very similar levels of activation of the enzyme.
On the other hand, the addition of a membrane-permeable DAG analogue or other activators of protein kinase C (PKC) (Ogita et al., 1990 ) to intact cells activates the H+-ATPase and at the same time causes a stimulation of proton extrusion from the cells (Brandão et al., 1994
). Both effects are reversed by the addition of staurosporine, a PKC inhibitor, which can cause a more potent inhibition of the enzyme activation when added together with calmidazolium, an inhibitor of Ca2+/calmodulin-dependent protein kinase (Brandão et al., 1994
). These results have suggested a model that explains glucose-induced activation of the enzyme via a phosphatidylinositol-type signalling pathway triggering phosphorylation of the enzyme by both PKC and Ca2+/calmodulin-dependent protein kinase. As shown in Fig. 5(a)
, the exogenous addition of DAG analogue could largely cancel the inhibitory effect of AMPC16 on cellular proton extrusion although its protective effect was not enhanced at a higher concentration (data not shown). The DAG analogue did not protect cells against the inhibition of glucose-induced proton extrusion by staurosporine (Fig. 5b
). These results agree with the above model in which AMPC16 acts as an antagonist of phosphatidylinositol-type signalling that mediates glucose-induced activation of plasma membrane H+-ATPase.
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Received 11 October 1999;
accepted 25 October 1999.
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