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Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa
Cell Biology International 29 (2005) 559e566 www.elsevier.com/locate/cellbi
Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa Lucielli Savegnago, Cristina W. Nogueira, Roselei Fachinetto, Joao Batista Teixeira Rocha * Departamento de Quimica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil Received 16 September 2004; revised 19 January 2005; accepted 8 March 2005
Abstract The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2C and Ca2C. The inhibitors ouabain (0.01e1 mM), N-ethylmaleimide (0.01e4 mM), levamisole (0.10e0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03e30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50e300 mM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2Cand Ca2C ATP/ ADPase at 100 mM. Lanthanum decreased Mg2C and Ca2C ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: NTPDase; Stomach; Extracellular ATP; Ectonucleotidases; Apyrase
1. Introduction Adenosine 5#-triphosphate (ATP) and its breakdown products, adenosine diphosphate (ADP) and adenosine, participate in many biological processes including smooth muscle contraction, gastric secretion, neurotransmission, immune response, inflammation, cardiac and platelet function, vasodilatation, liver glycogen metabolism and pain (Burnstock, 1990; Dubyak and El-Moatassim, 1993; Ralevic and Burnstock, 1988; Agteresh et al., 1999; Se´vigny et al., 1998; Bonan et al., 2001; Tasca et al., 2004).
* Corresponding author. Tel.: C55 55 220 8140; fax: C55 55 220 8978. E-mail address: [email protected] (J.B.T. Rocha).
ATP and its metabolites can be hydrolyzed by a variety of enzymes that are located on cell surfaces or soluble in the interstitial medium or within body fluids (Zimmermann, 2001). These enzymes are called ectonucleotidases. The ectonucleoside triphosphate diphosphohydrolase (ectonucleotidase E-NTPDase) family, ectonucleotide pyrophosphate phosphodiesterase (E-NPP) family and alkaline phosphatases may hydrolyze nucleoside 5#-tri-and-diphosphate. Nucleoside 5#-monophosphates are subject to hydrolysis by ecto5#-nucleotidase, as well as by alkaline phosphates and presumably also by some members of the E-NPP family (Zimmermann, 2001). E-NTPDase 1 hydrolyzes ATP and ADP almost equally well and thus is also referred to as an apyrase. E-NTPDase 2 has a 30-fold preference for the hydrolysis of ATP over ADP, whereas E-NTPDase 3, another
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.03.010
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apyrase-like enzyme, hydrolyzes ATP about three times faster than ADP (Zimmermann, 2001). Several studies have demonstrated the presence of ecto-ATP diphosphohydrolase (NTPDase 1, ecto-apyrase, ecto-CD39, EC 3.6.1.5), e.g. in Trichomonas vaginalis (Matos et al., 2001), salivary glands of the cat flea Ctenocephaides felis (Cheeseman, 1998), and in avian, pig, chicken, and mammalian liver (Leclerc et al., 2000; Vieira et al., 2001; Knowles et al., 2002). It has also been demonstrated in various other tissues, such as synaptosomes from the cerebral cortex of adult rats (Rocha et al., 1990, 1991; Battastini et al., 1991), rat and human placenta (Kettlun et al., 1994; Valenzuela et al., 1996), synovial membrane of equine metacarpo phalangeal joint (Jimenez et al., 2002), chicken gizzard and stomach (Lewis-Carl and Kirley, 1997), and pig gastrointestinal tract (stomach, intestine, pancreas and parotid gland) (Se´vigny et al., 1998). Several lines of evidence have indicated that extracellular purines play primordial roles in the gastrointestinal tract and these actions are mediated by purinergic receptors and modulated by still undefined nucleotidase activities. According to Se´vigny et al. (1998), ecto-apyrase influences gastric acid and pepsin secretion, mucus production, and contractility of the pig stomach, and this enzyme is mainly associated with parietal, chief and smooth muscle cells. The regulation of acid secretion is a complex process involving many cell types, hormones and mediators, but these processes converge in a final common step involving HC, KC, ATPase (Horn, 2000; Dunbar and Caplan, 2000). Furthermore, Lewis-Carl and Kirley (1997) also demonstrated that chicken ectoATPase and ecto-apyrase are compartmentalized to glandular and smooth muscle cells, respectively. These results are consistent with a function of the ecto-apyrase in secretory processes, and a function of the ecto-ATPase in termination of purinergic stimulation of smooth muscle. Based on the wide distribution of NTPDase in various tissues (Borges et al., 2004; Senger et al., 2004), and considering the variety of purine receptors associated with the digestive system, mainly in the stomach, the objective of the present study was to characterize the ATP and ADP hydrolyzing enzyme in rat gastric mucosa.
2.2. Animals
2. Materials and methods
2.5. Effect of pH
2.1. Materials
The apparent optimum pH was determined with 3 mM nucleotide in the substrate solution, containing 3 mM CaCl2 or MgCl2, and one of the following buffers at 50 mM TrisCMOPS and pH was adjusted to 6.0e 9.0. The mixture was incubated for 1 h at 37 C and the inorganic phosphate release was measured as previously described by Fiske and Subbarow (1925).
Nucleotides, Trizma base, sodium azide, ouabain, orthovanadate, N-ethylmaleimide (NEM) and adenyl (3,5)-adenosine pentaphosphate (Ap5A) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of the highest analytical grade.
Male Wistar rats (200e300 g) from our own breeding stock were maintained at 25 C on a 12 h light/12 h dark cycle, with free access to food and water. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sao Paulo, Brazil. 2.3. Stomach isolation The animals were killed by decapitation and the gastric mucosa was removed, placed on ice, and homogenized in ten volumes of cold buffer (250 mM sucrose, 1 mM EGTA and 5 mM TriseHCl buffer, pH 7.4). The homogenate was centrifuged at 1000!g for 10 min to yield a low-speed supernatant fraction (S1) that was used for enzyme assay. The material was prepared fresh daily and maintained at 0e4 C throughout the preparation. 2.4. Enzyme assay For NTPDase activity, the reaction mixture contained 80 mM TriseHCl buffer, pH 7.4, and 3 mM CaCl2 or MgCl2 in a final volume of 500 mL. A 25 mL aliquot of the enzyme preparation (100e200 mg of protein) was added to the reaction mixture and preincubated for 10 min at 37 C. The reaction was initiated by addition of substrate (ATP, ADP or other substrates as indicated) to a final concentration of 3 mM. The reaction was stopped by addition of 250 mL 10% trichloroacetic acid (TCA). The samples were centrifuged at 1800 rpm for 10 min and 500 mL aliquots were taken for the measurement of inorganic phosphate (Pi). Inorganic phosphate (Pi) release was determined as previously described by Fiske and Subbarow (1925). For all enzyme assays, incubation times and protein concentration were chosen to ensure the linearity of the reactions. All samples were run in duplicate. Controls with the addition of the enzyme preparation after mixing with TCA were used to correct for nonenzymatic hydrolysis of substrates. Enzyme activity was expressed as nmol of phosphate (Pi) released minÿ1 mg proteinÿ1.
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2.6. Substrate specificity
2.10. Protein determination
The substrate specificity was determined in a similar way as for the assay of NTPDase activity, with addition of ATP, ADP, AMP, b-glycerolphosphate and pyrophosphate (PPi) substrates.
Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.
To evaluate the correlation between the enzyme described in this study with other ATPases, various specific ATPase inhibitors were tested. The inhibitors tested were: (a) sodium azide, inhibitor of mitochondrial ATPase (with I50Z0.04 mM) (Pullman et al., 1960); (b) suramin, ecto-ATPase inhibitor at the low micromolar range (Martı´ et al., 1996); (c) orthovanadate, an inhibitor of transport ATPases, acid phosphatases, alkaline phosphatases, NaC/ KC-ATPase and phosphotyrosine phosphatases (Sorensen and Mahler, 1992; Cool and Blum, 1993); (d) N-ethylmaleimide (NEM), a Ca2C, Mg2C-ATPase and adenylate kinase inhibitor, (Mason and Saba, 1969; Russel et al., 1974); (e) ouabain, a specific inhibitor of NaC/KC-ATPase (Lebel et al., 1980); (f) lanthanum, a classical P-Type ATPase inhibitor (Battastini et al., 1991); there is also evidence that lanthanum can inhibit ecto-ATPases from urinary bladder (Ziganshin et al., 1995); (g) levamisole, a specific alkaline phosphatase inhibitor (Van Belle, 1972).
The values are expressed as meanGstandard deviation. The results were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s test. A value of P!0.05 was considered statistically significant.
3. Results The pH-activity curves of ATP and ADP hydrolysis coincide in the pH 8.0e8.5 range, in the presence of both Mg2C (Fig. 1a) and Ca2C (Fig. 1b). Like other NTPDases, the enzyme was shown to be divalent cation-dependent and its sensitivity to Ca2C and Mg2C is illustrated in Fig. 2a,b. ATP and ADP hydrolysis was similar with Ca2C or Mg2C as the activating divalent cation. The function of the cation is to form the metal ionenucleotide complex, which is the ATP
ADP
(a)
80
nmol Pi/ mg/ mim
2.7. Sensitivity to inhibitors
2.11. Statistical analysis
64 48 32 16 0
2.8. Pyrophosphatase and nonspecific phosphatase activities
6
7
8
ATP
2.9. Adenylate kinase activity For this assay the reaction medium was the same as for the assay of Ca2C and Mg2C-ATP diphosphohydrolase, except for the addition of a selective adenylate kinase inhibitor, P1,P5-di (adenosine 5#) pentaphosphate, Ap5A (Lienhard and Secemski, 1973), to the incubation system to determine hydrolysis of substrates.
ADP
(b)
80
nmol Pi/ mg/ mim
For these assays the reaction medium was the same as for the NTDPase assay except that the reaction was initiated by addition of one of these compounds (final concentration 3 mM): b-glycerolphosphate, pyrophosphate (PPi) or AMP, instead of ATP or ADP.
9
pH
64 48 32 16 0
6
7
8
9
pH Fig. 1. Effect of pH on Mg2CATP/ADPase (a) and Ca2CATP/ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
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L. Savegnago et al. / Cell Biology International 29 (2005) 559e566 ATP
ADP
(a)
nmol Pi/ mg/ mim
50
Substrate (3 mM)
40 30
Specific activity (nmol Pi minÿ1 mg proteinÿ1)
% Control activity
Mg2C
Ca2C
Mg2C
Ca2C
45.25G10.00 39.00G6.00 1.60G0.04 0 2.21G0.60
100 76 7 0 3
100 86 3 0 3
ATP 67.87G8.00 ADP 51.80G6.00 AMP 4.85G0.05 PPi 0 b-Glycerol2.05G0.07 phosphate
20 10 0
Mg
control
ATP
EDTA
Mg + EDTA
(b)
40 30 20 10 0 Ca
a Data represent meanGSD of at least three experiments and are expressed as percentage of control activity (ATP hydrolysis 100%).
ADP
50
nmol Pi/ mg/ mim
Table 1 Substrate specificity of NTPDase in the gastric mucosa of rats in the presence of Mg2Ca and Ca2Ca
control
EDTA
Ca + EDTA
Fig. 2. Effect of MgCl2 (a) or CaCl2 (b) on ATP and ADP hydrolysis promoted by NTPDase in the rat gastric mucosa. The control values were 4.0G1.5 and 7.0G3 nmol Pi minÿ1 mg proteinÿ1 for Mg2C/ Ca2CATPase and Mg2C/Ca2CADPase, respectively. Results are expressed as nmol Pi minÿ1 mg proteinÿ1. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
true substrate for the reaction (Valenzuela et al., 1989). EDTA (10 mM) decreased NTPDase activity in the presence of Ca2C and Mg2C, when the substrate was ATP or ADP. In the absence of Ca2C and Mg2C, rates of hydrolysis of ATP and ADP were very low (Fig. 2a,b), indicating a strict dependence on divalent cations. These results show that the gastric mucosa is able to hydrolyze di- and triphosphate nucleosides (Table 1). ATP was a better substrate than ADP. Some AMP hydrolysis was probably due to 5#-nucleotidase activity present as an ecto-enzyme in the gastric mucosa (Table 1). In the presence of 3 mM CaCl2, AMP hydrolysis was 1.6G0.04 nmol Pi minÿ1 mg proteinÿ1. However, MgCl2 was a better activator, since AMP hydrolysis with 3 mM MgCl2 corresponds to 4.85G0.05 nmol Pi minÿ1 mg proteinÿ1. Further studies will be required to conclude that the enzyme that promotes AMP hydrolysis in rat gastric mucosa is an ecto-5#-nucleotidase. Our results also showed no significant hydrolysis of AMP and b-glycerolphosphate (Table 1). Since there was no significant hydrolysis when b-glycerolphosphate and AMP were used as substrates instead of ATP or
ADP, the possibility that ATP and ADP were being hydrolyzed by the action of nonspecific phosphatases in the gastric mucosal preparation was excluded (Table 1). The possibility of the combined action of an ATP pyrophosphohydrolase and an inorganic pyrophosphatase was excluded. In fact, no Pi was released when PPi (pyrophosphate) was incubated at a final concentration of 3 mM instead of ATP or ADP (Table 1). The specific alkaline phosphatase inhibitor, levamisole, did not alter ATP or ADP hydrolysis in the presence of Mg2C and Ca2C (Table 2) in the gastric mucosa of rats. Thus, this result excludes alkaline phosphatase as a contaminant. The possibility that ADP hydrolysis occurs by prior conversion to ATP, catalyzed by adenylate kinase and later hydrolysis by an ATP-specific enzyme, should be ruled out since this enzyme combination could mimic apyrase activity. The influence of a contaminating adenylate kinase in our assay conditions was excluded, since Ap5A, a selective adenylate kinase inhibitor, did
Table 2 Effect of levamisole and Ap5A on Mg2Cand Ca2CATP/ADPase activity from rat gastric mucosa Inhibitor
Concentration (mM)
Enzyme activity (% of control) ATPase 2C
Levamisole Ap5A
0.10 0.20 0.03
ADPase 2C
Mg
Ca
Mg2C
Ca2C
103G4 105G4 99G3
106G2 98G2 97G1
109G8 102G2 96G4
109G8 102G2 96G4
The control values (without levamisole) were 75G10 and 58G10 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively, and 43G8 and 37G3 nmol Pi minÿ1 mg proteinÿ1, respectively, for Ca2C ATPase/ADPase. The control values (without Ap5A) were 71G5 and 68G10 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively, and 44G6 and 36G3 nmol Pi minÿ1 mg proteinÿ1 for Ca2CATPase/ADPase, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
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ADP
120
(a) % of control
96 72 48 24 0
0
0.05
0.10
0.15
0.20
0.25
0.30
[mM] Suramin ATP
ADP
120
(b) 96
% of control
not affect ATP or ADP hydrolysis in rat gastric mucosa (Table 2). Sodium azide, tested in the 0.01e30 mM range, inhibited ATP and ADP hydrolysis in the presence of either Mg2C (Fig. 3a) or Ca2C (Fig. 3b). When sodium azide was tested at high concentrations, in the presence of both cations, a parallel hydrolysis inhibition of ATP and ADP occurred. The maximal inhibitory effect obtained was at 30 mM, giving approximately 83% and 71% inhibition for Mg2CATP/ADPase, respectively (Fig. 3a). The specific enzyme activity was less sensitive to azide in the presence of Ca2C as the activating ion of ATP and ADP hydrolysis. In fact, the maximum inhibitory effects for the hydrolysis of these nucleotides were about 48% and 55%, respectively (Fig. 3b). Suramin (0.05e0.3 mM) inhibited ATP and ADP hydrolysis in the presence of Mg2C and Ca2C. The maximal inhibitory effect appeared at 0.3 mM and the specific enzyme activity was 60% for Mg2C/Ca2C ATPase (Fig. 4a) and 52% for Mg2C/Ca2CADPase hydrolysis (Fig. 4b).
72 48 24
ATP
ADP
120
0
(a)
0
0.05
0.10
0.15
0.20
0.25
0.30
[mM] Suramim
% of control
96 72 48 24 0 0
0.01
0.1
1
10
40
[mM] Azide ATP
ADP
120
(b)
% of control
96 72 48 24 0 0
0.01
0.1
1
10
40
[mM] Azide Fig. 3. Effect of sodium azide on Mg2CATP/ADPase (a) and Ca2CATP/ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Sodium azide was used in 0.01e30 mM range. The control values were approximately 86G5 and 65G3 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase and Mg2CADPase, respectively. Control values of Ca2C ATPase and Ca2CADPase activities (100%) were 66G6 and 61G7 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were tested by one-way ANOVA followed by Duncan’s test.
Fig. 4. Effect of suramin on Mg2CATP/ADPase (a) and Ca2CATP/ ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Suramin was used in 0.05e0.3 mM range. The control values were approximately 70G4 and 53G6 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively. Control values for Ca2CATPase and Ca2CADPase activities (100%) were 44G7 and 39G5 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
Orthovanadate, a P-type ATPase and alkaline phosphatase inhibitor, caused a maximal inhibitory effect of about 15% at 0.1 mM on Mg2CATP/ADPase. The inhibition determined in the presence of Ca2C, as activating divalent cation, was similar to that observed with Mg2C (data not shown). The inhibitor of Ca2C/Mg2CATPase and adenylate kinase, N-ethylmaleimide, had no effect on NTPDase activity (data not shown). The NaC/KC ATPase inhibitor, ouabain, tested at 0.01e1 mM, did not affect ATP or ADP hydrolysis in the presence of both cations (data not shown). The typical P-type ATPase and ecto-ATPases inhibitor, lanthanum, decreased the specific activity of Mg2CATP/ADPase (Fig. 5a) and presented a maximal inhibitory effect at 1 mM (w64% for ATP and w42% for ADP hydrolysis). ATP and ADP hydrolysis measured in the presence of Ca2C were less affected by La3C (w15% for ATP and 20% for ADP hydrolysis; Fig. 5b).
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(c) lack of inhibition by several inhibitors, such as P-type, F-type, V-type ATPases, alkaline phosphatases and adenylate kinase.
ADP
120
(a)
% of control
96 72 48 24 0 0
0.04
0.1
1
[mM] Lanthanun ATP
ADP
120
(b)
% of Control
96 72 48 24 0 0
0.04
0.1
1
[mM] Lanthanun Fig. 5. Effect of lanthanum on Mg2CATP/ADPase (a) and Ca2CATP/ ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Lanthanum was used in 0.04e1 mM range. The control values were 50G9 and 45G6 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively. Control values for Ca2CATPase and Ca2CADPase activities (100%) were 44G10 and 43G15 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
4. Discussion The results of the present study suggest the presence of NTPDase activity in gastric mucosal preparations. The enzyme described here has the following general properties that characterize an NTPDase: (a) optimum pH range from 8.0 to 8.5, in the presence of both Mg2C and Ca2C. These results are similar to those of salivary apyrase of Aedes aegypty (Ribeiro et al., 1984), salivary glands of the Bed Bug Cimex lectularis (Valenzuela et al., 1996), salivary glands of the cat flea Ctenocephalides felis (Cheeseman, 1998), pig pancreas (Lebel et al., 1980), and ATP diphosphohydrolase in synaptosomes from cerebral cortex of adult rats (Battastini et al., 1991). (b) activation by either Ca2C or Mg2C alone. Cation dependence was confirmed by a dramatic decrease in ATP and ADP hydrolysis in the absence of cation or in the presence of cation plus 10 mM EDTA (Fig. 2).
The concentrations of sodium azide necessary to inhibit mitochondrial ATPase and alkaline phosphatases are in the mM range. In this study, low concentrations of sodium azide did not affect NTPDase activity. Accordingly, Battastini et al. (1991) demonstrated inhibition of NTPDase only when azide was used at high concentrations (>100 mM). We have also shown that sodium azide significantly inhibited ATP and ADP hydrolysis using both cations (Mg2C and Ca2C). In addition, our results with sodium azide in the presence of Ca2C, as activating divalent cation, were similar to those of Se´vigny et al. (1998) in pig stomach. Unlike sodium azide, suramin can significantly inactivate NTPDase when used at low (M concentrations. Suramin inhibits ecto-ATPase activity from urinary bladder (Hourani and Chown, 1989), blood cells (Beukers et al., 1995) and endothelial cells (Meghji and Burnstock, 1995). Our results clearly demonstrate that suramin is one of the most potent inhibitors of NTPDase in the rat gastric mucosa (Fig. 4). When NaC, KC-ATPase inhibitors were studied, ouabain did not affect the hydrolysis of either substrate, but, in contrast, orthovanadate (0.1e100 mM) slightly reduced the hydrolysis (approximately 15%) of both substrates. This effect could be due to a minor contamination with ATPase type P or V. Moreover, in the presence of Mg2C, La3C inhibited the hydrolysis of ATP and ADP to a similar extent. A similar phenomenon was observed when Ca2C was used as the activator ion; however, the maximum inhibitory effect was lower than Mg2C. The parallel inhibition of ATP and ADP hydrolysis strongly suggests that La3C is inhibiting NTPDase in the gastric mucosa. In the same way, Ziganshin et al. (1995) reported that La3C inhibits ecto-ATPase activity in the guinea-pig urinary bladder. Levamisole, an inhibitor of alkaline phosphatase, did not change the hydrolysis of either substrate tested, in the presence of Mg2C and Ca2C (Table 2). This result rules out alkaline phosphatase as a contaminant in the gastric mucosa preparation. Three sets of experimental data rule out enzyme combinations as mimics of NTPDase activity: (1) Adenylate kinase inhibitors Ap5A (Table 2) and NEM had no effect, thus, ADP hydrolysis was not due to the participation of the combination of adenylate kinase/ATPase. (2) The possible participation of other enzymes that could hydrolyze ATP or ADP (such as ATPases, ADPases, nonspecific phosphatases or pyrophosphatases) was ruled out by the following results: insensitivity to classical ATPase inhibitors, lack of
L. Savegnago et al. / Cell Biology International 29 (2005) 559e566
activity with phosphate esters and pyrophosphate (PPi) (Table 1). (3) ATP and ADP are hydrolyzed in a similar way. NTPDase 2 hydrolyzes ATP faster (30-fold) than ADP (Zimmermann, 2001; Knowles and Chiang, 2003), and NTPDase 3 also hydrolyzes ATP faster (3-fold) than ADP (Zimmermann, 2001). On the basis of these characteristics, we propose that the hydrolysis of ATP and ADP by gastric mucosa may be due to NTPDase activity, possibly type 1. Furthermore, the parallelism of the kinetic behavior in relation to cation and pH dependence and sensitivity to different inhibitors reinforces the hypothesis that only one enzyme is acting on the two substrates (ATP and ADP). However, further experiments will be required in order to determine the member of the NTPDase family involved in ATP and ADP hydrolysis in this preparation. ATP and metabolites, in addition to their role in energy metabolism, play an important role as extracellular regulatory and signaling molecules for many different cell types (Vlajkovic et al., 1996; Inscho et al., 1994), including gastric mucosa (Kwok et al., 1990). In line with this, reports have demonstrated the presence of P2Y purinoceptors in rabbit gastric glands (Gilrodrigo et al., 1996; Vallejo et al., 1996). In fact, the antisecretory effect of ATP on isolated rabbit parietal cells may be mediated via P2Y purinoceptors. ATP-sensitive potassium channels have been implicated in several physiological functions of the gastrointestinal tract, such as contractility, acid secretion and regulation of the gastric blood flow, as well as some pathological conditions (Rahgozar et al., 2001). Like ATP, adenosine modulates the secretion of a variety of gastric hormones in the antral and corporeal mucosa, and adenosine receptors are expressed in both regions (Harty and Franklin, 1984; Heldsinger et al., 1986; Puurunen et al., 1987; Yip and Kwok, 2004). Thus, parietal apyrase (NTPDase) can modulate gastric function by metabolizing ATP and, indirectly, by influencing the synthesis of adenosine. In conclusion, we have described an enzyme in the gastric mucosa that shares several kinetic properties with NTPDase.
Acknowledgements This work was supported by CNPq, FAPERGS, CAPES and UFSM (FIPE).
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Kwok YN, McIntosh C, Brown J. Augmentation of release of gastric somatostatin-like immunoreactivity by adenosine, adenosine triphosphate and their analogs. Journal of Pharmacology and Experimental Therapeutics 1990;255:781e8. Lebel D, Poirier GG, Phaneuf S, St Jean P, Laliberte JF, Beaudoin AR. Characterization and purification of a calciumsensitive ATP diphosphohydrolase from pig pancreas. Journal of Biological Chemistry 1980;255:1227e33. Leclerc MC, Grondin G, Gendron F, Se´vigny J, Beaudoin RA. Identification, characterization and inmunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver. Archives of Biochemistry and Biophysics 2000;377:372e8. Lewis-Carl S, Kirley LT. Inmunolocalization of the ecto-ATPase and ecto-apyrase in chicken gizzard and stomach. Journal of Biological Chemistry 1997;272:23645e52. Lienhard GE, Secemski I. P1.P5-Di (adenosine-5#) pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. Journal of Biological Chemistry 1973;248:1121e3. Lowry HD, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 1951;193:265e75. Matos JAA, Borges FP, Tasca T, Bogo MR, De Carli GA, Fauth MG, et al. Characterization of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis. International Journal of Parasitology 2001;31:770e5. Martı´ E, Cantı´ C, Go´mez I, Miralles F, Solsona C. Action of suramin upon ecto-apyrase and synaptic depression of Torpedo electric organ. British Journal Pharmacology 1996;18:1232e6. Mason RG, Saba SR. Platelet ATPase activities. I. Ecto-ATPases of intact platelets and their possible role in aggregation. American Journal of Pathology 1969;55:215e30. Meghji P, Burnstock G. Inhibition of extracellular ATP degradation in endothelial cells. Life Sciences 1995;57:763e71. Pullman ME, Penefsky HS, Datta A, Racker E. Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphate. Journal of Biological Chemistry 1960;235:3322e9. Puurunen J, Ruoff HJ, Schwabe U. Lack of direct effect of adenosine on the parietal cell function in the rat. Pharmacology & Toxicology 1987;60:315e7. Rahgozar M, Pazokitoroudi H, Bakhtiarian A, Djahanguiri B. Diazoxide, a K-ATP opener, accelerates restitution of ethanol or indomethacin-induced gastric ulceration in rats independent of polyamines. Journal of Gastroenterology and Hepatology 2001;16: 290e6. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Journal of Pharmacology and Experimental Therapeutics 1988;50: 413e92. Ribeiro JMC, Sarkis JJF, Rossignol PA, Spielman A. Salivary apyrase of Aedes aegypti characterization and secretory fate. Comparative Biochemistry and Physiology B 1984;79:81e6. Rocha JBT, Mello CF, Sarkis JJF, Dias RD. Undernutrition during the preweaning period changes calcium ATPase and ADPase activities of synaptosomal fraction of weanling rats. British Journal of Nutrition 1990;63:273e83.
Rocha JBT, Battastini AMO, Sarkis JJF, Dias RD. Effects of undernutrition during suckling on ATP and ADP hydrolysis by synaptosomes from the cerebral cortex of adult rats. Brazilian Journal of Medical and Biological Research 1991;24:515e26. Russel PJ, Horenstein JM, Goins L, Jones D, Laver M. Adenylate kinase in human tissues. Journal of Biological Chemistry 1974;249: 1874e9. Senger MR, Rico EP, Dias RD, Bogo MR, Bonan CD. Ecto-5#nucleotidase activity in brain membranes of zebrafish (Danio rerio). Comparative Biochemistry and Physiology B 2004;139: 203e7. Se´vigny J, Grondin G, Gendron F, Roy J, Beaudoin RA. Demonstration and immunolocalization of ATP diphosphohydrolase in the pig digestive system. American Journal of Physiology 1998;275: 473e82. Sorensen RG, Mahler HR. Localization of endogenous ATPases at nerve terminal. Journal of Bioenergetics and Biomembranes 1992;14:527e46. Tasca T, Bonan CD, De Carli GA, Sarkis JJF. Trichomonas vaginalis: cytochemical localization of a NTPDase1 and an ecto-5#-nucleotidase and effects of adenine nucleotides on cellular viability. Parasitology Research 2004;93:300e3. Valenzuela MA, Lo´pez J, Depix M, Mancilla M, Ketlun AM, Catala´n L, et al. Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase. Comparative Biochemistry and Physiology 1989;93: 911e9. Valenzuela MA, Kettlun AM, Sandoval S, Garcı´ a L, Mancilla M, Neckelmann G, et al. Comparison of the human placenta and rat regulation and functional of ATP-diphosphohydrolase from human placenta and rat kidney. Brazilian Journal of Medical and Biological Research 1996;29:589e97. Vallejo AI, Bo XN, Burnstock G. P2Y purinoceptors in gastric gland plasma membranes. European Journal of Pharmacology 1996;312: 209e14. Van Belle H. Kinetics and inhibition of alkaline phosphatase from canine tissues. Biochimica et Biophysica Acta 1972;289:158e68. Vieira VP, Rocha JBT, Stefanello FM, Balz D, Morsch VM, Schetinger MRC. Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney enriched fractions. International Journal of Biochemistry & Cell Biology 2001;33: 1193e201. Vlajkovic SM, Thorne PR, Munoz DJB, Housley GD. Ectonucleotidase activity in the perilymphatic compartment of the guinea pig cochlea. Hearing Research 1996;99:31e7. Ziganshin AU, Ziganshina LE, Hoyle CHV, Burnstock G. Effects of divalent-cations and La3C on contractility and ecto-ATPase activity in the guinea-pig urinary bladder. British Journal of Pharmacology 1995;114:632e9. Zimmermann H. Ectonucleotidases: some recent developments and a note on nomenclature. Drug Development Research 2001;52: 44e56. Yip L, Kwok YN. Role of adenosine A2A receptor in the regulation of gastric somatostatin release. Journal of Pharmacology and Experimental Therapeutics 2004;309:804e15.
Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa Lucielli Savegnago, Cristina W. Nogueira, Roselei Fachinetto, Joao Batista Teixeira Rocha * Departamento de Quimica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil Received 16 September 2004; revised 19 January 2005; accepted 8 March 2005
Abstract The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2C and Ca2C. The inhibitors ouabain (0.01e1 mM), N-ethylmaleimide (0.01e4 mM), levamisole (0.10e0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03e30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50e300 mM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2Cand Ca2C ATP/ ADPase at 100 mM. Lanthanum decreased Mg2C and Ca2C ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: NTPDase; Stomach; Extracellular ATP; Ectonucleotidases; Apyrase
1. Introduction Adenosine 5#-triphosphate (ATP) and its breakdown products, adenosine diphosphate (ADP) and adenosine, participate in many biological processes including smooth muscle contraction, gastric secretion, neurotransmission, immune response, inflammation, cardiac and platelet function, vasodilatation, liver glycogen metabolism and pain (Burnstock, 1990; Dubyak and El-Moatassim, 1993; Ralevic and Burnstock, 1988; Agteresh et al., 1999; Se´vigny et al., 1998; Bonan et al., 2001; Tasca et al., 2004).
* Corresponding author. Tel.: C55 55 220 8140; fax: C55 55 220 8978. E-mail address: [email protected] (J.B.T. Rocha).
ATP and its metabolites can be hydrolyzed by a variety of enzymes that are located on cell surfaces or soluble in the interstitial medium or within body fluids (Zimmermann, 2001). These enzymes are called ectonucleotidases. The ectonucleoside triphosphate diphosphohydrolase (ectonucleotidase E-NTPDase) family, ectonucleotide pyrophosphate phosphodiesterase (E-NPP) family and alkaline phosphatases may hydrolyze nucleoside 5#-tri-and-diphosphate. Nucleoside 5#-monophosphates are subject to hydrolysis by ecto5#-nucleotidase, as well as by alkaline phosphates and presumably also by some members of the E-NPP family (Zimmermann, 2001). E-NTPDase 1 hydrolyzes ATP and ADP almost equally well and thus is also referred to as an apyrase. E-NTPDase 2 has a 30-fold preference for the hydrolysis of ATP over ADP, whereas E-NTPDase 3, another
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.03.010
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apyrase-like enzyme, hydrolyzes ATP about three times faster than ADP (Zimmermann, 2001). Several studies have demonstrated the presence of ecto-ATP diphosphohydrolase (NTPDase 1, ecto-apyrase, ecto-CD39, EC 3.6.1.5), e.g. in Trichomonas vaginalis (Matos et al., 2001), salivary glands of the cat flea Ctenocephaides felis (Cheeseman, 1998), and in avian, pig, chicken, and mammalian liver (Leclerc et al., 2000; Vieira et al., 2001; Knowles et al., 2002). It has also been demonstrated in various other tissues, such as synaptosomes from the cerebral cortex of adult rats (Rocha et al., 1990, 1991; Battastini et al., 1991), rat and human placenta (Kettlun et al., 1994; Valenzuela et al., 1996), synovial membrane of equine metacarpo phalangeal joint (Jimenez et al., 2002), chicken gizzard and stomach (Lewis-Carl and Kirley, 1997), and pig gastrointestinal tract (stomach, intestine, pancreas and parotid gland) (Se´vigny et al., 1998). Several lines of evidence have indicated that extracellular purines play primordial roles in the gastrointestinal tract and these actions are mediated by purinergic receptors and modulated by still undefined nucleotidase activities. According to Se´vigny et al. (1998), ecto-apyrase influences gastric acid and pepsin secretion, mucus production, and contractility of the pig stomach, and this enzyme is mainly associated with parietal, chief and smooth muscle cells. The regulation of acid secretion is a complex process involving many cell types, hormones and mediators, but these processes converge in a final common step involving HC, KC, ATPase (Horn, 2000; Dunbar and Caplan, 2000). Furthermore, Lewis-Carl and Kirley (1997) also demonstrated that chicken ectoATPase and ecto-apyrase are compartmentalized to glandular and smooth muscle cells, respectively. These results are consistent with a function of the ecto-apyrase in secretory processes, and a function of the ecto-ATPase in termination of purinergic stimulation of smooth muscle. Based on the wide distribution of NTPDase in various tissues (Borges et al., 2004; Senger et al., 2004), and considering the variety of purine receptors associated with the digestive system, mainly in the stomach, the objective of the present study was to characterize the ATP and ADP hydrolyzing enzyme in rat gastric mucosa.
2.2. Animals
2. Materials and methods
2.5. Effect of pH
2.1. Materials
The apparent optimum pH was determined with 3 mM nucleotide in the substrate solution, containing 3 mM CaCl2 or MgCl2, and one of the following buffers at 50 mM TrisCMOPS and pH was adjusted to 6.0e 9.0. The mixture was incubated for 1 h at 37 C and the inorganic phosphate release was measured as previously described by Fiske and Subbarow (1925).
Nucleotides, Trizma base, sodium azide, ouabain, orthovanadate, N-ethylmaleimide (NEM) and adenyl (3,5)-adenosine pentaphosphate (Ap5A) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of the highest analytical grade.
Male Wistar rats (200e300 g) from our own breeding stock were maintained at 25 C on a 12 h light/12 h dark cycle, with free access to food and water. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of Sao Paulo, Brazil. 2.3. Stomach isolation The animals were killed by decapitation and the gastric mucosa was removed, placed on ice, and homogenized in ten volumes of cold buffer (250 mM sucrose, 1 mM EGTA and 5 mM TriseHCl buffer, pH 7.4). The homogenate was centrifuged at 1000!g for 10 min to yield a low-speed supernatant fraction (S1) that was used for enzyme assay. The material was prepared fresh daily and maintained at 0e4 C throughout the preparation. 2.4. Enzyme assay For NTPDase activity, the reaction mixture contained 80 mM TriseHCl buffer, pH 7.4, and 3 mM CaCl2 or MgCl2 in a final volume of 500 mL. A 25 mL aliquot of the enzyme preparation (100e200 mg of protein) was added to the reaction mixture and preincubated for 10 min at 37 C. The reaction was initiated by addition of substrate (ATP, ADP or other substrates as indicated) to a final concentration of 3 mM. The reaction was stopped by addition of 250 mL 10% trichloroacetic acid (TCA). The samples were centrifuged at 1800 rpm for 10 min and 500 mL aliquots were taken for the measurement of inorganic phosphate (Pi). Inorganic phosphate (Pi) release was determined as previously described by Fiske and Subbarow (1925). For all enzyme assays, incubation times and protein concentration were chosen to ensure the linearity of the reactions. All samples were run in duplicate. Controls with the addition of the enzyme preparation after mixing with TCA were used to correct for nonenzymatic hydrolysis of substrates. Enzyme activity was expressed as nmol of phosphate (Pi) released minÿ1 mg proteinÿ1.
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2.6. Substrate specificity
2.10. Protein determination
The substrate specificity was determined in a similar way as for the assay of NTPDase activity, with addition of ATP, ADP, AMP, b-glycerolphosphate and pyrophosphate (PPi) substrates.
Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.
To evaluate the correlation between the enzyme described in this study with other ATPases, various specific ATPase inhibitors were tested. The inhibitors tested were: (a) sodium azide, inhibitor of mitochondrial ATPase (with I50Z0.04 mM) (Pullman et al., 1960); (b) suramin, ecto-ATPase inhibitor at the low micromolar range (Martı´ et al., 1996); (c) orthovanadate, an inhibitor of transport ATPases, acid phosphatases, alkaline phosphatases, NaC/ KC-ATPase and phosphotyrosine phosphatases (Sorensen and Mahler, 1992; Cool and Blum, 1993); (d) N-ethylmaleimide (NEM), a Ca2C, Mg2C-ATPase and adenylate kinase inhibitor, (Mason and Saba, 1969; Russel et al., 1974); (e) ouabain, a specific inhibitor of NaC/KC-ATPase (Lebel et al., 1980); (f) lanthanum, a classical P-Type ATPase inhibitor (Battastini et al., 1991); there is also evidence that lanthanum can inhibit ecto-ATPases from urinary bladder (Ziganshin et al., 1995); (g) levamisole, a specific alkaline phosphatase inhibitor (Van Belle, 1972).
The values are expressed as meanGstandard deviation. The results were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s test. A value of P!0.05 was considered statistically significant.
3. Results The pH-activity curves of ATP and ADP hydrolysis coincide in the pH 8.0e8.5 range, in the presence of both Mg2C (Fig. 1a) and Ca2C (Fig. 1b). Like other NTPDases, the enzyme was shown to be divalent cation-dependent and its sensitivity to Ca2C and Mg2C is illustrated in Fig. 2a,b. ATP and ADP hydrolysis was similar with Ca2C or Mg2C as the activating divalent cation. The function of the cation is to form the metal ionenucleotide complex, which is the ATP
ADP
(a)
80
nmol Pi/ mg/ mim
2.7. Sensitivity to inhibitors
2.11. Statistical analysis
64 48 32 16 0
2.8. Pyrophosphatase and nonspecific phosphatase activities
6
7
8
ATP
2.9. Adenylate kinase activity For this assay the reaction medium was the same as for the assay of Ca2C and Mg2C-ATP diphosphohydrolase, except for the addition of a selective adenylate kinase inhibitor, P1,P5-di (adenosine 5#) pentaphosphate, Ap5A (Lienhard and Secemski, 1973), to the incubation system to determine hydrolysis of substrates.
ADP
(b)
80
nmol Pi/ mg/ mim
For these assays the reaction medium was the same as for the NTDPase assay except that the reaction was initiated by addition of one of these compounds (final concentration 3 mM): b-glycerolphosphate, pyrophosphate (PPi) or AMP, instead of ATP or ADP.
9
pH
64 48 32 16 0
6
7
8
9
pH Fig. 1. Effect of pH on Mg2CATP/ADPase (a) and Ca2CATP/ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
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ADP
(a)
nmol Pi/ mg/ mim
50
Substrate (3 mM)
40 30
Specific activity (nmol Pi minÿ1 mg proteinÿ1)
% Control activity
Mg2C
Ca2C
Mg2C
Ca2C
45.25G10.00 39.00G6.00 1.60G0.04 0 2.21G0.60
100 76 7 0 3
100 86 3 0 3
ATP 67.87G8.00 ADP 51.80G6.00 AMP 4.85G0.05 PPi 0 b-Glycerol2.05G0.07 phosphate
20 10 0
Mg
control
ATP
EDTA
Mg + EDTA
(b)
40 30 20 10 0 Ca
a Data represent meanGSD of at least three experiments and are expressed as percentage of control activity (ATP hydrolysis 100%).
ADP
50
nmol Pi/ mg/ mim
Table 1 Substrate specificity of NTPDase in the gastric mucosa of rats in the presence of Mg2Ca and Ca2Ca
control
EDTA
Ca + EDTA
Fig. 2. Effect of MgCl2 (a) or CaCl2 (b) on ATP and ADP hydrolysis promoted by NTPDase in the rat gastric mucosa. The control values were 4.0G1.5 and 7.0G3 nmol Pi minÿ1 mg proteinÿ1 for Mg2C/ Ca2CATPase and Mg2C/Ca2CADPase, respectively. Results are expressed as nmol Pi minÿ1 mg proteinÿ1. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
true substrate for the reaction (Valenzuela et al., 1989). EDTA (10 mM) decreased NTPDase activity in the presence of Ca2C and Mg2C, when the substrate was ATP or ADP. In the absence of Ca2C and Mg2C, rates of hydrolysis of ATP and ADP were very low (Fig. 2a,b), indicating a strict dependence on divalent cations. These results show that the gastric mucosa is able to hydrolyze di- and triphosphate nucleosides (Table 1). ATP was a better substrate than ADP. Some AMP hydrolysis was probably due to 5#-nucleotidase activity present as an ecto-enzyme in the gastric mucosa (Table 1). In the presence of 3 mM CaCl2, AMP hydrolysis was 1.6G0.04 nmol Pi minÿ1 mg proteinÿ1. However, MgCl2 was a better activator, since AMP hydrolysis with 3 mM MgCl2 corresponds to 4.85G0.05 nmol Pi minÿ1 mg proteinÿ1. Further studies will be required to conclude that the enzyme that promotes AMP hydrolysis in rat gastric mucosa is an ecto-5#-nucleotidase. Our results also showed no significant hydrolysis of AMP and b-glycerolphosphate (Table 1). Since there was no significant hydrolysis when b-glycerolphosphate and AMP were used as substrates instead of ATP or
ADP, the possibility that ATP and ADP were being hydrolyzed by the action of nonspecific phosphatases in the gastric mucosal preparation was excluded (Table 1). The possibility of the combined action of an ATP pyrophosphohydrolase and an inorganic pyrophosphatase was excluded. In fact, no Pi was released when PPi (pyrophosphate) was incubated at a final concentration of 3 mM instead of ATP or ADP (Table 1). The specific alkaline phosphatase inhibitor, levamisole, did not alter ATP or ADP hydrolysis in the presence of Mg2C and Ca2C (Table 2) in the gastric mucosa of rats. Thus, this result excludes alkaline phosphatase as a contaminant. The possibility that ADP hydrolysis occurs by prior conversion to ATP, catalyzed by adenylate kinase and later hydrolysis by an ATP-specific enzyme, should be ruled out since this enzyme combination could mimic apyrase activity. The influence of a contaminating adenylate kinase in our assay conditions was excluded, since Ap5A, a selective adenylate kinase inhibitor, did
Table 2 Effect of levamisole and Ap5A on Mg2Cand Ca2CATP/ADPase activity from rat gastric mucosa Inhibitor
Concentration (mM)
Enzyme activity (% of control) ATPase 2C
Levamisole Ap5A
0.10 0.20 0.03
ADPase 2C
Mg
Ca
Mg2C
Ca2C
103G4 105G4 99G3
106G2 98G2 97G1
109G8 102G2 96G4
109G8 102G2 96G4
The control values (without levamisole) were 75G10 and 58G10 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively, and 43G8 and 37G3 nmol Pi minÿ1 mg proteinÿ1, respectively, for Ca2C ATPase/ADPase. The control values (without Ap5A) were 71G5 and 68G10 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively, and 44G6 and 36G3 nmol Pi minÿ1 mg proteinÿ1 for Ca2CATPase/ADPase, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
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ADP
120
(a) % of control
96 72 48 24 0
0
0.05
0.10
0.15
0.20
0.25
0.30
[mM] Suramin ATP
ADP
120
(b) 96
% of control
not affect ATP or ADP hydrolysis in rat gastric mucosa (Table 2). Sodium azide, tested in the 0.01e30 mM range, inhibited ATP and ADP hydrolysis in the presence of either Mg2C (Fig. 3a) or Ca2C (Fig. 3b). When sodium azide was tested at high concentrations, in the presence of both cations, a parallel hydrolysis inhibition of ATP and ADP occurred. The maximal inhibitory effect obtained was at 30 mM, giving approximately 83% and 71% inhibition for Mg2CATP/ADPase, respectively (Fig. 3a). The specific enzyme activity was less sensitive to azide in the presence of Ca2C as the activating ion of ATP and ADP hydrolysis. In fact, the maximum inhibitory effects for the hydrolysis of these nucleotides were about 48% and 55%, respectively (Fig. 3b). Suramin (0.05e0.3 mM) inhibited ATP and ADP hydrolysis in the presence of Mg2C and Ca2C. The maximal inhibitory effect appeared at 0.3 mM and the specific enzyme activity was 60% for Mg2C/Ca2C ATPase (Fig. 4a) and 52% for Mg2C/Ca2CADPase hydrolysis (Fig. 4b).
72 48 24
ATP
ADP
120
0
(a)
0
0.05
0.10
0.15
0.20
0.25
0.30
[mM] Suramim
% of control
96 72 48 24 0 0
0.01
0.1
1
10
40
[mM] Azide ATP
ADP
120
(b)
% of control
96 72 48 24 0 0
0.01
0.1
1
10
40
[mM] Azide Fig. 3. Effect of sodium azide on Mg2CATP/ADPase (a) and Ca2CATP/ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Sodium azide was used in 0.01e30 mM range. The control values were approximately 86G5 and 65G3 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase and Mg2CADPase, respectively. Control values of Ca2C ATPase and Ca2CADPase activities (100%) were 66G6 and 61G7 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were tested by one-way ANOVA followed by Duncan’s test.
Fig. 4. Effect of suramin on Mg2CATP/ADPase (a) and Ca2CATP/ ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Suramin was used in 0.05e0.3 mM range. The control values were approximately 70G4 and 53G6 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively. Control values for Ca2CATPase and Ca2CADPase activities (100%) were 44G7 and 39G5 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
Orthovanadate, a P-type ATPase and alkaline phosphatase inhibitor, caused a maximal inhibitory effect of about 15% at 0.1 mM on Mg2CATP/ADPase. The inhibition determined in the presence of Ca2C, as activating divalent cation, was similar to that observed with Mg2C (data not shown). The inhibitor of Ca2C/Mg2CATPase and adenylate kinase, N-ethylmaleimide, had no effect on NTPDase activity (data not shown). The NaC/KC ATPase inhibitor, ouabain, tested at 0.01e1 mM, did not affect ATP or ADP hydrolysis in the presence of both cations (data not shown). The typical P-type ATPase and ecto-ATPases inhibitor, lanthanum, decreased the specific activity of Mg2CATP/ADPase (Fig. 5a) and presented a maximal inhibitory effect at 1 mM (w64% for ATP and w42% for ADP hydrolysis). ATP and ADP hydrolysis measured in the presence of Ca2C were less affected by La3C (w15% for ATP and 20% for ADP hydrolysis; Fig. 5b).
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(c) lack of inhibition by several inhibitors, such as P-type, F-type, V-type ATPases, alkaline phosphatases and adenylate kinase.
ADP
120
(a)
% of control
96 72 48 24 0 0
0.04
0.1
1
[mM] Lanthanun ATP
ADP
120
(b)
% of Control
96 72 48 24 0 0
0.04
0.1
1
[mM] Lanthanun Fig. 5. Effect of lanthanum on Mg2CATP/ADPase (a) and Ca2CATP/ ADPase (b) from rat gastric mucosa with ATP (B) or ADP () as substrate. Lanthanum was used in 0.04e1 mM range. The control values were 50G9 and 45G6 nmol Pi minÿ1 mg proteinÿ1 for Mg2CATPase/ADPase, respectively. Control values for Ca2CATPase and Ca2CADPase activities (100%) were 44G10 and 43G15 nmol Pi minÿ1 mg proteinÿ1, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the meanGSD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan’s test.
4. Discussion The results of the present study suggest the presence of NTPDase activity in gastric mucosal preparations. The enzyme described here has the following general properties that characterize an NTPDase: (a) optimum pH range from 8.0 to 8.5, in the presence of both Mg2C and Ca2C. These results are similar to those of salivary apyrase of Aedes aegypty (Ribeiro et al., 1984), salivary glands of the Bed Bug Cimex lectularis (Valenzuela et al., 1996), salivary glands of the cat flea Ctenocephalides felis (Cheeseman, 1998), pig pancreas (Lebel et al., 1980), and ATP diphosphohydrolase in synaptosomes from cerebral cortex of adult rats (Battastini et al., 1991). (b) activation by either Ca2C or Mg2C alone. Cation dependence was confirmed by a dramatic decrease in ATP and ADP hydrolysis in the absence of cation or in the presence of cation plus 10 mM EDTA (Fig. 2).
The concentrations of sodium azide necessary to inhibit mitochondrial ATPase and alkaline phosphatases are in the mM range. In this study, low concentrations of sodium azide did not affect NTPDase activity. Accordingly, Battastini et al. (1991) demonstrated inhibition of NTPDase only when azide was used at high concentrations (>100 mM). We have also shown that sodium azide significantly inhibited ATP and ADP hydrolysis using both cations (Mg2C and Ca2C). In addition, our results with sodium azide in the presence of Ca2C, as activating divalent cation, were similar to those of Se´vigny et al. (1998) in pig stomach. Unlike sodium azide, suramin can significantly inactivate NTPDase when used at low (M concentrations. Suramin inhibits ecto-ATPase activity from urinary bladder (Hourani and Chown, 1989), blood cells (Beukers et al., 1995) and endothelial cells (Meghji and Burnstock, 1995). Our results clearly demonstrate that suramin is one of the most potent inhibitors of NTPDase in the rat gastric mucosa (Fig. 4). When NaC, KC-ATPase inhibitors were studied, ouabain did not affect the hydrolysis of either substrate, but, in contrast, orthovanadate (0.1e100 mM) slightly reduced the hydrolysis (approximately 15%) of both substrates. This effect could be due to a minor contamination with ATPase type P or V. Moreover, in the presence of Mg2C, La3C inhibited the hydrolysis of ATP and ADP to a similar extent. A similar phenomenon was observed when Ca2C was used as the activator ion; however, the maximum inhibitory effect was lower than Mg2C. The parallel inhibition of ATP and ADP hydrolysis strongly suggests that La3C is inhibiting NTPDase in the gastric mucosa. In the same way, Ziganshin et al. (1995) reported that La3C inhibits ecto-ATPase activity in the guinea-pig urinary bladder. Levamisole, an inhibitor of alkaline phosphatase, did not change the hydrolysis of either substrate tested, in the presence of Mg2C and Ca2C (Table 2). This result rules out alkaline phosphatase as a contaminant in the gastric mucosa preparation. Three sets of experimental data rule out enzyme combinations as mimics of NTPDase activity: (1) Adenylate kinase inhibitors Ap5A (Table 2) and NEM had no effect, thus, ADP hydrolysis was not due to the participation of the combination of adenylate kinase/ATPase. (2) The possible participation of other enzymes that could hydrolyze ATP or ADP (such as ATPases, ADPases, nonspecific phosphatases or pyrophosphatases) was ruled out by the following results: insensitivity to classical ATPase inhibitors, lack of
L. Savegnago et al. / Cell Biology International 29 (2005) 559e566
activity with phosphate esters and pyrophosphate (PPi) (Table 1). (3) ATP and ADP are hydrolyzed in a similar way. NTPDase 2 hydrolyzes ATP faster (30-fold) than ADP (Zimmermann, 2001; Knowles and Chiang, 2003), and NTPDase 3 also hydrolyzes ATP faster (3-fold) than ADP (Zimmermann, 2001). On the basis of these characteristics, we propose that the hydrolysis of ATP and ADP by gastric mucosa may be due to NTPDase activity, possibly type 1. Furthermore, the parallelism of the kinetic behavior in relation to cation and pH dependence and sensitivity to different inhibitors reinforces the hypothesis that only one enzyme is acting on the two substrates (ATP and ADP). However, further experiments will be required in order to determine the member of the NTPDase family involved in ATP and ADP hydrolysis in this preparation. ATP and metabolites, in addition to their role in energy metabolism, play an important role as extracellular regulatory and signaling molecules for many different cell types (Vlajkovic et al., 1996; Inscho et al., 1994), including gastric mucosa (Kwok et al., 1990). In line with this, reports have demonstrated the presence of P2Y purinoceptors in rabbit gastric glands (Gilrodrigo et al., 1996; Vallejo et al., 1996). In fact, the antisecretory effect of ATP on isolated rabbit parietal cells may be mediated via P2Y purinoceptors. ATP-sensitive potassium channels have been implicated in several physiological functions of the gastrointestinal tract, such as contractility, acid secretion and regulation of the gastric blood flow, as well as some pathological conditions (Rahgozar et al., 2001). Like ATP, adenosine modulates the secretion of a variety of gastric hormones in the antral and corporeal mucosa, and adenosine receptors are expressed in both regions (Harty and Franklin, 1984; Heldsinger et al., 1986; Puurunen et al., 1987; Yip and Kwok, 2004). Thus, parietal apyrase (NTPDase) can modulate gastric function by metabolizing ATP and, indirectly, by influencing the synthesis of adenosine. In conclusion, we have described an enzyme in the gastric mucosa that shares several kinetic properties with NTPDase.
Acknowledgements This work was supported by CNPq, FAPERGS, CAPES and UFSM (FIPE).
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