Cell Biology International (2005) 29, 559-566 - Lucielli Savegnago and others - Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

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Cell Biology International (2005) 29, 559–566 (Printed in Great Britain)

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Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

Lucielli Savegnago, Cristina W. Nogueira, Roselei Fachinetto and 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

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 Mg²⁺ and Ca²⁺. The inhibitors ouabain (0.01–1 mM), N-ethylmaleimide (0.01–4 mM), levamisole (0.10–0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03–30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50–300 μM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg²⁺and Ca²⁺ ATP/ADPase at 100 μM. Lanthanum decreased Mg²⁺ and Ca²⁺ 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.

Keywords: NTPDase, Stomach, Extracellular ATP, Ectonucleotidases, Apyrase.

^*Corresponding author. Tel.: +55 55 220 8140; fax: +55 55 220 8978.

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; Sévigny et al., 1998; Bonan et al., 2001; Tasca et al., 2004).

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 ecto-5′-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 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) (Sévigny 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 Sévigny 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 H⁺, K⁺, ATPase (Horn, 2000; Dunbar and Caplan, 2000). Furthermore, Lewis-Carl and Kirley (1997) also demonstrated that chicken ecto-ATPase 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 Materials and methods

2.1 Materials

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.

2.2 Animals

Male Wistar rats (200–300 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 Tris–HCl 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 0–4 °C throughout the preparation.

2.4 Enzyme assay

For NTPDase activity, the reaction mixture contained 80 mM Tris–HCl buffer, pH 7.4, and 3 mM CaCl2 or MgCl2 in a final volume of 500 μL. A 25 μL aliquot of the enzyme preparation (100–200 μg 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 μL 10% trichloroacetic acid (TCA). The samples were centrifuged at 1800 rpm for 10 min and 500 μL 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 non-enzymatic hydrolysis of substrates. Enzyme activity was expressed as nmol of phosphate (Pi) released min⁻¹ mg protein⁻¹.

2.5 Effect of pH

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 Tris+MOPS and pH was adjusted to 6.0–9.0. 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).

2.6 Substrate specificity

The substrate specificity was determined in a similar way as for the assay of NTPDase activity, with addition of ATP, ADP, AMP, β-glycerolphosphate and pyrophosphate (PPi) substrates.

2.7 Sensitivity to inhibitors

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 I50=0.04 mM) (Pullman et al., 1960);

(b)

suramin, ecto-ATPase inhibitor at the low micromolar range (Martí et al., 1996);

(c)

orthovanadate, an inhibitor of transport ATPases, acid phosphatases, alkaline phosphatases, Na⁺/K⁺-ATPase and phosphotyrosine phosphatases (Sorensen and Mahler, 1992; Cool and Blum, 1993);

(d)

N-ethylmaleimide (NEM), a Ca²⁺, Mg²⁺-ATPase and adenylate kinase inhibitor, (Mason and Saba, 1969; Russel et al., 1974);

(e)

ouabain, a specific inhibitor of Na⁺/K⁺-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).

2.8 Pyrophosphatase and nonspecific phosphatase activities

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): β-glycerolphosphate, pyrophosphate (PPi) or AMP, instead of ATP or ADP.

2.9 Adenylate kinase activity

For this assay the reaction medium was the same as for the assay of Ca²⁺ and Mg²⁺-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.

2.10 Protein determination

Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.

2.11 Statistical analysis

The values are expressed as mean±standard 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 8.0–8.5 range, in the presence of both Mg²⁺ (Fig. 1a) and Ca²⁺ (Fig. 1b).

Fig. 1

Effect of pH on Mg²⁺ATP/ADPase (a) and Ca²⁺ATP/ADPase (b) from rat gastric mucosa with ATP (○) or ADP (•) as substrate. Data represent the mean±SD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan's test.

Like other NTPDases, the enzyme was shown to be divalent cation-dependent and its sensitivity to Ca²⁺ and Mg²⁺ is illustrated in Fig. 2a,b. ATP and ADP hydrolysis was similar with Ca²⁺ or Mg²⁺ as the activating divalent cation. The function of the cation is to form the metal ion–nucleotide complex, which is the true substrate for the reaction (Valenzuela et al., 1989). EDTA (10 mM) decreased NTPDase activity in the presence of Ca²⁺ and Mg²⁺, when the substrate was ATP or ADP. In the absence of Ca²⁺ and Mg²⁺, rates of hydrolysis of ATP and ADP were very low (Fig. 2a,b), indicating a strict dependence on divalent cations.

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 4.0±1.5 and 7.0±3 nmol Pi min⁻¹ mg protein⁻¹ for Mg²⁺/Ca²⁺ATPase and Mg²⁺/Ca²⁺ADPase, respectively. Results are expressed as nmol Pi min⁻¹ mg protein⁻¹. Data represent the mean±SD of three different experiments and were statistically tested by one-way ANOVA followed by Duncan's test.

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 1.6±0.04 nmol Pi min⁻¹ mg protein⁻¹. However, MgCl2 was a better activator, since AMP hydrolysis with 3 mM MgCl2 corresponds to 4.85±0.05 nmol Pi min⁻¹ mg protein⁻¹. Further studies will be required to conclude that the enzyme that promotes AMP hydrolysis in rat gastric mucosa is an ecto-5′-nucleotidase.

Table 1.

Substrate specificity of NTPDase in the gastric mucosa of rats in the presence of Mg^2+a and Ca²⁺^a

Substrate (3 mM)	Specific activity (nmol Pi min⁻¹ mg protein⁻¹)		% Control activity
	Mg²⁺	Ca²⁺	Mg²⁺	Ca²⁺
ATP	67.87±8.00	45.25±10.00	100	100
ADP	51.80±6.00	39.00±6.00	76	86
AMP	4.85±0.05	1.60±0.04	7	3
PPi	0	0	0	0
β-Glycerol-phosphate	2.05±0.07	2.21±0.60	3	3

^a Data represent mean±SD of at least three experiments and are expressed as percentage of control activity (ATP hydrolysis 100%).

Our results also showed no significant hydrolysis of AMP and β-glycerolphosphate (Table 1). Since there was no significant hydrolysis when β-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 Mg²⁺ and Ca²⁺ (Table 2) in the gastric mucosa of rats. Thus, this result excludes alkaline phosphatase as a contaminant.

Table 2.

Effect of levamisole and Ap₅A on Mg²⁺and Ca²⁺ATP/ADPase activity from rat gastric mucosa

Inhibitor	Concentration (mM)	Enzyme activity (% of control)
		ATPase		ADPase
		Mg²⁺	Ca²⁺	Mg²⁺	Ca²⁺
Levamisole	0.10	103±4	106±2	109±8	109±8
	0.20	105±4	98±2	102±2	102±2
Ap₅A	0.03	99±3	97±1	96±4	96±4

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 not affect ATP or ADP hydrolysis in rat gastric mucosa (Table 2).

Sodium azide, tested in the 0.01–30 mM range, inhibited ATP and ADP hydrolysis in the presence of either Mg²⁺ (Fig. 3a) or Ca²⁺ (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 Mg²⁺ATP/ADPase, respectively (Fig. 3a). The specific enzyme activity was less sensitive to azide in the presence of Ca²⁺ 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).

Fig. 3

Effect of sodium azide on Mg²⁺ATP/ADPase (a) and Ca²⁺ATP/ADPase (b) from rat gastric mucosa with ATP (○) or ADP (•) as substrate. Sodium azide was used in 0.01–30 mM range. The control values were approximately 86±5 and 65±3 nmol Pi min⁻¹ mg protein⁻¹ for Mg²⁺ATPase and Mg²⁺ADPase, respectively. Control values of Ca²⁺ ATPase and Ca²⁺ADPase activities (100%) were 66±6 and 61±7 nmol Pi min⁻¹ mg protein⁻¹, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the mean±SD of three different experiments and were tested by one-way ANOVA followed by Duncan's test.

Suramin (0.05–0.3 mM) inhibited ATP and ADP hydrolysis in the presence of Mg²⁺ and Ca²⁺. The maximal inhibitory effect appeared at 0.3 mM and the specific enzyme activity was 60% for Mg²⁺/Ca²⁺ATPase (Fig. 4a) and 52% for Mg²⁺/Ca²⁺ADPase hydrolysis (Fig. 4b).

Fig. 4

Effect of suramin on Mg²⁺ATP/ADPase (a) and Ca²⁺ATP/ADPase (b) from rat gastric mucosa with ATP (○) or ADP (•) as substrate. Suramin was used in 0.05–0.3 mM range. The control values were approximately 70±4 and 53±6 nmol Pi min⁻¹ mg protein⁻¹ for Mg²⁺ATPase/ADPase, respectively. Control values for Ca²⁺ATPase and Ca²⁺ADPase activities (100%) were 44±7 and 39±5 nmol Pi min⁻¹ mg protein⁻¹, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the mean±SD 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 Mg²⁺ATP/ADPase. The inhibition determined in the presence of Ca²⁺, as activating divalent cation, was similar to that observed with Mg²⁺ (data not shown).

The inhibitor of Ca²⁺/Mg²⁺ATPase and adenylate kinase, N-ethylmaleimide, had no effect on NTPDase activity (data not shown).

The Na⁺/K⁺ ATPase inhibitor, ouabain, tested at 0.01–1 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 Mg²⁺ATP/ADPase (Fig. 5a) and presented a maximal inhibitory effect at 1 mM (∼64% for ATP and ∼42% for ADP hydrolysis). ATP and ADP hydrolysis measured in the presence of Ca²⁺ were less affected by La³⁺ (∼15% for ATP and 20% for ADP hydrolysis; Fig. 5b).

Fig. 5

Effect of lanthanum on Mg²⁺ATP/ADPase (a) and Ca²⁺ATP/ADPase (b) from rat gastric mucosa with ATP (○) or ADP (•) as substrate. Lanthanum was used in 0.04–1 mM range. The control values were 50±9 and 45±6 nmol Pi min⁻¹ mg protein⁻¹ for Mg²⁺ATPase/ADPase, respectively. Control values for Ca²⁺ATPase and Ca²⁺ADPase activities (100%) were 44±10 and 43±15 nmol Pi min⁻¹ mg protein⁻¹, respectively. Results are expressed as percentage of control activity in the absence of inhibitor. Data represent the mean±SD 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 Mg²⁺ and Ca²⁺. 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 Ca²⁺ or Mg²⁺ 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).

(c)

lack of inhibition by several inhibitors, such as P-type, F-type, V-type ATPases, alkaline phosphatases and adenylate kinase.

The concentrations of sodium azide necessary to inhibit mitochondrial ATPase and alkaline phosphatases are in the μM 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 μM). We have also shown that sodium azide significantly inhibited ATP and ADP hydrolysis using both cations (Mg²⁺ and Ca²⁺). In addition, our results with sodium azide in the presence of Ca²⁺, as activating divalent cation, were similar to those of Sévigny 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 Na⁺, K⁺-ATPase inhibitors were studied, ouabain did not affect the hydrolysis of either substrate, but, in contrast, orthovanadate (0.1–100 μM) 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 Mg²⁺, La3+ inhibited the hydrolysis of ATP and ADP to a similar extent. A similar phenomenon was observed when Ca²⁺ was used as the activator ion; however, the maximum inhibitory effect was lower than Mg²⁺. The parallel inhibition of ATP and ADP hydrolysis strongly suggests that La³⁺ is inhibiting NTPDase in the gastric mucosa. In the same way, Ziganshin et al. (1995) reported that La³⁺ 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 Mg²⁺ and Ca²⁺ (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 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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Received 16 September 2004/19 January 2005; accepted 8 March 2005

doi:10.1016/j.cellbi.2005.03.010

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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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