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Characterization of the effects of adenosine, adenosine 5′-triphosphate and related purines on acid secretion in isolated rabbit gastric glands
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CHARACTERIZATION OF THE EFFECTS OF ADENOSINE, ADENOSINE 5' TRIPHOSPHATE AND RELATED PURINES ON ACID SECRETION IN ISOLATED RABBIT GASTRIC GLANDS C.E . GIL RODRIGO*, B. GALDIZ, J .M. GANDARIAS, R. GOMEZ and L.F. AINZ Department of Physiology, Biochemistry and Biophysics, Faculty of Medicine, University of the Basque Country ( UPV-EHU ), P.O. Box 699, Bilbao, Spain Received in final form 20 September 1989
SUMMARY The influence of adenosine, AMP, ADP, ATP, the adenosine analogue L-PIA and the ATP analogue ß,y-methylene ATP, on gastric acid secretion, as measured by the aminopyrine accumulation method, in resting and histamine-stimulated glands isolated from rabbit gastric mucosa was studied . In resting glands, adenosine and its analogue L. PIA (10 ,um-1 mm) caused significant concentration-related increases of the basal H+ secretion, whereas no changes were obtained in response to the other purines tested . In histamine-stimulated glands, adenosine, L-PIA and AMP (10 um-1 mm) induced concentration-related increases of the H + secretory rate, whereas ATP, ß,y-methylene ATP and ADP (10 uM-1 mM) produced concentration-related decreases of the H + raised rate . The rank order of potency of the purine compounds in increasing the stimulated H+ secretion was : adenosine > L-P1A> AMP, and in decreasing it was : ATP> ADP> ß,y-methylene ATP. The stimulatory responses to adenosine were inhibited by theophylline (10 uM-100 uM) and caffeine (10 um-1 mm); whereas, the inhibitory responses to ATP were significantly reduced by the well known prostaglandin synthesis inhibitor indomethacin (1 um-100 um). From the results it is concluded that in isolated rabbit gastric glands, purine compounds are effective modulators of the gastric H+ secretory process . The pattern of purine activity obtained suggests that the stimulatory responses, inhibited by methylxanthines, may be mediated via P,purinoceptors, while the inhibitory responses, reduced by indomethacin, may be mediated via P,-purinoceptors . KEY WORDS:
adenosine, ATP, purines, acid secretion, rabbit gastric glands .
*To whom correspondence should be addressed . 1043-6618/90/020103-11/$03.00/0
© 1990 The Italian Pharmacological Society
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INTRODUCTION Adenosine and adenine nucleotides exert potent extracellular actions on a wide range of physiological systems (see [1, 2] for review). Most of these actions are mediated via specific purine receptors at the target cell . A division of purine receptors was proposed by Burnstock [3] : P,-purinoceptors were more responsive to adenosine than ATP and were antagonized by methylxanthines such as theophylline and caffeine, while P,-purinoceptors were more responsive to ATP than adenosine and in some cases seemed to evoke prostaglandin biosynthesis . Adenosine (P,-) receptors have been subdivided into subtypes A, and A, [4] or Ri and Ra [5] on the basis of the effects on the enzyme adenylate cyclase : activation of the A, (or Ri)-receptor inhibits this enzyme, whereas activation of the A, (or Ra)receptor causes stimulation [2] . A further subclassification of the P 2 -purinoceptor into subtypes P2i, and Pty has also been proposed [6, 7]. There is now considerable evidence in support of the relevant influence of purines in the control of gastrointestinal smooth muscle activity [2, 8] . Studying the involvement of mechanisms other than adrenergic or cholinergic mechanisms to explain the effects displayed by a water-soluble splenic material on gastrointestinal preparations [9-11] including purinergic mechanisms, we found that adenosine and adenine nucleotides caused significant changes on acid secretion in whole stomach preparations isolated from rats and based on this a regulatory role for purines in the gastric acid secretory process was proposed [12] . The effects of these purines on gastric acid secretion, particularly those concerning adenosine and its analogues, have been controversial . For example, in gastric preparations from rats adenosine and/or its derivatives have been described to cause stimulation [13], inhibition [14, 15], both stimulation and inhibition depending on the presence and the absence, respectively, of adrenergic and cholinergic blocking agents [16] and no effects [17] on acid secretion . Furthermore, Heldsinger et al. [18] have described dual concentration-dependent effects, stimulatory and inhibitory, for adenosine in guinea-pig gastric mucosa . In dogs, adenosine has been reported to inhibit gastric acid secretion [19, 20] ; whereas in rabbits, our preliminary results showed [21] that adenosine stimulates acid secretion in isolated gastric glands . Some conflicting results have also been reported for ATP [16, 18, 22, 23] . The aim of the present work has been to investigate the effects of adenosine (ADO), the ADO analogue L-N'-phenylisopropyladenosine (L-PIA), adenosine 5'monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'triphosphate (ATP) and the ATP analogue ß,y-methylene ATP on acid secretion in resting and histamine-stimulated glands isolated from rabbit gastric mucosa . In addition, the influence of theophylline and caffeine, described as selective competitive antagonists to P,-purinoceptors [24], on the H+ secretory responses to ADO and that of the well known prostaglandin synthesis inhibitor indomethacin on the responses elicited by ATP have been examined . Since acid secretion cannot be measured directly in glandular preparations, secretory responsiveness in the glands has been assessed by the measurement of changes in the accumulation of the radiolabelled weak base [` 4 C]aminopyrine (AP) [25] . As has been mentioned [21], a preliminary report of some of this work was presented to the Federation of European Biochemical Societies .
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MATERIALS AND METHODS Fed New Zealand white rabbits weighing 2 . 5-4 kg were killed by cervical fracture/ dislocation and the stomach was perfused as described by Berglindh and Obrink [26] . The buffer solutions for perfusion, disaggregation of glands and AP accumulation studies (respiratory medium) and the general assay procedure were performed essentially as reported by Sack and Spenney [25] . Briefly, aliquots of 0 . 1 ml of the stock gland suspension were placed in 1 . 5 ml preweighed sealed conical polypropylene Eppendorf tubes containing 0 . 01 uCi of AP, agents to be tested in adequate quantity to achieve the desired final concentration and respiratory medium up to complete a volume of 1 ml . The tubes were then sealed and incubated by submersion and horizontal mixing in a shaking incubator at 37°C for 30 min. After the incubation, the tubes were centrifuged for 5 min . Aliquots of 0 . 1 ml of the supernatant from each tube were placed in vials containing 10 ml of a suitable scintillation cocktail and counted . The remaining supernatant was discarded and the gland pellets were dried at 90 °C for 60 min. The tubes containing dry pellets were then reweighed, resuspended in 0 . 1 ml of 1 M KOH, dissolved by heating at 90 ° C for 15 min and quantitatively transferred and counted in 10 ml of suitable scintillation cocktail . The AP accumulation values were estimated as the ratio, Rap=AP intraglandular/AP medium, applying the expression [25] : Rap = pellet cpm/(2,ul/mg) (mg dry wt) (medium cpm/ml) Each preparation was tested for viability by trypan blue dye exclusion . Normally, a cellular viability greater than 90% was obtained . In addition, each experimental batch with gastric glands from separate rabbits was assayed with histamine (1 ,um-100µM) in order to control the secretory responsiveness . All agent concentrations given in the text are expressed as final molar tube concentrations . The secretory responses are given in terms of AP ratio quotient (QRap)=Rap (basal or agent)/Rap (basal) . The basal acid secretion was defined as the aminopyrine accumulation ratio in the absence of any drug and therefore its QRap = 1 . All experiments were carried out in triplicate for each data point . Statistical evaluation of the results, expressed as means ± SE mean (n = number of experiments performed with glandular preparations from different rabbits), was calculated by Student's t-test . The agents used were : histamine, adenosine, ATP, ADP, AMP and indomethacin purchased from Merck, L-PIA, ß,)A-methylene ATP, theophylline and caffeine from Sigma and [' -`C]aminopyrine from Amersham . All other chemicals used were of the highest grade available . M RESULTS Assays in resting glands
In preparations of resting glands, increasing concentrations (10 µM-1 mm) of both ADO and its analogue L-PIA caused significant (P< 0 . 05) and concentration-
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related increases of the basal acid secretory rate . The stimulation of H+ secretion in response to ADO and L-PIA (100 µM) was close to the 40% of that obtained with an equivalent concentration (100 /4M, the highest used) of histamine . The slopes and the theoretical maximal responses stimated from the double-reciprocal plot of the concentration-response curves to ADO and L-PIA were not significantly different . In addition, the apparent dissociation constant value for ADO (7 . 8 ,um ) . This provides a first was not significantly different to that of L-PIA (10 .1 µm) indication suggesting that the secretagogue activity displayed by ADO and L-PIA is mediated by the same purinergic receptor. On the other hand, assays carried out using theophylline (0 . 1 um-100 µm) and caffeine (1 um-1 mm) showed that both methylxanthines in concentrations of 1 um and 10 um respectively caused noticeable but not significant inhibitory effects on the H+ stimulatory responses to ADO, while concentrations of 10 ,um for theophylline and 100 µM for caffeine caused significant potentiation of the ADO secretory responses (Fig . 1) . At these concentrations either theophylline or caffeine alone caused no significant modification on basal H + secretion. Purine compounds other than ADO and L-P1A did not significantly affect the basal acid secretion . 4 QRap
3
2
1 -5 .5 -5 -4.5 -4 -3 .5 -3 -2 .5 log [adenosine] M
Fig. 1 . Effects of theophylline (A) 10 µ.M and caffeine (Y) 100,um on the concentration-response curve to adenosine (•) in rabbit resting glands . The gastric acid secretory responses are expressed in terms of aminopyrine ratio quotient (QRap)=Rap agents/Rap basal, thus QRap basal= 1 . Each point represents the mean of at least four experiments ; vertical bars indicate st mean .
Assays in histamine-stimulated gastric glands Effect of purines. Several studies led to the conclusion that histamine, acting via
H,-receptors, plays a central role in the physiological regulation of gastric acid secretion [27] ; therefore, histamine stimulation of glands was not merely used as a procedure to obtain a working-raised level of H+ secretion but to study a possible interdependence between the purinergic and the histaminergic gastric mechanisms . In this way, the effects of the purine compounds on stimulated-glands were examined and their influence on the histamine concentration-response curve
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studied . In these histamine-stimulated glandular preparations, the effects displayed by ADO, L-PIA and AMP were opposite to those of ATP, ß,y-methylene ATP and ADP. The first group of purines, headed by ADO, induced stimulation of the acid secretion and the second group of purines, headed by ATP, induced inhibition . ,um-100 ADO (10 um-100 uM ), L-PIA (10 µM) and AMP (10 µM-1 mm) caused a noticeable and qualitatively similar leftward shift in the histamine (1 um -100 'um) concentration-response curve indicating that the histamine secretory responses were potentiated by this group of purine compounds (Fig . 2, a and c) . The progressive potentiation displayed by ADO, L-PIA and AMP, became significant (P< 0 . 05) at concentrations of 30 JAM for ADO and 100 JAM for L-PIA, being close to the limit of significance for AMP 1 mm . Based on the data from concentration-response curves (Fig . 2, a and c), the rank order of potency of these three purine derivatives in increasing the histamine raised H+ secretory rate was : ADO > L,-PIA> AMP. Construction of the double-reciprocal plots of the histamine concentration-response curves in the presence of ADO (10 um-100 JM) lead to the suggestion that the potentiation induced by ADO is of non-competitive nature (Fig . 2d). Similar double-reciprocal plots were obtained with L-PIA and AMP. This provides some evidence in favour of a relationship between gastric mechanisms mediating ADO, L-PIA and AMP responses and those mediating histamine responses . Conversely to the secretagogue activity reported above for ADO, L-PIA and AMP ; ATP, ß,y-methylene ATP and ADP, in concentrations ranging from 10 um to 1 mm, caused concentration-dependent decreases of the histamine-stimulated H + secretory rate (Fig . 2, b and c) that reached significant levels at concentrations of 10 JAM for ATP, 100 um for ADP, being not significant for ß,y-methylene ATP . According to the data from concentration-response curves (Fig . 2, b and c), the rank order of potency of these purine compounds in decreasing the histamine raised H + secretory rate was: ATP> ADP > ß,y-methylene ATP . The double-reciprocal plots of the histamine concentration-response curves in the presence of ATP (10 ,um-1 mm) suggests that the inhibitory effect of ATP is of uncompetitive nature (Fig . 2e). A similar double reciprocal plot was obtained with ADP, but not with ß, y-methylene ATP where the double-reciprocal plot relationship suggested an interaction of non-competitive nature . Despite this last case, these findings provide some indication that, at least in early stages, the gastric mechanisms mediating ATP and ADP responses are not directly related to those mediating histamine responses . Effects of theophylline and caffeine on the stimulation of the histamine raised H+ secretion induced by ADO. Experiments carried out to examine the influence of
theophylline (1 um-100 ,um) and caffeine (10 ,um-1 mm) on the secretory responses to histamine (1 um-100 ,um) revealed that the histamine raised H+ secretory rate was not significantly modified by either theophylline (1 um and 10 JM) or caffeine (10 um and 100 um) ; whereas, both methylxanthines in concentrations of 100 JAM for theophylline and 1 mm for caffeine caused significant potentiation on the secretory responses to histamine (Fig . 3a) . In this context, the stimulatory responses to ADO 100 ,um were significantly inhibited by theophylline 1 JAM and 10 um but not by theophylline 100 JAM (Fig. 3b).
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3 2 I -6 .5 -6 -5.5 -5 -4.5 -4 -3.5 log [histamine] M
5 QRap
(b)
4
3
2
-6 .5 -6 -5 .5 -5 -4.5 -4 -3.5 log [histamine] M 8 QRap
(c)
7
6 5 4 3 2 1 -6 .5 -6 -5 .5 -5 -4.5 -4 -3.5 log [histamine] M
Fig . 2 . Effects of (a) ADO 10 µM (∎), 30 µM (V) and 100 µM (A), (b) ATP 10µM (∎), 100µM (V) and 1 mm (A), (c) L-PIA 100 µM ( • ), AMP 1 mm (0), ß, y-methylene ATP 1 mm (V) and ADP 1 mm (A) on the concentration-response curve to histamine (0) in rabbit gastric glands . Responses are expressed in terms of aminopyrine ratio quotient (QRap) =Rap agents/Rap basal, thus QRap basal= 1 . Each point represents the mean of at least four experiments ; vertical bars indicate SE mean . Graphs d and e show the doublereciprocal plots of the histamine concentration-response curves in the presence of ADO (data in graph a) and ATP (data in graph b) respectively.
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-6.5 -6 -5.5 -5 -4 .5 -4 -3 .5 -6 .5 -6 -5 .5 -5 -4.5 -4 -3 .5 log [histamine] M log [histamine] M
Fig. 3.
Effects of theophylline (A) 1 ,um, (D) 100 ,um and caffeine (Y) 10 ,um, ( •) 1 mm on the concentration-response curves (a) to histamine ( •) and (b) to histamine plus adenosine 100 um (∎) in isolated rabbit gastric glands . The concentration-response curve to histamine alone (•) is included as control in graph b . The H+ secretory responses are expressed in terms of aminopyrine ratio quotient (QRap) = Rap agents/Rap basal, thus QRap basal = 1 . Each point represents the mean of at least four experiments ; vertical bars indicate SE mean .
The order of potency of theophylline (THEO) concentrations in producing inhibition of the ADO induced acid secretory responses was : THEO 1 µM > THEO 10 µM > THEO 100 µM . Similar results were obtained with caffeine (CAF, Fig . 3b). In this case the order of potency was : CAF 10µm,>-CAF 100 µM > CAF 1 mm . Effect of indomethacin on the ATP-induced inhibition of the histamine-stimulated
It has been proposed that ATP is a potent agonist for P,purinoceptors and that occupation of these may lead to prostaglandin synthesis . Prostaglandins have been reported to be inhibitors of gastric acid secretion [28-30] . Based on this, inhibition of the histamine-stimulated H+ secretory rate in response to ATP (see above) may be mediated through prostaglandin biosynthesis . Experiments with histamine-stimulated glands preincubated with indomethacin (1 µM-100µM) for 10 min before addition of ATP were carried out to examine this possibility. Under our experimental conditions, indomethacin alone did not significantly potentiate the histamine-raised H + secretion. As is shown in Fig . 4, indomethacin (1 µm-100 µM) caused graded decreases of the inhibitory responses to ATP (1 mm) . This suggests that the inhibitory effects of ATP are mediated, at least in part, via prostaglandin production . H+ secretion.
DISCUSSION The important role of histamine, acetylcholine and gastrin in the physiological regulation of gastric acid secretion is well documented [27] . In addition, several other agents have been considered as possible effective modulators of the gastric H+ secretory function. In this way, purine nucleosides and nucleotides have been
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-6 .5 -6 -5.5 -5 -4 .5 -4 -3.5 log [histamine] M
Fig . 4 . Effect of graded concentrations of indomethacin (Y) 1 µm, (∎) 10 ,um and (A) 100 µM on the concentration-response curve to histamine plus ATP 1 mm ( •) in gastric glands from rabbit . The concentration-response curve to histamine alone ( •) is included as control . The gastric acid secretory responses are expressed in terms of aminopyrine ratio quotient (QRap) = Rap agents/Rap basal, thus QRap basal = 1 . Each point represents the mean of at least four experiments ; vertical bars indicate sL mean .
proposed to be involved in the physiological regulation of this process [12] . In support of this proposition, the present results show that purines exert relevant effects on acid secretion in isolated rabbit gastric glands . From the activities found here, the H + secretion was stimulated by ADO, L-PIA and AMP and inhibited by ATP, ß,y-methylene ATP and ADP . Our experimental approach on the mechanism by which ADO stimulates H + secretion led to the following findings and considerations : 1 . The range (µM) of apparent Kd values obtained here fitted better to those reported for ADO at the A 2/Ra receptor (low affinity) than those at the A,/Ri receptor (high affinity) [31] . 2 . The rank order of potency : ADO > L-PIA> AMP, was similar to that described in other tissues in which purines cause adenylate cyclase activation [24] . In addition, increases of cAMP in gastric tissue have been correlated with the stimulation of acid secretion in response to histamine and some other secretagogues [32-35] . 3 . The apparent relationship found here between gastric mechanisms mediating ADO responses and those mediating histamine responses, agrees with that of a previous work [36] in which potentiation of the adenosine A2 /Ra receptormediated accumulation of cAMP by histamine, acting via H,-receptors, in guineapig cerebral cortical slices has been reported . 4 . Theophylline and caffeine behave as selective antagonists at the adenosine (P,-) receptors (see above) but also as phosphodiesterase inhibitors . At high concentrations both methylxanthines act mainly as phosphodiesterase inhibitors, whereas, at low concentrations they act mainly as adenosine receptor antagonists
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[8] . Based on this, the pattern of activity reported here for theophylline and caffeine on the H+ secretory responses elicited by ADO in both resting and histaminestimulated glands can be interpreted as the result of an antagonist effect at the adenosine receptor and its inhibitory effect on phosphodiesterase activity . In this way, at the lower concentrations used theophylline and caffeine act mainly as adenosine receptor antagonists, whereas at the higher concentrations used the adenosine receptor antagonist effect appears to be masked by the effect due to phosphodiesterase inhibition . These findings are consistent with the involvement of adenosine (P,-) receptors and adenylate cyclase activation (A 2/Ra-receptors) in the acid secretory responses to ADO and its related derivatives . In agreement with our previous results [21] and those described here, Ota et al. [37] have recently reported that ADO and its analogues cause stimulation on acid secretion in rabbit parietal cells and they have suggested that this effect is mediated via adenosine A,_receptors . From the present results and those previously reported on the effects of ADO and its derivatives on acid secretion in gastric preparations such as isolated glands and isolated parietal cells, three main lines of evidence have emerged . One of them supports that these purines exert inhibitory effects on acid secretion in canine parietal cells and suggests that adenosine A,- (or Ri)- receptors are involved [20, 38] ; another suggests the absence of adenosine receptors in rat parietal cells on the basis of the lack of effect of ADO on acid secretion in this preparation [17] ; and finally, the third one supports that ADO and its derivatives stimulate acid secretion in rabbit gastric glands ([21], the present work) and parietal cells [37] and suggests that adenosine A,- (or Ra)- receptors are involved . We cannot explain this variety of results with any relevant difference in the experimental procedure ; thus it seems that the action of ADO on gastric acid secretion depends on the species used . Conversely to the secretagogue activity obtained from assays with ADO, ATP caused inhibition of the histamine-stimulated H + secretion in gastric glands . In previous works with supraepithelial preparations, ATP has been reported to cause inhibition [22, 23], moderate stimulation and/or no effect [16, 18] on gastric acid secretion. The influence of ATP on acid secretion has also been studied in isolated gastric glands, but from a metabolic point of view [39]. In these studies gastric glands have been reported to be subjected to a permeabilization process to facilitate the incorporation of the exogenous ATP into the glands . As could be expected, under these conditions ATP enhances acid secretion probably by increasing the activity of H+-transporting ATPase . Assuming the purinergic receptor classification criteria proposed by Burnstock [3], our present results indicate that the P 2-purinoceptors may mediate the inhibitory effect of ATP on H+ secretion . This purinoceptor has been suggested not to be linked to the adenylate cyclase system, being related in some cases to the prostaglandin biosynthesis [24] . Prostaglandins have been reported to be inhibitors of gastric acid secretion [28-30] . In support of this possibility, the results show that the inhibitory effect of ATP was significantly reduced by the prostaglandin synthesis inhibitor indomethacin and that the interaction between gastric mechanisms, mediating ATP, ADP and probably ß,y-methylene ATP responses and those mediating histamine responses was of uncompetitive nature . On the other
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hand, the rank order of potency obtained here, ATP> ADP >> fl, y-methylene ATP, evokes the order of potency described for purines at the P2Ypurinoceptor [6, 7] . In conclusion, the present findings support the view that ADO, ATP and related purines act as effective modulators in the regulation of gastric acid secretion and provide evidence indicating that the P,-purinoceptor via adenylate cyclase activation and the P2-purinoceptor via stimulation of prostaglandin production may mediate the stimulatory and the inhibitory effects, respectively, displayed by these purines on gastric H+ secretion in isolated rabbit gastric glands . ACKNOWLEDGEMENTS This study was supported in part by the Department of Health of the Government of the Basque Country . REFERENCES 1 . Gordon GL . Extracellular ATP: effects, sources and fate . Biochem J 1986 ; 233 : 309-19 . 2 . Williams M . Purine receptors in mammalian tissues : pharmacology and functional significance . Ann Rev Pharmacol Toxicol 1987 ; 27 : 315-45 . 3 . Burnstock G. A basis for distinguishing two types of purinergic receptor. In : Straub RW, Bolis L, eds. Cell membrane receptors for drugs and hormones . New York : Raven Press, 1978 :107-18 . 4 . Van Calker D, Muller M, Hamprecht B . Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells . J Neurochem 1979 ; 33 :999-1005 . 5 . Londos C, Cooper DMF, Wolff J . Subclasses of external adenosine receptors . Proc Natl Acad Sci USA 1980 ; 77 :2551-4 . 6 . Burnstock G, Kennedy C . Is there a basis for distinguishing two types of P2purinoceptor? Gen Pharmacol1985 ; 16 : 433-40 . 7 . Burnstock G, Warland JJI. PZ purinoceptors of two subtypes in the rabbit mesenteric artery : reactive blue 2 selectively inhibits responses mediated via the P,- but not the Ppurinoceptor. BrJPharmacol1987 ; 90 : 383-91 . 8 . Burnstock G . Purinergic transmitters and receptors : new directions . In : Stefanovich V, Rudolphi K, Schubert P, eds. Adenosine : receptors and modulation of cell function . Oxford : IRL Press, 1985 : 3-13 . 9 . Gandarias JM, Ainz LF, Fernândez B, Goiriena JJ, Lacort M, Rabanal S . Effects of water soluble splenic extracts on intestinal smooth muscle . Arch Farmac Toxicol 1978 ; IV: 331-8 . 10 . Gil-Rodrigo CE . Mecanismo de action de algunos extractos tisulares hidrosolubles sobre miisculo liso intestinal . [Doctoral Thesis 1980] . Madrid : Universidad Complutense de Madrid, 1981 : 324 p. 11 . Ainz LF, Casis E, Gandarias JM, Gil-Rodrigo CE, Goiriena JJ . H,-histamine receptors may mediate the contractile response of guinea-pig ileum to 'histamine-free' splenic extracts . BrJPharmacol1983 ; 79 : 373-8 . 12 . Gandarias JM, Ainz LF, Gil-Rodrigo CE, Martinez I, Goiriena JJ . Efecto de algunas purinas sobre la secretion g$strica de âcido . [abstract] . V11 Reunion Nacional de la Asociacion Espanola de Farmacologos . Salamanca, 1982 :196 . 13 . Puurunen J, Aittakumpu R, Tanskanen T . Vagally mediated stimulation of gastric acid secretion by intravenously administered adenosine derivatives in anaesthetized rats . Acta Pharmacol Toxicol 1986 ; 58 : 265-71 . 14 . Glavin GB, Westerberg VS, Geiger JD. Modulation of gastric acid secretion by adenosine in conscious rats . Can J Physiol Pharmacol 1987 ; 65 : 1182-5 . 15 . Scarpignato C, Tramacere R, Zappia L, Del Soldato P . Inhibition of gastric acid secretion by adenosine receptor stimulation in rat . Pharmacology 1987 ; 34 : 264-8 .
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16 . Gandarias JM, Ainz LF, Gil-Rodrigo CE, Goiriena JJ, Gomez R, Martinez 1 . Efecto de algunos derivados de la adenina sobre la secrecion gâstrica de âcido en est6mago aislado de rata . Rev Esp Fisiol 1985 ; 41 : 83-8. 17 . Puurunen J, Ruoff HJ, Schwabe U . Lack of direct effect of adenosine on the parietal cell function in the rat . Pharmacol Toxicol 1987 ; 60 : 315-17 . 18 . Heldsinger AA, Vinik AI, Fox IH. Inhibition of guinea-pig oxyntic cell function by adenosine and prostaglandins . JPharmacol Exp Ther 1986 ; 237 : 351-6 . 19 . Gerber JG, Fadul S, Payne NA, Nies AS . Adenosine : a modulator of gastric acid secretion in vivo. J Pharmacol Exp Ther 1984 ; 231 : 109-13 . 20 . Gerber JG, Nies AS, Ann Payne N . Adenosine receptors on canine parietal cells modulate gastric acid secretion to histamine . J Pharmacol Exp Ther 1985 ; 233 : 623-7 . 21 . Gandarias JM, Gil-Rodrigo CE, Ainz LF, Requejo D, Malillos M, Gomez R . Stimulation by adenosine of H+ secretion in rabbit gastric glands : influence of some methylxanthines . [abstract] . 18th FEBS Meeting. Ljubljana 1987 : 174 . 22 . Kidder GW. The effects of exogenous ATP on gastric secretion in frog gastric mucosa . Am J Physiol 197 1 ; 221 : 421-6 . 23 . Sanders SS, Butler CF, O'Callaghan J, Rehm WS . Inhibition of H+ secretion in the frog gastric mucosa by ATP and related compounds . Am JPhysiol 1976 ; 230 : 1688-94 . 24 . Burnstock G, Buckley NJ . The classification of receptors for adenosine and adenine nucleotides . In : Paton DM, ed . Methods in pharmacology, vol 6 . London: Plenum, 1985 : 193-212 . 25 . Sack J, Spenney JG . Aminopyrine accumulation by mammalian gastric glands : an analysis of the technique . Am JPhysiol 1982 ; 243 : G313-19 . 26 . Berglindh T, Obrink KJ . A method for preparing isolated glands from the rabbit gastric mucosa . Acta Physiol Scand 1976 ; 96 : 150-9 . 27 . Sanders MJ, Soil AH . Characterization of receptors regulating secretory function in the fundic mucosa. Ann Rev Physiol 1986 ; 48 : 89-101 . 28 . Soll AH, Walsh JH . Regulation of gastric acid secretion . Ann Rev Physiol 1979 ; 41 : 35-53 . 29 . Soll AH . Specific inhibition by prostaglandin E, and I, of histamine-stimulated [ "C]aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells . J Clin Invest 1980 ; 65 : 1222-9 . 30 . Seidler U, Beinborn M, Sewing K-F. Inhibition of acid formation in rabbit parietal cells by prostaglandins is mediated by the prostaglandin E . receptor. Gastroenterology 1989 : 96 : 314-20 . 31 . Schwabe U . Classification of adenosine receptors . In: Stefanovich V, Rudolphi K, Schubert P, eds . Adenosine : receptors and modulation of cell function. Oxford : IRL Press, 1985 : 15-28 . 32 . Forte JG, Machen l'E, Obrink KJ. Mechanisms of gastric H+ and Cl - transport . Ann Rev Physiol 1980 ; 42 : 111-26 . 33 . Chew CS, Hersey SJ, Sachs G, Berglindh T . Histamine responsiveness of isolated gastric glands . Am J Physiol 1980 ; 238 : G312-20 . 34 . Sachs G, Berglindh T . Physiology of the parietal cell . In : Johnson LR, ed . Physiology of the gastrointestinal tract. New York : Raven Press, 1981 : 567-602 . 35 . Mardh S, Song YH, Carlsson C, Bjorkman T . Mechanisms of stimulation of acid production in parietal cell isolated from the pig gastric mucosa. Acta Physiol Scand 1987;131 :589-98 . 36 . Hollingsworth EB, De la Cruz RA, Daly JW . Accumulation of inositol phosphates and cyclic AMP in brain slices : synergistic interactions of histamine and 2-chloroadenosine . EurJPharmacoll986 ; 122 :45-50 . 37 . Ota S, Hiraishi H, Terano A, et al. The effect of adenosine (ADO) and adenosine analogues on ["C]aminopyrine (AP) uptake of rabbit parietal cells . [Abstract] . Gastroenterology 1988 ; 94(5) : A337 . 38 . Gerber JG, Ann Payne N . Endogenous adenosine modulates gastric acid secretion to histamine in canine parietal cells. J Pharmacol Exp Ther 1988 ; 244 : 190-4 . 39 . Berglindh T . The mammalian gastric parietal cell in vitro. Ann Rev Physiol 1984; 46 : 377-92 .
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CHARACTERIZATION OF THE EFFECTS OF ADENOSINE, ADENOSINE 5' TRIPHOSPHATE AND RELATED PURINES ON ACID SECRETION IN ISOLATED RABBIT GASTRIC GLANDS C.E . GIL RODRIGO*, B. GALDIZ, J .M. GANDARIAS, R. GOMEZ and L.F. AINZ Department of Physiology, Biochemistry and Biophysics, Faculty of Medicine, University of the Basque Country ( UPV-EHU ), P.O. Box 699, Bilbao, Spain Received in final form 20 September 1989
SUMMARY The influence of adenosine, AMP, ADP, ATP, the adenosine analogue L-PIA and the ATP analogue ß,y-methylene ATP, on gastric acid secretion, as measured by the aminopyrine accumulation method, in resting and histamine-stimulated glands isolated from rabbit gastric mucosa was studied . In resting glands, adenosine and its analogue L. PIA (10 ,um-1 mm) caused significant concentration-related increases of the basal H+ secretion, whereas no changes were obtained in response to the other purines tested . In histamine-stimulated glands, adenosine, L-PIA and AMP (10 um-1 mm) induced concentration-related increases of the H + secretory rate, whereas ATP, ß,y-methylene ATP and ADP (10 uM-1 mM) produced concentration-related decreases of the H + raised rate . The rank order of potency of the purine compounds in increasing the stimulated H+ secretion was : adenosine > L-P1A> AMP, and in decreasing it was : ATP> ADP> ß,y-methylene ATP. The stimulatory responses to adenosine were inhibited by theophylline (10 uM-100 uM) and caffeine (10 um-1 mm); whereas, the inhibitory responses to ATP were significantly reduced by the well known prostaglandin synthesis inhibitor indomethacin (1 um-100 um). From the results it is concluded that in isolated rabbit gastric glands, purine compounds are effective modulators of the gastric H+ secretory process . The pattern of purine activity obtained suggests that the stimulatory responses, inhibited by methylxanthines, may be mediated via P,purinoceptors, while the inhibitory responses, reduced by indomethacin, may be mediated via P,-purinoceptors . KEY WORDS:
adenosine, ATP, purines, acid secretion, rabbit gastric glands .
*To whom correspondence should be addressed . 1043-6618/90/020103-11/$03.00/0
© 1990 The Italian Pharmacological Society
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INTRODUCTION Adenosine and adenine nucleotides exert potent extracellular actions on a wide range of physiological systems (see [1, 2] for review). Most of these actions are mediated via specific purine receptors at the target cell . A division of purine receptors was proposed by Burnstock [3] : P,-purinoceptors were more responsive to adenosine than ATP and were antagonized by methylxanthines such as theophylline and caffeine, while P,-purinoceptors were more responsive to ATP than adenosine and in some cases seemed to evoke prostaglandin biosynthesis . Adenosine (P,-) receptors have been subdivided into subtypes A, and A, [4] or Ri and Ra [5] on the basis of the effects on the enzyme adenylate cyclase : activation of the A, (or Ri)-receptor inhibits this enzyme, whereas activation of the A, (or Ra)receptor causes stimulation [2] . A further subclassification of the P 2 -purinoceptor into subtypes P2i, and Pty has also been proposed [6, 7]. There is now considerable evidence in support of the relevant influence of purines in the control of gastrointestinal smooth muscle activity [2, 8] . Studying the involvement of mechanisms other than adrenergic or cholinergic mechanisms to explain the effects displayed by a water-soluble splenic material on gastrointestinal preparations [9-11] including purinergic mechanisms, we found that adenosine and adenine nucleotides caused significant changes on acid secretion in whole stomach preparations isolated from rats and based on this a regulatory role for purines in the gastric acid secretory process was proposed [12] . The effects of these purines on gastric acid secretion, particularly those concerning adenosine and its analogues, have been controversial . For example, in gastric preparations from rats adenosine and/or its derivatives have been described to cause stimulation [13], inhibition [14, 15], both stimulation and inhibition depending on the presence and the absence, respectively, of adrenergic and cholinergic blocking agents [16] and no effects [17] on acid secretion . Furthermore, Heldsinger et al. [18] have described dual concentration-dependent effects, stimulatory and inhibitory, for adenosine in guinea-pig gastric mucosa . In dogs, adenosine has been reported to inhibit gastric acid secretion [19, 20] ; whereas in rabbits, our preliminary results showed [21] that adenosine stimulates acid secretion in isolated gastric glands . Some conflicting results have also been reported for ATP [16, 18, 22, 23] . The aim of the present work has been to investigate the effects of adenosine (ADO), the ADO analogue L-N'-phenylisopropyladenosine (L-PIA), adenosine 5'monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'triphosphate (ATP) and the ATP analogue ß,y-methylene ATP on acid secretion in resting and histamine-stimulated glands isolated from rabbit gastric mucosa . In addition, the influence of theophylline and caffeine, described as selective competitive antagonists to P,-purinoceptors [24], on the H+ secretory responses to ADO and that of the well known prostaglandin synthesis inhibitor indomethacin on the responses elicited by ATP have been examined . Since acid secretion cannot be measured directly in glandular preparations, secretory responsiveness in the glands has been assessed by the measurement of changes in the accumulation of the radiolabelled weak base [` 4 C]aminopyrine (AP) [25] . As has been mentioned [21], a preliminary report of some of this work was presented to the Federation of European Biochemical Societies .
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MATERIALS AND METHODS Fed New Zealand white rabbits weighing 2 . 5-4 kg were killed by cervical fracture/ dislocation and the stomach was perfused as described by Berglindh and Obrink [26] . The buffer solutions for perfusion, disaggregation of glands and AP accumulation studies (respiratory medium) and the general assay procedure were performed essentially as reported by Sack and Spenney [25] . Briefly, aliquots of 0 . 1 ml of the stock gland suspension were placed in 1 . 5 ml preweighed sealed conical polypropylene Eppendorf tubes containing 0 . 01 uCi of AP, agents to be tested in adequate quantity to achieve the desired final concentration and respiratory medium up to complete a volume of 1 ml . The tubes were then sealed and incubated by submersion and horizontal mixing in a shaking incubator at 37°C for 30 min. After the incubation, the tubes were centrifuged for 5 min . Aliquots of 0 . 1 ml of the supernatant from each tube were placed in vials containing 10 ml of a suitable scintillation cocktail and counted . The remaining supernatant was discarded and the gland pellets were dried at 90 °C for 60 min. The tubes containing dry pellets were then reweighed, resuspended in 0 . 1 ml of 1 M KOH, dissolved by heating at 90 ° C for 15 min and quantitatively transferred and counted in 10 ml of suitable scintillation cocktail . The AP accumulation values were estimated as the ratio, Rap=AP intraglandular/AP medium, applying the expression [25] : Rap = pellet cpm/(2,ul/mg) (mg dry wt) (medium cpm/ml) Each preparation was tested for viability by trypan blue dye exclusion . Normally, a cellular viability greater than 90% was obtained . In addition, each experimental batch with gastric glands from separate rabbits was assayed with histamine (1 ,um-100µM) in order to control the secretory responsiveness . All agent concentrations given in the text are expressed as final molar tube concentrations . The secretory responses are given in terms of AP ratio quotient (QRap)=Rap (basal or agent)/Rap (basal) . The basal acid secretion was defined as the aminopyrine accumulation ratio in the absence of any drug and therefore its QRap = 1 . All experiments were carried out in triplicate for each data point . Statistical evaluation of the results, expressed as means ± SE mean (n = number of experiments performed with glandular preparations from different rabbits), was calculated by Student's t-test . The agents used were : histamine, adenosine, ATP, ADP, AMP and indomethacin purchased from Merck, L-PIA, ß,)A-methylene ATP, theophylline and caffeine from Sigma and [' -`C]aminopyrine from Amersham . All other chemicals used were of the highest grade available . M RESULTS Assays in resting glands
In preparations of resting glands, increasing concentrations (10 µM-1 mm) of both ADO and its analogue L-PIA caused significant (P< 0 . 05) and concentration-
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related increases of the basal acid secretory rate . The stimulation of H+ secretion in response to ADO and L-PIA (100 µM) was close to the 40% of that obtained with an equivalent concentration (100 /4M, the highest used) of histamine . The slopes and the theoretical maximal responses stimated from the double-reciprocal plot of the concentration-response curves to ADO and L-PIA were not significantly different . In addition, the apparent dissociation constant value for ADO (7 . 8 ,um ) . This provides a first was not significantly different to that of L-PIA (10 .1 µm) indication suggesting that the secretagogue activity displayed by ADO and L-PIA is mediated by the same purinergic receptor. On the other hand, assays carried out using theophylline (0 . 1 um-100 µm) and caffeine (1 um-1 mm) showed that both methylxanthines in concentrations of 1 um and 10 um respectively caused noticeable but not significant inhibitory effects on the H+ stimulatory responses to ADO, while concentrations of 10 ,um for theophylline and 100 µM for caffeine caused significant potentiation of the ADO secretory responses (Fig . 1) . At these concentrations either theophylline or caffeine alone caused no significant modification on basal H + secretion. Purine compounds other than ADO and L-P1A did not significantly affect the basal acid secretion . 4 QRap
3
2
1 -5 .5 -5 -4.5 -4 -3 .5 -3 -2 .5 log [adenosine] M
Fig. 1 . Effects of theophylline (A) 10 µ.M and caffeine (Y) 100,um on the concentration-response curve to adenosine (•) in rabbit resting glands . The gastric acid secretory responses are expressed in terms of aminopyrine ratio quotient (QRap)=Rap agents/Rap basal, thus QRap basal= 1 . Each point represents the mean of at least four experiments ; vertical bars indicate st mean .
Assays in histamine-stimulated gastric glands Effect of purines. Several studies led to the conclusion that histamine, acting via
H,-receptors, plays a central role in the physiological regulation of gastric acid secretion [27] ; therefore, histamine stimulation of glands was not merely used as a procedure to obtain a working-raised level of H+ secretion but to study a possible interdependence between the purinergic and the histaminergic gastric mechanisms . In this way, the effects of the purine compounds on stimulated-glands were examined and their influence on the histamine concentration-response curve
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studied . In these histamine-stimulated glandular preparations, the effects displayed by ADO, L-PIA and AMP were opposite to those of ATP, ß,y-methylene ATP and ADP. The first group of purines, headed by ADO, induced stimulation of the acid secretion and the second group of purines, headed by ATP, induced inhibition . ,um-100 ADO (10 um-100 uM ), L-PIA (10 µM) and AMP (10 µM-1 mm) caused a noticeable and qualitatively similar leftward shift in the histamine (1 um -100 'um) concentration-response curve indicating that the histamine secretory responses were potentiated by this group of purine compounds (Fig . 2, a and c) . The progressive potentiation displayed by ADO, L-PIA and AMP, became significant (P< 0 . 05) at concentrations of 30 JAM for ADO and 100 JAM for L-PIA, being close to the limit of significance for AMP 1 mm . Based on the data from concentration-response curves (Fig . 2, a and c), the rank order of potency of these three purine derivatives in increasing the histamine raised H+ secretory rate was : ADO > L,-PIA> AMP. Construction of the double-reciprocal plots of the histamine concentration-response curves in the presence of ADO (10 um-100 JM) lead to the suggestion that the potentiation induced by ADO is of non-competitive nature (Fig . 2d). Similar double-reciprocal plots were obtained with L-PIA and AMP. This provides some evidence in favour of a relationship between gastric mechanisms mediating ADO, L-PIA and AMP responses and those mediating histamine responses . Conversely to the secretagogue activity reported above for ADO, L-PIA and AMP ; ATP, ß,y-methylene ATP and ADP, in concentrations ranging from 10 um to 1 mm, caused concentration-dependent decreases of the histamine-stimulated H + secretory rate (Fig . 2, b and c) that reached significant levels at concentrations of 10 JAM for ATP, 100 um for ADP, being not significant for ß,y-methylene ATP . According to the data from concentration-response curves (Fig . 2, b and c), the rank order of potency of these purine compounds in decreasing the histamine raised H + secretory rate was: ATP> ADP > ß,y-methylene ATP . The double-reciprocal plots of the histamine concentration-response curves in the presence of ATP (10 ,um-1 mm) suggests that the inhibitory effect of ATP is of uncompetitive nature (Fig . 2e). A similar double reciprocal plot was obtained with ADP, but not with ß, y-methylene ATP where the double-reciprocal plot relationship suggested an interaction of non-competitive nature . Despite this last case, these findings provide some indication that, at least in early stages, the gastric mechanisms mediating ATP and ADP responses are not directly related to those mediating histamine responses . Effects of theophylline and caffeine on the stimulation of the histamine raised H+ secretion induced by ADO. Experiments carried out to examine the influence of
theophylline (1 um-100 ,um) and caffeine (10 ,um-1 mm) on the secretory responses to histamine (1 um-100 ,um) revealed that the histamine raised H+ secretory rate was not significantly modified by either theophylline (1 um and 10 JM) or caffeine (10 um and 100 um) ; whereas, both methylxanthines in concentrations of 100 JAM for theophylline and 1 mm for caffeine caused significant potentiation on the secretory responses to histamine (Fig . 3a) . In this context, the stimulatory responses to ADO 100 ,um were significantly inhibited by theophylline 1 JAM and 10 um but not by theophylline 100 JAM (Fig. 3b).
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3 2 I -6 .5 -6 -5.5 -5 -4.5 -4 -3.5 log [histamine] M
5 QRap
(b)
4
3
2
-6 .5 -6 -5 .5 -5 -4.5 -4 -3.5 log [histamine] M 8 QRap
(c)
7
6 5 4 3 2 1 -6 .5 -6 -5 .5 -5 -4.5 -4 -3.5 log [histamine] M
Fig . 2 . Effects of (a) ADO 10 µM (∎), 30 µM (V) and 100 µM (A), (b) ATP 10µM (∎), 100µM (V) and 1 mm (A), (c) L-PIA 100 µM ( • ), AMP 1 mm (0), ß, y-methylene ATP 1 mm (V) and ADP 1 mm (A) on the concentration-response curve to histamine (0) in rabbit gastric glands . Responses are expressed in terms of aminopyrine ratio quotient (QRap) =Rap agents/Rap basal, thus QRap basal= 1 . Each point represents the mean of at least four experiments ; vertical bars indicate SE mean . Graphs d and e show the doublereciprocal plots of the histamine concentration-response curves in the presence of ADO (data in graph a) and ATP (data in graph b) respectively.
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-6.5 -6 -5.5 -5 -4 .5 -4 -3 .5 -6 .5 -6 -5 .5 -5 -4.5 -4 -3 .5 log [histamine] M log [histamine] M
Fig. 3.
Effects of theophylline (A) 1 ,um, (D) 100 ,um and caffeine (Y) 10 ,um, ( •) 1 mm on the concentration-response curves (a) to histamine ( •) and (b) to histamine plus adenosine 100 um (∎) in isolated rabbit gastric glands . The concentration-response curve to histamine alone (•) is included as control in graph b . The H+ secretory responses are expressed in terms of aminopyrine ratio quotient (QRap) = Rap agents/Rap basal, thus QRap basal = 1 . Each point represents the mean of at least four experiments ; vertical bars indicate SE mean .
The order of potency of theophylline (THEO) concentrations in producing inhibition of the ADO induced acid secretory responses was : THEO 1 µM > THEO 10 µM > THEO 100 µM . Similar results were obtained with caffeine (CAF, Fig . 3b). In this case the order of potency was : CAF 10µm,>-CAF 100 µM > CAF 1 mm . Effect of indomethacin on the ATP-induced inhibition of the histamine-stimulated
It has been proposed that ATP is a potent agonist for P,purinoceptors and that occupation of these may lead to prostaglandin synthesis . Prostaglandins have been reported to be inhibitors of gastric acid secretion [28-30] . Based on this, inhibition of the histamine-stimulated H+ secretory rate in response to ATP (see above) may be mediated through prostaglandin biosynthesis . Experiments with histamine-stimulated glands preincubated with indomethacin (1 µM-100µM) for 10 min before addition of ATP were carried out to examine this possibility. Under our experimental conditions, indomethacin alone did not significantly potentiate the histamine-raised H + secretion. As is shown in Fig . 4, indomethacin (1 µm-100 µM) caused graded decreases of the inhibitory responses to ATP (1 mm) . This suggests that the inhibitory effects of ATP are mediated, at least in part, via prostaglandin production . H+ secretion.
DISCUSSION The important role of histamine, acetylcholine and gastrin in the physiological regulation of gastric acid secretion is well documented [27] . In addition, several other agents have been considered as possible effective modulators of the gastric H+ secretory function. In this way, purine nucleosides and nucleotides have been
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-6 .5 -6 -5.5 -5 -4 .5 -4 -3.5 log [histamine] M
Fig . 4 . Effect of graded concentrations of indomethacin (Y) 1 µm, (∎) 10 ,um and (A) 100 µM on the concentration-response curve to histamine plus ATP 1 mm ( •) in gastric glands from rabbit . The concentration-response curve to histamine alone ( •) is included as control . The gastric acid secretory responses are expressed in terms of aminopyrine ratio quotient (QRap) = Rap agents/Rap basal, thus QRap basal = 1 . Each point represents the mean of at least four experiments ; vertical bars indicate sL mean .
proposed to be involved in the physiological regulation of this process [12] . In support of this proposition, the present results show that purines exert relevant effects on acid secretion in isolated rabbit gastric glands . From the activities found here, the H + secretion was stimulated by ADO, L-PIA and AMP and inhibited by ATP, ß,y-methylene ATP and ADP . Our experimental approach on the mechanism by which ADO stimulates H + secretion led to the following findings and considerations : 1 . The range (µM) of apparent Kd values obtained here fitted better to those reported for ADO at the A 2/Ra receptor (low affinity) than those at the A,/Ri receptor (high affinity) [31] . 2 . The rank order of potency : ADO > L-PIA> AMP, was similar to that described in other tissues in which purines cause adenylate cyclase activation [24] . In addition, increases of cAMP in gastric tissue have been correlated with the stimulation of acid secretion in response to histamine and some other secretagogues [32-35] . 3 . The apparent relationship found here between gastric mechanisms mediating ADO responses and those mediating histamine responses, agrees with that of a previous work [36] in which potentiation of the adenosine A2 /Ra receptormediated accumulation of cAMP by histamine, acting via H,-receptors, in guineapig cerebral cortical slices has been reported . 4 . Theophylline and caffeine behave as selective antagonists at the adenosine (P,-) receptors (see above) but also as phosphodiesterase inhibitors . At high concentrations both methylxanthines act mainly as phosphodiesterase inhibitors, whereas, at low concentrations they act mainly as adenosine receptor antagonists
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[8] . Based on this, the pattern of activity reported here for theophylline and caffeine on the H+ secretory responses elicited by ADO in both resting and histaminestimulated glands can be interpreted as the result of an antagonist effect at the adenosine receptor and its inhibitory effect on phosphodiesterase activity . In this way, at the lower concentrations used theophylline and caffeine act mainly as adenosine receptor antagonists, whereas at the higher concentrations used the adenosine receptor antagonist effect appears to be masked by the effect due to phosphodiesterase inhibition . These findings are consistent with the involvement of adenosine (P,-) receptors and adenylate cyclase activation (A 2/Ra-receptors) in the acid secretory responses to ADO and its related derivatives . In agreement with our previous results [21] and those described here, Ota et al. [37] have recently reported that ADO and its analogues cause stimulation on acid secretion in rabbit parietal cells and they have suggested that this effect is mediated via adenosine A,_receptors . From the present results and those previously reported on the effects of ADO and its derivatives on acid secretion in gastric preparations such as isolated glands and isolated parietal cells, three main lines of evidence have emerged . One of them supports that these purines exert inhibitory effects on acid secretion in canine parietal cells and suggests that adenosine A,- (or Ri)- receptors are involved [20, 38] ; another suggests the absence of adenosine receptors in rat parietal cells on the basis of the lack of effect of ADO on acid secretion in this preparation [17] ; and finally, the third one supports that ADO and its derivatives stimulate acid secretion in rabbit gastric glands ([21], the present work) and parietal cells [37] and suggests that adenosine A,- (or Ra)- receptors are involved . We cannot explain this variety of results with any relevant difference in the experimental procedure ; thus it seems that the action of ADO on gastric acid secretion depends on the species used . Conversely to the secretagogue activity obtained from assays with ADO, ATP caused inhibition of the histamine-stimulated H + secretion in gastric glands . In previous works with supraepithelial preparations, ATP has been reported to cause inhibition [22, 23], moderate stimulation and/or no effect [16, 18] on gastric acid secretion. The influence of ATP on acid secretion has also been studied in isolated gastric glands, but from a metabolic point of view [39]. In these studies gastric glands have been reported to be subjected to a permeabilization process to facilitate the incorporation of the exogenous ATP into the glands . As could be expected, under these conditions ATP enhances acid secretion probably by increasing the activity of H+-transporting ATPase . Assuming the purinergic receptor classification criteria proposed by Burnstock [3], our present results indicate that the P 2-purinoceptors may mediate the inhibitory effect of ATP on H+ secretion . This purinoceptor has been suggested not to be linked to the adenylate cyclase system, being related in some cases to the prostaglandin biosynthesis [24] . Prostaglandins have been reported to be inhibitors of gastric acid secretion [28-30] . In support of this possibility, the results show that the inhibitory effect of ATP was significantly reduced by the prostaglandin synthesis inhibitor indomethacin and that the interaction between gastric mechanisms, mediating ATP, ADP and probably ß,y-methylene ATP responses and those mediating histamine responses was of uncompetitive nature . On the other
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hand, the rank order of potency obtained here, ATP> ADP >> fl, y-methylene ATP, evokes the order of potency described for purines at the P2Ypurinoceptor [6, 7] . In conclusion, the present findings support the view that ADO, ATP and related purines act as effective modulators in the regulation of gastric acid secretion and provide evidence indicating that the P,-purinoceptor via adenylate cyclase activation and the P2-purinoceptor via stimulation of prostaglandin production may mediate the stimulatory and the inhibitory effects, respectively, displayed by these purines on gastric H+ secretion in isolated rabbit gastric glands . ACKNOWLEDGEMENTS This study was supported in part by the Department of Health of the Government of the Basque Country . REFERENCES 1 . Gordon GL . Extracellular ATP: effects, sources and fate . Biochem J 1986 ; 233 : 309-19 . 2 . Williams M . Purine receptors in mammalian tissues : pharmacology and functional significance . Ann Rev Pharmacol Toxicol 1987 ; 27 : 315-45 . 3 . Burnstock G. A basis for distinguishing two types of purinergic receptor. In : Straub RW, Bolis L, eds. Cell membrane receptors for drugs and hormones . New York : Raven Press, 1978 :107-18 . 4 . Van Calker D, Muller M, Hamprecht B . Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells . J Neurochem 1979 ; 33 :999-1005 . 5 . Londos C, Cooper DMF, Wolff J . Subclasses of external adenosine receptors . Proc Natl Acad Sci USA 1980 ; 77 :2551-4 . 6 . Burnstock G, Kennedy C . Is there a basis for distinguishing two types of P2purinoceptor? Gen Pharmacol1985 ; 16 : 433-40 . 7 . Burnstock G, Warland JJI. PZ purinoceptors of two subtypes in the rabbit mesenteric artery : reactive blue 2 selectively inhibits responses mediated via the P,- but not the Ppurinoceptor. BrJPharmacol1987 ; 90 : 383-91 . 8 . Burnstock G . Purinergic transmitters and receptors : new directions . In : Stefanovich V, Rudolphi K, Schubert P, eds. Adenosine : receptors and modulation of cell function . Oxford : IRL Press, 1985 : 3-13 . 9 . Gandarias JM, Ainz LF, Fernândez B, Goiriena JJ, Lacort M, Rabanal S . Effects of water soluble splenic extracts on intestinal smooth muscle . Arch Farmac Toxicol 1978 ; IV: 331-8 . 10 . Gil-Rodrigo CE . Mecanismo de action de algunos extractos tisulares hidrosolubles sobre miisculo liso intestinal . [Doctoral Thesis 1980] . Madrid : Universidad Complutense de Madrid, 1981 : 324 p. 11 . Ainz LF, Casis E, Gandarias JM, Gil-Rodrigo CE, Goiriena JJ . H,-histamine receptors may mediate the contractile response of guinea-pig ileum to 'histamine-free' splenic extracts . BrJPharmacol1983 ; 79 : 373-8 . 12 . Gandarias JM, Ainz LF, Gil-Rodrigo CE, Martinez I, Goiriena JJ . Efecto de algunas purinas sobre la secretion g$strica de âcido . [abstract] . V11 Reunion Nacional de la Asociacion Espanola de Farmacologos . Salamanca, 1982 :196 . 13 . Puurunen J, Aittakumpu R, Tanskanen T . Vagally mediated stimulation of gastric acid secretion by intravenously administered adenosine derivatives in anaesthetized rats . Acta Pharmacol Toxicol 1986 ; 58 : 265-71 . 14 . Glavin GB, Westerberg VS, Geiger JD. Modulation of gastric acid secretion by adenosine in conscious rats . Can J Physiol Pharmacol 1987 ; 65 : 1182-5 . 15 . Scarpignato C, Tramacere R, Zappia L, Del Soldato P . Inhibition of gastric acid secretion by adenosine receptor stimulation in rat . Pharmacology 1987 ; 34 : 264-8 .
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