Absolute Dependence on Chloride for Acid Secretion in Isolated Gastric Glands

73:874-880, 1977 Copyright © 1977 by the American Gastroenterological Association

Vol. 73, No. 4, Part2 Printed in U.S A.

GASTROENTEROLOGY

ABSOLUTE DEPENDENCE ON CHLORIDE FOR ACID SECRETION IN ISOLATED GASTRIC GLANDS THOMAS BERGLINDH, PH.D.

Department of Physiology and Medical Biophysics, Biomedical Center, University of Uppsala, Uppsala , Sweden

Isolated glands from the rabbit gastric mucosa lost their ability to accumulate H+ ions when incubated in Cl- -free solutions, as measured by accumulation of the weak base aminopyrine (AP). Simultaneously, basal oxygen consumption was significantly increased (+56%). Addition of histamine or dibutyryl cyclic AMP (db-cAMP) further increased the respiration but only a minor increment in the AP accumulation was seen. Addition of Cl- dose-dependently increased the AP accumulation back to normal basal values, a function which was coupled with a progressive decrease in oxygen consumption down to the normal level. ED5o for AP accumulation was 15.0 mM Cl-. seN- also decreased the respiratory rate but dH not restore AP accumulation. Increasing the K+ concentration of the Cl--free solution 10-fold did not per se affect the AP ratio, but facilitated the rise in AP accumulation upon addition ofCl- . At standard Cl- concentrations the final AP ratio was significantly increased above that of glands incubated at normal K+ concentrations. Addition of Br- to the chloride-free medium induced responses which were indistinguishable from those ofCl- , whereas I- showed less affinity and was inhibitory at higher concentrations (above 20 mM). Studies on AP accumulation kinetics showed that addition of Cl- to glands pretreated with db-cAMP did not induce a more rapid accumulation than that seen in glands to which db-cAMP and Cl- were added simultaneously. This might indicate that db-cAMP does not induce a morphological transformation of the parietal cell into a secreting state in the absence of Cl- . As one explanation for the results obtained, a Cl- -HC03 - exchange mechanism in the secretory membrane is proposed which implies that HC0 3- handling in the secretory membrane is impaired in the absence ofCl- and that this could lead to a neutralization of the H+ ions formed. Chloride has been shown to be actively transported by the gastric mucosa, giving rise to a negative potential on the mucosal side with respect to the blood side. 1 The existence of the potential is not dependent on the acid secretory rate of the mucosa. Measurement of chloride fluxes in amphibian or piglet gastric mucosa clearly shows that upon stimulation of acid secretion the chloride transport from blood to mucosa increases with a value corresponding to the H+ secretion. 1• 2 This indicates that two types of chloride transport exist, one nonacidic and one coupled to the H+ ions. 3 The site of the nonacidic chloride pump has not been determined, even though most investigators appear to believe it to be situated in the parietal cell. 4 In any case, this cell is with little doubt the site for the acidic chloride transport.

Much effort has been put into the question of whether the H+ ion pump is electrogenic or whether HCl secretion is neutral.:; If the frog gastric mucosa is bathed in media where all Cl- has been replaced by SO/- , a positive potential is obtained on the mucosal side, which seems to correspond to the small acid secretion seen in those situations. H. 7 In some other preparations, like the Necturus gastric mucosa, 8 the lizard gastric mucosa, 9 and the isolated piglet gastric mucosa, 2 no acid secretion was obtained when incubated in a chloride-free environment. To obtain information about the nature of chloride transport and its interactions with H + secretion in the mammalian parietal cell, it was of interest to investigate the behavior of isolated gastric glands from the rabbit 10 in chloride-free situations.

This study was supported by the Swedish Medical Research Council (Project 151) and the Magnus Bergwall Foundation . Address requests for reprints to: Dr. Thomas Berglindh, Department of Physiology and Medical Biophysics, Biomedical Center, University of Uppsala, P.O. Box 572 , S-751 23 Uppsala, Sweden. The author wishes to thank Miss Elisabet Bergqvist for her invaluable assistance and Dr. K. J. Obrink for his constructive criticism and general support.

874

Materials and Methods Isolated glands from the rabbit gastric mucosa were obtained by the method of Berglindh and Obrink. 10 Chemicals. Nn-02 -dibutyryl cyclic adenosine 3',5'-monophosphoric acid (db-cAMP, grade II) as sodium salt, rabbit albumin, and histamine diphosphate were obtained from Sigma Chemical Company, St. Louis, Missouri. Sodium thiocyanate and glucose were products of Mallinckrodt Chemical

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CHLORIDE AND ISOLATED GASTRIC GLANDS

Works, St. Louis, Missouri. All chemicals used in making media were of the highest grade. Aminopyrine (N-4-dimethyl14C) (AP), with a specific activity of 4.1 mCi per mmole, was obtained from New England Nuclear Chemicals, Dreieichenhain, West Germany. Solutions. Table 1 presents the composition of the different incubation media used. In those cases when Br- or I- was investigated the composition was the same as for the Naglucuronate solution but the glucuronate was exchanged for 137.8 mM NaBr or Nal. The lower osmolarity of the Cl- -free media was chosen to compensate somewhat for the Donnan effect of the nonpenetrating ions. After gland separation the glandular suspension was divided into several portions and each was washed five times during 45 min at room temperature in the medium in which following study was to be performed. For Br- or I- substitution, glands were washed three times in glucuronate solution and twice in the respective media. Oxygen consumption and AP accumulation. The glandular respiratory rates were determined by the Warburg technique and drugs were added at the start of inmbation, as described earlier. 10• 11 Intermediate Cl- , Br- , and I- concentrations were obtained by mixing proper amounts of normal medium with the corresponding glucuronate solutions. The oxygen consumption was usually followed for 60 min. To the Warburg flasks , each containing 3 ml of glandular suspension, AP, 0.1 ~-tCi per ml, was added. At the end of the experiment the glands were spun down and the AP content in the glands and in the supernatants was determined according to the technique described before. 11 • 12 The results were exTABLE

1. Composition of the solutions•

Compound

NaCl, mM Na,so., mM Na glucuronate, mM Ca acetate, mM MgS0 4 , mM KCl,mM K2HP0 4 , mM Na2HP04 , mM NaH,P0 4 , mM K,so., mM Sucrose, mM Glucose, mM Rabbit albumin, g/liter Phenol red, mg/liter a

Normal medium

Normal Cl -free medium medium, Na 2- Na glucu- High K+ high K+ so, ronate

132.4

83.8 66.2 1.0 1.2

132.4 1.0 1.2

83 .8 1.0 1.2

5.0 1.0

1.0 1.2 48.6 2.7 2.3 1.0

2.7 2.3 1.0

2.7 2.3 1.0

11.1 2 10

10.8 11.1 2 10

66.2 11.1 2 10

11.1 2 10

2.7 2.3 1.0 24.3 24.3 11.1 2 10

1.0 1.2 5.4

pH for all solutions was adjusted to 7.4.

875

pressed as the ratio of AP in intraglandular water to AP in extraglandular water. 11 • 12 For studies on AP accumulation kinetics, changes in AP content of the extraglandular fluid were determined by frequent sampling. For methodological details see Berglindh. 12 Electrolyte content. The electrolyte content of glands incu" bated in Cl- -free solution was studied using (14 C) inulin as extracellular marker (The Radiochemical Centre, Amersham England). Dried glandular pellets were digested by 0.2 ml of HN0 3 , whereupon water was added to 1.0 ml. Potassium and sodium were determined in an Eppendorf flame photometer. For the determination of intracellular Cl- in glands incubated in normal medium, the glands were treated with 3.0 ml of distilled water at 70°C for 20 hr and the chloride was determined by a CMT 10 chloride titrator (Radiometer, Copenhagen Denmark). Statistical analyses were made by Student's t-test for paired samples.

Results Isolated glands from the same preparations were incubated in normal, as well as el- -free SOl- medium. Glandular oxygen consumption and AP accumulation . were investigated in the presence of histamine, I0- 4 M, db-cAMP, I0- 3 M, and NaSeN, I0- 2 M. For the el--free glands the responses to a mixture of secretagogue and SeN- were also tested. The results, listed in table 2, show the normal response for the glands incubated in el--containing medium; 1 t. 12 i.e., oxygen consumption and AP accumulation increase upon histamine or dbcAMP stimulation, whereas the glandular AP accumulation was drastically inhibited in the presence of SeN-. 12 During incubation in el--free solution, however, several intriguing results were obtained. The basal oxygen consumption increased substantially (+56%), and upon . addition of secretagogues this rate was further and significantly increased. However no difference in response between histamine or db-cAMP stimulation could be seen, in contrast to what was observed in normal glands. Addition of SeN- brought the basal rate back to the value in normal control glands. Secretagogue-induced respiration was likewise lowered in the presence of seN- . The basal oxygen consumption of el--free glands was drastically lowered toward normal values by addition of a small amount of el- . Thus, 11.5 mM ellowered the consumption to 16.0 ± 1.0 p,l of02 per mg of dry weight per hr (mean ± SE, n = 5).

2. Oxygen consumption and aminopyrine (AP) accumulation ratio for glands from the same preparations, incubated in normal and Cl--free so;- medium; the glands were incubated for a total of90 min and oxygen consumption was determined during the last 60 min (mean ± SE, n = 5)

TABLE

CJ- -free medium

Normal medium 0 2 consumption (J.Ll/mg dry wt/hr)

Control Histamine, 10-• M Db-cAMP, I0- 3 M NaSCN, I0- 2 M Histamine, IQ- • M + NaSCN, I0- 2 M Db-cAMP I0- 3 M + N aSCN I0- 2 M a b

14.4 23.2 31.8 12.4

± 1.5 ± 2.4b ± 3.3b

± 1.0

AP accumulation ratio

53 .2 100.5 131.6 1.6

± ± ± ±

8.5 15.0b 20.2b 0.1b

The same treatment differed significantly (P < 0.05) between the two media. Significantly different (P < 0.05) from the respective control.

0 2 consumption (J.Ll/mg dry wt/hr)

22 .5 28.9 28.4 14.9 17.9 21.6

± ± ± ± ±

2.0° 2.9• -b 2.0b 0.5b l.Ob

± 2.7

AP accumulation ratio

1.8 3.5 5.0 1.4 1.5 1.8

± ± ± ± ± ±

0.2° 0.2•- b 0.8• -b 0.1b 0.1b 0.2

876

BERGLINDH

The normal basal AP accumulation was almost totally abolished by the Cl-free conditions and only a very small increment was obtained by secretagogue addition, an increase which was sensitive to SCN- . In the frog gastric mucosa, substituting glucuronate for Cl- leads to a more pronounced inhibition of acid secretion than SOl- substitution. 13 For this reason the present studies were also performed in glucuronate medium; this, however, gave exactly the same type of responses as described above. Dose-response relationship. Progressively increasing the Cl- concentration made the glands accumulate more AP in a dose-dependent way up to the level of the normal, basal AP ratio. Figure 1 shows such a Cltitration curve where the AP ratios are expressed as percentages of normal accumulation. The AP ratio of Cl--free, glucuronate-incubated glands was 1.9 ± 0.2 and that from the normal medium was 44.9 ± 5.9 (mean ± SE, n =5). The dose-response curve showed an apparent ED50 of 15.0 mM Cl- . Increasing the Cl- concentration also decreased the oxygen consumption progressively, back to the normal basal value. High K +- medium. Because Cl- normally is passively distributed in cells, depending on the membrane potential, the effect of diminishing the potential in order to facilitate Cl- entrance in the cells was investigated. Thus, the extracellular K+ concentration was increased 10-fold to 54 mM. Glands from the same preparation were incubated in a Cl- -free, high K+ medium, and as control also in normal medium with standard or high K+. The dose-response curve obtained after addition of increasing concentrations ofCl- to the high K+ glands is presented in figure 2. Increasing the K+ concentration •j. of

Vol. 73, No.4, Part 2

in a Cl- -free medium did not per se significantly affect the low AP accumulation seen with normal K+, giving a ratio of 4.3 ± 1.5 (mean± SE, n = 4). On the other hand, at a normal Cl- concentration (132, 4 mM), the high K+ appreciably increased the ratio above that of the normal K+ medium; the values for control and high K+ were 48.2 ± 6.5 and 78.9 ± 10.6, respectively. In the high K+ medium, the Cl- concentration needed to obtain half maximal response (ED 50) was calculated to be 15.2 mM (fig. 2). This value was not sig11ificantly different from that seen at normal K+ concentrations (fig. 1). However, because the maximal response was significantly increased, the high K+ environment clearly seemed to facilitate the increase in AP accumulation upon addition of Cl- . In normal medium the oxygen consumption was not significantly affected by increasing the K+ concentration ( -4.8 ± 3.8% of control, mean ± SE, n = 4), whereas glands incubated in Cl- -free, high K+ medium showed an increase in the respiratory rate of 35.6 ± 6.0 % (mean ± SE, n = 4, P < 0.01) . This increase was not different from that obtained in Cl- -free normal K+ glucuronate medium indicating that the effect was mediated by the lack of Cl- . Thus, addition of Cl- dosedependantly decreased the response to 9.2 ± 4.2 % at 44 mM Cl- . Other halogens . The gastric mucosa has been shown to be capable of transporting bromide and iodide. 13 • 14 Incubating the glands in media where all Cl- had been exchanged for NaBr or Nai showed that the glands possessed the ability to handle those ions. The AP ratio obtained after Br- incubation was of the same magnitude as after Cl- incubation, whereas Nai gave a value which was 20% of the control. Figure 3 presents typical

control

•t. of control 200

80 c

.. ~

c

·~ 60 ;;

~

E

"

"

~

.

ED 50 : 15.0 mM

v v

"'

11.

Control AP rat io :48.2

100

~

Q.

<

40

<(

50

20 oL---~~~~~~----L-----~--~

1

10

20

50 Log

10

20

50

100

[Ci·]

mM

FIG. 1. Increase in aminopyrine (AP) ratio upon addition ofCl- to glands incubated in Cl- -free glucuronate solution, expressed as percentage of the normal AP ratio obtained in standard Cl- -solution. The vertical bars represent mean :t SE, n = 5. The curve was drawn according to the integral of the normal probability curve obtained by probit analysis. The star denotes the apparent ED50 for AP accumulation, which was 15.0 mM Cl- .

100

[c1·]

mM

FIG. 2. Increase in aminopyrine (AP) ratio upon addition of Cl- to glands incubated in Cl- -free glucuronate solution, containing 54 mM K+. The response is presented as percentage of control AP accumulation found in glands from the same preparation, incubated in normal medium. The dashed lines show the respective SE at the electrolyte compositions indicated in the figure . Vertical bars represent mean :t SE, n = 4. The Cl--concentration which produced half-maximal response was 15.2 mM , as indicated by the star. The curve is drawn as in figure 1.

October 1977

CHLORIDE AND ISOLATED GASTRIC GLANDS

AP ratio ICWJEcw

70

50

5

10

50

100

500

Dose mM

FIG. 3. Representative example of the aminopyrine (AP) accumulation induced by addition of increasing concentrations ofBr- and rto glands incubated in Cl- -free glucuronate medium. The accumulation ratio± SE for Cl- -free-incubated glands and glands incubated in normal Cl- medium are also shown. The lower and upper bar represent the AP accumulation ± SE, n = 3, at standard concentrations of Nal and NaBr, respectively. The curves are drawn by eye.

877

addition of Cl- . The direct effect Of Cl- addition to untreated glands was a rapid increase in accumulation reaching a steady state value 30 min after addition. More interesting, however, was that Cl- addition to dbcAMP-treated glands or simultaneous addition of dbcAMP and Cl- to untreated glands, gave responses which followed a common stimulatory pattern in every instance. In accordance with the reasoning above, this similar time-response behavior of the two db-cAMPtreated preparations, could indicate that no morphological transformation had taken place: in the db-cAMPpretreated glands in the absence of Cl-. Electrolyte content. Intracellular electrolyte contents in glands incubated in Cl- -free glucuronate solutions (mean± SE, n = 6) were Na+ = 24.1 ± 4.8 mM and K+ = 115.7 ± 1.3 mM. The Cl- content in glands incubated in normal medium was 80.7 ± 1.4 mM, whereas that of glands incubated in Cl-free solutions was not detectable with the method here used.

Discussion Isolated gastric glands have been shown to be a good working model for studies on mammalian gastric acid secretion. 1()- 12 They should also provide a suitable tool dose-response curves obtained upon addition ofBr- or I- for investigations on electrolyte substitution, because to glucuronate-incubated glands. As seen, the Br- curve the glandular cells expose such a large surface to the is of the same type as the normal Cl- curve (fig. 1), extracellular medium, making ion exchange much simwhereas iodide-induced accumulation reached a peak pler. Glands incubated in normal medium had an intravalue around 15 mM, followed by a decline. Addition of cellular Cl- concentration of 80.7 mM. After incubation Br- affected oxygen consumption in the same way as in Cl- -free solution that value was less than 1 mM (i.e., Cl- did, whereas iodide at high concentration appeared undetectable with the analytical method used here). to decrease the rate below the normal basal value. AP accumulation kinetics. The AP accumulation is a x 'C!- 22 .0 mM reflection of acid concentration and the space where the I o 'C1- 22 .0 mM accumulation takes place. The latter should give some f t., C( 22 .0 mM • db-cAMP 1 mM kind of indirect determination of the parietal cell morphology.12 Because no or very small AP accumulation was obtained in Cl- -free medium regardless of treat30 ment, the question arose whether the secretagogues still induced their normal morphological transformation of the parietal cell 11 in a Cl- -free situation. 20 A parietal cell in its secretory state, i.e., filled with intracellular canaliculi, although secreting very poorly in the absence of Cl- , might be expected rapidly to turn 10 on the secretion upon addition of Cl-, in contrast to a AP ratio !CW / ECW resting cell where a time consuming morphological 60 transformation has to take place. Thus, to study the parietal cell morphology indirectly, the following exper40 iment was designed. Glands from the same preparation 20 were divided into three portions and transferred to separate flasks. To one, db-cAMP, IQ- 3 M, was added from the start, and the glands were incubated for 45 min at 37°C; at this point db-cAMP, IQ- 3 M, + 22 mM Cl- was 30 60 90 mm added to a second flask, whereas the other two only FIG. 4. Aminopyrine (AP) accumulation kinetics determined as received 22 mM Cl- . Incubation was then continued for changes in the extraglandular AP content, for glands incubated in another 60 min. During the whole incubation period, Cl- -free glucuronate solution. At time 0 AP was added to three flasks samples were frequently taken for determination of the containing glands from the same preparation, to one 0 - - 0 1 mM extracellular AP content. A representative experiment db-cAMP was added simultaneously. Cl- and Cl- + 1 mM db-cAMP of this kind is presented in figure 4. Although a small were added to the different flasks at the time shown by the upper accumulation was induced by db-cAMP, no substantial arrows . At times indicated by the bottom arrows the AP accumuladifference was obtained among the three curves before tion ratios in the glands were determined.

878

Vol. 73, No.4 , Part 2

BERGLINDH

The K+ and Na+ concentrations however, were not significantly altered from the normal composition. 10 Incubation of the glands in a chloride-free solution had a striking effect on the acid formation as measured in terms of AP accumulation. The theoretical accumulation value for nonacid-containing cells is 1 (at an intracellular pH of 7) . The accumulation found here of 1.8 thus is very close to unity. Such a low figure has only been shown before with thiocyanate treatment (table 2). 12 In fact, addition of SCN- to chloride-free glands further significantly (P < 0.01) lowered the ratio to 1.4. Addition of histamine or db-cAMP induced a small SCN- -sensitive increase in accumulation which, however, at the most was only 10% of the normal basal AP ratio. These results appear to be in contrast to the finding from the frog gastric mucosa, where a small acid secretion persists in a chloride-free situation, giving rise to a positive potential. 5-7That finding has been taken as one evidence for the electrogenicity of the H+ pump. 5 For the Necturus gastric mucosa, Shoemaker et al. 8 did not find any residual secretion; the same finding was obtained for the lizard. 9 As an explanation for these discrepancies, a lower SOl- permeability for the latter two species, as compared to the frog, has been proposed.8· 9 Turning to the mammalian field , much less information is available. In one of the more promising isolated preparations, piglet gastric mucosa,2 acid secretion was abolished without chloride in the serosal solution. The present findings of the direct relationship between addition of chloride and increase in glandular AP accumulation, forming the sigmoid curve presented in figure 1, indicate a very strong coupling between H+ secretion mechanisms and chloride in the glands and could per se indicate that the H+ ion pump would fail to work in the absence of chloride. In attempting to define the H+ pump, the finding and characterization of a K+-activated adenosine triphosphatase found in the gastric mucosa 15-18 is of special interest. This adenosine triphosphatase has been shown to translocate H+ ions for K+ ions over vesicular membranes in a nonelectrogenic way. 17· 18 This translocation does not seem to be absolutely dependent on the presence of chloride, because only a 40% reduction was seen in SOl - medium. 18As for the glands, increasing the K+ con-:-::ntration of the chloride-free medium did not per se induce an increased accumulation, a finding which again supports the dependence between Cl- and H +. With increasing Clconcentrations, however, the accumulation was facilitated by high K+ , and at normal CI- concentrations the accumulation was significantly increased above basal level. It is unlikely that this increase depends to a major extent on a facilitated entrance of Cl- ' owing to the decrease in membrane potential, because the effect of Cl- showed saturation phenomena already at normal K + concentration, as shown in figure 1. In spite of a high K+ content of the gastric mucosal cells, 10· 19 it is well known that only a small fraction of this is needed for acid secretion (Reference 19 and T. Berglindh, unpublished observations). This could mean that an increase in the medium K+ concentration would also increase the

available amount of exchange K+ inside the parietal cell, leading to an increase in acid formation. Comparing the results from glands incubated in normal and high K+, a situation where both preparations ought to have the same intracellular AP accumulation space, 12 the increase in AP accumulation in the high K+ state could be seen as an increased concentration of H+ ions. This K+-induced accumulation was, however, not coupled to an increased oxygen consumption. It thus appears that if the K+-activated adenosine triphosphatase is the H+ pump, a separate Cl- pathway may be necessary to produce acid in the intact parietal cells. On the nutrient membrane of the gastric mucosa there is a neutral Cl- -HC03- exchange. 20 It is well known that the amount of bicarbonate appearing on the nutrient side exactly matches the amount of hydrogen ions produced.21 Because no HC03- conductance pathway has been found on the nutrient side,5 this means that taking Cl- away would stop the outflow of HC03-, thus leading to an intracellular accumulation of this ion. The expected increase in intracellular pH by such a mechanism could perhaps per se inhibit H + ion transport and/or induce nonspecific side effects, such as large metabolic shifts. In the secretory membrane H+ ions and base are formed. The base has to be kept apart from the H+ ions, and one way to do this is to postulate a Cl--base exchange mechanism in that membrane. In a chloride-free situation, the base would accumulate in the secretory membrane and thus be able to reunite with the hydrogen ions and severely impair the H+ ion secretion. A model for H+ secretion involving such a mechanism is shown in figure 5. The model proposed in the figure implies that acid formation is still taking place in the absence of intracellular Cl- , but that the H+ ions subsequently are neutralized by HC03-. IfCl- was directly coupled to the H + pump, this should be inhibited in a Cl--free environment and thus oxygen consumption should be lowered. For the frog gastric mucosa no change in basal oxygen consumption was obtained, nor was the respiration inNutrient Membrane

Secretory Membrane

1. Neutral exchange 2 . Forced exchange 3. ATP :ase

Fra. 5. A tentative model for HCl secretion, to illustrate a possible relationship between Cl- and HC03 - in the secretory membrane. The dashed line in the secretory membrane indicates the barrier necessary to keep H+ ions separated from the base. The figure is selfexplanatory.

October 1977

CHLORIDE AND ISOLATED GASTRIC GLANDS

creased upon stimulation with histamine, 22 • 23 when bathed in el--free solutions. In contrast to this, in the present studies basal glandular oxygen consumption was significantly increased and the addition of secretagogues stimulated the respiration further. This ability to stimulate parietal cell metabolism supports the concept that H+ formation still takes place in the e1--free parietal cell, and accordingly there could be separate sites for H+ and e1- transport. 5 Furthermore, the glands have been shown to possess a basal AP accumulation ratio, which is not inhibited by burimamide or atropine (table 2), 11 • 12 indicating an acid formation already in a resting state. If this basal H+ gradient is destroyed by neutralization one might expect the H + formation to increase, leading to the rise in respiration obtained here. Alternatively, an increase in intracellular pH might nonspecificely affect oxygen consumption. SeN- significantly lowered the increase in respiration induced by the el--free state, a result which could be explained by the halogen characteristics of that ion; i.e., SeN- has been shown to be transported in elchannels.4· 13 Thus, the effect of SeN- on glandular respiration was the same as after addition of a corresponding amount of e1- . seN- has been shown totally to inhibit glandular AP accumulation, 12 and a neutralizing mechanism has also been suggested for that effect. 12 However, a SeN- -induced neutralization could be of a different kind from the one proposed here, because that inhibition took place in e1- solutions and thus the tentative model would allow a el- -He03- exchange at the secretory membrane. A very interesting contribution to the understanding of the SeN- effect has been presented by Sanders et al., who showed that the weak base imidazole can reverse the SeN- inhibition. 24 This finding might be interpreted as indicating that imidiazole, being un-ionized at physiological pH and thus lipid-soluble, can diffuse to the acid-producing site, take up an H + ion, and thus, by some means, protect it from an SeN--induced neutralization. The glands were shown to handle bromide in a manner similar to that of chloride, and, as shown before for several different preparations, both mammalian 14 and amphibian, 13 as well as for isolated vesicles, 18 the gastric mucosa has a much higher affinity for bromide than iodide. In fact, iodide in higher concentration appeared to have an inhibitory action on the glandular acid production mechanism. Addition of db-cAMP has previously been shown to change the intracellular morphology of the glandular parietal cell into a more secretory state. 11 If db-cAMP were added to glands in a chloride-free solution, would one expect the intracellular morphology to be changed in a similar manner in spite of the apparent inhibition of H + secretion? The final answer to that question, i.e., whether H + secretion and morphology could be uncoupled in a e1- -free situation, must await a detailed ultrastructural investigation. At present, one way to interpret the AP accumulation kinetics found here is to favor the possibility that no morphological transformation takes place in a e1--free environment.

879

In conclusion, the results presented here could be interpreted to mean that H+ ions and e1- are transported at separate sites, but that e1:.. is essential for keeping the H+ ions apart from the base formed, as indicated by Obrink in 1948. 25 REFERENCES 1. Hogben CAM: Active transport of chloride by isolated frog gastric epithelium: origin of gastric mucosal potential. Am J Physiol 180:641-649, 1955 2. Forte JG, Machen TE: Transport and electrical phlmomena in resting and secreting piglet gastric mucosa'. J Physiol 244:33-51, 1975 3. Durbin RP, HeinzE: .Electromotive chloride transport and gastric acid secretion in the frog. J Gen Physiol41:1035-1047, 1958 4. Durbin RP, Kasbekar DK: Adenosine triphosphate and active transport by the stomach. Fed Proc 24:1377-1381, 1965 5. Rehm WS, Sanders SS: Implications of the neutral carrier Cl- HC03- exchange mechanism in gastric mucosa. Ann NY Acad Sci 246:442-455, 1975 6. H einz E, Durbin R: Evidence for an independent hydrogen-ion pump in the stomach . Biochim Biophys Acta 31:246-247 , 1959 7. Rehm WS, Davis TL, Chandler C, et al: Frog gastric mucosa bathed in chloride-free solutions. Am J Physiol 204:233-242, 1963 8. Shoemaker RL, Hirschowitz BI, Sachs G: Hormonal stimulation ofnecturus gastric mucosa in vitro. Am J Physiol212:1013-1016, 1967 9. Hansen T, Slegers JFG, Banting SL: Gastric acid secretion in the lizard, ionic requirements and effects of inhibitors. Biochim Biophys Acta 382:590-608, 1975 10. Berglindh T, Obrink KJ: A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol Scand 96:150-159, 1976 11. Berglindh T, Helander HF, Obrink KJ: Effects of secretagogues on oxygen consumption, a minopyrine accumulation and morphology in isolated gastric glands. Acta Physiol Scand 97:401414, 1976 12. Berglindh T: Effects of common inhibitors of gastric acid secretion on secretagogue-induced respiration and aminopyrine accumulation in isolated gastric gland. Biochim Biophys Acta 464:217-233, 1977 13. Durbin RP: Anion requirements for gastric acid secretion. J Gen Physiol 47:735-748, 1964 14. HeinzE, Obrink KJ, Ulfendahl H: The secretion of halogens into the gastric juice. Gastroenterology 27:98-112 , 1954 15. Ganser AL, Forte JG: K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells. Biochim Biophys Acta 307:169180, 1973 16. Ganser AL, Forte JG: Ionophoretic stimulation ofK+-ATPase of oxyntic cell microsomes. Biochem Biophys Res Commun 54:690696, 1973 17. Lee J , Simpson G, Scholes P: An ATPase from dog gastric mucosa: changes of outer pH in suspensions of membrane vesicles accompanying ATP hydrolysis. Biochem Biophys Res Commun 60:825-832, 1974 18. Chang H , Saccomani G, Rabon E , et al: Proton transport by gastric membrane vesicles. Biochim Biophys Acta 464:313-327, 1977 19. Rehm WS, Sanders SS, Rutledge JR, et al : Effect of removal of external K+ on frog's stomach in Cl--free solutions. Am J Physiol 210:689-693, 1966 20. Rehm WS: Ion permeability and electrical resistance of the frog's gastric mucosa. Fed Proc 26:1303-1313, 1967 21. Teorell T: The acid-base balance of the secreting isolated gastric mucosa. J Physiol 114:267-276, 1951

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22. Alonso D, Harris JB: Effect of xanthines and histamine on ion transport and respiration by frog gastric mucosa. Am J Physiol 208:18-23, 1965 23. Bannister WH: Acid secretion by frog gastric mucosae incubated in a chloride-free medium. Am J Physiol 210:211-215, 1966

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24. Sanders SS, Pirkle JA, Shoemaker RL, et al: Reversal of thiocyanate inhibition of H + secretion in frog gastric mucosa by imidazole. Physiologist 19(3):350, 1976 25. Obrink KJ: Studies on the kinetics of the parietal secretion of the stomach. Acta Physiol Scand 15(suppl 51):1-106, 1948