(Davenport, 1952; Davenport & Chavre, 1953). Similar experiments on. of this ratio is hampered by the fact that in general it is only possible

Transcription

1 J. Physiol. (1965), 177, pp With 3 text-figures Printed in Great Britain THE RELATION BETWEEN ACID SECRETION AND OXYGEN UPTAKE BY GASTRIC MUCOSA OF THE FROG BY W. H. BANNISTER* From the N'uffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford (Received 10 September 1964) Several attempts have been made to determine the quantitative relation between acid secretion by gastric mucosa and the oxygen uptake involved in the secretory process (Crane & Davies, 1948, 1951; Davenport, 1952; Davenport & Chavre, 1953). Interest has largely centred on whether the ratio of acid secretion to the associated oxygen uptake has a value greater than four, which is the maximum value predicted by the redox theory of acid secretion (Conway & Brady, 1948; Crane & Davies, 1948). The determination of this ratio is hampered by the fact that in general it is only possible to measure the total oxygen uptake of isolated gastric mucosa. Crane & Davies (1948, 1951) assumed that the increment in oxygen uptake which they observed in frog gastric mucosae stimulated with histamine to secrete acid, gave the oxygen uptake involved in the secretory process. The ratio of acid secretion to the increment in oxygen uptake was found to have values which varied from 1-5 to Only a third of the ratios obtained in this way had values less than four. Davenport (1952) observed a linear correlation between acid secretion and oxygen uptake in a large number of experiments on sacs of frog gastric mucosa. The coefficient of regression of acid secretion on oxygen uptake, which was considered to give the mean value of the ratio between acid secretion and the associated oxygen uptake, had a value of 2-15 and its 99 % confidence limits were 0-95 and Attempts by Davenport (1952) to determine the ratio between acid secretion and the associated oxygen uptake in a single frog gastric mucosa were not successful. In these experiments a sac of mucosa was incubated twice, and the ratio AqH+/AqO2 (qh+ = rate of acid secretion and qo2 = rate of oxygen uptake) given by the results for the two incubations was determined. The values obtained varied from -46 to 50 (Davenport, 1952; Davenport & Chavre, 1953). Similar experiments on whole mouse stomachs, however, gave values which varied from 1-2 to 4-8 (Davenport & Chavre, 1953). * Present address: Department of Physiology, Royal University of Malta, Valletta, Malta.

2 430 W. H. BANNISTER Followinig the work of Davenport (1952) and Davenport & Chavre (1953) it has been widely considered that the value of the ratio between acid secretion and the associated oxygen uptake by gastric mucosa is not greater than four (Heinz & Obrink, 1954; Conway, 1959; Robertson, 1960). It has been implied that Crane & Davies (1948, 1951) obtained higher values for this ratio because part of the oxygen uptake before addition of histamine in their experiments might have become available for acid secretion (Conway, 1953; Heinz & Obrink, 1954), though this was doubted by Crane & Davies (1951). The problem of determining the ratio between acid secretion and the associated oxygen uptake in a single frog gastric mucosa was re-investigated by making several determinations of acid secretion and the corresponding oxygen uptake on a single mucosa so that the relation between these variables could be examined. An everted sac technique was devised for this purpose. It is proposed to describe this technique and the results which were obtained with it in the present paper. A preliminary account of this work has been published (Bannister, 1963). METHODS Preparation of everted sacs offrog gastric mucosa. Gastric mucosa was obtained from starved frogs of the species Rana temporaria and R. esculenta after injection of histamine intravenously. The frog was pithed. The skin of the ventral abdominal wall was incised in the mid line, and the muscle wall and peritoneum paramedially. The medial edge of the muscle wound was picked up with an artery forceps and reflected over the tip of an index finger. The anterior abdominal vein was thereby exposed. A 26 S.W.G. hypodermic needle was used to inject very slowly 0-5 ml. of a 0-12M-NaCl solution containing 0-2 mg histamine per ml. The vein was clamped at the point of injection to prevent subsequent bleeding. An everted sac of mucosa was prepared after an interval of min. A piece of polythene tubing (bore 1 mm) was inserted into the tube of mucosa obtained after removing the muscle coat of the stomach, until it just emerged from the pyloric end, and this end was tied over the tubing. The mucosa was everted by rolling it gently over the tied-on end. The oesophageal end was ligated. The polythene tubing attached to the pyloric end served for filling the sac with about 0.5 ml. incubating medium by means of a 1 ml. syringe. It was shortened as required and was closed with a small plug after filling the sac. The preparation of the mucosa took about 10 min. Incubation medium. The incubating medium had the following primary composition (mm): NaCl 112-2, KCI 45, CaCi2 1-1, MgSO4 0-6, sodium phosphate buffer (ph 7.4) 0-7. Substrates were added to this medium in the concentrations indicated subsequently. With the exception of glucose, which was added in the solid form (in a concn. of 0-2 % wt/vol.) these substrates were added as the sodium salt in solution isosmotic with 0 12M-NaCl, and an osmotically equivalent amount of NaCl was left out of the medium. Measurement of oxygen uptake and acid secretion. Oxygen uptake was measured with a Warburg manometer at 25 C. The gas phase was air or oxygen. Each mucosa was incubated in 4 ml. of medium in a specially designed flask of about 20 ml. capacity (Fig. 1). The design of this flask facilitated the insertion of the mucosal sac, and permitted free movement of the sac as the manometer was shaken. A number of successive incubations of each mucosa were made. Each period of incubation

3 ACID SECRETION AND OXYGEN UPTAKE 431 lasted 45 min. The medium was then replaced and the manometer re-equilibrated. There was an interval of 15 min between successive periods of incubation. Two thermobarometers were employed, and the changes in their readings agreed to within 1 mm. An uncertainty of this order would have caused an error of about 13 % in the lowest, and an error of about 2 % in the highest oxygen uptake measured. Fig. 1. Diagram of the Warburg flask used for the incubation of an everted sac of frog gastric mucosa. Two-thirds actual size. Acid secretion was determined for each period of incubation by titrating 3 ml. of the incubating medium with 0 1 N-NaOH to the ph observed in a control flask. The alkali was delivered with an Agla micrometer syringe. The ph was measured at room temperature with a C33B ph Measuring Unit and 33B Vibron electrometer (Electronic Instruments Ltd., Surrey) to the nearest 0.01 ph unit. A screened glas electrode and a saturated KCI calomel electrode with a ceramic plug liquid junction were employed. The electrode system was standardized at ph 4 00 with 0 05m-potassium hydrogen phthalate, which was also used to standardize the NaOH. The electrometric titrations were found to have an error of about 3 % in determinations of a known amount of acid added to the incubating medium. The determinations of the rates of acid secretion might have been affected by two additional errors of an indeterminate nature resulting from the fact that a layer of adherent mucoid secretion formed on the everted sac in some experiments and was not recovered with the incubating medium, and the possibility that acid secretion occurred before each period of incubation during equilibration of the manometer. Weight of dried ti8sue. This weight was obtained after drying the mucosae at 110 C for 24 hr. Calculation of result. The rate of oxygen uptake (qo2) and the rate of acid secretion (qh+) for each incubation period were calculated as follows: O observed oxygen uptake (/d.) x 60 /-Molehr, q02= ~~22.4 x 45 ~ mlsh~ qh+ = /s-equiv H+ measured x 60 g-equiv H+/hr. 45

4 432 W. H. BANNISTER RESULTS Oxygen uptake and acid secretion with and without added substrate IOxygen uptake and acid secretion were measured with and without the addition of a substrate to the incubating medium. There was wide variation in the qo2 and the qh+ from mucosa to mucosa, which was independent of Rates of oxygen uptake (qo2) and acid secretion (qh+) by everted sacs of frog TAmr.z 1. gastric mucosa during four successive incubations under various experimental conditions Wt. of Incubation periods qo2 (,u-moles/hr) qh+ (,u-equiv/hr) dried Expt. and substrate tissue no. added (1) (2) (3) (4) (1) (2) (3) (4) (mg) 1) (1)-(4), none 1i121 1X *16 0 0* * f *92 3*17 3* *42 3*00 2* (1), (2), none ' * *56 0*82 13*5 4 (3), (4), 11 mm *83 4*01 4*06 3* *92 4* pyruvate 5 (1), (2), none (3), (4), 11 mm- 2X glucose 7 (1), (2), none (3), (4), 7X5 mm succinate 9 (1), (2), 15 mm succinate 10 (3), (4) 11 m - 3* glucose 11 (1)-(4), 11 mm pyruvate mM-L-malate (1)-(4), 11 mm pyruvate 14) + 1 mm-bicarbonate (1)-(4), 11mM pyruvate + 5 mmoxaloacetate 16 (1)-(4), 11 mm pyruvate 17 (oxaloacetate i.v.) the addition of substrate. The effect of substrate could, however, be observed in a single experiment. Table 1 shows the results obtained with those mucosae which manifested the lowest and highest qo2 respectively during the first period of incubation under a particular set of conditions. The addition of glucose or pyruvate to the incubating medium appeared to increase the qo2 and the qh+. This effect was not apparent with succinate. Succinate, however, increased the oxygen uptake of a homogenate of mucosa in three experiments. Mucosae incubated initially in the presence of succinate showed an increase in the qo2 and the qh+ when subsequently provided with glucose.

5 ACID SECRETION AND OXYGEN UPTAKE 433 The qo2 manifested a peculiar behaviour with time in many experiments. It showed a tendency to decrease during the second period of incubation, and to increase subsequently. The qh+ was generally low during the first period of incubation but tended to increase after this period, and followed linearly the increase in the q02 when this occurred. These effects were observed in mucosae incubated without added substrate, and in mucosae provided with glucose or pyruvate (Table 1 and Fig. 2). 1-5 (6) (5) I-, 1.0 a (4) 0-5 (3) o I 05 1*0 qo, (#-moles/hr) Fig. 2. The relation between the rate of acid secretion (qh+) and the rate of oxygen uptake (qo2) in an everted sac of frog gastric mucosa. Numbers (1) to (6) refer to period of incubation. Each period lasted 45 min. The medium contained 11 mmpyruvate. Data of Expt. 3, Table 3. There was a progressive increase in the qo2 and the qh+ after the first period of incubation in some experiments in which glucose was added to the incubating medium (e.g. Table 1, Expt. 5) and in a series of experiments in which the mucosae were provided with 11 mm-pyruvate plus 9 mm-lmalate, or were obtained from frogs which had been injected with 05 ml. of a 05M-solution of oxaloacetate (ph 7.0) intravenously (Table 1). In three experiments the addition of oxaloacetate in a concentration of 2'5, 5 and 1O mm respectively to incubating medium containing 11 mm-pyruvate, 1 5 (1)

6 434 W. H. BANNISTER caused a progressive decrease in the qo2 and the qh+ (Table 1). The addition of 1 mm-bicarbonate to medium containing 11 mm-pyruvate produced a progressive increase in the qo2 in two experiments (Table 1); acid secretion was not measured under this condition of incubation. TABLE 2. Mean rate of acid secretion (qh+) and oxygen uptake (q02) by everted sacs of frog gastric mucosa, and the constants qo' and 8 of the linear relation qh+ = s(qo2- qo); r = coefficient of correlation between the qh+ and the qo2. Experiments without added substrate throughout or initially Wt. of qh+ q02 qo/ dried Expt. Substrate and no. of (,u-equiv/ (,u-moles/ (,-moles/ tissue no. incubations hr) hr) hr) 8 r (mg) 1 None, None, None, None, None, 1; glucose, None, 1; glucose, None, 2; glucose, None, 2; glucose, None, 1; pyruvate, None, 1; pyruvate, None, 1; pyruvate, None, 1; pyruvate, None, 1; pyruvate, None, 1; succinate, TABLE 3. Mean rate of acid secretion (qh+) and oxygen uptake (q02) by everted sacs of frog gastric mucosa, and the constants qo' and 8 of the linear relation qh+ = 8(qO2-qO'); r = coefficient of correlation between the qh+ and the qo2. Experiments with added substrate Wt. of qh+ qq2 qo' dried Expt. Substrate and no. of (t-equiv/ (1amoles/ (ymoles/ tissue no. incubations hr) hr) hr) 8 r (mg) 1 Pyruvate, Pyruvate, 3 1V Pyruvate, O Pyruvate, Pyruvate, Pyruvate, Pyruvate, Pyruvate + L-malate, Pyruvate + L-malate, Pyruvate+L-malate, Pyruvate+L-malate, Pyruvate+L-malate, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose, Glucose,

7 ACID SECRETION AND OXYGEN UPTAKE 435 The relation between acid 8ecretion and oxygen uptake A good linear relation between the qo2 and the qh+ was observed in thirty-nine experiments (out of a total of fifty-nine), when the qh+ was plotted as a function of the q02. The relation qh+ = s(qo2 -qo2), where s is the slope and qo' is the intercept on the abscissa, was calculated for each of these experiments by the method of least squares. In most experiments this relation was calculated from the values of the qh+ and the q02 for the periods of incubation after the first period, because a linear relation between the two variables appeared to exist only after this period (Figs. 2 and 3). The values obtained for the constants s and qo' are given in Tables 2 and 3 together with the mean qh+, the mean qo2, and the coefficient of correlation between the qh+ and the qo2. Table 2 gives the data from experiments in which a substrate was added to the medium after preliminary incubations without substrate. The addition of substrate to the incubating medium did not appear to alter the relation between the qh+ and the q02. In experiments in which two incubations without added substrate were followed by two or three incubations in the presence of glucose or pyruvate, the qo2 and the qh+ for the second incubation without substrate gave a point which appeared to fall on the same line as the points given by the rates for the incubations made in the presence of substrate when the qh+ was plotted as a function of the q02 (Fig. 3). DISCUSSION The data presented in this paper indicate the probable existence of a linear relation between the qh+ and the q02 in a single frog gastric mucosa in the secretory state (Figs. 2 and 3; Tables 2 and 3). This relation tended to manifest itself after the first period of incubation, i.e. after an hour of incubation. In twenty-two of the experiments for which data are given in Tables 2 and 3, there was a fall in the q02 during the second period of incubation. This fall in the q02 might have been due to deficient carboxylation of pyruvate and inability to maintain the tissue level of oxaloacetate, which is a rate-limiting factor in oxidative metabolism (Krebs & Kornberg, 1957), under the experimental conditions which were employed to measure oxygen uptake. In fact it did not occur in experiments in which L-malate or bicarbonate was provided in addition to pyruvate in the incubating medium, and in experiments in which the mucosae were obtained from frogs which had been injected with a large amount of oxaloacetate (Table 1). Presumably the fall in the q02 was transitory because of retention of metabolic CO2 in the medium and inside the sacs (as bicarbonate). The linear relation which was observed between the qh+ and the q02 28 Physiol. 177

8 436 W. H. BANNISTER permitted the determination of the ratio of acid secretion to the associated oxygen uptake, and the value of the non-acid-producing oxygen consumption in a single mucosa. In the equation qh+ = s(qo2 -qo2), which was employed to express this relation, 8 (the slope) represents the ratio of acid secretion to the associated oxygen uptake, i.e. AqH+/AqO2, and qo' (the x-intercept) represents the non-acid-producing oxygen consumption. The latter is the component of the oxygen uptake in the secretory state which is not utilized for acid secretion. (4) 15 0o (5) + (2) mr fo0(3) 05s 0~~~~~~~~ / ~~~~~~(1) q02 (iu-moles/hr) Fig. 3. The relation between the rate of acid secretion (qh+) and the rate of oxygen uptake (qo2) in an everted sac of frog gastric mucosa. Numbers (1) to (5) refer to period of incubation. Each period lasted 45 min. The medium contained no added substrate (-) in periods (1) and (2), and 11 mm-pyruvate (0) in periods (3) to (5). Data of Expt. 13, Table 2. The present work indicates that mucosae which are not secreting acid may have an oxygen uptake (resting oxygen uptake) higher than their calculated non-acid-producing oxygen consumption. No acid secretion was detected during the first period of incubation in four experiments and during the first and second periods in five experiments. The resting oxygen

9 ACID SECRETION AND OXYGEN UPTAKE 437 uptake was higher than the non-acid-producing oxygen consumption in seven and equal to the latter in two of these experiments (Table 4). When the resting oxygen uptake is higher than the non-acid-producing oxygen consumption, the former would appear to contain a component of oxidative metabolism which may become available for acid secretion. Presumably this component was involved when a fall in the q02 occurred during the second period of incubation in the present experiments. (see Fig. 2), and it apparently re-appeared in the q02 with the occurrence of acid secretion after this period. TABLE 4. Rate of oxygen uptake by everted sacs of frog gastric mucosa in a resting state with regard to acid secretion (resting qo,) and the calculated non-acid-producing oxygen consumption Calc. non-acid- Incubation producing period and oxygen resting qo2 consumption Source of data Substrate (it-moles/hr) (/z-moles/hr) Expt. 3, Table 2 None (1) 1V Expt. 4, Table 2 None (1) Expt. 3, Table 3 11 mm-pyruvate (1) (2) 1-16 Expt. 5, Table 3 11 mm-pyruvate (1) Expt. 9, Table 3 11 mm-pyruvate+9 mm-l-malate (1) Expt. 10, Table 3 11 mm-pyruvate+9 mm-l-malate (1) (2) 1-90 Expt. 11, Table 3 11 mm-pyruvate+9 mm-l-malate (1) (2) 0-97 Expt. 12, Table 3 11 mm-pyruvate + 9 mm-l-malate (1) (2) 0-92 Expt. 17, Table 3 11 mm-glucose (1) Crane & Davies (1948, 1951) assumed that the resting oxygen uptake was identical with the non-acid-producing oxygen consumption in their determinations of the ratio between acid secretion and the associated oxygen uptake. It would appear from the present findings that this assumption cannot be made for every mucosa in a resting state with regard to acid secretion. A part of the oxygen uptake in this state may become available for acid secretion when the latter occurs. Crane & Davies (1948, 1951) did not take this into account in their determinations of the ratio between acid secretion and the associated oxygen uptake, and might have obtained ratios greater than four for this reason. Similarly, it is possible that in two-stage experiments on frog gastric mucosae reported by Davenport (1952) a component of the oxygen uptake in the first stage became available for acid secretion in the second stage, and consequently high values were obtained for the ratio AqH+/AqO2. Negative values for this ratio were also obtained by Davenport (1952), because in some experiments there was a fall in the rate of oxygen uptake in the second stage and a concomitant rise in the rate 28-2

10 438 W. H. BANNISTER of acid secretion. This effect was also observed in the present work, but the fall in oxygen uptake was transitory, and it was followed by a spontaneous progressive increase in oxygen uptake and a concomitant linear increase in acid secretion. A good linear relation between the qh+ and qo2 was observed in 66 % (39 out of 59) of the experiments performed. In two experiments without added substrate, and in three experiments with oxaloacetate in the incubating medium, there was a progressive decrease in the qo2 and a low qh+ which also tended to decrease. In one experiment without added substrate no acid secretion was observed, and there was a progressive decrease in the qo2. These experiments did not give a correlation between acid secretion and oxygen uptake. Fourteen other experiments were also not useful for analysis of the relation between acid secretion and oxygen uptake either because of bunching together of the points on the plot of the qh+ against the qo2, or because of scatter. Failure to observe a good correlation between acid secretion and oxygen uptake occurred usually in the experiments in which either no substrate was added at some stage to the incubating medium, or succinate was added. Thus a linear relation between the qh+ and the qo2 was evident in thirty-four of forty-three experiments (79 %) with glucose or pyruvate, but only in five of sixteen experiments (31 %) without substrate or with succinate. The latter is apparently not absorbed in sufficient quantities by gastric mucosa to act as a substrate of oxidative metabolism (Davenport, Chavr6 & Davenport, 1956). The values of the ratio of acid secretion to the associated oxygen uptake observed in the present work varied from 07 to 4-8 (Tables 2 and 3). The finding of three ratios greater than four, viz. 4*3, 4.7, and 4*8 (Table 3), would appear to be due to a cumulative effect of the errors in the measurements of acid secretion and oxygen uptake. The mean of the ratios given in Tables 2 and 3 is (mean + S.D.), and does not differ significantly (t test) from 4 0 (assigned value with zero variance). It would therefore appear that the value of the ratio of acid secretion to the associated oxygen uptake in isolated frog gastric mucosa has a maximum value of four within the limits of experimental error. This finding is in agreement with the work of Davenport (1952). On its basis there is experimental justification for the redox theory of acid secretion which sets the ratio of acid secretion to the associated oxygen uptake by gastric mucosa an upper limit of four (Conway & Brady, 1948; Crane & Davies, 1948). SUMMARY 1. A technique was devised for making several observations of the rate of acid secretion and the rate of oxygen uptake of an everted sac of frog gastric mucosa.

11 ACID SECRETION AND OXYGEN UPTAKE A linear relation between the rate of acid secretion and the rate of oxygen uptake was observed in a single mucosa, generally after an initial transitory fall in the rate of oxygen uptake. 3. The ratio of acid secretion to the associated oxygen uptake was found to have values which varied from 0-7 to 4-8, and a mean value of (S.D.). 4. Experimental evidence was obtained which suggested that part of the oxygen uptake of mucosae which are not secreting acid may be diverted into the process of acid secretion. The author gratefully acknowledges the advice and guidance of Mr J. R. P. O'Brien in whose Department this work was carried out and wishes to thank Mr G. E. Newman for helpful comments on the manuscript and Mr P. J. Tosh for making the special Warburg flasks. REFERENCES BANNISTER, W. H. (1963). Acid secretion and oxygen consumption by isolated frog gastric mucosa. Biochem. J. 89, 62 P. CONWAY, E. J. (1953). The Biochemistry of Gastric Acid Secretion, p Springfield Thomas; Oxford: Blackwell. CONWAY, E. J. (1959). The redox pump theory and present evidence. In The Method of Isotopic Tracers Applied to the Study of Active Ion Transport. London: Pergamon Press. CONWAY, E. J. & BRADY, T. G. (1948). Source of the hydrogen ions in gastric juice. Nature, Lond., 162, CRANE, E. E. & DAVIES, R. E. (1948). Chemical energy relations in gastric mucosa. Biochem. J. 43, xliii. CRANE, E. E. & DAVIES, R. E. (1951). Chemical and electrical energy relations for the stomach. Biochem. J. 49, DAVENPORT, H. W. (1952). Substrate and oxygen consumption during gastric secretion. Fed. Proc. 11, DAVENPORT, H. W. & CHAVRIE, V. J. (1953). Acid secretion and oxygen consumption by mouse stomachs in vitro. Amer. J. Physiol. 174, DAVENPORT, H. W., CHAVRE, V. J. & DAVENPORT, V. D. (1956). Sulfhydryl groups and gastric acid secretion. Amer. J. Physiol. 184, HEINZ, E. & OBRINK, K. J. (1954). Acid formation and acidity control in the stomach. Physiol. Rev. 34, KREBS, H. A. & KORNBERG, H. L. (1957). A survey of the energy transformations in living matter. Ergebn. Physiol. 49, ROBERTSON, R. N. (1960). Ion transport and respiration. Biol. Rev. 35,