Effect of Krebs cycle intermediates and inhibitors on toad gastric mucosa JESUS CHACfN, R. RINCON, D, INCIARTE, A. CARIZALES, G. MARTINEZ, AND DARWIN ALONSO Department of Physiology, School of Medicine, University of Zulia, Maracaibo, Venezuela; Medical Service, San Francisco General Hospital Medical Center, and University of California School of Medicine, San Francisco, California
A. CANIZALES, G. of Krebs cycle intermediates and inhibiLors on toad gastric mucosa. Am. J. Physiol. 236(6): E692-E700, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(6): E692-E700, 1979.-An attempt to increase the permeability of gastric mucosa to exogenous Krebs cycle intermediates seemed advisable for a better understanding their relationship with acid secretion. At pH 7.4, citrate, oxoglutarate, fumarate, and malate had no significant effect on oxygen uptake (Qo.,) nor on acid secretion (QH+) by toad gastric mucosa; succinaie increased Qo, slightly and had no effect on QH+; but at pH 5.0, oxoglutarate and succinate increased Qo, by 18 and 21%, respectively. 14C02 evolved by gastric mucosa incubated with [ 14C]oxoglutarate, succinate, malate, or citrate was 155, 92, 128, and 353%, respectively, greater at pH 5. Citrate, oxoglutarate, succinate, fumarate, and malate increased Q El+ by theophylline-stimulated mucosa at pH 5.0 by 25, 39, 35, 17, and 28%, respectively. Oxoglutarate-dependent respiration was shown to correlate with oxoglutarate oxidation. Malonate and arsenite inhibited Qo, and QH+ ; malonate inhibition was reversed by washout or by succinate. Arsenite was reversed by washout and accelerated by addition of lipoate immediately after washout. The results suggest that the Krebs cycle has concomitant roles in the regulation ofQH+ and oxidative metabolism in the toad gastric mucosa.
with QH+ in gastric mucosa. Davenport and Chavre (4,5, 7) reported that KC intermediates added to mouse gastric mucosa do not affect QH+, but they suggested that the KC supplies part of the energy, because fluoroacetate inhibits QH+ (7). Sachs et al, (22) found that malonate, an inhibitor of succinate oxidation, did not inhibit QH+ by frog gastric mocusa. Kasbekar (17) also found that KC intermediates did not affect QH+ by frog gastric mucosa. The negative results with KC intermediates and malonate have been attributed to a permeability problem. Sachs et al. (21, 23) and Sarau et al. (25) recently reported that KC intermediates are significantly increased in dog gastric mucosa after stimulation by histamine, a result that suggests increased KC activity associated with acid secretion. We (2) presented evidence suggesting that oxalacetate concentration is a key factor in controlling Qo, and QH+in the frog gastric mucosa. We therefore examined in further detail KC activity and its relations with QH+. The effects of KC intermediates and inhibitors on oxygen uptake (Qo,) andQH+ of toad gastric mucosa are reported herein; some of these data were included in a preliminary report (3).
gastric secretion;
MATERIALS
CNACIN, MARTINEZ,
JESUS, R. RINC~N, D. INCIARTE, AND DARWIN ALONSO. Effect
oxidative
metabolic
activity
in Bufu marinus
SOME OF THE CURRENT PROBLEMS of gastric physiology are the question of the metabolic pathway(s) that provide
the energy for acid secretion (QH+) and the relations between secretion and intermediary metabolism. The aerobic phases of intermediary metabolism are essential for normal rates of QH+ by the gastric mucosa in vitro (6). Glycolysis supports secretion only transiently under anaerobic conditions or contributes a small fraction to it under aerobic conditions (10, II, 13, 20). Fatty acid oxidation and the oxidative metabolism of pyruvate play a role in QH+ of amphibian (1, 14-16) and canine (21, 25) gastric mucosa. Sernka and Harris (26) reported that the pentose phosphate shunt makes an important contribution of energy to the spontaneous QH+ in vitro by the gastric mucosa of rat.
The Krebs cycle (KC) is a major source of energy in animal cells. Until recently little attention has been given to the metabolism of KC intermediates and its relations E692
AND METHODS
The experiments were performed in vitro on gastric mucosa from fasted Bufo marinus (Venezuelan toads). Hydrogen ion secretion was measured as reported previously (2, 14), using the pH stat method. The end point of titration was 5.6 when the pH of the nutrient solution was 7.4 and 5.0 when the pH of the nutrient solution was 5.0. The buffers used in the nutrient side were 17 mM Ntris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) at pH 7.4 or 17 mM phosphate buffer at pH 5.0, depending on the experiment. One hundred percent oxygen was used on both sides. The compounds used were a number of KC intermediates; the inhibitors, arsenite (NaAsOz) and malonate; the stimulants, histamine and theophylline; the fatty acid salt (Na), butyrate, and the cofactor, lipoic acid. The compounds tested were added to the nutrient solution in the various experiments at the times indicated in the results. Oxygen uptake was measured in a Gilson respirometer as described previously (2, 14), using 17 mM TES at 7.4 or 17 mM phosphate at pH 5.0 as buffers. In the Qo,
0363-6100/79/OUOO-0000$01.25
Copyright
0 1979 the American
Physiological
Society
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KREBS
CYCLE
AND
ACID
E693
SECRETION
experiments most of the above compounds used were added to the reaction mixture as indicated in the results. Production of 14C02 from labeled KC intermediates was measured as follows: preweighed paired slices of tissue were incubated for 2 h at 30°C with 2 ml of 17 mM TES nutrient solution at pH 7.4 or 2 ml of 17 mM phosphate buffer solution at pH 5.0, containing 0.2 @i of the labeled intermediate (New England Nuclear) at a final concentration of 10 or 20 mM. The incubation flasks were sealed after gassing with 100% 02; the evolved 14C02 was trapped in 0.2 ml of Hyamine (New England Nuclear), in a small glass vial placed in the center well of the flask. The incubation was terminated by an injection of 1 ml 2 M H$04, and the flask was shaken for 90 min. The vial was then transferred to a counting vial with 18 ml Aquasol (New England Nuclear) for liquid scintillation counting (Nuclear Chicago, model Mark III). Counts of a control flask without tissue, which was run simultaneously, were subtracted from the total counts per min of the flask with tissue. Oxygen uptake and 14C02 production from a labeled intermediate were measured simultaneously in the same preparation as follows: tissue was prepared and incubated with a labeled intermediate as previously described (2, 14) for standard respiration studies in a Gilson respirometer; the tissues were placed in Warburg flasks, gassed with 100% 02, and Qo, was measured at 30-min intervals for 2 h. The incubation was terminated by addition of 0.5 ml of 6% (wt/vol) perchloric acid from the side-arm to the main compartment of the flasks and by further shaking for 30 min. The content of the center well with the absorbed 14C02 by the Hyamine was then transferred to a scintillation vial with 18 ml Aquasol for counting. The tissues were recovered and heated overnight at 80°C for dry weight determinations. Aliquots of the incubation medium were taken for specific activity measurements and apropriate quench corrections were applied. The results are expressed as means t standard error. Statistical significance of the differences was determined by Student’s t test, paired or unpaired. P values c 0.05 were considered significant.
14C02 at pH 5.0 was greater than that at pH 7.4 with labeled oxoglutarate, succinate, citrate, and malate (155, 92, 353, and 128%, respectively) (Fig. I). The rate of 14C02 production from labeled oxoglutarate at pH 7.4 was not increased by stimulating respiration with 5 mM theophylline (Table 1), suggesting that permeability is rate limiting at pH 7.4. Similar results were obtained with other KC intermediates (citrate, two experiments; succinate, two experiments; malate, two experiments). In contrast, the rate of oxidation of labeled glucose, a substrate whose oxidation is assumed not to be rate limited by permeability, was significantly stimulated by theophylline at that pH (Table 1). Increased activity of KC at pH 5.0 was also suggested by the Qo, results in the nonstimulated gastric mucosa (Table 2): the percentage stimulation of Qo, at pH 5 was twice that at pH 7.4 with succinate as the substrate; at pH 5, oxoglutarate stimulated Qo, by 15% compared with no stimulatidn at pH 7.4. The above results prompted us to study the effects of various KC intermediates on QH+ at pH 5.0. Citrate, oxoglutarate, and succinate at 10 mM increased QH+ of nonstimulated gastric mucosa by 24, 20, and 22%, respectively; malate and fumarate had no effect. Citrate, oxoglutarate, succinate, fumarate, and malate increased QH+ of theophylline-stimulated mucosa by 25, 39, 35, 17, and 28%, respectively. These results are summarized in
PH
kzzl
7. 4
m
50
I
RESULTS
Effect of Krebs cycle intermediates. The QH+ and Qo, of toad gastric mucosa were not significantly affected by addition of 10 mM citrate, oxoglutarate, fumarate, or malate at pH 7.4. In nine experiments 10 mM succinate increased &a, only by 7.19 t 1.4% ( P < 0.05) and had no effect on QH+. The lack of stimulation of Qo, and QH+ when KC intermediates were added at pH 7.4 may have merely reflected a failure of the compounds to penetrate the cells in sufficient concentration. Because penetration of the epithelial cells by weak acids may be increased by decreasing pH, we investigated the effects and metabolism of KC intermediates by employing gastric mucosa bathed in phosphate nutrient solution at pH 5.0. Increased oxidation of exogenous KC intermediates at pH 5.0 with respect to that at pH 7.4 was demonstrated by measuring the rates of 14C02 production from exogenously supplied 14C-labeled intermediates. The evolved
(10)
(81 *
OXOGLUTARAT - l-1°C FIG.
diates. theses
SUCCINATE
-1,4-w
CITRATE
-1,5
- ‘4c
I r!f!m MALATE -t]J4c
1. Effect of pH on ‘4CO2 production from labeled KC intermeCompounds were at 20 mM concentration. Numbers in parenare numbers of experiments in each case. * P < 0.05.
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E694
CHACIN
1. Effect of theophylline
TABLE
oxidation
and on respiration
ET
AL.
3. Effect of KC intermediates on QH+ of theuphylline-stimulated and nonstimulated
on substrate at pH 7,4
TABLE
gastric mucosa at pH 5.0 Labeled
Substrates
No, of Expts
Treatment
Qol
“C02,
QH+,
nmoU(2
[I- 14C]oxoglutarate
0
6
Theophylline
6
h-mg) dry wt
Pretreatment
2.38 t0.28 2.44 &0.26
86.48 kg.96 137.55 t11.30*
5.18 zkl.16 11.54 *1.44*
49.38 + 13.01 112.01 t 16.04*
None
Addition
0 Citrate
[U-14C]glucose
0
3
Theophylline
3
Values are means -+ SE. Oxoglutarate I mM, and theophylline at 5 mM.
was used at 2 mM, * P < 0.05.
2. Effect ofpH on mucosal
TABLE
to succinate
glucose
at
Theophylline Addition
PH
7.4 (17 mM TES)
5.0 (17 mM PO4)
0
16
Succinate
9
Oxoglutarate
6
0
26
Succinate
14
Oxoglutarate
10
02, @(h-mg)
Succinate
6
Malate
6
0
Dry Wt
Before (6090 min)
After ( 12% 150 min)
Change’
1.53 &0.05 1.39 kO.06 1.50 kO.08
1.54 kO.05 1.50 kO.05 1.45 kO.04
0.6 k2.4 7.9 *1.4* -3.3 k3.3
1.49 kO.12 1.46 kO.12 I.26 kO.13
1.44 to. 10 1.72 *o. 12 1.44 kO.11
-3.4 k2.1 17.8 ~2.7 * 15*0 *5.5*
Citrate
’
10 8
and oxoglutarate No. of Expts
7
Oxoglutarate
Fumarate
Qo, responses
No. of Expts
Oxoglutarate Succinate Fumarate Malate
6
pq/km%
Before (90120 min)
After (GO180 min)
Lll k0.24 1.36 k0.28 1.15 kO.19 2.11 k0.27 1.11 to.08 0.83 kO.20
1.06 k0.28 1.64 to.32 1.34 kO.25 2.59 kO.36 1.15
2.48 kO.17 2.91 +o. 10 1.96 -to.43 2.50 k0.38 3*70 k0.48 4.29 kO.28
2.36 kO.14 3.64 kO.20 2.61
kO.12 0.77 kO.14
to.36 3.32 k0.53 4.32 kO.59
5.47 to.32
Values are means k SE. Theophylline (10 mM) was added 0 and the Krebs cycle intermediates (10 mM) at 120 min.
0.05.
Change,
%
-8.7 k5.6 23.7 &6.9* 19.6 &4.2* 21.7 &7,2" 2.8 k5.8 -7.0 kf1.4 -4.2 u.7 25J *5.3* 38.7 *11.9* 34.9 k6.1” 16.8 t4.5* 28.0 t6.1* at tine
“PC
significant correlation coefficient (r = 0.89, P < 0.01). These results strongly suggest, but do not prove, that KC intermediates stimulate QH+ and &o, via their metabolism. Effect of inhibitors of Krebs cycle. Malonate, a known inhibitor of succinate oxidation in other tissues, when Table 3. In five experiments at pH 5, 10 mM citrate added at pH 7.4 significantly reduced the Qo, of both increased QH+ of histamine-stimulated mucosa by 30%. nontreated and theophylline-treated gastric mucosa (Fig, In six experiments, 10 mM aspartate, a dicarboxylic 4). In six experiments, at pH 5, malonate also decreased amino acid, had no effect at pH 5. the 14C02that evolved from [1,4-14C]succinate from 7.6 The degree of QH+ stimulation by different KC intert 0.8 pmo1/(2 ho g) wet wt in the control mucosal slices mediates at pH 5.0 in the theophylline-treated gastric to 4.3 t 1.2 in the experimental mucosal slices, an inhimucosa was related to the pKa values and the proportion bition of 45 t 11% ( P < 0.01 by paired t test). of the less charged molecules of these carboxylic acids, A These results indicate that malonate also inhibits sucgreater degree of stimulation was associated with a higher cinate oxidation by the toad gastric mucosa and led us to proportion of the unionized and singly charged molecules investigate the effects of malonate on QH+, The results of the acids. This result is illustrated in Fig. 2. A greater are presented in Table 4. At pH 7.4, malonate (10 mM) degree of QH+ stimulation was also associated with a decreased QH+in theophylline-stimulated mucosa by 10% higher rate of oxidation of the substrates, suggesting that (P c 0.05), compared with that in the untreated mucosa; they must be metabolized to exert their stimulating ef- at pH 5.0, the decreases were 25% in the theophyllinefects. To further test this possibility we examined the stimulated and 27% in the nonstimulated mucosa. The relationship between oxoglutarate oxidation and oxoglu- inhibitory effect of malonate was reversed by washing tarate-stimulated respiration at pH 5.0 in the theophylout the nutrient-malonate solution or by adding 10 mM line-treated gastric mucosa, It was reasonably assumed succinate at pH 5 (Fig. 5). that Qo, was directly related to the stimulation of QH+, Arsenite (NaAsOz), another inhibitor of the Krebs In these experiments Qo, and 14C02 production from cycle (19), inhibited Qo, of toad gastric mucosa more labeled oxoglutarate were measured simultaneously in than malonate. The response of Qo, to arsenite concenthe same preparation, and respiration was stimulated tration is shown in Fig. 6; inhibition increased with with different doses of oxoglutarate. The relationship concentration. A similar effect of arsenite on Qo2 by between AQo, and A14C02 is shown in Fig. 3. The two theophylline-stimulated and nonstimulated mucosa is ilparameters showed a positive correlation with a highly lustrated in Fig. 7. Values are means k SE. Compounds (10 mM) were added at 90 min. The rates in the second 30-min period after the addition were compared to those in the 30-min period prior to the addition and expressed as percent of change. * (P < 0*05).
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KREBS
CYCLE
AND
ACID
E695
SECRETION
I A
FIG. 2. Relationship between degree of QH+ stimulation by several KC intermediates and proportion of unionized acid ( C), and of singly (B) and doubly plus trebly (A) charged anions in theophylline-stimulated gastric mucosa at pH 5.0.
20
40
60
60
CONCENTRATION
Y = 20.48X 140 r
q
0.89
100
0
2
OF CITRIC
ACIDS
6
4
8
12
10
14
( % OF 10 mM >
t14.19 P ~0.01
120
100
1.2L 1 0 30
1 60
1 90
1 120
1 150
I 180
1 210
MIN
of malonate on Qo, of nontreated and theophyllinetreated gastric mucosae in TES at pH 7.4. Theophylhne (10 mM) was added to (n-a) and (A -----A) of 4 mucosal aliquots at tine 0, At 90 min malonate (10 mM) was added to a theophylhne-treated (A-----A) and a nontreated (o----- O) mucosa; buffer was added at this time to the mucosae. Values are means, and number other 2 (n-n) and (e) of experiments are given in parentheses. * P < 0.05, FIG.
/ a
1
1
I
1
I
1
1
2
3
4
5
6
A CO2
3. Relationship between “C02 production from labeled oxoglutarate and oxoglutarate-induced increase in respiration in theophylline (5 mM)-treat e d gastric mucosa at pH 5.0. Increments in respiration and ‘4C02 production are relative to control tissues exposed to 0.5 mM oxoglutarate. Values were obtained by varying oxoglutarate concentration over range of l-10 mM. Units are nmol/(Z h’mg) dry wt, FIG.
Furthermore, in eight experiments, arsenite (2 mM) reduced the 14C02that evolved from labeled oxoglutarate from 5.2 * 0.9 pmol/(2 h g) wet wt in the control slices l
4. Effects
to 2.4 t 0,4 in the experimental slices, a decrease of 47% t- 9 ( P c 0.005 by paired t test). Arsenite also inhibited QH+ of stimulated mucosa (Fig. 8). The QH+was stimulated to maximal rates by addition of 10 mM theophylline plus 10 mM butyrate, and arsenite was added at the peak of the response; the dose of arsenite that produced about 50% inhibition in the second 30-min period after addition was 2 mM. In one experiment, lb mM arsenite inhibited QH+ by 94%. Arsenite also inhibited the QH+ of both theophylline- and histamine-stimulated gastric mucosa in the absence of exogenous substrate. The time-course effect of 0.5 mM arsenite on QH+ of theophylline plus butyrate-stimulated mucosa is illustrated in Fig. 9. In the presence of arsenite, QH+ could not be restored to pretreatment values by the addition of the following compounds: 10 mM oxoglutarate at pH 5.0 (2 experi-
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E696
CHACfN
ments), lipoate 1 and 10 mM (4 experiments), 10 mM succinate at pH 5.0 (2 experiments), 10 mM propionate (2 experiments), 10 mM pyruvate with and without I mM lipoate (4 experiments), 10 mM malate at pH 5.0 (2 experiments), and 5 mM adenosine triphosphate at pH 5.0 (2 experiments), However, the inhibition was reversed by removing arsenite from the nutrient side by washing 2 or 3 times and replacing with fresh nutrient solution without arsenite. Because the inhibitory effect of arsenite on KC has been attributed to blocking the SH groups of the lipoyl moieties in the pyruvate and oxoglutarate dehydrogenase enzyme complexes (19), the effect of adTABLE
different .--
4. Effects of malonate pH levels
2.0 r
Addition
Theophylhne
No. of Expts
Theophylline
2.27 rts0.15 2.13 to.18
2.17 kO.16 kO.14
-15.0 &2.7*
1.11 kO.24
1.06 kO.28 0,82
-8.7 k5.6 -35.7
5
1*30
8
Malonate
7
-4.8
t1.9
1.79
to.18
to. 13
2.48 kO.17 2.52 kO.45
2.36 kO.14 1.97 k0.46
Values are means t SE. Gastric mucosa was bathed in solution, pH 7.4, or in phosphate nutrient solution, pH were pretreated with 10 mM theophylline; malonate added at 120 min. * The difference between control QH+ is statistically significant ( P < 0.05).
I
Change, %
After ( 180240 min)
7
0
h)
Before (90120 min)
12
0
None
QH+, pq/(cm’-
10
0
Malonate 5.0
dition of lipoate on the inhibitory effect of arsenite was examined. The results are shown in Fig. 10. In these experiments, QH+ was stimulated by theophylline plus butyrate and 2 mM arsenite was added at the peak of QH+ (in general, 60 min after addition of the stimulants). When the inhibitory effect was very evident, in general between 50 and 60 min after addition of the inhibitor, arsenite was washed out and replaced with fresh nutrient solution containing the stimulants; 1 mM lipoate was added to one mucosa immediately after washing, and nothing was added to the other one, which served as control. In the presence of lipoate, the restoration of
on QH+ at
Malonate
5.0
AL.
ARSENITE Pretreatment
7.4
ET
k6.2 * -4,2 kI.7 -29.6 t7.8"
TES nutrient 5.0. Mucosae (10 mM) was and malonate
0.5 30
1 60
1 90
1 120
1 150
I 180
I 210
MN
6. Typical experiment showing effect of various concentrations of arsenite (NaAs&) on Qo,. At 90 min, the indicated concentrations of arsenite were added to respective mucosal aliquots. Each point is mean of 2 dunlicate exseriments. Suspending medium was TES buffer, pH FIG,
a
7.4.
THEOPHYLLINE 10 mM MALONATE 10 mM i SUCCINATE
*
10 mM
FIG. 5. Effect of succinate on malonate inhibition of the QH+ by theophylline-stimulated gastric mucosa. The buffer was 17 mM phosphate, pH 5.0.
2
3
PERIODS
4
5
6
7
OF 20 MINUTES
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KRFBS
CYCLE
AND
ACID
E697
SECRETION -2323.8
“12 -39
(141
T I
T
7+90/o*
(6)
2.4 -9.422.5
T: & 2.2 + F + 2
ARSENITE
'10
(6)
\
i
\
T
T
\ \
2.0
1,8
3 1.6 1.4
1.2
I ‘30 0’ /’
60 I
90
120
L
1
150 1
180I
MIN 7. Effects of arsenite on Qu., of nontreated and theophyllinetreated gastric mucosae. At time 0, theophylline (10 mM) was added to (c- - -o) and (n ) of 4 mucosal aliquots. At 90 min, 0.5 mM arsenite was added to a treated (o- - a) and nontreated (n- - -a) mucosae, and TES buffer, pH 7.4, was added to other two, (w---+ ) and (A-A). Each point is mean of 6 or more experiments. * P < 0.05. FIG.
QH+ was much more rapid and more complete than in the absence of lipoate. This effect of lipoate seemed to be specific because two other SH donor reagents, Lcysteine and reduced glutathione at 2 mM, did not significantly affect the time-course of the restoration of QH+ (Fig. 10). DISCUSSION
In contrast to earlier findings (4, 5, 7), the present experiments demonstrate that several KC intermediates are efficiently oxidized by the toad gastric mucosa and can stimulate QH+under appropriate conditions of nutrient solution. Stimulation of QH+ on serosal addition of KC intermediates was observed when phosphate buffer of pH 5.0 was the nutrient solution, but not with a nutrient solution of pH 7.4. The high pH of the bathing solutions used by Davenport and Chavrh (4, 5, 7) probably accounts for the observed lack of response of mouse gastric mucosa to KC intermediates. Cell membranes are, in general, more permeable to uncharged than to charged molecules (9). Because the KC intermediates are tri- and dicarboxylic acids with pK, between 3 and 5.61 at 25°C (30), the charged (unprotonated) molecules are the predominant forms of the acids at pH 7.4. At pH 5.0, the concentrations in the nutrient solution of the less charged carboxylic acids are significantly higher than those at pH 7.4. It is therefore likely that the stimulation of QH+ observed on addition of KC intermediates at pH 5.0 is simply attributable to a higher permeability rate of acidic substrates into the oxyntic cells at this pH. A similar mechanism has been postulated by Taylor, Hess, and Maffly (29) to explain the stimula-
0
0
0.5
0
10
2.0
ARSENIT E CONC. Inb 1 8. Effect of arsenite on QH+- The concentration of arsenite is shown at the base of each set of experiments. Experiments were carried out with paired mucosae in TES buffer, PI-I 7.4. Values over 2nd bar in each group is calculated percent of change of arsenite-treated with respect to the nontreated mucosae, Numbers in parentheses are the numbers of experiments in each case. * P < 0.05. FIG.
2.8 2.4 -
0.4 ++ LL 0 30
11 60
I 90
I 150
I 120
LL 180
11 210
11 240
MN
9. Time-course response of QH+ to arsenite. TES (17 mM), pH 7.4, was nutrient solution. Theophylline plus butyrate (10 mM each) and arsenite (0.5 mM) were added at time shown. FIG.
tion of sodium transport (short-circuit current) produced by several KC intermediates in toad bladder bathed in phosphate Ringer’s solution at pH 6.4 but not at pH levels above 7.4 (12, 27). Our experiments showed that several exogenously supplied KC intermediates stimulate Qo,, 14C02evolution, and QH+ at pH 5 in contrast to a small, or lack of, effect at pH 7.4, thereby supporting the increased permeability mechanism. Moreover, the oxidation rates of exogenous KC intermediates and the
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E698
CHACIN
THEOPHYLLINE 10 mM t ARSENITE 2mM WASHOUT
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
10. Effects
AL.
10 mM
+ ADDITIONS
-1
PERIODS of washing plus lipoate and washing or reduced glutathione on arsenite inhibition of QH+ plus butyrate-stimulated mucosa. Buffer was 17 mM QH+ is expressed as % of pretreatment value immediately of arsenite (control value, 100%). (v) At arrow, FIG.
BUTYRATE
ET
0
1
OF
plus L-cysteine by theophylline TES, pH 7.4. before addition lipoate (1 mM)
degree of QH+ stimulation at pH 5.0 in the theophyllinestimulated gastric mucosa were dependent on the p& values and on the proportion or concentration of the unionized and singly charged molecules of the acids (Figs. 1 and Z), suggesting that these are the molecular forms that predominantly penetrate into the cells, The above correlation was not found using the QH+ values of the nonstimulated gastric mucosa at pH 5.0. Secretagogue availability is rate limiting for QH+ and Qo, in this preparation and might account for these unexpected results. The fact that the rate of oxidation of oxoglutarate and other KC intermediates at pH 7.4 was not stimulated by increasing respiration with theophylline (Table 1) is also consistent with the point of view that permeability is rate limiting at pH 7.4 for KC intermediates. As expected, the rate of glucose oxidation, which is assumed not to be limited by permeability, was significantly stimulated by increasing respiration with theophylline at pH 7.4. It seems unlikely that the stimulation of QH+ by KC intermediates at pH 5.0 was due to diffusion of the protonated forms of the acids from the nutrient side to the secretory side and releasing H+ to the secretory solution because there was no pH gradient. The following experiment ruled out significant diffusion: when 1 PCi of labeled citrate (10 mM) was added on the nutrient side (58, 407 cpm/ml of nutrient solution), the radioactivity of the secretory solution (I ml) was 18, 21, 36, 26, 44, and 56 cpm at 10, 20, 30, 40, 50, and 60 min after addition of citrate, respectively; the background was 22 cpm. In this experiment, Q H+ was stimulated by 30%, and the peak response was observed at 30 min after addition of citrate. It is also possible that the low pH affects other variables that might influence the rate of substrate oxidation, the coupling of substrate oxidation to transport, or the transport process itself.
2
3
4
5
6
7
0
9
10
II
12
13
14
15
10 MINUTES was added immediately after washout (6 expts); (X-X) at arrow, 2 mM L-cysteine (3 expts) or 2 mM reduced glutathione (3 expts) was added immediately after washout; ( G---O) at arrow, washout only (6 experiments). Bars represent I SE. * P < 0.05, compared to corresponding values of X-X and m.
Another possible explanation is that KC intermediates at low pH could stimulate QH+ by neutralizing bases in the oxyntic cells or by increasing the rate of recycling of endogenous COz at the nutrient barrier membrane in a situation in which CO2 concentration is a limiting factor for H+ secretion. This mechanism was first postulated by Sanders, Hayne, and Rehm (24) and supported later by Kidder and Montgomery (18) and Silen, Machen, and Forte (28). However, the following results argue against this possibility. I) Malonate, a dicarboxylic acid with p&s of 2.83 and 5.69 at 25°C (30), inhibited Qoz, 14C02 production, and Q H+ at low pH; this inhibition is reasonably explained by the known inhibitory effect of malonate on succinate oxidation and KC activity. 2) Aspartic acid, a dicarboxylic amino acid whose p&s for the carboxylic groups are 2.10 and 3.86 at 25*C (30), did not affect QW at pH 5; this result is in accordance with Davenport and Jensen (8) and Alonso et al. (1) who reported that amino acids are not utilized by the H’ transport process. We therefore favor the hypothesis that the stimulation of Qn+ by KC intermediates at low pH depends on their oxidation in the oxyntic cells. The highly significant correlation between oxoglutarate oxidation and oxoglutarate-dependent respiration (Fig. 3) is consistent with, but does not prove, this point of view. Increased KC activity can also stimulate the oxidation of other substrates (fatty acids and/or carbohydrates) that can be mobilized during the secretory process (15, 16). If the amount of 14C02liberated is used to calculate the amount of oxygen required to oxidize oxoglutarate to COZ, assuming complete oxidation in the KC, the slope of the regression line correlating oxoglutarate oxidation and oxoglutarate-dependent respiration is converted from a value of 20 to a value of 5. This value indicates that oxoglutarate oxidation accounts for only 20% of the increase in respi-
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KREBS
CYCLE
AND
ACID
E699
SECRETION
ration and leaves room for the possibility that oxoglutarate stimulates the oxidation of other endogenous substrates. This is not surprising because the catalytic effect of KC intermediates on respiration in animal tissues is well known. The increased substrate oxidation would stimulate QH+ by providing reducing equivalents and/or high energy compounds (such as adenosine triphosphate) to the H+ transport mechanism. The present studies also showed that inhibitors of KC, such as malonate and arsenite, can reduce QH+. The inhibitory effect of malonate was more evident at low pH. This result can be due to a higher rate of penetration of the compound into the cells at this pH. The lack of effect of malonate reported by Sachs et al. (22) probably resulted from insufficient penetration of the compound at the relatively high pH of the nutrient solution. However, other factors including species difference cannot be ruled out. The inhibitory effects of both malonate and arsenite on QH+ were reversed by washing, and the reversal was accelerated by addition of succinate and lipoate, respectively. The accelerated reversal of the inhibitory effect or arsenite by lipoate, but not by L-cysteine and reduced glutathione, supports the point of view that arsenite acts by blocking the -SH groups of the lipoyl moieties in the oxoglutarateand pyruvate-dehydrogenase enzyme complexes (19). Although malonate and arsenite could affect other sites involved in the H+ transport process, it seems that their inhibitory effects on QH+ are reasonably explained by inhibition of the KC because of the inhibition of the succinic- and the pyruvic- and oxoglutaric-dehydrogenase systems, respectively. The results with KC intermediates and inhibitors together suggest that KC activity plays a central role in
the regulation of oxidative metabolism and acid secretion by the toad gastric mucosa. Davenport and Chavrb (7) suggested that in the mouse stomach the KC supplies part of the en ,ergy of the H+ transport because flu&o acetate inhibits acid secretion. The situation seems to be different in rat stomach in vitro, because Sernka and Harris (26) found that in this animal QH+ is supported by substrates that can be oxidized through the pentose phosphate shunt but not by substrates that require the KC for their oxidation. However, low permeability of the latter substrates was not ruled out. The fact that all the KC intermediates tested at pH 5.0 stimulated QH + and that inhibition of QH+ was obtained by using inhibitors that act at two different steps of the KC suggest that QH+ in the toad gastric mucosa is not linked to any specific step of the KC. The entire cycle seemsto be necessary, In dog gastric mucosa, Sarau et al. (25) and Sachs et al. (21, 23) found an increase in the levels of the citric acid cycle intermediates during stimulation with histamine, indicating increased KC activity. This result could be a general metabolic response to secretagogues and could result from mobilization of substrates (15, 16, 21, 23, 25) and/or activation of some of the KC enzymes (21, 23, 25). This investigation was supported in part by CONDES of L*U.Z. and by CONICIT Grant Sl-0455, Venezuela, and National Institutes of Health Grant AM 12606. The authors acknowledge the efforts and help of Ms. Annabell Castillo, Mrs. Zoraida de Jaime, Mr. Jesus Anez, and Mr. Ernest0 Ssnchez in this work. Present address of J. Chacin and G. Martinez: Unidad de Investigaciones Biolbgicas, Facultad de Medicina, Universidad de1 Zulia, Maracaibo, Venezuela. Received
20 July
1978; accepted
in final
form
2 January
1979.
REFERENCES 1. ALONSO, D., K. NIGON, I. DORR, AND J. B. HARRIS. Energy sources for gastric secretion: substrates. Am, J. PhysioZ. 212: 992-K@O, 1967. 2. CHAC~N, J., 0. H. PARK, J. B. HARRIS, AND D. ALONSO. Role of oxalacetate in the lipoate effect on frog gastric mucosa. Am. J. Physiol. 231: 209-215, 1976. 3, CIIAC~N, J., R. RINC~N, D, INCIARTE, AND A. CA~~IZALES. Krebs cycle and H+ secretion in amphibian gastric mucosa. Biophys. J. 16: 129a, 1976. 4. DAVENPORT, H. W., AND V. 3. CHAVR&. Conditions affecting acid secretion by mouse stomachs in vitro. Gastroenterology 15: 467480, 1950. 5, DAVENPORT, H. W., AND V. J. CHAVR~. Acid secretion and acetoacetate utilization in mouse stomachs in vitro. Federation Proc. 10: 33, 1951. 6* DAVENPORT, H. W,, AND V. J. CHAVRB. Relation between substrate disappearance and acid secretion in mouse stomach in vitro. Am. J. Physiol. 166: 456-461, 1951. 7, DAVENPORT, H. W., AND V. J. CHAVRI?. Acid secretion and oxygen consumption by mouse stomachs in vitro, Am, J, PhysioZ, 174: 203208, 1953. 8. DAVENPORT, H. W., AND V, JENSEN. Observations on the secretion of acid by the mouse stomach in vitro, Gastroenterology 12: 630636, 1949. 9. DAVSON, H., AND J. F. DANIELLI. The Permeability of NaturaL Membranes. Cambridge: Cambridge Univ. Press, 1952, p. 186, 10. DURBIN, R, P. Utilization of high-energy phosphate compounds by stomach. J. Gen. Physiol. 51: 233+239s, 1968, 11. DURBIN, R. P., AND F. MICHELANGELI. High-energy phosphate compounds in frog gastric mucosa. In: Gastric Secretion, edited by G. Sachs, E. Heinz, and K. J. Ullrich. New York: Academic, 1972, p. 307-318.
12. FIMOGNARI, G. M., G. A. PORTER, AND 1, S. EDELMAN. The role of the tricarboxylic acid cycle in the action of aldosterone on sodium transport, Biochim. Biophys, Acta. 135: 89, 1967. 13, FORTE, J. G., P. H. ADAMS, AND R. E. DAVIES. Acid secretion and phosphate metabolism in bullfrog gastric mucosa. Biochim. Biophys. Acta. 104: 25-38, 1965. 14. HARRIS, J. B,, D. ALONSO, 0. IS. PARK, D. CORNFIELD, AND cl, CHAC~N. Lipoate effect on carbohydrate and lipid metabolism and gastric H’ secretion. Am. J. PhysioZ. 228: 964-971, 1975. 15. HERSEY, S. J. Metabolic changes associated with gastric stimulation. Gastroenterology 73: 914-919, 1977. 16. HIGH, W. L., AND S. J, HERSEY. Mechanism of theophylline stimulation of acid secretion by frog gastric mucosa, Am. J. Physiol. 226: 1408-1412, 1974. 17. KASBEKAR, D. K. Gastric H* secretion in presence of substrates: absolute dependence on secretagogues. Am. J. Physiol, 231: 522528, 1976. 18. KIDDER, G, W., AND C. W. MONTGOMERY. CO2 diffusion into frog gastric mucosa as rate-limiting factor in acid secretion. Am. J. Physiol. 227: 300-304, 1974. 19. LEHNINGER, A. L. Biochemistry. New York: Worth, 1970, p. 346. 20. REHM, W. S. The metabolic state and the response of the potential of frog gastric mucosa to changes in external ion concentrations. J. Gen. Physiol, 51: 25Os-26Os, 1968. 21. SACHS, G., H. C, CHANC, E. RABON, R. SHACKMAN, H. M, SARAU, AND G. SACCOMANI. Metabolic and membrane aspects of gastric H+ transport. Gastroenterology 73: 931-940, 1977. 22. SACHS, G., R. H. COLLIER, R. L. SHOEMAKER, AND B. I. HIRSCHOWITZ. The energy source for gastric H* secretion. Biochim. Biophys. Acta. 162: 210-219, 1968. 23. SACHS, CL, E. RABON, G. SACCOMANI, AND H. W. SARAU. Redox and ATP in acid secretion. Ann. NY Acad. Sci. 264: 456-475, 1975.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 11, 2019.
E700 SANDERS, S. S., V. 8, HAYNE, JR., AND W. S, REHM. Normal H’ rates in frog stomach in absence of exogenous CO2 and a note on pH stat method. Am. J. PhysioZ. 225: 1311-1321, 1973. 25. SARAU, H. M., J. J. FOLEY, G. MOONSAMMY, AND G. SACHS. Metabolism of dog gastric mucosa. Levels of glycolytic, citric acid cycle and other intermediates. J. Biol. Chem, 252: 8572-8581, 1977. 26. SERNKA, T. J., AND J, B. HARRIS. Yentose phosphate shunt and gastric acid secretion in the rat. Am. J. PhysioZ. 222: 25-32, 1972. 27. SHARP, G. W. G., AND A. LEAF. The central role of pyruvate in the stimulation of sodium transport by aldosterone. Proc. IVat. Acad. 24.
CHACIN
ET
AL.
Sci. USA 52: 1114, 1964. 28. SILEN, W., T. E. MACHEN, AND J. G. FORTE. Acid-base balance in amphibian gastric mucosa, Am. J. Physiol. 229: 721-730, 1975. 29. TAYLOR, A., J. J. HESS, AND R. H. MAFFLY. On the effects of tricarboxylic acid cycle intermediates on sodium transport, by the toad bladder. J. Membrane Biol. 15: 319-329, 1974. 30. WEAST, R, W., S. M. SELBY, AND C. D. HODGMAN. Handbook of Chemistry and Physics (46th ed,)* Cleveland: Chemical Rubber, 1965, p. D-78,
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A. CANIZALES, G. of Krebs cycle intermediates and inhibiLors on toad gastric mucosa. Am. J. Physiol. 236(6): E692-E700, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(6): E692-E700, 1979.-An attempt to increase the permeability of gastric mucosa to exogenous Krebs cycle intermediates seemed advisable for a better understanding their relationship with acid secretion. At pH 7.4, citrate, oxoglutarate, fumarate, and malate had no significant effect on oxygen uptake (Qo.,) nor on acid secretion (QH+) by toad gastric mucosa; succinaie increased Qo, slightly and had no effect on QH+; but at pH 5.0, oxoglutarate and succinate increased Qo, by 18 and 21%, respectively. 14C02 evolved by gastric mucosa incubated with [ 14C]oxoglutarate, succinate, malate, or citrate was 155, 92, 128, and 353%, respectively, greater at pH 5. Citrate, oxoglutarate, succinate, fumarate, and malate increased Q El+ by theophylline-stimulated mucosa at pH 5.0 by 25, 39, 35, 17, and 28%, respectively. Oxoglutarate-dependent respiration was shown to correlate with oxoglutarate oxidation. Malonate and arsenite inhibited Qo, and QH+ ; malonate inhibition was reversed by washout or by succinate. Arsenite was reversed by washout and accelerated by addition of lipoate immediately after washout. The results suggest that the Krebs cycle has concomitant roles in the regulation ofQH+ and oxidative metabolism in the toad gastric mucosa.
with QH+ in gastric mucosa. Davenport and Chavre (4,5, 7) reported that KC intermediates added to mouse gastric mucosa do not affect QH+, but they suggested that the KC supplies part of the energy, because fluoroacetate inhibits QH+ (7). Sachs et al, (22) found that malonate, an inhibitor of succinate oxidation, did not inhibit QH+ by frog gastric mocusa. Kasbekar (17) also found that KC intermediates did not affect QH+ by frog gastric mucosa. The negative results with KC intermediates and malonate have been attributed to a permeability problem. Sachs et al. (21, 23) and Sarau et al. (25) recently reported that KC intermediates are significantly increased in dog gastric mucosa after stimulation by histamine, a result that suggests increased KC activity associated with acid secretion. We (2) presented evidence suggesting that oxalacetate concentration is a key factor in controlling Qo, and QH+in the frog gastric mucosa. We therefore examined in further detail KC activity and its relations with QH+. The effects of KC intermediates and inhibitors on oxygen uptake (Qo,) andQH+ of toad gastric mucosa are reported herein; some of these data were included in a preliminary report (3).
gastric secretion;
MATERIALS
CNACIN, MARTINEZ,
JESUS, R. RINC~N, D. INCIARTE, AND DARWIN ALONSO. Effect
oxidative
metabolic
activity
in Bufu marinus
SOME OF THE CURRENT PROBLEMS of gastric physiology are the question of the metabolic pathway(s) that provide
the energy for acid secretion (QH+) and the relations between secretion and intermediary metabolism. The aerobic phases of intermediary metabolism are essential for normal rates of QH+ by the gastric mucosa in vitro (6). Glycolysis supports secretion only transiently under anaerobic conditions or contributes a small fraction to it under aerobic conditions (10, II, 13, 20). Fatty acid oxidation and the oxidative metabolism of pyruvate play a role in QH+ of amphibian (1, 14-16) and canine (21, 25) gastric mucosa. Sernka and Harris (26) reported that the pentose phosphate shunt makes an important contribution of energy to the spontaneous QH+ in vitro by the gastric mucosa of rat.
The Krebs cycle (KC) is a major source of energy in animal cells. Until recently little attention has been given to the metabolism of KC intermediates and its relations E692
AND METHODS
The experiments were performed in vitro on gastric mucosa from fasted Bufo marinus (Venezuelan toads). Hydrogen ion secretion was measured as reported previously (2, 14), using the pH stat method. The end point of titration was 5.6 when the pH of the nutrient solution was 7.4 and 5.0 when the pH of the nutrient solution was 5.0. The buffers used in the nutrient side were 17 mM Ntris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) at pH 7.4 or 17 mM phosphate buffer at pH 5.0, depending on the experiment. One hundred percent oxygen was used on both sides. The compounds used were a number of KC intermediates; the inhibitors, arsenite (NaAsOz) and malonate; the stimulants, histamine and theophylline; the fatty acid salt (Na), butyrate, and the cofactor, lipoic acid. The compounds tested were added to the nutrient solution in the various experiments at the times indicated in the results. Oxygen uptake was measured in a Gilson respirometer as described previously (2, 14), using 17 mM TES at 7.4 or 17 mM phosphate at pH 5.0 as buffers. In the Qo,
0363-6100/79/OUOO-0000$01.25
Copyright
0 1979 the American
Physiological
Society
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KREBS
CYCLE
AND
ACID
E693
SECRETION
experiments most of the above compounds used were added to the reaction mixture as indicated in the results. Production of 14C02 from labeled KC intermediates was measured as follows: preweighed paired slices of tissue were incubated for 2 h at 30°C with 2 ml of 17 mM TES nutrient solution at pH 7.4 or 2 ml of 17 mM phosphate buffer solution at pH 5.0, containing 0.2 @i of the labeled intermediate (New England Nuclear) at a final concentration of 10 or 20 mM. The incubation flasks were sealed after gassing with 100% 02; the evolved 14C02 was trapped in 0.2 ml of Hyamine (New England Nuclear), in a small glass vial placed in the center well of the flask. The incubation was terminated by an injection of 1 ml 2 M H$04, and the flask was shaken for 90 min. The vial was then transferred to a counting vial with 18 ml Aquasol (New England Nuclear) for liquid scintillation counting (Nuclear Chicago, model Mark III). Counts of a control flask without tissue, which was run simultaneously, were subtracted from the total counts per min of the flask with tissue. Oxygen uptake and 14C02 production from a labeled intermediate were measured simultaneously in the same preparation as follows: tissue was prepared and incubated with a labeled intermediate as previously described (2, 14) for standard respiration studies in a Gilson respirometer; the tissues were placed in Warburg flasks, gassed with 100% 02, and Qo, was measured at 30-min intervals for 2 h. The incubation was terminated by addition of 0.5 ml of 6% (wt/vol) perchloric acid from the side-arm to the main compartment of the flasks and by further shaking for 30 min. The content of the center well with the absorbed 14C02 by the Hyamine was then transferred to a scintillation vial with 18 ml Aquasol for counting. The tissues were recovered and heated overnight at 80°C for dry weight determinations. Aliquots of the incubation medium were taken for specific activity measurements and apropriate quench corrections were applied. The results are expressed as means t standard error. Statistical significance of the differences was determined by Student’s t test, paired or unpaired. P values c 0.05 were considered significant.
14C02 at pH 5.0 was greater than that at pH 7.4 with labeled oxoglutarate, succinate, citrate, and malate (155, 92, 353, and 128%, respectively) (Fig. I). The rate of 14C02 production from labeled oxoglutarate at pH 7.4 was not increased by stimulating respiration with 5 mM theophylline (Table 1), suggesting that permeability is rate limiting at pH 7.4. Similar results were obtained with other KC intermediates (citrate, two experiments; succinate, two experiments; malate, two experiments). In contrast, the rate of oxidation of labeled glucose, a substrate whose oxidation is assumed not to be rate limited by permeability, was significantly stimulated by theophylline at that pH (Table 1). Increased activity of KC at pH 5.0 was also suggested by the Qo, results in the nonstimulated gastric mucosa (Table 2): the percentage stimulation of Qo, at pH 5 was twice that at pH 7.4 with succinate as the substrate; at pH 5, oxoglutarate stimulated Qo, by 15% compared with no stimulatidn at pH 7.4. The above results prompted us to study the effects of various KC intermediates on QH+ at pH 5.0. Citrate, oxoglutarate, and succinate at 10 mM increased QH+ of nonstimulated gastric mucosa by 24, 20, and 22%, respectively; malate and fumarate had no effect. Citrate, oxoglutarate, succinate, fumarate, and malate increased QH+ of theophylline-stimulated mucosa by 25, 39, 35, 17, and 28%, respectively. These results are summarized in
PH
kzzl
7. 4
m
50
I
RESULTS
Effect of Krebs cycle intermediates. The QH+ and Qo, of toad gastric mucosa were not significantly affected by addition of 10 mM citrate, oxoglutarate, fumarate, or malate at pH 7.4. In nine experiments 10 mM succinate increased &a, only by 7.19 t 1.4% ( P < 0.05) and had no effect on QH+. The lack of stimulation of Qo, and QH+ when KC intermediates were added at pH 7.4 may have merely reflected a failure of the compounds to penetrate the cells in sufficient concentration. Because penetration of the epithelial cells by weak acids may be increased by decreasing pH, we investigated the effects and metabolism of KC intermediates by employing gastric mucosa bathed in phosphate nutrient solution at pH 5.0. Increased oxidation of exogenous KC intermediates at pH 5.0 with respect to that at pH 7.4 was demonstrated by measuring the rates of 14C02 production from exogenously supplied 14C-labeled intermediates. The evolved
(10)
(81 *
OXOGLUTARAT - l-1°C FIG.
diates. theses
SUCCINATE
-1,4-w
CITRATE
-1,5
- ‘4c
I r!f!m MALATE -t]J4c
1. Effect of pH on ‘4CO2 production from labeled KC intermeCompounds were at 20 mM concentration. Numbers in parenare numbers of experiments in each case. * P < 0.05.
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E694
CHACIN
1. Effect of theophylline
TABLE
oxidation
and on respiration
ET
AL.
3. Effect of KC intermediates on QH+ of theuphylline-stimulated and nonstimulated
on substrate at pH 7,4
TABLE
gastric mucosa at pH 5.0 Labeled
Substrates
No, of Expts
Treatment
Qol
“C02,
QH+,
nmoU(2
[I- 14C]oxoglutarate
0
6
Theophylline
6
h-mg) dry wt
Pretreatment
2.38 t0.28 2.44 &0.26
86.48 kg.96 137.55 t11.30*
5.18 zkl.16 11.54 *1.44*
49.38 + 13.01 112.01 t 16.04*
None
Addition
0 Citrate
[U-14C]glucose
0
3
Theophylline
3
Values are means -+ SE. Oxoglutarate I mM, and theophylline at 5 mM.
was used at 2 mM, * P < 0.05.
2. Effect ofpH on mucosal
TABLE
to succinate
glucose
at
Theophylline Addition
PH
7.4 (17 mM TES)
5.0 (17 mM PO4)
0
16
Succinate
9
Oxoglutarate
6
0
26
Succinate
14
Oxoglutarate
10
02, @(h-mg)
Succinate
6
Malate
6
0
Dry Wt
Before (6090 min)
After ( 12% 150 min)
Change’
1.53 &0.05 1.39 kO.06 1.50 kO.08
1.54 kO.05 1.50 kO.05 1.45 kO.04
0.6 k2.4 7.9 *1.4* -3.3 k3.3
1.49 kO.12 1.46 kO.12 I.26 kO.13
1.44 to. 10 1.72 *o. 12 1.44 kO.11
-3.4 k2.1 17.8 ~2.7 * 15*0 *5.5*
Citrate
’
10 8
and oxoglutarate No. of Expts
7
Oxoglutarate
Fumarate
Qo, responses
No. of Expts
Oxoglutarate Succinate Fumarate Malate
6
pq/km%
Before (90120 min)
After (GO180 min)
Lll k0.24 1.36 k0.28 1.15 kO.19 2.11 k0.27 1.11 to.08 0.83 kO.20
1.06 k0.28 1.64 to.32 1.34 kO.25 2.59 kO.36 1.15
2.48 kO.17 2.91 +o. 10 1.96 -to.43 2.50 k0.38 3*70 k0.48 4.29 kO.28
2.36 kO.14 3.64 kO.20 2.61
kO.12 0.77 kO.14
to.36 3.32 k0.53 4.32 kO.59
5.47 to.32
Values are means k SE. Theophylline (10 mM) was added 0 and the Krebs cycle intermediates (10 mM) at 120 min.
0.05.
Change,
%
-8.7 k5.6 23.7 &6.9* 19.6 &4.2* 21.7 &7,2" 2.8 k5.8 -7.0 kf1.4 -4.2 u.7 25J *5.3* 38.7 *11.9* 34.9 k6.1” 16.8 t4.5* 28.0 t6.1* at tine
“PC
significant correlation coefficient (r = 0.89, P < 0.01). These results strongly suggest, but do not prove, that KC intermediates stimulate QH+ and &o, via their metabolism. Effect of inhibitors of Krebs cycle. Malonate, a known inhibitor of succinate oxidation in other tissues, when Table 3. In five experiments at pH 5, 10 mM citrate added at pH 7.4 significantly reduced the Qo, of both increased QH+ of histamine-stimulated mucosa by 30%. nontreated and theophylline-treated gastric mucosa (Fig, In six experiments, 10 mM aspartate, a dicarboxylic 4). In six experiments, at pH 5, malonate also decreased amino acid, had no effect at pH 5. the 14C02that evolved from [1,4-14C]succinate from 7.6 The degree of QH+ stimulation by different KC intert 0.8 pmo1/(2 ho g) wet wt in the control mucosal slices mediates at pH 5.0 in the theophylline-treated gastric to 4.3 t 1.2 in the experimental mucosal slices, an inhimucosa was related to the pKa values and the proportion bition of 45 t 11% ( P < 0.01 by paired t test). of the less charged molecules of these carboxylic acids, A These results indicate that malonate also inhibits sucgreater degree of stimulation was associated with a higher cinate oxidation by the toad gastric mucosa and led us to proportion of the unionized and singly charged molecules investigate the effects of malonate on QH+, The results of the acids. This result is illustrated in Fig. 2. A greater are presented in Table 4. At pH 7.4, malonate (10 mM) degree of QH+ stimulation was also associated with a decreased QH+in theophylline-stimulated mucosa by 10% higher rate of oxidation of the substrates, suggesting that (P c 0.05), compared with that in the untreated mucosa; they must be metabolized to exert their stimulating ef- at pH 5.0, the decreases were 25% in the theophyllinefects. To further test this possibility we examined the stimulated and 27% in the nonstimulated mucosa. The relationship between oxoglutarate oxidation and oxoglu- inhibitory effect of malonate was reversed by washing tarate-stimulated respiration at pH 5.0 in the theophylout the nutrient-malonate solution or by adding 10 mM line-treated gastric mucosa, It was reasonably assumed succinate at pH 5 (Fig. 5). that Qo, was directly related to the stimulation of QH+, Arsenite (NaAsOz), another inhibitor of the Krebs In these experiments Qo, and 14C02 production from cycle (19), inhibited Qo, of toad gastric mucosa more labeled oxoglutarate were measured simultaneously in than malonate. The response of Qo, to arsenite concenthe same preparation, and respiration was stimulated tration is shown in Fig. 6; inhibition increased with with different doses of oxoglutarate. The relationship concentration. A similar effect of arsenite on Qo2 by between AQo, and A14C02 is shown in Fig. 3. The two theophylline-stimulated and nonstimulated mucosa is ilparameters showed a positive correlation with a highly lustrated in Fig. 7. Values are means k SE. Compounds (10 mM) were added at 90 min. The rates in the second 30-min period after the addition were compared to those in the 30-min period prior to the addition and expressed as percent of change. * (P < 0*05).
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KREBS
CYCLE
AND
ACID
E695
SECRETION
I A
FIG. 2. Relationship between degree of QH+ stimulation by several KC intermediates and proportion of unionized acid ( C), and of singly (B) and doubly plus trebly (A) charged anions in theophylline-stimulated gastric mucosa at pH 5.0.
20
40
60
60
CONCENTRATION
Y = 20.48X 140 r
q
0.89
100
0
2
OF CITRIC
ACIDS
6
4
8
12
10
14
( % OF 10 mM >
t14.19 P ~0.01
120
100
1.2L 1 0 30
1 60
1 90
1 120
1 150
I 180
1 210
MIN
of malonate on Qo, of nontreated and theophyllinetreated gastric mucosae in TES at pH 7.4. Theophylhne (10 mM) was added to (n-a) and (A -----A) of 4 mucosal aliquots at tine 0, At 90 min malonate (10 mM) was added to a theophylhne-treated (A-----A) and a nontreated (o----- O) mucosa; buffer was added at this time to the mucosae. Values are means, and number other 2 (n-n) and (e) of experiments are given in parentheses. * P < 0.05, FIG.
/ a
1
1
I
1
I
1
1
2
3
4
5
6
A CO2
3. Relationship between “C02 production from labeled oxoglutarate and oxoglutarate-induced increase in respiration in theophylline (5 mM)-treat e d gastric mucosa at pH 5.0. Increments in respiration and ‘4C02 production are relative to control tissues exposed to 0.5 mM oxoglutarate. Values were obtained by varying oxoglutarate concentration over range of l-10 mM. Units are nmol/(Z h’mg) dry wt, FIG.
Furthermore, in eight experiments, arsenite (2 mM) reduced the 14C02that evolved from labeled oxoglutarate from 5.2 * 0.9 pmol/(2 h g) wet wt in the control slices l
4. Effects
to 2.4 t 0,4 in the experimental slices, a decrease of 47% t- 9 ( P c 0.005 by paired t test). Arsenite also inhibited QH+ of stimulated mucosa (Fig. 8). The QH+was stimulated to maximal rates by addition of 10 mM theophylline plus 10 mM butyrate, and arsenite was added at the peak of the response; the dose of arsenite that produced about 50% inhibition in the second 30-min period after addition was 2 mM. In one experiment, lb mM arsenite inhibited QH+ by 94%. Arsenite also inhibited the QH+ of both theophylline- and histamine-stimulated gastric mucosa in the absence of exogenous substrate. The time-course effect of 0.5 mM arsenite on QH+ of theophylline plus butyrate-stimulated mucosa is illustrated in Fig. 9. In the presence of arsenite, QH+ could not be restored to pretreatment values by the addition of the following compounds: 10 mM oxoglutarate at pH 5.0 (2 experi-
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E696
CHACfN
ments), lipoate 1 and 10 mM (4 experiments), 10 mM succinate at pH 5.0 (2 experiments), 10 mM propionate (2 experiments), 10 mM pyruvate with and without I mM lipoate (4 experiments), 10 mM malate at pH 5.0 (2 experiments), and 5 mM adenosine triphosphate at pH 5.0 (2 experiments), However, the inhibition was reversed by removing arsenite from the nutrient side by washing 2 or 3 times and replacing with fresh nutrient solution without arsenite. Because the inhibitory effect of arsenite on KC has been attributed to blocking the SH groups of the lipoyl moieties in the pyruvate and oxoglutarate dehydrogenase enzyme complexes (19), the effect of adTABLE
different .--
4. Effects of malonate pH levels
2.0 r
Addition
Theophylhne
No. of Expts
Theophylline
2.27 rts0.15 2.13 to.18
2.17 kO.16 kO.14
-15.0 &2.7*
1.11 kO.24
1.06 kO.28 0,82
-8.7 k5.6 -35.7
5
1*30
8
Malonate
7
-4.8
t1.9
1.79
to.18
to. 13
2.48 kO.17 2.52 kO.45
2.36 kO.14 1.97 k0.46
Values are means t SE. Gastric mucosa was bathed in solution, pH 7.4, or in phosphate nutrient solution, pH were pretreated with 10 mM theophylline; malonate added at 120 min. * The difference between control QH+ is statistically significant ( P < 0.05).
I
Change, %
After ( 180240 min)
7
0
h)
Before (90120 min)
12
0
None
QH+, pq/(cm’-
10
0
Malonate 5.0
dition of lipoate on the inhibitory effect of arsenite was examined. The results are shown in Fig. 10. In these experiments, QH+ was stimulated by theophylline plus butyrate and 2 mM arsenite was added at the peak of QH+ (in general, 60 min after addition of the stimulants). When the inhibitory effect was very evident, in general between 50 and 60 min after addition of the inhibitor, arsenite was washed out and replaced with fresh nutrient solution containing the stimulants; 1 mM lipoate was added to one mucosa immediately after washing, and nothing was added to the other one, which served as control. In the presence of lipoate, the restoration of
on QH+ at
Malonate
5.0
AL.
ARSENITE Pretreatment
7.4
ET
k6.2 * -4,2 kI.7 -29.6 t7.8"
TES nutrient 5.0. Mucosae (10 mM) was and malonate
0.5 30
1 60
1 90
1 120
1 150
I 180
I 210
MN
6. Typical experiment showing effect of various concentrations of arsenite (NaAs&) on Qo,. At 90 min, the indicated concentrations of arsenite were added to respective mucosal aliquots. Each point is mean of 2 dunlicate exseriments. Suspending medium was TES buffer, pH FIG,
a
7.4.
THEOPHYLLINE 10 mM MALONATE 10 mM i SUCCINATE
*
10 mM
FIG. 5. Effect of succinate on malonate inhibition of the QH+ by theophylline-stimulated gastric mucosa. The buffer was 17 mM phosphate, pH 5.0.
2
3
PERIODS
4
5
6
7
OF 20 MINUTES
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KRFBS
CYCLE
AND
ACID
E697
SECRETION -2323.8
“12 -39
(141
T I
T
7+90/o*
(6)
2.4 -9.422.5
T: & 2.2 + F + 2
ARSENITE
'10
(6)
\
i
\
T
T
\ \
2.0
1,8
3 1.6 1.4
1.2
I ‘30 0’ /’
60 I
90
120
L
1
150 1
180I
MIN 7. Effects of arsenite on Qu., of nontreated and theophyllinetreated gastric mucosae. At time 0, theophylline (10 mM) was added to (c- - -o) and (n ) of 4 mucosal aliquots. At 90 min, 0.5 mM arsenite was added to a treated (o- - a) and nontreated (n- - -a) mucosae, and TES buffer, pH 7.4, was added to other two, (w---+ ) and (A-A). Each point is mean of 6 or more experiments. * P < 0.05. FIG.
QH+ was much more rapid and more complete than in the absence of lipoate. This effect of lipoate seemed to be specific because two other SH donor reagents, Lcysteine and reduced glutathione at 2 mM, did not significantly affect the time-course of the restoration of QH+ (Fig. 10). DISCUSSION
In contrast to earlier findings (4, 5, 7), the present experiments demonstrate that several KC intermediates are efficiently oxidized by the toad gastric mucosa and can stimulate QH+under appropriate conditions of nutrient solution. Stimulation of QH+ on serosal addition of KC intermediates was observed when phosphate buffer of pH 5.0 was the nutrient solution, but not with a nutrient solution of pH 7.4. The high pH of the bathing solutions used by Davenport and Chavrh (4, 5, 7) probably accounts for the observed lack of response of mouse gastric mucosa to KC intermediates. Cell membranes are, in general, more permeable to uncharged than to charged molecules (9). Because the KC intermediates are tri- and dicarboxylic acids with pK, between 3 and 5.61 at 25°C (30), the charged (unprotonated) molecules are the predominant forms of the acids at pH 7.4. At pH 5.0, the concentrations in the nutrient solution of the less charged carboxylic acids are significantly higher than those at pH 7.4. It is therefore likely that the stimulation of QH+ observed on addition of KC intermediates at pH 5.0 is simply attributable to a higher permeability rate of acidic substrates into the oxyntic cells at this pH. A similar mechanism has been postulated by Taylor, Hess, and Maffly (29) to explain the stimula-
0
0
0.5
0
10
2.0
ARSENIT E CONC. Inb 1 8. Effect of arsenite on QH+- The concentration of arsenite is shown at the base of each set of experiments. Experiments were carried out with paired mucosae in TES buffer, PI-I 7.4. Values over 2nd bar in each group is calculated percent of change of arsenite-treated with respect to the nontreated mucosae, Numbers in parentheses are the numbers of experiments in each case. * P < 0.05. FIG.
2.8 2.4 -
0.4 ++ LL 0 30
11 60
I 90
I 150
I 120
LL 180
11 210
11 240
MN
9. Time-course response of QH+ to arsenite. TES (17 mM), pH 7.4, was nutrient solution. Theophylline plus butyrate (10 mM each) and arsenite (0.5 mM) were added at time shown. FIG.
tion of sodium transport (short-circuit current) produced by several KC intermediates in toad bladder bathed in phosphate Ringer’s solution at pH 6.4 but not at pH levels above 7.4 (12, 27). Our experiments showed that several exogenously supplied KC intermediates stimulate Qo,, 14C02evolution, and QH+ at pH 5 in contrast to a small, or lack of, effect at pH 7.4, thereby supporting the increased permeability mechanism. Moreover, the oxidation rates of exogenous KC intermediates and the
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E698
CHACIN
THEOPHYLLINE 10 mM t ARSENITE 2mM WASHOUT
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
10. Effects
AL.
10 mM
+ ADDITIONS
-1
PERIODS of washing plus lipoate and washing or reduced glutathione on arsenite inhibition of QH+ plus butyrate-stimulated mucosa. Buffer was 17 mM QH+ is expressed as % of pretreatment value immediately of arsenite (control value, 100%). (v) At arrow, FIG.
BUTYRATE
ET
0
1
OF
plus L-cysteine by theophylline TES, pH 7.4. before addition lipoate (1 mM)
degree of QH+ stimulation at pH 5.0 in the theophyllinestimulated gastric mucosa were dependent on the p& values and on the proportion or concentration of the unionized and singly charged molecules of the acids (Figs. 1 and Z), suggesting that these are the molecular forms that predominantly penetrate into the cells, The above correlation was not found using the QH+ values of the nonstimulated gastric mucosa at pH 5.0. Secretagogue availability is rate limiting for QH+ and Qo, in this preparation and might account for these unexpected results. The fact that the rate of oxidation of oxoglutarate and other KC intermediates at pH 7.4 was not stimulated by increasing respiration with theophylline (Table 1) is also consistent with the point of view that permeability is rate limiting at pH 7.4 for KC intermediates. As expected, the rate of glucose oxidation, which is assumed not to be limited by permeability, was significantly stimulated by increasing respiration with theophylline at pH 7.4. It seems unlikely that the stimulation of QH+ by KC intermediates at pH 5.0 was due to diffusion of the protonated forms of the acids from the nutrient side to the secretory side and releasing H+ to the secretory solution because there was no pH gradient. The following experiment ruled out significant diffusion: when 1 PCi of labeled citrate (10 mM) was added on the nutrient side (58, 407 cpm/ml of nutrient solution), the radioactivity of the secretory solution (I ml) was 18, 21, 36, 26, 44, and 56 cpm at 10, 20, 30, 40, 50, and 60 min after addition of citrate, respectively; the background was 22 cpm. In this experiment, Q H+ was stimulated by 30%, and the peak response was observed at 30 min after addition of citrate. It is also possible that the low pH affects other variables that might influence the rate of substrate oxidation, the coupling of substrate oxidation to transport, or the transport process itself.
2
3
4
5
6
7
0
9
10
II
12
13
14
15
10 MINUTES was added immediately after washout (6 expts); (X-X) at arrow, 2 mM L-cysteine (3 expts) or 2 mM reduced glutathione (3 expts) was added immediately after washout; ( G---O) at arrow, washout only (6 experiments). Bars represent I SE. * P < 0.05, compared to corresponding values of X-X and m.
Another possible explanation is that KC intermediates at low pH could stimulate QH+ by neutralizing bases in the oxyntic cells or by increasing the rate of recycling of endogenous COz at the nutrient barrier membrane in a situation in which CO2 concentration is a limiting factor for H+ secretion. This mechanism was first postulated by Sanders, Hayne, and Rehm (24) and supported later by Kidder and Montgomery (18) and Silen, Machen, and Forte (28). However, the following results argue against this possibility. I) Malonate, a dicarboxylic acid with p&s of 2.83 and 5.69 at 25°C (30), inhibited Qoz, 14C02 production, and Q H+ at low pH; this inhibition is reasonably explained by the known inhibitory effect of malonate on succinate oxidation and KC activity. 2) Aspartic acid, a dicarboxylic amino acid whose p&s for the carboxylic groups are 2.10 and 3.86 at 25*C (30), did not affect QW at pH 5; this result is in accordance with Davenport and Jensen (8) and Alonso et al. (1) who reported that amino acids are not utilized by the H’ transport process. We therefore favor the hypothesis that the stimulation of Qn+ by KC intermediates at low pH depends on their oxidation in the oxyntic cells. The highly significant correlation between oxoglutarate oxidation and oxoglutarate-dependent respiration (Fig. 3) is consistent with, but does not prove, this point of view. Increased KC activity can also stimulate the oxidation of other substrates (fatty acids and/or carbohydrates) that can be mobilized during the secretory process (15, 16). If the amount of 14C02liberated is used to calculate the amount of oxygen required to oxidize oxoglutarate to COZ, assuming complete oxidation in the KC, the slope of the regression line correlating oxoglutarate oxidation and oxoglutarate-dependent respiration is converted from a value of 20 to a value of 5. This value indicates that oxoglutarate oxidation accounts for only 20% of the increase in respi-
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KREBS
CYCLE
AND
ACID
E699
SECRETION
ration and leaves room for the possibility that oxoglutarate stimulates the oxidation of other endogenous substrates. This is not surprising because the catalytic effect of KC intermediates on respiration in animal tissues is well known. The increased substrate oxidation would stimulate QH+ by providing reducing equivalents and/or high energy compounds (such as adenosine triphosphate) to the H+ transport mechanism. The present studies also showed that inhibitors of KC, such as malonate and arsenite, can reduce QH+. The inhibitory effect of malonate was more evident at low pH. This result can be due to a higher rate of penetration of the compound into the cells at this pH. The lack of effect of malonate reported by Sachs et al. (22) probably resulted from insufficient penetration of the compound at the relatively high pH of the nutrient solution. However, other factors including species difference cannot be ruled out. The inhibitory effects of both malonate and arsenite on QH+ were reversed by washing, and the reversal was accelerated by addition of succinate and lipoate, respectively. The accelerated reversal of the inhibitory effect or arsenite by lipoate, but not by L-cysteine and reduced glutathione, supports the point of view that arsenite acts by blocking the -SH groups of the lipoyl moieties in the oxoglutarateand pyruvate-dehydrogenase enzyme complexes (19). Although malonate and arsenite could affect other sites involved in the H+ transport process, it seems that their inhibitory effects on QH+ are reasonably explained by inhibition of the KC because of the inhibition of the succinic- and the pyruvic- and oxoglutaric-dehydrogenase systems, respectively. The results with KC intermediates and inhibitors together suggest that KC activity plays a central role in
the regulation of oxidative metabolism and acid secretion by the toad gastric mucosa. Davenport and Chavrb (7) suggested that in the mouse stomach the KC supplies part of the en ,ergy of the H+ transport because flu&o acetate inhibits acid secretion. The situation seems to be different in rat stomach in vitro, because Sernka and Harris (26) found that in this animal QH+ is supported by substrates that can be oxidized through the pentose phosphate shunt but not by substrates that require the KC for their oxidation. However, low permeability of the latter substrates was not ruled out. The fact that all the KC intermediates tested at pH 5.0 stimulated QH + and that inhibition of QH+ was obtained by using inhibitors that act at two different steps of the KC suggest that QH+ in the toad gastric mucosa is not linked to any specific step of the KC. The entire cycle seemsto be necessary, In dog gastric mucosa, Sarau et al. (25) and Sachs et al. (21, 23) found an increase in the levels of the citric acid cycle intermediates during stimulation with histamine, indicating increased KC activity. This result could be a general metabolic response to secretagogues and could result from mobilization of substrates (15, 16, 21, 23, 25) and/or activation of some of the KC enzymes (21, 23, 25). This investigation was supported in part by CONDES of L*U.Z. and by CONICIT Grant Sl-0455, Venezuela, and National Institutes of Health Grant AM 12606. The authors acknowledge the efforts and help of Ms. Annabell Castillo, Mrs. Zoraida de Jaime, Mr. Jesus Anez, and Mr. Ernest0 Ssnchez in this work. Present address of J. Chacin and G. Martinez: Unidad de Investigaciones Biolbgicas, Facultad de Medicina, Universidad de1 Zulia, Maracaibo, Venezuela. Received
20 July
1978; accepted
in final
form
2 January
1979.
REFERENCES 1. ALONSO, D., K. NIGON, I. DORR, AND J. B. HARRIS. Energy sources for gastric secretion: substrates. Am, J. PhysioZ. 212: 992-K@O, 1967. 2. CHAC~N, J., 0. H. PARK, J. B. HARRIS, AND D. ALONSO. Role of oxalacetate in the lipoate effect on frog gastric mucosa. Am. J. Physiol. 231: 209-215, 1976. 3, CIIAC~N, J., R. RINC~N, D, INCIARTE, AND A. CA~~IZALES. Krebs cycle and H+ secretion in amphibian gastric mucosa. Biophys. J. 16: 129a, 1976. 4. DAVENPORT, H. W., AND V. 3. CHAVR&. Conditions affecting acid secretion by mouse stomachs in vitro. Gastroenterology 15: 467480, 1950. 5, DAVENPORT, H. W., AND V. J. CHAVR~. Acid secretion and acetoacetate utilization in mouse stomachs in vitro. Federation Proc. 10: 33, 1951. 6* DAVENPORT, H. W,, AND V. J. CHAVRB. Relation between substrate disappearance and acid secretion in mouse stomach in vitro. Am. J. Physiol. 166: 456-461, 1951. 7, DAVENPORT, H. W., AND V. J. CHAVRI?. Acid secretion and oxygen consumption by mouse stomachs in vitro, Am, J, PhysioZ, 174: 203208, 1953. 8. DAVENPORT, H. W., AND V, JENSEN. Observations on the secretion of acid by the mouse stomach in vitro, Gastroenterology 12: 630636, 1949. 9. DAVSON, H., AND J. F. DANIELLI. The Permeability of NaturaL Membranes. Cambridge: Cambridge Univ. Press, 1952, p. 186, 10. DURBIN, R, P. Utilization of high-energy phosphate compounds by stomach. J. Gen. Physiol. 51: 233+239s, 1968, 11. DURBIN, R. P., AND F. MICHELANGELI. High-energy phosphate compounds in frog gastric mucosa. In: Gastric Secretion, edited by G. Sachs, E. Heinz, and K. J. Ullrich. New York: Academic, 1972, p. 307-318.
12. FIMOGNARI, G. M., G. A. PORTER, AND 1, S. EDELMAN. The role of the tricarboxylic acid cycle in the action of aldosterone on sodium transport, Biochim. Biophys, Acta. 135: 89, 1967. 13, FORTE, J. G., P. H. ADAMS, AND R. E. DAVIES. Acid secretion and phosphate metabolism in bullfrog gastric mucosa. Biochim. Biophys. Acta. 104: 25-38, 1965. 14. HARRIS, J. B,, D. ALONSO, 0. IS. PARK, D. CORNFIELD, AND cl, CHAC~N. Lipoate effect on carbohydrate and lipid metabolism and gastric H’ secretion. Am. J. PhysioZ. 228: 964-971, 1975. 15. HERSEY, S. J. Metabolic changes associated with gastric stimulation. Gastroenterology 73: 914-919, 1977. 16. HIGH, W. L., AND S. J, HERSEY. Mechanism of theophylline stimulation of acid secretion by frog gastric mucosa, Am. J. Physiol. 226: 1408-1412, 1974. 17. KASBEKAR, D. K. Gastric H* secretion in presence of substrates: absolute dependence on secretagogues. Am. J. Physiol, 231: 522528, 1976. 18. KIDDER, G, W., AND C. W. MONTGOMERY. CO2 diffusion into frog gastric mucosa as rate-limiting factor in acid secretion. Am. J. Physiol. 227: 300-304, 1974. 19. LEHNINGER, A. L. Biochemistry. New York: Worth, 1970, p. 346. 20. REHM, W. S. The metabolic state and the response of the potential of frog gastric mucosa to changes in external ion concentrations. J. Gen. Physiol, 51: 25Os-26Os, 1968. 21. SACHS, G., H. C, CHANC, E. RABON, R. SHACKMAN, H. M, SARAU, AND G. SACCOMANI. Metabolic and membrane aspects of gastric H+ transport. Gastroenterology 73: 931-940, 1977. 22. SACHS, G., R. H. COLLIER, R. L. SHOEMAKER, AND B. I. HIRSCHOWITZ. The energy source for gastric H* secretion. Biochim. Biophys. Acta. 162: 210-219, 1968. 23. SACHS, CL, E. RABON, G. SACCOMANI, AND H. W. SARAU. Redox and ATP in acid secretion. Ann. NY Acad. Sci. 264: 456-475, 1975.
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E700 SANDERS, S. S., V. 8, HAYNE, JR., AND W. S, REHM. Normal H’ rates in frog stomach in absence of exogenous CO2 and a note on pH stat method. Am. J. PhysioZ. 225: 1311-1321, 1973. 25. SARAU, H. M., J. J. FOLEY, G. MOONSAMMY, AND G. SACHS. Metabolism of dog gastric mucosa. Levels of glycolytic, citric acid cycle and other intermediates. J. Biol. Chem, 252: 8572-8581, 1977. 26. SERNKA, T. J., AND J, B. HARRIS. Yentose phosphate shunt and gastric acid secretion in the rat. Am. J. PhysioZ. 222: 25-32, 1972. 27. SHARP, G. W. G., AND A. LEAF. The central role of pyruvate in the stimulation of sodium transport by aldosterone. Proc. IVat. Acad. 24.
CHACIN
ET
AL.
Sci. USA 52: 1114, 1964. 28. SILEN, W., T. E. MACHEN, AND J. G. FORTE. Acid-base balance in amphibian gastric mucosa, Am. J. Physiol. 229: 721-730, 1975. 29. TAYLOR, A., J. J. HESS, AND R. H. MAFFLY. On the effects of tricarboxylic acid cycle intermediates on sodium transport, by the toad bladder. J. Membrane Biol. 15: 319-329, 1974. 30. WEAST, R, W., S. M. SELBY, AND C. D. HODGMAN. Handbook of Chemistry and Physics (46th ed,)* Cleveland: Chemical Rubber, 1965, p. D-78,
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