Redox changes in the respiratory chain related to acid secretion by the intact gastric mucosa

Vol. 36, No. 2, 1969

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REDOX CHANGES IN THE RESPIRATORYCHAIN RELATED TO ACID SECRETION BY THE INTACT GASTRIC MUCOSA S. J. Hersey and F. F. Jbbsis Dept. of Physiology, Duke Univ., Durham, N. C.

27706

Received June 2, 1969 SUMMARY Spectrophotometric studies were carried out on the intact isolated gastric mucosa of bullfrog. The anoxic vs oxygenated difference spectrum revealed absorbtion maxima typical of respiratory chain components with some noteworthy special features. Large reductions of all the components occured upon stimulation of acid secretion by histamine while inhibition by thiocyanate was accompanied by oxidation of all components. The reductions accompanying histamine addition do not appear to be due to hypoxia.

Active secretion of hydrochloric upon oxidative

acid by the gastric mucosa is dependent

metabolism (1,2,3) and in turn exerts a controlling

respiration in this tissue (4,s). the concentration

effect on

This control is thought to arise from changes in

of ADP which supposedly depends upon the hydrolysis

during the secretory process (3,6).

According to this theory the mitochondria

within the tissue would be close to the resting metabolic state,

state 4 (7), when

hydrogen ion secretion is inhibited and approach state 3, the actively state, upon stimulation the steady-state

of secretion.

oxidation-reduction

(SCN).

metabolizing

To test this hypothesis we have examined levels of respiratory

intact gastric mucosa during stimulation by thiocyanate

of ATP

chain components in the

by histamine and inhibition

of secretion

The results have been such that we find it impossible to

describe them in terms of transitions

between states 4 and 3.

This work was supported by grants from U.S.P.H.S, Smith, Kline and French. 243

GM00929,

and Tom

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METHODS The stomachs of Bullfrogs (Rana catesbiana) were perfused with Ringer The mucosa was then separated from the

solution via the descending aorta. -

muscularis by sharp dissection and mounted as a diaphragm between two lucite half chambers with clear optical windows which served as cuvette system.

The

chambers served to separate two adjacent pieces of the same mucosa as well as to separate the mucosal and serosal surfaces.

The tissues were bathed in a

bicarbonate buffered amphibian Ringer solution and aereated with 95% 02 5% C02. For the measurement of hydrogen ion secretion the mucosal solution was replaced with an unbuffered solution having identical Acid secretion was measured by titrating a fixed end -point. pre-incubated

salt content and aereated with 100% 02.

samples of the mucosal bathing media to

In order to reduce their spontaneous secretion tissues were

for 12 to 18 hours in the cuvette system at 12’C (8).

solutions both during the pre-incubation with 10 mM pyuvate. wavelength-scanning,

Difference differential

Serosal

and during experiments were fortified

spectra were obtained using a “split-beam”, spectrophotometer

(9).

RESULTS Fig. 1 shows an anoxic difference obtained at 12’C.

spectrum of the intact gastric mucosa

The absorption maxima are identified

on the figure,

spectrum appears to be rather typical

of the respiratory

in mitochondria and in other tissues,

with two exceptions.

355 mu has not been completely identified.

in the 350 to 360 murange.

chain components found

In all probability

component of NADH (340 mu peak) and an additional The other peculiarity

This

First,

the peak at

it contains a

component with a maximum

is an apparent shift of the

cytochrome b-u band to 560 m!, from the usual peak at 564 mu . The differences noted go unexplained for the present. 244

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Figure 1. Anoxic difference spectrum of bullfrog gastric mucosa . Sample tissue was made anoxic by bubbing chambers with N2 , sealing the chamber with mineral oil and allowing the tissue to consume the residual 02 for 30 minutes. Reference tissue (baseline) was oxygenated. Temperature 120C. Note separate optical density scales for right and left parts of curve.

Further experiments were carried out to determine what changes, if any, occurred upon stimulating

or inhibiting

acid secretion.

These experiments were

performed at 18OC. Fig. 2A shows the change in optical density produced by the addition of histamine (lO+M) secreting base line.

as compared to the non-stimulated

but spontaneously

It can be clearly seen that the stimulation of acid secretion

is accompanied by large reductions of all of the respiratory

chain components.

Fig. 2B shows the effect of a subsequent addition of SCN (10 mM) to the same tissue. Since Fig. 2A and 2B represents experiments on the identical

sample, the magnitude

of the oxidation-reduction

Fig. 28 shows that

changes are directly

comparable.

SCN not only reverses the reductions produced by histamine but causes a further oxidation. beyond the level which is found in the spontaneously secreting condition.

It should be noted that in these experiments the peak absorption wave245

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AND BlOPh’YSlCAL RESEARCH COMMUNICATIONS

c7 n

A

niti*mlne c(*c,)a SW 1

P

e (*a.)

a

604 b ba

*

-a02

o.a-

-0.04

0.02o.o?L

-0.06

F

B

SCN

P :

\

\? -0.02

/

\ \

‘1

:

::

\

.

.

1

340 NADN T

I / !

‘\-

/

:

441 o,~*a)r

r l400

T

I

1

, 460

-

\

/’

‘1,

\

.I

1

-0.00

/ ‘\

/ ‘41

-0.04

t

’

IT . 1 Iwo Cl

‘J

I o.a-

\-I t 602 e(*eJ

ba

aOt0

t bso ebc,)a

0.03-

I

I

,

I

I

I

I

600

A (mp)

Figure 2. Spectral changes induced by histamine and thiocyanate. Curve A, optical density changes at 40 minutes after addition of histamine (10m4M) as compared with spontaneously secreting condition (baseline). Curve B, optical density changes at 20 minutes after addition of SCN(lO-2M); for this spectrum the histamine stimulated condition (Curve A) was used as baseline. Temperature, 18OC.

length in the NADH region of the difference

spectra occurs at approximately

not 355 mu as seen in the anoxic spectra.

It should also be noted that the

cytochrome bo peak still occurs at 560 mP rather than the usual 564 mu . In the experiment depicted in Fig. 2 histamine stimulation produced 246

340 mu

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a 200% increase in hydrogen ion secretion as compared to the

spontaneously secreting condition.

SCN in turn reduced the rate essentially

to zero.

For 8 similar experiments the mean increase in hydrogen ion secretion produced by histamine was 240% and in each case SCN reduced the secretory rate to zero. all of these experiments oxidation-reduction

changes in the respiratory

components accompanied the changes in acid secretion. at 10°C, no significant

chain

In experiments carried out

changes in acid secretion resulted from the addition of

histamine or SCN, nor were there any changes in the oxidation-reduction respiratory

In

state of the

chain components.

The massive reduction of all members of the respiratory

chain upon stimulation

by histamine is similar to the effects expected for hypoxia or local anoxic. to test this possibility

two types of experiments were performed.

In order

In the first,

acid

secretion and O2 consumption were increased by raising the temperature from 14’C to 29’C.

The observed percentage of cytochromes a3, c and b in the oxidized

plotted in Fig. 3 as a function of the temperature.

00 1 14

I I8

I

22

form is

If hypoxia occurred a continuous

I

26

I

30

OC

Effect of temperature on steady-state oxidation level of cytochromes. Figure 3. Tissues were in the spontaneously secreting condition. 60 minutes equilibration period was allowed after each temperature change. Total cytochrome compliment was determined as the difference between the anoxic and oxygenated conditions at 1ooc. 247

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decline should be evident.

This is clearly not the case, from 14-23’C a relatively

stable level is maintained.

The further decreases at temperatures beyond 23’C

probably signal the first occurrence of hypoxia.

In the second type of experiment,

the stomachs were not cleared of blood and hemoglobin was used as an indicater of the PO2 in the tissue.

At 18OC the reduction of cyt.

a3 accompanied histamine

stimulation without a change of the spectrum from Hb02 to Hb. intracapillary

Thus the

O2 pressure must remain above 100 mm Hg, a level that should be

fully adequate to provide O2 to the cells by diffusion in identical

fashion to the

normal occurrence in vivo.

DISCUSSION The difference normal respiratory

spectra of the intact gastric mucosa reveal the presence of the

chain components with some notable,

special features.

The

unexpected peak at 355 mu is probably composed of two separate components, NADH, and an unidentified The unidentified

compound which absorbs in the, 350-360 mu region.

component does not appear to be directly

linked with the respiratory

chain in as much as it does not undergo oxidation-reduction of the tissue with histamine or SCN.

Only the normal NADH peak at 340 mu seems

to be affected by changes in the rate of acid secretion. might similarly approximately

be caused by an additional 550-560 mV.

oxidation-reduction

changes upon treatment

The shift in the cyt bo peak

peak with a wavelength maximum of

Such an additional

component, however,

changes upon treatment with histamine and SCN.

experiments indicate that this component is not cytochrome b5.

must undergo Preliminary

Further identification

of the component would be of considerable interest owing to its possible involvement in acid secretion. The oxidation-reduction

changes produced by histamine and SCN are very

diffe.rent born those that would be expected if the mitochondria within the gastric 248

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mucosa were undergoing simple transitions

between state 4 and state 3.

Stimulation

of acid secretion by histamine results in an increased oxygen consumption (4,5) suggesting that the tissue ought to approach state 3. NADH, flavoprotein

In this case an oxidation of

and cyt b should be observed easily and perhaps a slight

reduction of cyt c and cyt a3 could be measured (7). all of the respiratory

The results actually

show that

chain components attain a more reduced steady-state.

Furthermore, the reduction seen in cyt a3 is a very large amount, often involving 20% or greater change of the total.

Inhibition

a

of acid secretion might be expected to

produce a transition towards state 4 and this would involve a reduction of most of the respiratory

chain components.

opposite changes actually inhibition

The results with SCN show that essentially

the

occur : all the components become more oxidized.

Since

of acid secretion by SCN does not affect the rate of 02 uptake (4,10,11,12,13),

the effect appears to be one of “uncoupling” presence of SCN the steady-state

the two tissue functions.

In fact in the

do resemble those of maximally respiring

mitochondria much more closely than when the tissue is secreting acid. The very large oxidation-reduction

changes of cyt a3 are remarkable since in

mitochondria changes of at most a few percent are observed in any transitions the exception The possibility

of a transition

with

between state 5 (anoxia) and one of the other states.

that reductions seen upon stimulation with histamine result from a local

hypoxia produced by the increased oxygen consumption seems unlikely three observations.

First SCN re-oxidizes

in view of

cyt a3 while it is known that a concomitant

decrease in oxygen consumption does not occur, at least not within the time span of these experiments (4,10,11,12,13). increase continuously finally

with increases in the rate of O2 consumption (Fig. 3).

the lack of a disoxygenation

existence of a critically

Secondly the degree of reduction of cyt a3 does not

of hemoglobin in the capillaries

low oxygen tension in the tissue.

249

And

denies the

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In summary, the results do not appear to be explainable of mitochondria within the tissue reflecting concentrations. and oxidative

changes in phosphate acceptor

A more detailed theory as to the relationship metabolism must therefore be advanced.

involve a much more direct relationship

simply on the basis

between acid secretion

Such a new theory may well

between respiration and secretion than is

found in the adenosinephosphate cycling mechanism.

REFERENCES 1. Davenport, H. W., In “Metabolic Aspects of Transport Across Cell Membranes” ed. Q. R. Murphy. Madison University Wisconsin Press. p. 295 (1957). 2, Rehm, W. S. and W. H. Dennis, Ibid. p. 303 (1957). 3. Forte, J. G., P. H. Adams, and R. E. Davies, Biochem. Biophys. Acta la: 25 (1965) 4. Forte, J. G. and R. E. Davies, Amer. 1. Physiol. 22: 218 (1964).5. Villgeas, L. and R. P. Durbin, Biochem., Biophys., Acta 44: 612 (1960). Acta 105 : 472 (1965). 6. Kasbekar, D. K., and R. P. Durbin, Biochem. , Biophys . , -7. Chance, B. and G. R. Williams, 1. Biol. Chem. 217 : 409 (1955). 8. Kasbekar, D. K., -Proc. Sot. Exptl.%l--A,-* Med 125 * 263 (1967). 9. Jl)bsis, F. F., I. Gen. Physiol. 46: 905 (1963). 10. Davies, R. E. andC.Terner, Biochem. L 44: 377 (1949). 11. Alonso, D., R. Rynes and J. B. Harris, G I. Physiol. 208: 1183 (1965). 12. Bannister, W. H., -w Nature 203 : 978 (1964). 13. Moody, F. G., Amer. 1. Physiol. 2&: 127 (1968).

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