ACTA HISTOCHEM. CYTOCHEM. Vol. 17, No. 4, 1984
ENZYMATIC RESPONSE TO HCL INHIBITION IN
OXYNTIC CELLS OF FASTING RATS
F. J. MORENO, J. PINERO, R. COVENAS AND J. L. LOPEZ-CAMPOS
Depa men s o Cy ology and His ology, Facul y o Biology, Se ice o Elec on
Mic oscopy, Se illa Uni e si y, Se illa, Spain
Recei ed o publica ion Feb ua y 8, 1983 and in e ised o m June 14, 1984
Mo phological modi ica ions and cy ochemical changes in he acid phos-
pha ase on oxin ic cells o he gas ic mucosa o a s dep i ed o ood o
a ying leng hs o ime we e s udied. Wi h long as ing pe iods a lysosomal
quali a i e inc ease and mi ochond ial mo phological al e a ion a e caused.
This las ac would make an ATP dec ease and an al e a ion in he ATPase
ac ua ion, so p e en ing he in e change o H+ o K+ and hus blocking in a-
esicula HCl o ma ion.
O he di e en cell ypes which o m he gas ic mucosa, he oxyn ic cells a e
hose esponsible o he sec e ion o hyd ochlo ic acid. This cell ype, desc ibed
by Heidenhain in 1870 (7), has been s udied equen ly, on accoun o bo h i s
cy ological s uc u e (1, 9), i s physiology (3, 16) and i s cy ochemical cha ac-
e iza ion (2).
S udies ela ing o he localiza ion o ac i e si es o enzymes belonging o he
K+/ H+ ATPase complex ha e been pe o med on di e en issues a he cell le el.
E idence o he p esence o hese enzymes in he cell memb anes and in o he
o ganelles has pa ly es ablished he ela ion be ween s uc u e and unc ion and
he e is e e inc easing e idence o his.
The objec o ou s udy was o es ablish a unc ional compa ison o he ac i i y
o acid phospha ase, using 9-glyce ophospha e as subs a e, in gas ic mucosa
oxyn ic cells o a s subjec ed o as ing and o he s wi h no mal die s.
The e ha e been se e al s udies ela ed o ul as uc u al modi ica ion o
oxyn ic cells in di e en sec e o y s a es (4, 5, 15) and he physiological HCl se-
c e o y mechanism has been demons a ed wi h enzyma ic, cy ochemical and
biochemical echniques (17, 18).
MATERIAL AND METHODS
Sp ague dawley a s, bo n and aised in ou labo a o y, we e used. One lo
o animals was isola ed and dep i ed o ood o pe iods o 82 and 342 h ; gas ec o-
mies we e pe o med and he s omachs hen sec ioned, selec ing he undus egion.
This same zone was also ob ained om animals whose die had been no mal.
Small pieces o gas ic undus we e ixed in 2.5% glu a aldehyde in 0.1 M
cacodyla e bu e a pH 7.2 o one h . Thick ozen sec ions we e cu on a c yos a
379
380 MORENO ET AL.
p e iously cooled o -20°C, and incuba ed in a medium con aining 9,-glyce ophos-
pha e and lead ni a e in a 0.5 M is-malea e bu e a pH 5 o one h a 37°C (11).
We also incuba ed sec ions wi hou ~-glyce ophospha e.
A e washing in cacodyla e bu e sec ions we e pos - ixed in 1 % osmium
e oxide in dis illed wa e o 1 h a 4°C. They we e hen embedded in Spu 's
esin. O he agmen s o gas ic undus we e ixed and embedded using con-
en ional elec on mic oscopic echniques, o no mal his ological s udy.
RESULTS
Elec on mic oscopic enzyma ic s udies o he ac i i y o acid phospha ase
ca ied ou on oxyn ic cells in gas ic mucosa o animals dep i ed o ood o di e en
pe iods using i-glyce ophospha e as subs a e, ga e di e en esul s when compa ed
o his enzyme ac i i y in no mal mucosa a he oxyn ic cell le el.
Figu e 1 shows hin sec ion o no mal mucosa oxyn ic cell, in which he dis i-
bu ion o s uc u al elemen s in a pe iod o sec e ion can be obse ed. When
compa ed wi h mucosa oxyn ic cells unde as , we can see lysosomes p esen in he
FIG. 1. Oxyn ic cell agmen o no mal gas ic mucosa in sec e ion phase. Obse e he dila ed
lumen o in acellula canaliculi (head a ow), he mode a e ubulo- esicula sys em (a ow) and
mi ochond ia wi h conse ed c es s. X 30,000
OXYNTIC CELL, FASTING, PHOSPHATASE, HCL SECRETION 381
p oximi y o he in acellula canaliculi (Fig. 2), g ow h o he ubula esicula
sys em; obli e a ion and de o ma ion o he in acellula canaliculi and mi o-
chond ial al e a ions (Fig. 3).
No mal gas ic mucosa oxyn ic cells show acid phospha ase ac i i y in
lysosomes, which a e localized in he basal zones o he cell (Fig. 4) ; howe e , he
numbe o lysosomes inc eased quali a i ely wi h longe pe iods o as ing, h ough
he whole o he cy oplasm (Fig. 5) ; hus, he oxyn ic cell, unde hese condi ions o
as ing, shows an inc ease in lysosomal enzymes.
In no mal mucosal oxyn ic cells, he exis ence o a mode a e ubula esicula
sys em (1), which is inc eased when he animals a e dep i ed o ood, is obse ed.
Wi h espec o ul as uc u al modi ica ions o he in acellula canaliculi o
con ol animals, we obse ed ha he acid phospha ase echnique esul ed in lead
phospha e p ecipi a e on he memb anes which make up he mic o illi o hese
canaliculi (Fig. 6).
Compa ison o he di e en g oups o animals unde s udy showed a dec ease
in enzyme ac i i y in he mic o illi along wi h he s uc u al modi ica ions o he
canaliculi. The loss o phospha ase ac i i y on he canaliculi was pa allely p o-
Fin. 2. Inc ease o ubulo- esicula sys em (a ow) and obli e a ed in acellula channels o an
oxyn ic cell o gas ic mucosa in as . No e lysosomes nex o obli e a ed in acellula canaliculi
(head a ow). Mi ochond ia p esen al e ed c es . X 32,000
382 MORENO ET AL.
po ional o he inc ease o as ing ime. Thus, we obse ed an inc ease o lysosomal
phospha ase ac i i y.
DISCUSSION
I o and Scho ield (10) desc ibed how, in gas ic pa ie al cells, one o he mos
no able de ails in connec ion wi h sec e ion inhibi ion is he p esence o a g ea
numbe o in acellula esicles and he sca ci y o su ace mic o illi in he canaliculi.
Howe e , he p esence o lysosome- ype elemen s and mul i esicula bodies has been
ela ed o inhibi o y ac ion o sec e o y ac i i y in oxyn ic cells (6, 14).
The de elopmen o he Golgi complex in oxyn ic cells has been ela ed o HC1
sec e ion inhibi ion (12) and o he de elopmen o new lysosomes in gas ic mucosa
o animals subjec ed o he ac ion o he d ug.
FIG. 4. Oxyn ic cell o no mal gas ic mucosa acid phospha ase eac ion in basal lysosomes (L).
Basal memb ane (a ow). x 8,000
FIG. 5. In ense acid phospha ase eac ion in lysosomal bodies o oxyn ic cells o gas ic mucosa unde
as . Unspeci ic p ecipi a e on mi ochond ia. Nucleus (N). x 15,000
Fic. 3. De ail o ubulo- esicula de elopmen and obli e a ion o oxyn ic cell canaliculi o gas ic
mucosa in as ing pe iod. 35,000
OXYNTIC CELL, FASTING, PHOSPHATASE, HCL SECRETION 383

384 MORENO ET AL.
We ha e no obse ed any conside able inc ease in he de elopmen o he
Golgi complex in as ing animals and o his eason we beli e ha he o igin o he
lysosomes and he e o e he syn hesis o lysosomal hyd olase, akes place in he
ibosomes associa ed wi h he memb anous cis e na o he endoplasma ic e iculum,
con inuing owa ds he GERL which p oduces (13), by genma ion, esicles which
o m lysosomes.
In an exhaus i e s udy on s uc u al and unc ional changes du ing he HC1
sec e o y cycle in oxyn ic cells (15), he s a ed mo phological changes a e co obo-
a ed, and a he same ime a unc ional in e p e a ion is gi en o he HCI sec e o y
mechanism, which akes place in he ubula esicula sys em; H+ o igina ing in
ATP hyd olisis a e p oduced, and in e change wi h K+ educing he concen a ion
o K+ ions in he in e io o he esicles, which, in he inhibi o y s a e o cell syn hesis
a e abundan in he cy oplasm and in whose he e is a high concen a ion o
po assium ions.
Fo e e a . (4) also poin ou he p esence o lysosomes in he p oximi y o hese
esicles, and sugges ha he localiza ion a hese cell le els could ha e an acciden al
o unc ional signi icance.
We ha e obse ed ha oxyn ic mi ochond ia degene a e wi h an inc ease in
as ing ime; his could be a ibu ed ini ially o an a i ac in he issue ixa ion
echnique, howe e , in con ol g oups unde he same condi ions no mi ochond ial
FIG. 6. In acellula mic o illi wi hh enzyma ic eac ion o acid phospha ase in oxyn ic cells o
no mal gas ic mucosa. x 40,000
OXYNTIC CELL, FASTING, PHOSPHATASE, HCL SECRETION 385
degene a ion ook place. On he o he hand, s udies ca ied ou by Helande and
Hi schowi z (8) om a mo phologic and s e eologic iewpoin ha e shown ha wi h
he use o chlo hyd ic sec e ion inhibi ion d ugs a dec ease o mi ochond ial olume
and an inc ease o he ubulo- esicula memb ane can be obse ed. Thus we could
es ablish a unc ional co ela ion be ween he deg ee o as ing, mi ochond ial
al e a ions and modi ica ions o he ubulo- esicula sys em and subsequen dec ease
o chlo hyd ic sec e ion.
Since in ou s udy we ob ained an inc ease o acid phospha ase and aking in o
accoun he abo e men ioned da a, we sugges a unc ional co ela ion be ween
ul as uc u al al e a ions and localiza ion o enzyme ac i i y. Thus, as a con-
sequence o mi ochond ial degene a ion, along wi h he p olonga ion o as ing
imes, he e is a dec ease o ATP concen a ion in he in acellula medium. This
ATP dec ease implies ha he hyd oly ic enzymes o he K+/H+-ATPase complex
sequen ially inhibi hei ac i i y as as ing ime inc eases.
On he o he hand he syn hesis o lysosomal enzymes ac ing on he K+/H+-
ATPase complex would be e ained a he lysosomal le el, he eby inc easing he
numbe o enzymes which would be dis ibu ed h ough he apical zones o he cell
nex o he occluded in acellula canaliculi.
Figu e 7 shows HCl p oduc ion in he esicles o he ubula esicula sys em o
FIGS. 7A, B. A. HCl p oduc ion mechanism in he ubula - esicula sys em o s imula ed oxyn ic
cells (acco ding o Fo e e al. (4)).
B. Mo phological al e a ions du ing long as ing pe iods sugges ha HCl p oduc ion is inhibi ed.
Hyaloplasmic ATP concen a ion dec eases owing o mi ochond ial al e a ion, and he e o e ATP
hyd olysis ca ied ou by he ATPase complex enzymes dec eases, p e en ing he in e change o
H+ o K+, and hus blocking in a esicula HCl o ma ion.
SEC RETORY STATE
386 MORENO ET AL.
s imula ed oxyn ic cells, acco ding o he model p oposed by Fo e e al. (4). On
he igh is he p oposed HCl sec e ion inhibi o y mechanism, p obed by ex eme
pe iods o as ing. In o de o con i m he abo e men ioned da a, biochemical and
cy ochemical s udies o mi ochond ia) ATPase ac i i y a e a p esen being ca ied
ou .
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