Enzymatic response to hcl inhibition in oxyntic cells of fasting rats

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 . REFERENCES 1. Beams, H. W. and King, R. L.: No es on he cy ology o he pa ie al cell o he s omach o he a . Ana . Rec. 53; 31, 1932. 2. Fo e, T. M, and Fo e, J. G.: His ochemical s aining and cha ac e iza ion o glycop o eins in acid sec e ing cells o og s omach. J. Cell. Biol. 47; 437, 1970. 3. Fo e, J. G. and Machen, T. E.: T anspo and elec ical phenomena in es ing and sec e ing pigle gas ic mucosa. J. Physiol. 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