HistochemicalJournal, 7 (I975), 4oi-4o9
Lactate dehydrogenase isoenzyme patterns in isolated cells of pig gastric mucosa B. G. TIMMS* and B. PROCHAZKA-PERTHEN Department of Biological Sciences, University of Aston in Birmingham, Gosta Green, Birmingham, UK
Received 13 August I974 and in revised form II April I975
Synopsis. A technique is described for obtaining a relatively pure suspension of parietal cells from pig gastric mucosa. All cell types were released from the tissue by treatment with pronase. Four cellular fractions were separated by centrifugation on a discontinuous Ficoll gradient. One fraction contained 68% (SD I3.3) parietal cells and revealed a high lactate dehydrogenase-i level compared to the other fractions.
Introduction
In complex cellular structures such as the glandular mucosa of the mammalian stomach, the physiological role of individual cell types is difficult to elucidate from tissue samples because of the heterogeneous cell population. Previous studies have involved techniques ofhistochemistry (Bradford & Davies, I95 o) and electron microscopy (Helander I962; Lillibridge I964; Rohrer et al. x965). Attempts to prepare a suspension of cells of one type from gastric mucosa were made by BreMiller & Davenport (x96I) and Walder & Lunseth (I963). Other investigations on isolated cells using similar techniques have also been described (Croft & Ingelfinger, I969; McDogual & DeCosse, I97o; Blum et al., I971; Lee, I972). These studies have concentrated on the isolation of parietal cells and the investigation of metabolic events involved in the process of hydrochloric acid secretion. Different modes of metabolism are reflected by changes in the patterns of lactate dehydrogenase (LDH) isoenzymes (Cahn et al., I962). The enzyme is a tetrameric molecule made up from two types of polypeptide subunits (H and M), each synthesized in the cell under separate genetic control. The subunits combine by random association to form five LDH isoenzymes: LDH-x (H4); LDH-2 (H3MI); LDH- 3 (H2M2); * Present address: Tenovus Institute for Cancer Research, The Heath, Cardiff, UK. Present address: Gesellschaft Ffirstrahlen und Umweltforschung, 8o42, Neuherberg bei Mfin-
chen. Ingolstadter Landstrasse I. 9 1975 Chapman and Hall Ltd
401
402
Timms and Prochazka-Perthen
LDH-4 (H1M3); LDH-5 (M4). The predominance of L D H - I , which is strongly inhibited by excess pyruvate, is indicative of a system requiring a steady supply of energy from oxidative metabolism, whereas a predominance of LDH-5 is associated with glycolysis and high pyruvate concentrations (Dawson et al., 1964). An increase in the L D H - I isoenzyme in the fundic mucosa of stomach was observed by Leese (1965), and attempts have been made to correlate this with the parietal cells. L D H isoenzyme patterns were investigated in samples of human normal and diseased gastric mucosa, and abrasions from the surface of the mucosa (Prochazka et al., I968b), and histochemical studies were made on gastric mucosa (Lojda & Fric, 197o), in an attempt to ascertain the contribution of individual cell types to the overall pattern of the gross tissue sample. Examination of individual cells or an homogenous preparation of cells is a prerequisite for understanding the biochemistry of cells and tissues in normal and pathological conditions. The purpose of this study is to provide more direct evidence, using isolated cells, to support the theory that the prevalence of L D H - I found in fundic mucosa is attributed to the presence of parietal cells. Materials and methods
Reagents Phosphate-buffered saline/glucose (PBSG) contained 137 mM NaC1; 2. 7 mM KC1; 8 mM Na2HPO4; I. 5 mM KH2POa; and I mg/ml glucose, pH 7.4 4- o.o5. Pronase (Merck Biochemicals) 7~ ooo PUK/g, o. x5 g/Ioo ml, prepared in PBSG. Ficoll (Pharmacia, G.B.) 2o%, 12.5%, lO% and 5%, prepaxed respectively in PBSG.
Preparation of isolated cells The technique used was a modification of the method described by Walder & Lunseth (I96I). Plastic apparatus was used throughout the procedure. Fresh, unopened pig stomachs were obtained from the abattoir within half an hour of the animal's death. A portion of the stomach, approximately 5 cm square was removed from the greater curvature of the fundus, the external muscle cut away, and the tissue rinsed several times in PBSG. The mucosa was placed upwards on a cork mat and blotted with a piece of filter paper to remove superficial mucus. Using the back edge of a scalpel blade, the surface of the mucosa was abraded several times to remove the mucous epithelium. The underlying muscularis mucosae and submucosae were separated from the mucosa by careful dissection. Approximately z g of the dissected and abraded mucosa was minced with scissors in 2-3 ml of PBSG, transferred to 2o ml of pronase solution (pre-warmed to 37 ~ C), in a IOO ml beaker, placed in an incubating bath at 37~ and stirred for 20 min at approximately 15o rpm, using a magnetic stirrer. The beaker was removed, to allow the tissue to settle and the pronase solution poured off and discarded. A further 5~ ml of fresh, pre-warmed pronase solution was added to the tissue, and stirred as before at 37~ for another 4o rain. The tissue and pronase were then filtered through a blood administration set filter (Baxter Division, Travenol Laboratories), the pronase and released cells being collected in a clean ioo ml beaker, and the tissue fragments in the nylon-mesh filter. 2-3 ml e f
L D H isoenzymes in pig gastric mucosa
403
fresh pronase solution was added to the filter set and retained by using a pressure clamp on the outlet tube. By gentle squeezing of the pliable filter set between thumb and index finger, isolated cells still retained within the gastric pit and enveloped by the framework of the lamina propria were released, and the resultant cloudy cell suspension was added to the incubating filtered pronase solution. After repeating this treatment twice, the pooled suspension was stirred as before for a final 30 min to disperse the mucus released by squeezing the tissue. The suspension was strained through a clean filter set into 5 • z5 ml conical centrifuge tubes, followed by centrifugation at 3oo g for 2 min. The cells were resuspended in 0. 5 ml of PBSG with the aid of a pasteur pipette and a further 4.5 ml of PBSG added before centrifuging at 300 g for i min. To four of the tubes, I ml of zz.5% Ficoll was added to resuspend the cells. The remaining cell plug was used as a sample of the mixed cell suspension for cytological examination and isoenzyme studies.
Preparation of cell fractions by density gradient centrifugation Each mixed cell suspension of Ficoll (4 • I ml) was subjected to centrifugation in a discontinuous Ficoll gradient, using a modification of the technique described by Hamberger et al. (I97I). The gradients were prepared in 5 ml tubes (7.4 • I.o cm), calibrated in o. 5 ml increments, in the following manner: 1.5 ml of 2o% Ficoll was added, followed by I ml of cell suspension in i z.5 % Ficoll, I ml of I o % Ficoll and finally o.5 ml of 5% Ficoll. The tubes were capped and centrifuged at IOOOg for 3o min (Fig. I). Cells were collected from the interphases between the Ficoll solutions and from the bottom of the gradient, and transferred to pre-weighed Eppendorf micro reaction tubes, which contained a small amount of PBSG. I f necessary, more PBSG was added to fill the tubes, after which the cell suspensions were mixed and centrifuged at 30o g
~Frbction
B
~-- Fraction' C
Fraction D
~ Fraction
Figure z. Ficoll density gradient used for the separation of isolated gastric mucosal cells. The mixed cell suspension was applied to the central part of the gradient in the I2. 5 % Ficoll. After centrifugation, the cell fractions appeared at the interphases of the Ficoll solutions.
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Timms and Prochazka-Perthen
for 5 min. The supernatant was removed as completely as possible and smears were made of the various fractions before the tubes and cells were re-weighed. All cytological preparations were fixed with 0.3% acetic acid in absolute alcohol, and both smears and histological sections were stained using a modified Zimmerman technique (Marks & Drysdale, 1957).
Homogenization and extraction of tissue and cell samples Weighed samples of fundic and pyloric mucosa (including muscularis mucosae and submucosae), prepared fundic mucosa and abrasion, together with the cell fractions from the same stomach, were homogenized* in barbitone buffer pH 8. 4 (ionic strength o.o5). The ratio of the wet tissue weight (rag) to the volume of buffer (ml) was I : 3, and for the cell fractions, I : I. The samples were centrifuged at I o o o g for 3o min, and duplicate aliquots of the supernatant used immediately for isoenzyme studies.
Determination o)r the L D H isoenzyme pattern The isoenzymes of L D H were separated by agar gel electophoresis using the method developed by Wieme (I 959). Location of L D H activity and quantitation of the individual isoenzymes was made using the reagents and techniques described by Prochazka & Wachsmuth (1972).
Results
Cell fractions Stained preparations of the cell fractions obtained after centrifuging the mixed cell suspension in the discontinuous Ficoll gradient were examined by light microscopy. Differential cell counts were performed to ascertain the contributions made by the various cell populations found in the fundic mucosa. Both mucous epithelial and mucous neck cells were included in the same group because the surface epithelium of the mucosa used for the experiments was abraded, and in most cases the number of epithelial cells present was very small, The results are summarized in Table 1. The cells obtained from the interphase of the 5% and IO% Fico11 layers (fraction B) were predominantly parietal cells of varying size. A typical sample of cells obtained from this fraction is illustrated in Fig. 2. Although a similar high proportion of parietal cells were found in fraction C, their morphological appearance differed from those in fraction B in that the former exhibited greater intensity of cytoplasmic staining, and the nuclei in some ceils were pyknotic and sometimes fragmented. The yield of chief cells was relatively low in all fractions due to the fact that the base of the fundic glands, where these cells predominate, was dissected away during preparation of the mucosa. In preliminary experiments using the dissected lower part of the gastric gland, it has been possible to concentrate the amount of chief cells to between * A micro tissue homogenizer was specially prepared for use with the small amount of cellular material. It was constructed from epoxy resin (Araldite) using an Eppendorfmicro reaction tube as a mould. A tube was half filled with resin and centrifuged briefly to remove air bubbles before inserting a glass rod for the handle. When the resin had set, the tube was cut away, and the surface of the resin was roughened with fine emery paper. After thorough washing with distilled water, the pestle was used to homogenize the cell samples contained in the micro tubes.
L D H isoenzymes in pig gastric mucosa
405
Table I. Differential cell counts. Fraction A, mixed cell suspension obtained from pig fundic mucosa by incubation with pronase for 90 min; fraction B, cells collected from the 5-IO% Ficoll interphase; fraction C, cells collected from the IO--I2.5 % Ficoll interphase; fraction D, cells collected from the 12.5-2o% Ficoll interphase; and fraction E, cells collected from the bottom of the gradient.
Fraction
Percentages of cell types No. of experiments Parietal Chief
Mucous
Erythroeytes Others*
A B C D E
12 IO 3 9 9
38.8 i 6.3 8.5 ~ 2.2 7.7 ~ 4.3 31.9 i 6.4 49.4 4- 8.2
22-3 • 6.7 5.0 • 2.6 I.O 4- 0. 7 12.6 4- 4.6 2o.5 4- 7 .0
11.6 • 1.6t 68. 3 4- 4.4 67.6 i 13.3 19.2 4- 3.6 5.7 4- 1.9
14.3 • 3.I 4.3 • 1.6 6. 7 • 2.6 17.o • 4.9 II.I 4- 3.3
13.o • 13.9 • 17.o • 19-3 413.3 4-
4.5 3.5 9.7 5.9 4.7
*Includes lymphocytes, leucocytes and connective tissue cells. Results expressed as mean i SEM.
~
iii~ ~
fi~
~
~
~w
~
Figure a. Fraction B: a relatively pure suspension of parietal cells, collected from the interphase of the 5% and lO% Ficoll. Modified Zimmerman stain.
30% and 60% b y centrifuging the cells obtained from fraction D in a second discontinuous gradient o f higher Ficoll concentrations. Because o f the variation in the percentage o f erythrocytes present in the different fractions, the observed data was catagorized under a separate group in view o f the possible significance on the interpretation o f the L D H isoenzyme patterns, with respect to L D H - I and L D H - 2 (Nerenberg & Pogojeff, 1969).
L D H isoenzyme patterns F r o m preliminary visual examination o f the electrophoretograms it was apparent than an outstanding difference existed between the pattern o f the fraction containing a high proportion o f parietal cells (fraction B), to those of the remaining cell fractions (Fig. 3). After quantitation b y densitometry, this difference proved statistically to be highly significant. I n T a b l e 2, the isoenzyme patterns are expressed as mean values o f the percentages o f individual isoenzymes.
Timms and Prochazka-Perthen
406
A constant feature of the patterns derived from fraction B was (a) the predominance of L D H - r with a progressive decrease in L D H - z and LDH-3, a virtual absence of L D H - 4 and a complete absence of LDH-5, even in samples exhibiting high enzyme activity; and (b) a greater ratio of monomer H subunits to M subunits, which was calculated from the mean percentages ofisoenzymes to be ~3 : ~ in contrast to the H: M ratio of 2: I for entire fundic mucosa.
Figure 3- L D H isoenzyme patterns of fractions A - E , and abraded surface epithelium of pig fundic mucosa. Fraction B, the cell fraction containing the highest percentage of parietal cells, exhibits an almost exclusive presence of the p u r e H 4 tetramer, L D H - 1 .
Table 2. Percentages o f L D H isoenzymes in fractions A - E , and abraded surface epithelium of pig fundic mucosa. L D H isoenzymes Fraction
No. of experiments
LDH-r
LDH-2
A B C D E Abrasion
12 lO 3 9 9 5
6o.I 79.7 77.2 44 .6 37.0 3o.4
16.2 12.5 zo,9 21,2 21.8 17. 7
• 4.6* ~: 1.6 i 4.9 • 3-9 -4- 4.0 ~z 4.9
• i ~ ~ • ::
2.2 1.5 0, 7 I. 9 2.0 2.2
LDH- 3
LDH- 4
LDH- 5
19.4 • 1.8 7.0 ~2 1.4 9,6 ~ 2.6 25.2 ~_ I. 9 31,2 i 2.6 27. 5 • 2.o
4.3 q--- z.z 0.8 -L 1.2 2. 3 ~: I. 7 8. 7 ~ 2.0 I0.0 ~ 3.2 23.9 • 4.1
---o.3 :~z 0,3 o. 5 • o.6
* Results expressed as m e a n -4- S E M .
The majority ofelectrophoretograms did not reveal the presence of LDH-5, but small amounts were detected in fraction D and in samples of the abraded surface layer. The abrasion pattern was characterized by the prevalence of L D H - 4 and a concomitant decrease in L D H - I , in comparison with the other fractions.
L D H isoenzymes in pig gastric mucosa
407
During the course of the study, samples of normal human fundic mucosa became available from two patients undergoing partial gastrectomy for duodenal ulcer. Cell fractions were obtained from the human tissue using the techniques described for pig stomach. Of particular interest was the L D H isoenzyme pattern of fraction B, which clearly exemplified the characteristic elevated L D H - I , and progressive decrease of LDH-2, L D H - 3 and LDH-4, that was found in fraction B from pig gastric mucosa.
Discussion Although the L D H isoenzyme patterns observed in fundic and pyloric regions of pig gastric mucosa (Table 3) do not exhibit the characteristic differences found in similar samples from rat stomach (Prochazka et al., I968a), and human stomach (Baume et al., 1966; Prochazka et al., I968b), the patterns of the cell fractions used in this study, revealed marked differences in individual isoenzyme levels when comparing one fraction with another. These differences appeared to be related to the proportion of individual cell populations present in the suspensions. Table 3. LDH isoenzyme percentages in the fundic and pyloric regions of pig stomach. LDH isoenzymes Tissue Fundicmucosa Fundic mucosa (free from muscularis mucosae and submucosae) Pyloric mucosa
No. of experiments
LDH-I
4
35.7 zk 3.9* 17.8 • 1.6 3o.9 -4- 1.3 14.6 • 2.4 1.o • 0.4
8 4
38.7 -- 4.0 24.5 d- 4.4
LDH-2
LDH-3
LDH-4
LDH-5
20.3 ~ 1.6 27.6 -- I.O 12.8 = 2.4 0.6 • 0.3 21.8 • 1.4 31.9 • 0.9 20.5 ~ 3.4 1.3 • 0.6
* Results expressed as mean i SEM.
Characteristic of the fraction enriched with parietal cells was the almost exclusive presence of the pure H 4 tetramer, L D H - I . These results indicate that the parietal cells have a high rate of synthesis of H monomer subunits, and the prevalance of L D H - I found in the fundic Inucosa can, therefore, be unequivocally attributed to the unique L D H isoenzyme pattern of these cell~. L D H - I is associated with aerobic oxidation of pyruvate and lactate in mitochondria, providing a constant supply of energy, and the demonstration of a high level o f L D H - I in the parietal cell fraction is in accordance with the energy demands required for the secretion of hydrochloric acid. In comparison, the remaining fractions, which contained a high proportion of mucous secreting cells, and few parietal cells, revealed a decrease in the H monomer production relative to the M monomer. In these fractions it is assumed that the L D H - I isoenzyme is derived partly from the parietal cells, erythrocytes and possibly chief cells, but because of the heterogeneity of the cell populations, it is difficult to correlate individual cell types with a characteristic iso-
408
Timms and Prochazka-Perthen
enzyme pattern, except in the case of the relatively pure parietal cell fraction and the abrasion sample. In a comparative qualitative study on the distribution o f L D H isoenzymes in human fundic mucosa made by Leese (I965), using freeze-dried tissue sections, it was found that L D H - I was elevated in sections taken from the upper gastric gland, ~nd was thought to be associated with the parietal cells. This assumption was further justified by subsequent experiments on diseased human gastric mucosa (Prochazka et alo, I968b) where it was found that progressive decreases in the number of parietal ceils, due to the presence of varying degrees of intestinal metaplasia, resulted in a parallel decrease in the amount of L D H - I . It was postulated by Prochazka et al. (I968a) that the isoenzyme pattern of homogenized gastric mucosa was the sum of the different isoenzyme patterns of the individual cell populations, and to test this hypothesis a simple calculation was made using results from this investigation. The L D H isoenzyme percentages in one random sample of pig fundic mucosa were: L D H - I , 39%, LDH-2, 240/0, LDH-3,260/0, and LDH-4, I2~ . The sum of the individual isoenzyme patterns of fractions B, D, and E (representative of a mixed cell population isolated from the fundic mucosa) and the abrasion, expressed as percentages were found to be: L D H - I , 44%, LDH-2, 21%, LDH-3, 24% , and LDH-4, I 1%. The two patterns are almost identical, and provide a novel demonstration to confirm the foregoing assumption.
Acknowledgements The authors are indebted to Mr A. C. Youngs for his skilled technical assistance. This investigation was supported by a grant from the Science Research Council.
References Isoenzymes of lactate dehydrogenasein human gastric mucosa and gastric carcinoma tissue. Gastroenterology 5o, 781-6.
BAUME, P. E., BUILDER, J. E., FENTON, B. H., IRVING, L. G. & PIPER, D. W. (I966).
BLUM,A. L.~ SHAH, G. T., WIEBELHAUS,V. D., BRENNAN,F. T., HELANDER,H. F., CEBALLOS,
R. & SACHS, G. (I971). Pronase method for isolation of viable cells from necturus gastric mucosa. Gastroenterology 6I, 189-2oo. BgADFORD, N. M. & DAVI ES, R. E. (1950). The site of hydrochloric acid production in the stomach as determined by indicators. Biochem. J. 46, 414-2o. BREMILLER,R. A. & DAVENPORT,H. W. (196I). Attempt to separate cells of the gastric mucosa. Gastroenterology 40, 798-802. CAHN, R. D., KAPLAN, N. O., LEVINE~ L. ~ ZWILLING, E. (1962). N a t u r e and development of lactate dehydrogenases. Science I36, 9 6 2 - 9 . c R 0 F T , D. N . ~: I N G L E F I N G E R, F. J. (1969). I so lated gastric parietal cells: oxygen consumption, electrolyte content and intracellular p H . Clin. Sci. 37, 491-5Ol. DAWSON, D. M., GOODFRIEND, T. L. • KAPLAN, N. O. (1964). Lactate dehydrogenases : functions of the two types. Science 143, 929-33. HAMBERGER s A., ERIKSSON, O. & NORRBY, K. (1971). Cell size distribution in brain suspensions and in fractions enriched with neuronal and glial cells. E x p l . C e l l R e s . 67, 38o-8. H EL AND ER, H. F. (1962). Ultrastructure of f u n d u s glands of the m o u s e gastric mucosa. J . U l t r a -
struct. Res. Suppl. 4, 1-123. LEE, S. H. (I 972). Isolation of parietal cells from glutaraldehyde-fixedrabbit stomach.J. Histoehem. Cytochem. 2o, 634-43.
L D H isoenzymes in pig gastric mucosa
4o9
L EES E, C. L. (1965). The histological distribution of lactate dehydrogenase isoenzymes in human normal and malignant gastric mucosa. Europ. J. Cancer, I, 21 I-I 6. LILLIBRIDGE~ C. B. (I964). The fine structure of normal human gastric mucosa Gastroenterology 47~ 269-90. L o J DA, Z. & FR I C, P. (1970). Enzymes and isoenzymes, structure, properties and rune tion. F.E.B.S. Symposium, I8~ pp. 185-94. London & New York: Academic Press. MARKS, I. ~. & DRYSDALE, K. M. (I957). A modification of Zimmerman's method for differential staining of gastric mucosa. Stain Teehnol. 32, 48. MCDOG~JAL, W. S. & DECOSSE, J. J. (1970). Method for determining differential secretory function of isolated cells in vitro: chloride movement in isolated parietal cells. Expl. Cell Res. 6x~ 2o3-6. NERENBERG~ S. T. • POGOJEFF, G. (1969). A rapid technic of tissue extraction for isoenzyme studies in normal and diseased tissues. A m . J . din. Path. 5I~ 9 5 - I o i . PROCHAZKA,B. JIRASEK~V.~BARTA~V.~KOHOUT~ J.~KORBOVA~L.~SCHLUPEK,A. ~: SLEZAK~ Z. (I968a). Differences in lactate dehydrogenase isoenzyrne patterns in fundus and pylorus of the rat stomach. Gastroenterology 54, 6o-4. PROCHAZKA,B. JIRASEK~V. BARTA~V.~KOHOUT~ J.~ KORBOVA~L.~ SCHLUPEK~ A. ~SLEZAK, Z. (I968b). Differences in lactate dehydrogenase isoenzyme patterns in various parts of the human stomach and in gastritis, peptic ulcer, and gastric carcinoma. Gastroenterology 54~ 65-7IPROCHAZKA~ B. & WACHSMUTH, E. D. (I972). Isoenzyme patterns of lactate dehydrogenase, creatine phosphokinase, phosphoglucomutase and aldolase of guinea pig tissues during ontogeny. J . exp. Zool. I82, 2oI-IO. ROHRER~ G. V., SCOTT~ J. R.~ JOEL, W. ~1WOLF~ S. (I965). The fine structure of human gastric parietal cells. A m . J . Dig. Dis. Io, I3-2I. WALD ER, A. I. & LU N SET H, J. B. (I963). A technic for separation of the cells of the gastric mucosa. Proe. Soc. exp. Biol. Aied. 112, 494-6. WIEmE, R. J. (I959). An improved technique for agar-gel clectrophorcsis on microscope slides. Clinica chim. Aeta. 4, 317-21-
Lactate dehydrogenase isoenzyme patterns in isolated cells of pig gastric mucosa B. G. TIMMS* and B. PROCHAZKA-PERTHEN Department of Biological Sciences, University of Aston in Birmingham, Gosta Green, Birmingham, UK
Received 13 August I974 and in revised form II April I975
Synopsis. A technique is described for obtaining a relatively pure suspension of parietal cells from pig gastric mucosa. All cell types were released from the tissue by treatment with pronase. Four cellular fractions were separated by centrifugation on a discontinuous Ficoll gradient. One fraction contained 68% (SD I3.3) parietal cells and revealed a high lactate dehydrogenase-i level compared to the other fractions.
Introduction
In complex cellular structures such as the glandular mucosa of the mammalian stomach, the physiological role of individual cell types is difficult to elucidate from tissue samples because of the heterogeneous cell population. Previous studies have involved techniques ofhistochemistry (Bradford & Davies, I95 o) and electron microscopy (Helander I962; Lillibridge I964; Rohrer et al. x965). Attempts to prepare a suspension of cells of one type from gastric mucosa were made by BreMiller & Davenport (x96I) and Walder & Lunseth (I963). Other investigations on isolated cells using similar techniques have also been described (Croft & Ingelfinger, I969; McDogual & DeCosse, I97o; Blum et al., I971; Lee, I972). These studies have concentrated on the isolation of parietal cells and the investigation of metabolic events involved in the process of hydrochloric acid secretion. Different modes of metabolism are reflected by changes in the patterns of lactate dehydrogenase (LDH) isoenzymes (Cahn et al., I962). The enzyme is a tetrameric molecule made up from two types of polypeptide subunits (H and M), each synthesized in the cell under separate genetic control. The subunits combine by random association to form five LDH isoenzymes: LDH-x (H4); LDH-2 (H3MI); LDH- 3 (H2M2); * Present address: Tenovus Institute for Cancer Research, The Heath, Cardiff, UK. Present address: Gesellschaft Ffirstrahlen und Umweltforschung, 8o42, Neuherberg bei Mfin-
chen. Ingolstadter Landstrasse I. 9 1975 Chapman and Hall Ltd
401
402
Timms and Prochazka-Perthen
LDH-4 (H1M3); LDH-5 (M4). The predominance of L D H - I , which is strongly inhibited by excess pyruvate, is indicative of a system requiring a steady supply of energy from oxidative metabolism, whereas a predominance of LDH-5 is associated with glycolysis and high pyruvate concentrations (Dawson et al., 1964). An increase in the L D H - I isoenzyme in the fundic mucosa of stomach was observed by Leese (1965), and attempts have been made to correlate this with the parietal cells. L D H isoenzyme patterns were investigated in samples of human normal and diseased gastric mucosa, and abrasions from the surface of the mucosa (Prochazka et al., I968b), and histochemical studies were made on gastric mucosa (Lojda & Fric, 197o), in an attempt to ascertain the contribution of individual cell types to the overall pattern of the gross tissue sample. Examination of individual cells or an homogenous preparation of cells is a prerequisite for understanding the biochemistry of cells and tissues in normal and pathological conditions. The purpose of this study is to provide more direct evidence, using isolated cells, to support the theory that the prevalence of L D H - I found in fundic mucosa is attributed to the presence of parietal cells. Materials and methods
Reagents Phosphate-buffered saline/glucose (PBSG) contained 137 mM NaC1; 2. 7 mM KC1; 8 mM Na2HPO4; I. 5 mM KH2POa; and I mg/ml glucose, pH 7.4 4- o.o5. Pronase (Merck Biochemicals) 7~ ooo PUK/g, o. x5 g/Ioo ml, prepared in PBSG. Ficoll (Pharmacia, G.B.) 2o%, 12.5%, lO% and 5%, prepaxed respectively in PBSG.
Preparation of isolated cells The technique used was a modification of the method described by Walder & Lunseth (I96I). Plastic apparatus was used throughout the procedure. Fresh, unopened pig stomachs were obtained from the abattoir within half an hour of the animal's death. A portion of the stomach, approximately 5 cm square was removed from the greater curvature of the fundus, the external muscle cut away, and the tissue rinsed several times in PBSG. The mucosa was placed upwards on a cork mat and blotted with a piece of filter paper to remove superficial mucus. Using the back edge of a scalpel blade, the surface of the mucosa was abraded several times to remove the mucous epithelium. The underlying muscularis mucosae and submucosae were separated from the mucosa by careful dissection. Approximately z g of the dissected and abraded mucosa was minced with scissors in 2-3 ml of PBSG, transferred to 2o ml of pronase solution (pre-warmed to 37 ~ C), in a IOO ml beaker, placed in an incubating bath at 37~ and stirred for 20 min at approximately 15o rpm, using a magnetic stirrer. The beaker was removed, to allow the tissue to settle and the pronase solution poured off and discarded. A further 5~ ml of fresh, pre-warmed pronase solution was added to the tissue, and stirred as before at 37~ for another 4o rain. The tissue and pronase were then filtered through a blood administration set filter (Baxter Division, Travenol Laboratories), the pronase and released cells being collected in a clean ioo ml beaker, and the tissue fragments in the nylon-mesh filter. 2-3 ml e f
L D H isoenzymes in pig gastric mucosa
403
fresh pronase solution was added to the filter set and retained by using a pressure clamp on the outlet tube. By gentle squeezing of the pliable filter set between thumb and index finger, isolated cells still retained within the gastric pit and enveloped by the framework of the lamina propria were released, and the resultant cloudy cell suspension was added to the incubating filtered pronase solution. After repeating this treatment twice, the pooled suspension was stirred as before for a final 30 min to disperse the mucus released by squeezing the tissue. The suspension was strained through a clean filter set into 5 • z5 ml conical centrifuge tubes, followed by centrifugation at 3oo g for 2 min. The cells were resuspended in 0. 5 ml of PBSG with the aid of a pasteur pipette and a further 4.5 ml of PBSG added before centrifuging at 300 g for i min. To four of the tubes, I ml of zz.5% Ficoll was added to resuspend the cells. The remaining cell plug was used as a sample of the mixed cell suspension for cytological examination and isoenzyme studies.
Preparation of cell fractions by density gradient centrifugation Each mixed cell suspension of Ficoll (4 • I ml) was subjected to centrifugation in a discontinuous Ficoll gradient, using a modification of the technique described by Hamberger et al. (I97I). The gradients were prepared in 5 ml tubes (7.4 • I.o cm), calibrated in o. 5 ml increments, in the following manner: 1.5 ml of 2o% Ficoll was added, followed by I ml of cell suspension in i z.5 % Ficoll, I ml of I o % Ficoll and finally o.5 ml of 5% Ficoll. The tubes were capped and centrifuged at IOOOg for 3o min (Fig. I). Cells were collected from the interphases between the Ficoll solutions and from the bottom of the gradient, and transferred to pre-weighed Eppendorf micro reaction tubes, which contained a small amount of PBSG. I f necessary, more PBSG was added to fill the tubes, after which the cell suspensions were mixed and centrifuged at 30o g
~Frbction
B
~-- Fraction' C
Fraction D
~ Fraction
Figure z. Ficoll density gradient used for the separation of isolated gastric mucosal cells. The mixed cell suspension was applied to the central part of the gradient in the I2. 5 % Ficoll. After centrifugation, the cell fractions appeared at the interphases of the Ficoll solutions.
404
Timms and Prochazka-Perthen
for 5 min. The supernatant was removed as completely as possible and smears were made of the various fractions before the tubes and cells were re-weighed. All cytological preparations were fixed with 0.3% acetic acid in absolute alcohol, and both smears and histological sections were stained using a modified Zimmerman technique (Marks & Drysdale, 1957).
Homogenization and extraction of tissue and cell samples Weighed samples of fundic and pyloric mucosa (including muscularis mucosae and submucosae), prepared fundic mucosa and abrasion, together with the cell fractions from the same stomach, were homogenized* in barbitone buffer pH 8. 4 (ionic strength o.o5). The ratio of the wet tissue weight (rag) to the volume of buffer (ml) was I : 3, and for the cell fractions, I : I. The samples were centrifuged at I o o o g for 3o min, and duplicate aliquots of the supernatant used immediately for isoenzyme studies.
Determination o)r the L D H isoenzyme pattern The isoenzymes of L D H were separated by agar gel electophoresis using the method developed by Wieme (I 959). Location of L D H activity and quantitation of the individual isoenzymes was made using the reagents and techniques described by Prochazka & Wachsmuth (1972).
Results
Cell fractions Stained preparations of the cell fractions obtained after centrifuging the mixed cell suspension in the discontinuous Ficoll gradient were examined by light microscopy. Differential cell counts were performed to ascertain the contributions made by the various cell populations found in the fundic mucosa. Both mucous epithelial and mucous neck cells were included in the same group because the surface epithelium of the mucosa used for the experiments was abraded, and in most cases the number of epithelial cells present was very small, The results are summarized in Table 1. The cells obtained from the interphase of the 5% and IO% Fico11 layers (fraction B) were predominantly parietal cells of varying size. A typical sample of cells obtained from this fraction is illustrated in Fig. 2. Although a similar high proportion of parietal cells were found in fraction C, their morphological appearance differed from those in fraction B in that the former exhibited greater intensity of cytoplasmic staining, and the nuclei in some ceils were pyknotic and sometimes fragmented. The yield of chief cells was relatively low in all fractions due to the fact that the base of the fundic glands, where these cells predominate, was dissected away during preparation of the mucosa. In preliminary experiments using the dissected lower part of the gastric gland, it has been possible to concentrate the amount of chief cells to between * A micro tissue homogenizer was specially prepared for use with the small amount of cellular material. It was constructed from epoxy resin (Araldite) using an Eppendorfmicro reaction tube as a mould. A tube was half filled with resin and centrifuged briefly to remove air bubbles before inserting a glass rod for the handle. When the resin had set, the tube was cut away, and the surface of the resin was roughened with fine emery paper. After thorough washing with distilled water, the pestle was used to homogenize the cell samples contained in the micro tubes.
L D H isoenzymes in pig gastric mucosa
405
Table I. Differential cell counts. Fraction A, mixed cell suspension obtained from pig fundic mucosa by incubation with pronase for 90 min; fraction B, cells collected from the 5-IO% Ficoll interphase; fraction C, cells collected from the IO--I2.5 % Ficoll interphase; fraction D, cells collected from the 12.5-2o% Ficoll interphase; and fraction E, cells collected from the bottom of the gradient.
Fraction
Percentages of cell types No. of experiments Parietal Chief
Mucous
Erythroeytes Others*
A B C D E
12 IO 3 9 9
38.8 i 6.3 8.5 ~ 2.2 7.7 ~ 4.3 31.9 i 6.4 49.4 4- 8.2
22-3 • 6.7 5.0 • 2.6 I.O 4- 0. 7 12.6 4- 4.6 2o.5 4- 7 .0
11.6 • 1.6t 68. 3 4- 4.4 67.6 i 13.3 19.2 4- 3.6 5.7 4- 1.9
14.3 • 3.I 4.3 • 1.6 6. 7 • 2.6 17.o • 4.9 II.I 4- 3.3
13.o • 13.9 • 17.o • 19-3 413.3 4-
4.5 3.5 9.7 5.9 4.7
*Includes lymphocytes, leucocytes and connective tissue cells. Results expressed as mean i SEM.
~
iii~ ~
fi~
~
~
~w
~
Figure a. Fraction B: a relatively pure suspension of parietal cells, collected from the interphase of the 5% and lO% Ficoll. Modified Zimmerman stain.
30% and 60% b y centrifuging the cells obtained from fraction D in a second discontinuous gradient o f higher Ficoll concentrations. Because o f the variation in the percentage o f erythrocytes present in the different fractions, the observed data was catagorized under a separate group in view o f the possible significance on the interpretation o f the L D H isoenzyme patterns, with respect to L D H - I and L D H - 2 (Nerenberg & Pogojeff, 1969).
L D H isoenzyme patterns F r o m preliminary visual examination o f the electrophoretograms it was apparent than an outstanding difference existed between the pattern o f the fraction containing a high proportion o f parietal cells (fraction B), to those of the remaining cell fractions (Fig. 3). After quantitation b y densitometry, this difference proved statistically to be highly significant. I n T a b l e 2, the isoenzyme patterns are expressed as mean values o f the percentages o f individual isoenzymes.
Timms and Prochazka-Perthen
406
A constant feature of the patterns derived from fraction B was (a) the predominance of L D H - r with a progressive decrease in L D H - z and LDH-3, a virtual absence of L D H - 4 and a complete absence of LDH-5, even in samples exhibiting high enzyme activity; and (b) a greater ratio of monomer H subunits to M subunits, which was calculated from the mean percentages ofisoenzymes to be ~3 : ~ in contrast to the H: M ratio of 2: I for entire fundic mucosa.
Figure 3- L D H isoenzyme patterns of fractions A - E , and abraded surface epithelium of pig fundic mucosa. Fraction B, the cell fraction containing the highest percentage of parietal cells, exhibits an almost exclusive presence of the p u r e H 4 tetramer, L D H - 1 .
Table 2. Percentages o f L D H isoenzymes in fractions A - E , and abraded surface epithelium of pig fundic mucosa. L D H isoenzymes Fraction
No. of experiments
LDH-r
LDH-2
A B C D E Abrasion
12 lO 3 9 9 5
6o.I 79.7 77.2 44 .6 37.0 3o.4
16.2 12.5 zo,9 21,2 21.8 17. 7
• 4.6* ~: 1.6 i 4.9 • 3-9 -4- 4.0 ~z 4.9
• i ~ ~ • ::
2.2 1.5 0, 7 I. 9 2.0 2.2
LDH- 3
LDH- 4
LDH- 5
19.4 • 1.8 7.0 ~2 1.4 9,6 ~ 2.6 25.2 ~_ I. 9 31,2 i 2.6 27. 5 • 2.o
4.3 q--- z.z 0.8 -L 1.2 2. 3 ~: I. 7 8. 7 ~ 2.0 I0.0 ~ 3.2 23.9 • 4.1
---o.3 :~z 0,3 o. 5 • o.6
* Results expressed as m e a n -4- S E M .
The majority ofelectrophoretograms did not reveal the presence of LDH-5, but small amounts were detected in fraction D and in samples of the abraded surface layer. The abrasion pattern was characterized by the prevalence of L D H - 4 and a concomitant decrease in L D H - I , in comparison with the other fractions.
L D H isoenzymes in pig gastric mucosa
407
During the course of the study, samples of normal human fundic mucosa became available from two patients undergoing partial gastrectomy for duodenal ulcer. Cell fractions were obtained from the human tissue using the techniques described for pig stomach. Of particular interest was the L D H isoenzyme pattern of fraction B, which clearly exemplified the characteristic elevated L D H - I , and progressive decrease of LDH-2, L D H - 3 and LDH-4, that was found in fraction B from pig gastric mucosa.
Discussion Although the L D H isoenzyme patterns observed in fundic and pyloric regions of pig gastric mucosa (Table 3) do not exhibit the characteristic differences found in similar samples from rat stomach (Prochazka et al., I968a), and human stomach (Baume et al., 1966; Prochazka et al., I968b), the patterns of the cell fractions used in this study, revealed marked differences in individual isoenzyme levels when comparing one fraction with another. These differences appeared to be related to the proportion of individual cell populations present in the suspensions. Table 3. LDH isoenzyme percentages in the fundic and pyloric regions of pig stomach. LDH isoenzymes Tissue Fundicmucosa Fundic mucosa (free from muscularis mucosae and submucosae) Pyloric mucosa
No. of experiments
LDH-I
4
35.7 zk 3.9* 17.8 • 1.6 3o.9 -4- 1.3 14.6 • 2.4 1.o • 0.4
8 4
38.7 -- 4.0 24.5 d- 4.4
LDH-2
LDH-3
LDH-4
LDH-5
20.3 ~ 1.6 27.6 -- I.O 12.8 = 2.4 0.6 • 0.3 21.8 • 1.4 31.9 • 0.9 20.5 ~ 3.4 1.3 • 0.6
* Results expressed as mean i SEM.
Characteristic of the fraction enriched with parietal cells was the almost exclusive presence of the pure H 4 tetramer, L D H - I . These results indicate that the parietal cells have a high rate of synthesis of H monomer subunits, and the prevalance of L D H - I found in the fundic Inucosa can, therefore, be unequivocally attributed to the unique L D H isoenzyme pattern of these cell~. L D H - I is associated with aerobic oxidation of pyruvate and lactate in mitochondria, providing a constant supply of energy, and the demonstration of a high level o f L D H - I in the parietal cell fraction is in accordance with the energy demands required for the secretion of hydrochloric acid. In comparison, the remaining fractions, which contained a high proportion of mucous secreting cells, and few parietal cells, revealed a decrease in the H monomer production relative to the M monomer. In these fractions it is assumed that the L D H - I isoenzyme is derived partly from the parietal cells, erythrocytes and possibly chief cells, but because of the heterogeneity of the cell populations, it is difficult to correlate individual cell types with a characteristic iso-
408
Timms and Prochazka-Perthen
enzyme pattern, except in the case of the relatively pure parietal cell fraction and the abrasion sample. In a comparative qualitative study on the distribution o f L D H isoenzymes in human fundic mucosa made by Leese (I965), using freeze-dried tissue sections, it was found that L D H - I was elevated in sections taken from the upper gastric gland, ~nd was thought to be associated with the parietal cells. This assumption was further justified by subsequent experiments on diseased human gastric mucosa (Prochazka et alo, I968b) where it was found that progressive decreases in the number of parietal ceils, due to the presence of varying degrees of intestinal metaplasia, resulted in a parallel decrease in the amount of L D H - I . It was postulated by Prochazka et al. (I968a) that the isoenzyme pattern of homogenized gastric mucosa was the sum of the different isoenzyme patterns of the individual cell populations, and to test this hypothesis a simple calculation was made using results from this investigation. The L D H isoenzyme percentages in one random sample of pig fundic mucosa were: L D H - I , 39%, LDH-2, 240/0, LDH-3,260/0, and LDH-4, I2~ . The sum of the individual isoenzyme patterns of fractions B, D, and E (representative of a mixed cell population isolated from the fundic mucosa) and the abrasion, expressed as percentages were found to be: L D H - I , 44%, LDH-2, 21%, LDH-3, 24% , and LDH-4, I 1%. The two patterns are almost identical, and provide a novel demonstration to confirm the foregoing assumption.
Acknowledgements The authors are indebted to Mr A. C. Youngs for his skilled technical assistance. This investigation was supported by a grant from the Science Research Council.
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L EES E, C. L. (1965). The histological distribution of lactate dehydrogenase isoenzymes in human normal and malignant gastric mucosa. Europ. J. Cancer, I, 21 I-I 6. LILLIBRIDGE~ C. B. (I964). The fine structure of normal human gastric mucosa Gastroenterology 47~ 269-90. L o J DA, Z. & FR I C, P. (1970). Enzymes and isoenzymes, structure, properties and rune tion. F.E.B.S. Symposium, I8~ pp. 185-94. London & New York: Academic Press. MARKS, I. ~. & DRYSDALE, K. M. (I957). A modification of Zimmerman's method for differential staining of gastric mucosa. Stain Teehnol. 32, 48. MCDOG~JAL, W. S. & DECOSSE, J. J. (1970). Method for determining differential secretory function of isolated cells in vitro: chloride movement in isolated parietal cells. Expl. Cell Res. 6x~ 2o3-6. NERENBERG~ S. T. • POGOJEFF, G. (1969). A rapid technic of tissue extraction for isoenzyme studies in normal and diseased tissues. A m . J . din. Path. 5I~ 9 5 - I o i . PROCHAZKA,B. JIRASEK~V.~BARTA~V.~KOHOUT~ J.~KORBOVA~L.~SCHLUPEK,A. ~: SLEZAK~ Z. (I968a). Differences in lactate dehydrogenase isoenzyrne patterns in fundus and pylorus of the rat stomach. Gastroenterology 54, 6o-4. PROCHAZKA,B. JIRASEK~V. BARTA~V.~KOHOUT~ J.~ KORBOVA~L.~ SCHLUPEK~ A. ~SLEZAK, Z. (I968b). Differences in lactate dehydrogenase isoenzyme patterns in various parts of the human stomach and in gastritis, peptic ulcer, and gastric carcinoma. Gastroenterology 54~ 65-7IPROCHAZKA~ B. & WACHSMUTH, E. D. (I972). Isoenzyme patterns of lactate dehydrogenase, creatine phosphokinase, phosphoglucomutase and aldolase of guinea pig tissues during ontogeny. J . exp. Zool. I82, 2oI-IO. ROHRER~ G. V., SCOTT~ J. R.~ JOEL, W. ~1WOLF~ S. (I965). The fine structure of human gastric parietal cells. A m . J . Dig. Dis. Io, I3-2I. WALD ER, A. I. & LU N SET H, J. B. (I963). A technic for separation of the cells of the gastric mucosa. Proe. Soc. exp. Biol. Aied. 112, 494-6. WIEmE, R. J. (I959). An improved technique for agar-gel clectrophorcsis on microscope slides. Clinica chim. Aeta. 4, 317-21-
