Cultured gastric parietal cells from the guinea pig: adherence, cell growth and stimulus coupling of Ca2+ and cyclic AMP

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Cultured gastric parietal cells from the guinea pig: adherence, cell growth and stimulus coupling of Ca2 + and cyclic AMP Jtirgen Giebel*, **, Gerhard Rechkemmer***, Ralf Reimer*, Karl-Friedrich Sewing*, Jochen Fanghiinel**, and Michael Scbwenk* *Depanment of General Pharmacology, Medical School, Konstanty-Gutschow-Stra13e 8, D-30625 Hannover, **Department of Anatomy, Ernst Moritz Arndt University, FriedrichLoeffler-Stral3e 23 c, D-17487 Greifswald, and ***Department of Physiology, Veterinary School , Bischofsholer Damm 15 , D-30173 Hannover

Summary. The aim of the study was to establish cell culture conditions for responsive guinea pig parietal cells. Parietal cells were isolated by a pronase/ collagenase method, enriched by counterflow elutriation and cultured on plastic culture dishes in minimum essential medium . Precoating with gelatine or collagen increased adherence; optimum fetal calf serum concentration was 10070. Parietal cells were cultured for up to 120 h. Intracellular calcium levels in cells cultured for 48 h were 150 nmol/ I and increased to 320 nmolll after stimulation with carbachol and to 250 nmolll after histamine stimulation as determined by video imaging microscopy. Intracellular cyclic AMP levels were increased 9-fold by histamine in cells cultured for 24 h and more than 30-fold in cells cultured for 48 h. The results show that guinea pig parietal cells grow in primary culture and are suitable for studying second messenger coupling. Key words: Cell culture - cyclic AMP - Gastric cells - Intracellular calcium - Parietal cells - Guinea pig

Introduction Isolated gastric parietal cells have been widely employed for the study of the mechanisms and regulation of acid production/ secretion. Though freshly isolated cells have provided important information, e. g. with regard to modulation of receptor-mediated second-messenger pathways (Batzri and Dyer 1981; Beil et al. 1980; Mardh et al. 1985) these preparations present some problems: the isolation procedure may cause loss of or damage to receptors, viability decreases with time, measurement in single cells is hardly possible and Correspondence to: J. Giebel , Greifswald

Ann Anat (1996) 178: 405 -- 4 ! 2 Gustav Fischer Verlag Jena

modulating effects of the matrix environment are excluded (Batzri and Gardner 1979; Chen et al. 1989). Therefore the most promising tool to overcome these difficulties might be parietal cell culture. The attachment and culturing of gastric parietal cells for several days was first described by Chew et al. (1989) and thereafter by Mangeat et aJ. (1990). These authors prepared enriched rabbit parietal cells which were attached to tissue culture dishes precoated with extracellular matrix (matrigel). The cultured cells showed morphological changes after stimulation with histamine (Chew et al. 1989). Ljungstr6m and Chew (1991) reported stimulation of intracellular calcium in cultured rabbit gastric cells after the application of carbachol or histamine. Successful adherence and culture of physiologically active (and highly enriched) parietal cells from other species, however, have not yet been described. This might be due to the fact that parietal cells are highly differentiated and require specific culture conditions adapted to the animal species. Recently, we have found that gastric mucosal cell cultures of the guinea pig contain mucous cells, chief cells and parietal cells (Giebel et al. 1991). In this study we investigated the culture conditions of guinea pig mucosal cells in more detail with special regard to parietal cells. We demonstrated that adherent parietal cells are suitable for studying cell-cell interactions, proliferation and stimulation of second messengers, e. g. Ca2 + and cyclic AMP.

Materials and methods Isolation and separation oj cells Dispersed gastric mucosal cells were obtained and enriched by counterflow elutriation in different cell fractions according to the

method described by Beinborn et aJ. (1993). For the following studies, crude cell fractions and enriched parietal cell fractions containing about 80070 parietal cells. 12% chief cells and 8% mucous cells were used.

ing or by immunocytochemistry using antibodies against pepsinogen or vimentin and subsequent detection with FITC-Iinked secondary antibodies (Sigma).

Detection oj extracellular matrix Cell culture For cell culture, 6 x 106 cells were spread in 2.5 ml culture medium [minimum essential medium (MEM, Serva, Heidelberg) containing Hank's salts supplemented with 20 mmolll HEPES, 25 mmolll NaHC0 3 , 10% fetal calf serum (FCS), 5 ~g/ml gentamycin (Serva) and 2.5 ~g/ml amphotericin B (Boehringer, Mannheim)] on 6 cm plastic culture dishes (Tissue culture quality; Greiner, Niirtingen). In parallel experiments, the cells were spread on culture dishes which were coated with one of the following matrices: matrigel (Collaborative Research, USA; 150 ~I were distributed on culture dishes; alternatively, 50 ~I matrigel was added to culture medium, or 100 ~I matrigel in 2.5 ml PBS were allowed to polymerise on the culture dishes before use): fibronectin (Petri dishes were incubated with a solution of 50 ~giml in PBS at 37°C for 90 min) or laminin (Petri dishes were incubated with 2 ml of a 10 ~g/ml dilution in 0.05 molll TRIS-buffer at 22°C for 45 min), gelatine or collagen type R as described previously (Giebel et al. 1991). Cells were incubated on the respective culture dishes at 37°C in an atmosphere of 5% CO 2 with 100°70 humidity. The culture medium was changed daily. Alternatively, cells were also incubated in bicarbonate-free MEM at 37°C or 22 °C respectively. To study the effect on cell attachment, different concentrations of FCS (0%, 1%, 10%) in MEM were tested. In all experiments, unseparated cell suspensions containing approximately 24% of parietal cells were cultured in parallel.

Pre-adherence oj other cell types A pre-adherence procedure was used as an alternative to e1utriation for the enrichment of parietal cells: the freshly isolated cells were suspended in MEM and 6x 106 cells!2.5 ml were spread on uncoated plastic Petri dishes and incubated at 37"C After 30, 60, 120 or 180 min respectively, the supernatant suspension containing non-adherent cells was collected, spread on another Petri dish and cultured. The primary dishes containing rapidly adhering cells (sediment culture) were immediately supplied with fresh medium and incubated. After 24 h or 48 h all dishes were analysed for the total cell number and number of parietal cells and fibroblasts.

Identification oj parietal cells After isolation, the parietal cells were identified by mitochondrial succinic dehydrogenase (SDH) reactivity or by the vital dye Janus green as previously described (Giebel et al. 1995). In parallel, guinea pig parietal cells were identified by the routine immunocytochemistry of methanol- or formaldehyde-fixed material with an antibody against the H' IK ATP-ase oj rat parietal cells, as formulated by Mercier et al. (1989). This was kindly provided by Dr. P. Mangeat (Montpellier, France). Ultramuclural investigations were made as described previously (Schw1cnk et al 1993).

Identification oj mucous cells. chief cells and subepithelial cells Identification of different cell types was achIeved as described previously (Beinborn et al. 1993; Giebel et al. 995 ) bv lectin bind-

For detection of extracellular matrix deposited by cultured cells on the dishes, cells were grown in MEM without serum on untreated tissue quality culture dishes. After 48 h culture, cells were dissolved with 0.1 % Triton in PBS. The culture plates were rinsed with PBS (3 x 5 min) and subjected to an indirect immunofluorescence test employing a primary antibody against fibronectin (Sigma) and a secondary FITC-conjugate.

Detection oj proliferating cells Proliferating cells were detected by the incorporation of 5-bromo2' -deoxyuridine (BrdU) into cell nuclei and subsequent immunocytochemical visualisation using FITC-immunofluorescence as previously described (Beinborn et al. 1993).

Immunocytochemical controls In all immunocytochemical assays, specificity of antibody binding was assessed in control experiments by 1) omission of the first antibody, 2) omission of the second antibody and 3) by untreated specimens. Moreover, binding of the fibronectin antibody was inhibited after preincubation of the antibody with 100 ng/ml fibronectin.

Detection of intracellular cyclic AMP For cyclic AMP-assay, primary cultures of parietal cells were incubated in MEM + 1% bovine serum albumin. Control experiments revealed that the addition of 10- 4 molll 3-isobutyl-l methylxanthine (IBMX) during incubation increased the intracellular cyclic AMP level more than 2-fold. Therefore all experiments were carried out with IBMX and with one of the following substances: histamine (10- 4 molll), carbachol (10- 4 molll), prostaglandin E2 (PGE 2)(10- 7 molll), forskolin (10- 7 molll), in the presence of 10- 4 molll IBMX. Control incubations were done without any substance, and with IBMX alone. After incubation for 10 min (37°C), the culture dishes were incubated at 96°C (5 min) to disrupt the cells. The supernatants were collected, centrifuged at 10,000 g and acetylated with a mixture (2: 1) of acetic acid anhydrate/methylamin. Samples (50 ~l) were incubated with 100 ~I anti-cyclic AMP-antibody (kindly provided by Dr. Heim, Department of General Pharmacology, Medical School, Hannover) and 50 ~I 125I-iodotyrosyl-cyclic AMP (5,000 -10,000 cpm) at 4°C. After 24 h, 100 ~I 0.8% y-globulin in RIA-buffer (4.1 gil Naacetate, pH 6.0) and 750 ~I polyethylene glycol 6,000 were added. After 10 min at 4°C, samples were centrifuged (5 min, 10,000 g), the supernatants were discarded, the sediments washed in RIA-buffer (2 x 5 min, 10,000 g) and counted in a y-counter.

Detection oj intracellular calcium Calcium in freshly isolated single cells or cultured cells was measured using a digital video imaging system (Nikon invert microscope, Hamamatsu video camera, image analyser). Freshly isolated cells were attached to poly-D-Iysine coated glass cover slides (20 ~I of a 1 mg/ml poly-D-Iysine solution was used per slide

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ferred to a second culture dish and then cultured for 24 h, the parietal cell content was approximately 50%. Subepithelial cells (endothelial cells and fibroblasts) adhered about as equally rapidly as did parietal cells.

and allowed to dry) or cells were cultured on gelatine-coated glass cover slides. Cells were labelled for 60 min at 20 'C with 5 Ilmolll Fura 2AM (Sigma) in a buffer containing 120 mmolll NaC!, 5.4 mmolll KCI, 0.5 mmolll MgCb, 0.2 mmoI/CaCl" 10 mmolll HEPES, 10 mmolll Glucose; pH 7.4), Determination oj protein

The protein content of cell cultures was determined with bicinchoninic acid according to the method of Redinbaugh and Turley (1986). Quantification oj cells ~ parietal cells

Cells were counted per visual field at the microscope level at 2S0-fold magnification. All experiments were carried out with 6 cellular preparations in duplicate. If not ot herwise indicated, results represent means ± SD.

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Fig. 1. FCS-dependent adhesion of gastric cells. Attachment was poor without FCS but significantly improved by increasing FCS concentrations. Mean of triplicate measurements from 4 cell preparations

Results Culture conditions The crude cell suspension of enzymatically isolated guinea pig gastric cells used in this study had the following composition: parietal cells: 20 - 28 0/0, chief cells: 15 - 20%, mucous cells: 41 - 540/0 and approximately 11 070 of other cell types, e. g. endocrine cells, undifferentiated cells, endothelial cells, fibroblasts, mast cells or phagocytes. When those suspensions were plated in MEM with 10% FCS the attachment on untreated standard quality culture dishes was poor, amounting to approximately 8 - 25 070 of initially seeded cells. Coating of these culture dishes with gelatine or collagen strongly improved adherence (68 - 92%), whereas fibronectin and laminin were less effective (47 --72%). On tissue culture quality Petri dishes, 60 - 82% of seeded cells adhered. Within 10 - 24 h cells flattened, and confluent patches were formed within 24 - 72 h. Matrix coating of these dishes did not improve attachment. On matrigel-coated dishes, attachment was less effective; the cells grew in clusters, did not flatten and had poor viability. Adherence of parietal cells and other cell types reached an optimum at about 100/0 FCS content of the medium and 5% CO 2 concentration (Fig. 1). As a result of these experiments, standard culture was carried out on untreated tissue culture quality culture dishes in MEM supplemented with 10% FCS in 50;'0 COc o When enriched parietal cells (after elutriation) were cultured, the cell culture contained about 50070 - 60% of parietal cells after 24 h of culture. When a suspension of unseparated gastric mucosal cells cOlltallling approximately 24% parietal cells was seeded, the monolayer contained about 18% parietal cells after 24 -- 48 h of incubation. Fig. 2 shows the time course for the adherence of total cells and parietal cells and the respective cell count in the supernatant when using the preadherence procedure, Whereas chief and mucous cells became attached predominantly within 4 h, parietal cells became attached after 4 ! 0 h When, after 4 h of preadherence, the supernatant cell suspension was trans-

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Fig. 2, Enrichment of parietal cells by time-dependent attachment of cells to culture dishes. Cells were allowed to attach for different times ("sediment culture"); after the respective time the supernatant cells which had not yet become attached were collected and cultured for 24 h as the sediment culture. Total cell count (TC) and number of parietal cells (PC) in the sediment cultures (A) and in the supernatant cultures (B).

Morphology oj parietal cells Freshly isolated parietal cells were distinguishable ultrastucturally by their abundance of mitochondria, intracellular canaliculi and their microvilli-like structured surface membrane (Fig. 3; A). After 48 h in culture, intracellular canaliculi and tubulovesicles seemed to disappear (Fig. 3; B), whereas the outer membrane preserved the microvillilike morphology. Parietal cells exhibited a variety of different shapes, depending on their environment: parietal cells had become attached among other cell types and flattened (Fig. 4; A, B). Other parietal cells were extended between two confluent patches and had an elongated shape (Fig. 4; A). Parietal cells which grew either isolated or in small parietal cell clusters were round and not flattened. Occasionally, parietal cells became attached to the tops of other flattened cells (Fig. 4; A).

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Fig. 3. Ultrastructure of parietal cells. A: freshly isolated parietal cell, showing intracellular canaliculi, abundant mitochondria and microvilli-like structured outer membrane. B: horizontal section of a cluster of parietal cells after 48 h culture. The cells were characterised by abundant mitochondria and their brush-border-like outer membranes. Bars represent 5 Jlm.

Immunoreactivity of H+ IK+ ATP-ase The monoclonal antibody against rat gastric H+ IK+ ATPase bound to single growing cells which had a round shape (Fig. 5; A), but it did not bind to flattened cells which were integrated in the monolayer or which grew at the periphery of these monolayers. Deposition of fibronectin In all the cultures examined, fibronectin was deposited on the dishes after 24 h. Fig. 5 (B) shows the immunocytochemical detection of a fibronectin-network in 48 hold parietal cell rich cultures grown in serum-free medium. In parallel, soluble fibronectin was also detected in the culture medium by ELISA (data not shown).

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Proliferation of parietal cells

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Fig. 4. Cultures of enriched parietal cells: A: 48 h old culture; parietal cells were stained black by SDH assay. Parietal cells grew flattened, associated with other cells or in small clusters of round and single attaching cells (in the upper right). Flattened parietal cells occasionally generate long protrusions; B, C: 72 h old culture; double labelling: detection of SDH activity (B) and visualisation of DNA-synthesis by immunocytochemical (immunofluorescence) detection of incorporated BrdU (C). BrdU-positive nuclei of parietal cells are numbered. D, E: 96 h old culture: demonstration of mitotic parietal cell by double-labelling. SDH-activity of mitotic parietal cell (arrow) localised at the periphery of the monolayer is shown in D, and detection of BrdU by immunofluorescence in the same mitotic parietal cell (arrows) is demonstrated in E.

During cell culture, the proliferation rate in unseparated cultures (containing 18070 parietal cells) increased from 5% on day 1 to about 27% on day 3 of culture. The number of proliferative cells then decreased to less than 5% on day 8. Similar results were observed with enriched parietal cell cultures. Double labelling experiments revealed that 48 - 72 h after plating, nuclei of parietal cells occasionally incorporated BrdU (Fig. 4; C, E). The number of BrdU-positive parietal cells was less than 1% of all BrdU-positive cells. Occasionally, mitosis of BrdU-positive parietal cells was observed in cultures older than 96 h (Fig. 4; D, E). Cyclic AMP Basal cyclic AMP production in parietal cell-rich cultures was stimulated 9-fold by histamine, 5-fold by PGE 2 and 2.5-fold by forskolin. After 48 h, higher basal cyclic AMP levels were detected. Histamine stimulated basal cyclic AMP production 18-fold, PGE 2 7.5-fold and forskolin 30-fold (Fig. 6). In control experiments, the stimulation profiles of non-parietal cell cultures (sediment cultures containing 75%

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Fig. 5. Immunocytochemical investigations on cultured parietal cells. A: detection of parietal cells by an antibody against proteins of H+ IK+ ATP-ase which is distributed in single growing parietal cells. There is no reaction in the nuclear region. Other cells were not stained. B: Detection of fibronectin in 48 h old enriched parietal cell cultures after detachment of cells. Fibronectin is deposited as an ir, regular "network". The cells were attached in serum-free medium to exclude deposition of serum fibronectin. Fluorescence excitation.

mucous cells and 200/0 chief cells) were assessed: basal cyclic AMP levels were stimulated IS-fold by histamine, 20-fold by PGE 2 and 25-fold by forskolin. After 48 h of culture, histamine increased basal cyclic AMP more than 50-fold, whereas PGE 2 stimulated 21-fold and torskolin 18-fold (Fig. 6). Carbachol had no effect at cyclic AMP levels. For cyclic AMP-assay, parietal cell cultures (50070 parietal cells) were obtained by the pre-adherence procedure.

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Intracellular calcium [Ca . L was measured in individual parietal cells. Basal calcium levels were about 150 nmol/l in 48 h old parietal cells. Carbachol raised rCa' 'L [0 350 nmol/1. PGE 2 had no effect (Fig. 7: A) The carbachol effect was blocked by atropine (Fig. 7; 8). Single growing, round but not flattened parietal cells could be stimulated. After measuring [Ca 2 - L, parietal cell identification was verified by vital staining with Janus green. In 24 hold parietal cells basal rCa? L levels were comparable with basal levels in 48 h old cultures. However. lhe [Cae], response was less than in older culture' 2

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Discussion This paper describes the morphological and functional characteristics of cultures of enriched responsive guinea pig parietal cells. Chew et al. (1989), Mangeat et al. (1990), and Ohtaki (1994) described the culture of parietal cells on matrigel and a [Ca 2 +1 i response to stimulation. Other authors found small numbers of parietal cells « 5070) as contaminants in rat (Hiraishi et al. 1991), rabbit (Ota et al. 1990) or guinea pig (Rattner et al. 1985) gastric cell cultures. After isolation, guinea pig parietal cells were characterised ultrastructurally by their abundance in mitochondria and their intracellular canaliculi as described previously for rabbit (Chew et al. 1989) or pig (Mardh et al. 1987). Parietal cells of the guinea pig had abundant microvilli on their outer membranes which persisted during culture (Fig. 3; B). Since these structures do not occur on guinea pig parietal cells in vivo and have not been described for other species, e. g. rabbit or pig (Chew et al. 1989; Mardh et al. 1987), this phenomenon may be a preparative artefact unique for the guinea pig. Compared to rabbit parietal cells which were seeded on matrigel-coated dishes, culturing of guinea pig parietal cells does not require special culture conditions. Guinea pig parietal cells readily adhered to uncoated tissue quality culture dishes. Although matrigel improves the attachment to glass slides, cells in our experiments were poorly attached to this matrix. Moreover, cells grown on this matrix were not suitable for functional studies. This IS in contrast to the findings of Chew et al. (1989) for rabbit parietal cells, who obtained responsive parietal cells when seeding on matrigel. That guinea pig parietal cells do not require any matrices for attachment might be due to the ability of these cells to synthesise extracellular matrix. As shown immunocytochemically (Fig. 5), fibronectin represents one component of the matrix which was deposited by gastric cells on the culture dishes, and fibronectin was also secreted into the medium (unpublished data). It was assumed that fibronectin may have been synthesised by epithelial cells, since fibroblasts made up only 1070 of growing cells. This is remarkable in that synthesis of fibronectin in vitro was reported mainly for fibroblasts and endothelial cells (Peters et al. 1990), tumour cells (Va rani et al. 1991) or macrophages (Rossman et al. (990). Deposition of solubilised fibronectin from the medium on the dishes can be excluded, since fibronectin synthesis took place when FCS-free medium was used. The fact that fibronectin synthesis occurred in enriched parietal cell cultures led to the assumption that parietal cells might be able to synthesise this matrix component. Since extracellular matrix is composed of several componnts, it is likely that other substances such as collagens or proteoglycans are also synthesised by gastric epithelial cells. It is currently unknown to what extent these matnx components contribute to the attachment of parietal cells. A variable morphology (round, flattened) of parietal cells was observed depending on their location in relation to other cell types in the monolayer. Since single grov\ ing round parietal

cells responded to stimuli and expressed H+ /K+ ATP-ase, whereas parietal cells which were flattened or integrated in the monolayer did not, a correlation between morphology and responsiveness is evident. These findings suggest that there might be different populations of parietal cells. This is in accordance with the observations of Chew et al. (1989) and Chew (1994) who described a different expression of the H+ /K+ ATP-ase in cultured rabbit parietal cells. Other studies showed different populations of parietal cells in the gastric mucosa deriving most probably from undifferentiated cells and occurring as pre-parietal cells (Karam 1991). The presence of different parietal cell populations is confirmed by the observation that some flattened parietal cells growing in the monolayers synthesised DNA or even underwent mitosis. Since there are controversial reports on the regeneration of parietal cells (Hunt and Hunt 1961; Lipkin 1987; Willems and Lehy 1975) in the gastric mucosa, one might conclude that, in the guinea pig, gastric mucosa parietal or pre-parietal cells (Karam 1991) with mitotic activity are present. Alternatively it seems possible that DNA synthesis in vitro reflects de-differentiation triggered by isolation and culturing. [Ca 2 +L of rabbit parietal cells could be elevated up to about 950 nmolll (Chew et al. 1989) with histamine and to 1 600 nmolll with carbachol. These cells underwent significant morphological changes (Chew and Ljungstrbm 1990). Guinea pig parietal cell basal [Ca2 +L was increased about 2-fold to 350 nmolli. This is in agreement with the results of Leaven et al. (1990) who described a similar [Ca2 +1 i rise in isolated rabbit parietal cells after carbachol stimulation. For isolated canine parietal cells, a 3-fold carbachol-mediated stimulation of basal [Ca2 +1 i has been reported (Delvalle et al. 1992). From these findings it is concluded that in cultured guinea pig parietal cells, basal [Ca 2 +L is not different from that in other species. No data are available on the generation of cyclic AMP in cultured parietal cells. Batzri and Gardner (1978) had shown that, in suspensions containing 70% guinea pig parietal cells, cyclic AMP was increased about to-fold by histamine. Similar results were obtained for suspended parietal cells from dog (Wollin et al. 1979), rat (Puurunen et al. 1987) and in our study for 24 h old cultures. After 48 h in culture, stimulation by histamine and forskolin was significantly more pronounced than in cells cultured for 24 h. This might be an indication for the regeneration or synthesis of histamine receptors and/or adenylate cyclase as previously suggested for canine chief cells in culture (Sanders et al. 1983). The hypothesis of cell/receptor regeneration is also confirmed by our calcium measurements, which worked out most satisfyingly in 48 h old cultures. In our experiments, guinea pig parietal cells easily became attached to culture dishes and did not require mat rigel for maintaining physiological properties in culture. However, there was no accumulation of the weak base aminopyrine used as a semiquantitative measure of parietal cell acid production (Berglindh et al. 1976) in response to secretagogues. This might be due to the 5% CO 2 containing atmosphere which was required for optimum growth and which inhibited

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aminopyrine accumulation as described previously (Chew et al. 1989). On the other hand, guinea pig parietal cell enriched cultures could be easily obtained by the preadherence method and do not require centrifugal elutriation and density centrifugation as used for rabbit parietal cells (Chew et aJ. 1994). Acknowledgements. The authors wish to thank Mrs. H, Arends for

outstanding technical work in preparing cell cultures. We are grateful to Dr. P. Mangeat (Montpellier, France) for providing the monoclonal antibody against antigen of the rat gastric H+ /K+ ATP-ase. Further thanks go to Dr. H.-K, Heim, Medical School Hannover for providing the polyclonal antibody against cyclic AMP. Supported by Deutsche Forschungsgemeinschafr, SFB 280.

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Willems G, Lehy T (1975) Radiographic and quantitative studies on parietal and peptic cell kinetics in the mouse. A selective effect of gastrin on parietal cell proliferation. Gastroenterology 69: 416-427 Wollin A, Soll AH, Samloff M (1979) Actions of histamine, secretin, and PGE 2 on cyclic AMP production by isolated canine fundic mucosal cells. Am J Physiol 237: E437 - E443

Sewing K-F, Harms P, Schulz G, Hannemann H (1983) Effect of substituted benzimidazoles on acid secretion in isolated and enriched guinea pig parietal cells. Gut 24: 55 7 - 560 Soli AH (1978) The action of secretagogues on oxygen uptake by isolated mammalian parietal cells. J Clin Invest 61 : 370 - 380 Varani J, Schuger L, Fligiel SEG, Inman DR, Chakrabarty S (1991) Production of fibronectin by human tumor cells and interaction with exogenous fibronectin: Comparison of cell lines obtained from colon adenocarcinomas and squamous carcinomas of the upper aero digestive tract. Int J Cancer 47: 421 - 425

Accepted June 20, 1996

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Buchbesprechungen Analysis of Vertebrate Structure. Von M_ Hildebrand, 4. Auf!. 1995. 657 Seiten, 415 Abbildungen. John Wiley & Sons, New York - Chichester - Brisbane - Toronto Singapore. Preis: £ 19,95, ISBN 0-471-11085-X

Thomas KerIe: Trockene Aussichten. 208 Seiten, Hardcover, 14,8x22 cm. edition q Verlags-GmbH Berlin 1994. DM 29,80. ISBN 3-86124-200-1

In seinem 1995 in der 4. Auf!age erschienenen Lehrbuch "Analysis of Vertebrate Structure" prasentiert Milton Hildebrand die Morphologie der Vertebraten als eine lebende Disziplin, indem er neben klassischen vergleichend anatomischen Lehrbeispielen auf ungelbste Probleme und aktuelle Trends im behandelten Fachgebiet aufmerksam macht. Das Buch ist in drei Abschnitte gegliedert: Nach einer kurzen Einfiihrung erlautert der Autor in den drei Kapiteln des 1. Abschnitts zunachst die Einteilung def Vertebraten in die groBen taxonomischen Einheiten. 1m 2. Abschnitt werden in sechzehn Kapiteln Phylogenie und Ontogenie der wesentlichen morphologischen Strukturen von Haut, Zahnen, Bewegungsapparat, Kreislauf, sowie des Eingeweide-, Atmungs-, Urogenital-, Nerven-, Sinnesund Endokrinen-Systems dargestellt. Der Abschnitt 3 gibt in zehn Kapiteln eine Ubersicht iiber die strukturelle Adaptation in Relation zur Anlage und zu Umweltbedingungen. Referenzlisten am Ende jedes Kapitels verweisen auf die Original- bzw. weiterfiihrende Literatur. Neben einem ausfiihrlichen Stichwortverzeichnis finden sich gesonden Erlaute- rungen zu mehr als 150 Fachbegriffen. Die iibersichtliche Gliederung der Fiille des vorgestellten Materials sowie reichliche und einpragsame schwarz-weif3 Zeichnungen zeugen ebenso wie eine gut faBbare sprachliche Darstellung von der groBen Kompetenz des Autors. Druck und Aufmachung des Buches sind gut. Das als Lehrbuch konzipierte Werk iSl slcherlich nicht nur fiir Studenten der Biologie interessant, sondem diirfte auch unter Veterinarmedizinern und Arzten interessierte Leser finden_

Dieser Debiitroman schildert die Entstehung und den VerI auf einer Alkoholabhangigkeit am Beispiel der Hauptfigur Hans Maurer. Jedoch will es dem Autor nicht recht gelingen, den Roman im Rahmen eines schliissigen Gesamtkonzeptes zu entwickeln. Kindheitsepi soden in Form von Riickblenden, die offenbar die spatere Trinkerkarriere erklaren sollen (da beziehungsreich mit der Uberschrift Erste Weichenstellung, Zweite Weichenstellung usw. versehen), sind in ihrer Banalitat als Ausloser der Sucht nicht nachvollziehbar. Der Charakter des iiberbehiiteten, selbstunsicheren, nach auBen hin aber arrogant agierenden Jungen und spateren Erwachsenen wird nicht sorgfaltig herausgearbeitet. Die Darstellung der inneren Befindlichkeit der Hauptperson ist rudimentar. Der Roman strap aziert den Leser durch Langatmigkeit, hOlzerne Dialoge und bemiihte Formulierungen. Textbeispiel: "Hans fiihlt sich trotz der Alkoholkonstanz in seinem Korper, deren Pegel hin und her schwankt, relativ wohl und besitzt wieder Selbstvertrauen, was ihm nach dem letzten Niederschlag gefehlt hat". Oder: "Martina bricht in Tranen aus. Sie bleibt allein mit ihren Wunden". Die in def zweiten Halfte des Buches aufgezeigten Uisungswege in Form der Inanspruchnahme einer Suchtberatung, einer Angehorigengruppe bzw. des stationaren Entzugs werden dem Leser betulich und belehrend nahegebracht. Alles in allem ein Buch, das als Roman zu oberf!achlich und mit vielen stilistischen Mangeln behaftet, als Reportage nicht dicht und spann end genug geschrieben ist.

\'1. Hanschke. Greifswald

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G. J. Wiedemann, Liibeck