Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin...

GASTROENTEROLOGY 1997;112:280-286

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Abnormal Gastric Histology and Decreased Acid Production in Cholecystokinin-B/Gastrin Receptor-Deficient Mice

NANCY LANGHANS,* GUIDO RINDI, t MARY CHIU, § JENS F. REHFELD,/I BLAIR ARDMAN, §

MARTIN BEINBORN, § and ALAN S. KOPIN § Departments of §Medicine and *Pediatrics, GRASP Digestive Disease Center, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts; *Department of Human Pathology, University of Pavia, Pavia, Italy; and IIDepartment of Clinical Biochemistry, University Hospital, Copenhagen, Denmark

Background & Aims: The cholecystokinin (CCK)-B/gastrin receptor is one of several regulators of gastric acid secretion and mucosal growth. To elucidate the contribution of this receptor relative to other trophic and secretory factors, mice that lack the CCK-B/gastrin receptor have been generated and studied. Methods: Both alleles of the CCK-B/gastrin receptor were inacti- vated by targeted gene disruption. Analysis of the mice included measurement of basal gastric pH and plasma gastrin levels. In addition, multiple gastric mucosal cell types were identified by immunostaining and quanti- fied. Results: Homozygous mutant mice were viable, fertile, and appeared grossly normal into adulthood. The receptor-deficient mice exhibited a marked increase in basal gastric pH (from 3.2 to 5.2) and an - lO- fo ld elevation in plasma gastrin concentration compared with wild-type controls. In the stomach of mutant animals, parietal and enterochromaffin-like cells were decreased, providing a likely explanation for the reduction in acid output. In the antrum, a decrease in somatostatin cell density and an increase in the gastrin cell number were observed, consistent with the concomitant elevation in circulating gastrin. Conclu- sions: Together, these findings demonstrate the importance of the CCK-B/gastrin receptor in maintaining the normal cellular composition and function of the gastric mucosa.

T he cholecystokinin B/gastrin receptor (CCK-BR) is a seven-transmembrane domain protein that is

abundantly expressed in mammalian stomach. This receptor subtype plays an important role in mediating gastric acid secretion and mucosal growth. Two endogenous ligands, CCK and gastrin, bind to the CCK-BR with subnanomolar affinity. In the stomach, gastrin is considered the primary physiological stimulus for CCK -BR- dependent function. 1

Multiple interacting mechanisms within the gastric mucosa determine the rate of acid secretion by parietal

cells. At least two of these pathways are regulated by CCK-B/gastrin receptors. A large portion of the gastrin- dependent acid secretion after food intake is mediated

mechamsm : gastrin stimulation of through an indirect • 1 CCK-BRs on enterochromaffin-like (ECL) cells results in histamine release, which in turn triggers acid secretion via parietal cell He receptors. In addition, gastrin acts directly on parietal cell CCK-BRs to stimulate the secretion of acid. However, the relative importance of the latter mechanism seems to vary significantly between different mammalian species. 2

As a secondary effect of gastrin-mediated acid secretion, the intraluminal pH influences the number of gastrin- and somatostatin-producing cells in the antral mucosa. 3-5 These endocrine cells in turn regulate the concentration of circulating gastrin, thus providing a feedback loop that counterregulates changes in acid production.

Gastrin is also a trophic hormone in the stomach and as such influences the rate of proliferation and/or differentiation of gastric mucosal cells. Hypergastrinemia in rodents resulting from administration of either proton pump inhibitors, histamine receptor antagonists, ~ or ex- ogenous gastrin v'8 invariably leads to an increase in parietal cell mass as well as ECL cell number. The growth- promoting effects of gastrin on ECL cells seem to be directly mediated through CCK-BRs. 9 In contrast, the gastrin-stimulated increase in parietal cells is generally considered to result from enhanced entry of progenitor cells into the parietal cell lineage, v4°

Unlike the well-documented effects of hypergastrinemia, the consequences of withdrawing normal physiological concentrations of gastrin have not yet been fully

Abbreviations used in this paper: CCK-BR, cholecystokinin-B/gastrin receptor; D, somatostatin; ECL, enterochromaffin-like; G, gastrin.

© 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

January 1997 CCK-B/GASTRIN RECEPTOR KNOCKOUT MICE 281

established. It has been difficult to clarify whether gastrin is essential in the maintenance of the normal gastric

mucosa and its ability to secrete hydrochloric acid. At- tempts to address this question have largely relied on animal studies in which the major site ofgastr in production, the gastric antrum, has been resected. H-15 Findings

in this model, however, may be affected by concomitant inflammation of the remaining stomach. 11 Moreover, the

possibilities that secretion of other growth factors is di- minished or that neural circuits are interrupted after

antral resection introduce additional confounding vari- ables in the interpretation of previous studies. Reflecting

the complexity of the antrectomy model, contradictory data have been reported regarding the effects of this surgical intervention on parietal cell density. 11-14

We report the generation of mice lacking functional

CCK-BRs, a new in vivo model to assess the mechanisms underlying gastrin-dependent cellular proliferation and

secretory function in the stomach. In the present study, we have begun to examine the relative contribution of

the CCK-BR in both of these processes.

Materials and Methods

Animals

C57B/6J and CBA/J mice were purchased from Ta- conic Farms (Gerrnantown, NY) and Jackson Laboratory (Bar Harbor, ME), respectively. Mice were housed in microisolator cages. Approval for animal experimentation was obtained from the Tufts-New England Medical Center Animal Research Committee (Boston, MA).

Construction of the CCK-BR Targeting Vector

Two contiguous genomic phage clones comprising the CCK-BR gene were isolated from a mouse PCC4 genomic

library (Stratagene). Restriction enzyme analysis and limited DNA sequencing were used to identify the protein coding exons. In the targeting construct, the region of the gene spanning a portion of exon 3, all of exon 4, and part of exon 5 was replaced with a neomycin resistance gene under the control of the phosphoglycerate kinase promoter (Figure 1). The segment of the mouse receptor gene which was removed encoded the fifth, sixth, and seventh transmembrane domains as well as the third intracellular and third extracellular loops of the CCK- BR.

Gene Targeting in Embryonic Stem Cells

The targeting construct was linearized with Xba I and electroporated into D3 embryonic stem cells (gift from Dr. Richard Hynes, Massachusetts Institute of Technology, Bos- ton, MA). 16J7 G418-resistant embryonic stem cells were screened by genomic Southern blot analysis for the appropriate targeting event. 18 With the occurrence of homologous recombination, the construct was designed to introduce an Sph I site into the CCK-BR locus. Introduction of this site enabled the 3' probe to distinguish the wild-type allele (5.8 kilobases [kb]) from the targeted allele (5.0 kb) in Sph I digested genomic DNA (Figure 1). Two positive clones of D3 embryonic stem cells were identified. Southern blot analysis of EcoRI digested genomic DNA was used to reconfirm homologous recombination. The 3' probe recognized an ~ 11-kb fragment (wild-type allele) and an ~6.5-kb band (targeted allele). The loss of ~4.5- kb from the wild-type allele verified the correct targeting event (data not shown).

Generation of CCK-BR-Deficient Mice

Two different clones of positive D3 embryonic stem cells were injected into blastocysts obtained from C57B6/J mice. ~7 Embryos were transferred to pseudopregnant CBA mice. Four chimeric males were identified by coat color; one of the chimeras gave germline transmission of the targeted allele. Heterozygous offspring, identified by genomic Southern

A E S

WT G e n e ..... = - - - X 3 4 5

Targe t ing E E S Cons t ruc t N E O

N S B I I

/ / ~ N 5,8 k b - , ' 5.0 kb ~

N S L J

Probe

S R e c o m b 5.0 kb S I !

i S Wild Type 5.ekb S

I I

Figure 1. Disruption of the CCK-BR gene. (A) Homologous recombination strategy. A partial restriction map shows the mouse wild-type CCK- BR gene and the corresponding targeting construct. Numbers correspond to exons that were altered in the targeting vector. A neomycin resistance gene replaced all of exon 4 and part of exons 3 and 5 along with the intervening intron sequences. E, EcoRI; S, Sph I; N, Nco I; NEO, neomycin resistance gene, (B) Genomic Southern blot analysis shows correct targeting in mutant mice. DNA isolated from mouse tails was digested with Sph I and hybridized with a probe corresponding to sequence outside the 3' end of the targeting construct. Wild-type (+) and mutant ( - ) alleles resulted in 5.8- and 5,0-kb hybridization signals, respectively.

282 LANGHANS ET AL. GASTROENTEROLOGY Vol. 112, No. 1

blot analysis of tail DNA, were bred to produce mice homozygous for the disrupted CCK-BR gene. The animals used for study were between 2 and 9 months of age. Within this age range, there was no obvious age-dependence to the expression of the mutant phenotype.

Pharmacological Confirmation of the Absence of the CCK-BR

The density of 1251 CCK-8 binding sites was compared between brain membranes isolated from wild-type and homozygous mutant mice. Low-speed supernatant (600g; 10 minutes) of brain homogenates was centrifuged at 100,000g for 1 hour, washed, and resuspended for binding experiments in Hank's balanced salt solution supplemented with 1.2 mmol/ L ethylene glycol-bis(~-aminoethyl ether)-N,N,N',N'-tetra- acetic acid, 4.2 mmol/L MgCI2, 25 mmol/L HEPES, pH 6.5, 150 btmol/L phenylmethylsulfonyl fluoride, 250 btg/mL baci- tracin, and 0.2% bovine serum albumin. Aliquots of 100 btg protein in 273 btL of buffer solution were incubated for 100 minutes at 37°C in the presence of 25 pmol/L 125I CCK-8 with increasing concentrations of unlabeled CCK-8 (up to 1 btmol/L) or gastrin-17-I (up to 3 ~mol/L) as competitors. Free radioligand was removed by vacuum filtration (GF/B; What- man, Clifton, NJ) at 7 psi. Bound radioligand retained on the filter was determined by gamma-counting (Beckman GAM 5500; Beckman Instruments, Fullerton, CA). Nonlinear curve fitting was used to determine the density of 125I CCK-8 binding sites as well as the dissociation constants for CCK-8 and gastrin-17-I (Scapre/Scafit software, version 4.7 for Macintosh computers).

Gastrin Radioimmunoassay

A gastrin-specific radioimmunoassay using antiserum 2604 was used to measure bioactive, carboxyamidated gastrins. The antiserum binds all bioactive gastrins (sulfated as well as nonsulfated) with equimolar potency. The cross-reactivity with cholecystokinin is negligible. 19

Measurement of Fasting Gastric pH

CCK-BR-deficient and wild-type control mice were fasted overnight in metabolic cages. Gastric pH was assessed using a modification of the method of Takagi et al. 2° Animals were anesthetized with an intraperitoneal injection of 2% tribromoethanol. The abdominal cavity was opened, and the stomach was exposed. The esophagus (just above the gastro- esophageal junction) and the pylorus were ligated. The stomach was excised, and 0.5 mL of saline injected into the gastric lumen. The fluid contents were collected, and the pH of the effluent was measured using a Radiometer PMH 83 Autocal pH meter (Radiometer, Copenhagen, Denmark).

Histology and Immunohistochemistry

Stomachs of mutant and age- and sex-matched wild- type mice (eight pairs) were compared. Mice were anesthetized with 2% tribromoethanol, bled by cardiac puncture, and killed. Blood was centrifuged at room temperature, and plasma

was stored at -80°C until assayed for gastrin concentration. Serial sections (3-5 btm) were stained with H&E for conven- tional histology and periodic acid-Schiff/alcian blue :1 as a marker for mucin. Immunohistochemical tests were performed using the avidin-biotin-peroxidase complex method (SPA, Mi- lan, Italy). 22 The following antisera were used: rabbit anti C- terminal o~ subunit (HK0cC2) of pig gastric H+,K +-adenosine triphosphatase (ATPase) (1:t000; A.J. Smolka, Charleston, SC), 23 rabbit anti-rat chromogranin A (1:2000; H. Winkler, Innsbruck, Austria), 24 rabbit a n t i - N terminus of gastrin 34 (1:2000, AC90; Cambridge Research Biochemicals, Cam- bridge, England), and rabbit antisomatostatin 2811-14] (1:5000, CA-08-330; Cambridge Research Biochemicals).

Morphometric Analysis

Only samples in which mucosal glands were oriented perpendicularly to the gastric lumen were used for cell quanti- fication. In longitudinal sections, fundic ECL cells immunoreactive for chromogranin A as well as antral cells immunoreactive for either gastrin (G cells), or somatostatin (D cells) were counted per millimeter of stomach length] 5 The length of the mucosa suitable for counts in each mouse ranged from 0.32 to 3.5 mm (mean value, 1.54 mm). H+,K+-ATPase-immuno - reactive cells were counted only in oxyntic glands showing the lumen throughout their entire thickness; results were expressed as number of H+,K+-ATPase-positive cells per gland unit. Gland units were counted in the zymogenic region of the gastric mucosa, paying particular attention to avoid the mucosa of the oxyntic/antral junction where parietal cells tend to be more scattered. Ten gland units were counted per mouse san> pie.

Statistical Analysis

Data were expressed as mean -4- SEM and compared by nonparametric statistical tests (Mann-Whitney). A P value of <0.05 was considered statistically significant.

Results and Discussion

The homozygous CCK-BR-de f i c i en t mice are vi-

able and fertile and have now been observed for periods

exceeding 12 months. Their overall appearance and be-

havior appear indistinguishable from wild-type lit-

termates. Consistent with these observations, no abnor-

malities in brain morphology were evident. To ensure

that the receptor in the mutan t mice was nonfunctional,

the targeting construct was designed to replace exon

sequences encoding a structurally essential region of the

receptor (spanning transmembrane domain V - V I I ) with

a neomycin resistance gene. Genetic ablation of the recep-

tor was shown by genomic Southern blot analysis of tail

D N A (Figure 1). Pharmacological confirmation that the knockout mice

did not, in fact, express functional receptors was obtained by 125I CCK-8 binding experiments using cerebral

plasma membranes, one of the major sites of CCK-BR

January 1 9 9 7 C C K - B / G A S T R I N RECEPTOR KNOCKOUT MICE 2 8 3

8 . 1 5 0 0 - 0.03 O

6" 0 -- 5 " o~ 1 0 0 0 -

0 . 0 2 o O 4..__~ 8 3 - 500- o

,} 2 - 0.01 - ~ o

, I I , i 1 "oo -11 -9 -7 -5 WT KO W T K O

[Competitor], log (M)

Figure 2. Pharmacological and functional alterations resulting from CCK-BR deletion. (A) Absence of displaceable 12Sl CCK-8 binding to brain membranes from knockout (KO) mice. Radioligand competition experiments using membranes from wild-type (WT) mice showed binding sites with high affinity for both CCK-8 (0; dissociation constant, 0.66 - 0.07 nmol/L) and gastrin (&; K~ = 3.71 + 1.29 nmol/L). The density of 12Sl CCK-8 binding sites in brain membranes from wild-type mice, calculated from homologous competition experiments, was 36.3 +_ 5.8 fmol/ mg protein. In contrast, using CCK-8 as competitor, there was no displaceable radioligand binding to brain membranes from knockout mice (0). Data represent averages + SEM from 3 independent experiments. (B) CCK-BR-deficient mice have reduced basal acid production. The mean pH of gastric effluents from fasted age- and sex-matched pairs of wild-type and knockout mice was 3.2 _+ 0.4 and 5.2 _+ 0.4, respectively (P < 0.01). Each circle represents the value corresponding to an individual animal. The horizontal bar reflects the mean. (C) Plasma gastrin levels are elevated in CCK-BR knockout mice. The mean gastrin concentrations were 59.3 _+ 8.3 and 612 +_ 135 pmol/L in age- and sex- matched pairs of wild-type and mutant animals, respectively (P < 0.0001). Each circle represents the value corresponding to an individual animal. The horizontal bar reflects the mean.

expression. Because it is well established that the central nervous system and stomach receptors are the product of a single CCK-BR gene, 26'-~v brain membranes provided a reasonable surrogate marker for gastric receptor expression. 1251 CCK-8 competition binding experiments showed that wild-type mouse brain membranes bound CCK-8 (dissociation constant, 0.66 ± 0.07 nmol/L) and gastrin (inhibition constant [Ki] 3.71 ± 1.29 nmol/L) with affinities in the nanomolar range, as expected for a CCK-BR (Figure 2A). In contrast, parallel experiments using membranes isolated from CCK-BR-deficient mice showed no displaceable binding of 125I CCK-8. Thus, by both pharmacological and genetic criteria, the homozygous knockout mice lack CCK-BR.

To assess the consequences of receptor inactivation in

the gastrointestinal tract, basal acid production in the CCK-BR-deficient mice and in wild-type controls was compared. After an overnight fast, the gastric pH in the mutant mice was significantly higher than in the wild- type animals, 5.2 + 0.4 vs. 3.2 ± 0.4, respectively (P < 0.01; Figure 2B and Table 1). As in other animal models • 4 6 9 9 8 • • of hypochlorhydna, ' ' '- low gastric acid concentrations in the receptor-deficient mice resulted in hypergastrinemia (Figure 2C and Table 1). However, this counterregulatory response to high gastric pH is ineffec- tive in receptor-deficient mice. The absence of CCK-BRs prevents gastrin from increasing acid secretion; the pH of the stomach therefore remains elevated.

In an attempt to identify anatomic abnormalities that could potentially explain the large difference in acid se-

Table 1. Comparison of Intraluminal pH, Plasma Gastrin Concentrat ions, and Mucosal Cell Counts Between Age- and Sex- Matched Wild-type and CCK-BR-Def ic ient Mice

pH Gastrin (pmol/L) Parietal ECL D cells G cells G/D ratio

Wild-type mice 3.2 + 0.4 59 _+ 8 19 + 2 49 _+ 6 14 +_ 1 74 + 8 5.3 + 0.6 Knockout mice 5.2 _+ 0.4 ° 612 _+ 135 ° 13 _+ I b 24 _+ 6 a 8 _+ I a 113 _+ 14 a 15.8 _+ 1.8 ° Difference t 63% 1" 937% $ 32% $ 51% $ 43% t 53% 1" 198% Reference studies 4's'34 1" t 1" i" ~ 1̀ t

NOTE. Parietal cell numbers are expressed per gland unit; ECL and D- and G-cell numbers are expressed per millimeter of stomach length (see Materials and Methods). Data from the literature on rodents treated with H +, K+-ATPase inhibitors are shown for comparison as reference studies. 4'~,34 Increases or decreases are denoted by corresponding arrows. ap < 0.05. Op < 0.01.

284 LANGHANS ET AL. GASTROENTEROLOGY Vol. 112, No. I

cretion between wild-type and knockout mice, a detailed immunohistochemical assessment of the cell populations that modulate acid production was undertaken. In the gastric mucosa of CCK-BR-deficient mice, there was a marked decrease in the densities of parietal cells (Figures 3A and B) as well as chromogranin A-immunoreact ive ECL cells (Figure 3C), both of which are important in determining acid output.

The numbers of parietal and ECL cells in the oxyntic mucosa of homozygous mutant mice were decreased by 32% and 51%, respectively, compared with wild-type control values (Table 1). It is of note that the reduction in ECL and parietal cell densities occurred in the setting ofhypergastrinemia. This is in marked contrast to normal mice in which pharmacologically induced hypergastrinemia invariably results in increased ECL cell density and parietal cell mass 6 8 (Table 1). Despite elevated circulating gastrin levels in the knockout mice, the absence of CCK-BRs renders the mice functionally "agastri- nemic." The resulting decrease in the number of ECL and parietal cells in the gastric glands of knockout mice allows us to conclude that a minimum level of CCK- BR-media ted stimulation is required to maintain these cell populations at normal densities.

In receptor-deficient mice, a decrease in the number of antral somatostatin-producing D cells was also observed (Table 1). Reduced D-cell density is a normal physiological response to long-term elevation of gastric pH. This occurs in rodents after partial fundectomy (selective removal of acid secreting mucosa) or as a result of chronic application of acid-inhibitory drugs. 4'5'28 Unlike changes

in the number of parietal and ECL cells, this shift in antral D-cell density is postulated to be pH dependent and to occur irrespective of circulating gastrin levels) '4'e8

This concept is supported by comparison of our data on CCK-BR-deficient mice with data obtained in earlier studies in which rodents were treated with H+,K + -ATPase inhibitors. 4'5 In both cases, hypochlorhydria-

Figure 3. Altered gastric mucosal cell populations in CCK-BR-deficient mice. Comparison of immunohistochemical findings in the gastric mucosa from age- and sex-matched pairs of wild-type (left) and knockout animals (right). (A) Loss of H+,K+-ATPase-immunoreactive (brown) parietal cells in a CCK-BR-deficient mouse (right) compared with the gastric mucosa from a wild-type control (left) (magnification x150). (B) High-power view of H+,K+-ATPase-immunoreactive (brown) cells shows a less regular distribution of parietal cells per gland unit in a CCK-BR-deficient mouse (right) compared with a wild- type control (left) (magnification ×600). (C) Gastric mucosa from a mutant mouse (right) shows a marked reduction in chromogranin A- immunoreactive (brown) ECL cells compared with a wild-type control (left) (magnification ×250). (D) The gastrin-immunoreactive (brown) G cells of a knockout mouse (right) are increased in number and extend further from the base of the gland than the corresponding cells in a wild-type control (left) (magnification x200).

January 1997 CCK-B/GASTRIN RECEPTOR KNOCKOUT MICE 285

induced hypergastrinemia occurs in association with a decrease in antral D-cell density (Table 1). The fact that the knockout mice lack the target receptor for gastrin provides further evidence that the alteration in D-cell numbers must be independent of the elevation in gastrin level. Whether the reduced D-cell density is a direct consequence of the elevated intraluminal pH in the stomach or an indirect effect modulated by a pH-sensitive mucosal growth factor remains to be determined.

In the homozygous mutant mice, the decrease in antral D cell numbers is accompanied by an increase in gastrin- producing G ceils (Figure 3D and Table 1). It has been previously established in studies of normal rodents that changes in these two populations are inversely correlated; somatostatin release by D cells is postulated to inhibit the growth of antral G cells. 1 Therefore, in the knockout mice, the increase in G cell number may be a direct consequence of the decrease in D cell density. These opposite changes in antral G and D cells lead to a mark- edly increased G/D cell ratio (vs. wild-type mice).

The antral G/D cell ratio is considered a sensitive indicator of gastric pH. 4'29 The pH dependence of the

G/D cell ratio was initially proposed based on comparison of humans with decreased gastric pH resulting from gas- trinomas (G/D cell ratio, 1.6 + 0.2) and increased gastric pH resulting from gastric atrophy (G/D cell ratio, 16.3 + 4.1). 3 Because hypergastrinemia is found in both these groups, it was concluded that the G/D cell ratio varied as a function of pH rather than of gastrin levels. In the knockout mice, the elevation in gastric pH coincided with a marked increase in G/D cell ratio relative to the wild-type control values, 15.8 + 1.8 vs. 5.3 + 0.6, respectively (mean + SEM; P < 0.001). This is consistent with the previously proposed model of antral endocrine cell regulation 4'5 and provides an additional histo-

logical correlate of the elevated pH in receptor-deficient animals.

Finally, it seems that the overall organization and distribution of individual cell types within the acid-secreting mucosa of receptor-deficient mice is abnormal. The oxyntic glands of mutant animals are, to a variable degree, distinguished from corresponding wild-type con- trois by an increased number of H+,K+-ATPase-nega - tive cells (Figure 3A) and a concomitant irregular distribution of parietal cells (Figure 3B). Preliminary electron-microscopic observations (not shown) suggest that the expanded population of H+,K+-ATPase-nega - t ire intervening cells observed in the mutant animals (Figure 3B) are epithelial cell precursors, as defined by the classification system of Karam and LeBlonde. 3° These investigators have suggested that 11 distinct epithelial cell types arise from a common stem cell in the mouse

gastric mucosa. Accumulating evidence suggests that the proliferation and differentiation programs of these cells are highly interdependent. 3~-~3 It is intriguing to specu- late that the absence of CCK-BRs leads to a develop- mental arrest or slowdown in the parietal and enteroendo- crine cell lineages and a redirection of cellular differentiation into alternative pathways. This transition may occur as a direct consequence of receptor absence on one or more stem cell derivative(s). Alternatively, the observed changes may result as a secondary effect due to the loss of a gastrin-dependent growth factor that is required for normal gastric differentiation.

In summary, the CCK-BR-deficient mouse model shows how deficiency of a single gene product can disrupt the intricate balance that determines the distribution of endocrine and acid-producing cells in the stomach. The changes observed in the stomach of CCK-BR-deficient mice are consistent with current theories of the role of gastrin as a gastric mucosal secretagogue and growth factor. Our results extend this model by establishing the physiological importance of the CCK-BR and its normal gastrin-stimulated activity in maintaining mucosal cyto- architecture and function. Significant numbers of parietal and ECL cells persist despite the absence of the CCK- BR. This suggests that some degree of functional re- dundancy may exist between gastrin and other trophic stimulants. Alternatively, gastrin may modulate the con- stitutive production of differentiated cell types within the gastric mucosa. The CCK-BR-deficient mouse model provides an important step toward understanding the complex interactions that underlie gastric differentiation as well as function.

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Received July 25, 1996. Accepted October 8, 1996. Address requests for reprints to: Alan S. Kopin, M.D., New England

Medical Center, 750 Washington Street, Box 239, Boston, Massa- chusetts 02111 . Fax: (617) 636-4207.

Supported by American Home Products, a Charles A. King Trust research grant (to M.B.), a Charles H. Hood foundation grant (to N.L.), and an Italian Health Ministry to I.R.C.C.S. Policlinico San Matteo grant 030RCR96 /4 (to G.R.).

Part of this work was performed in the Molecular Biology and Microbiology Core facilities of the GRASP Digestive Disease Center (P30-DK34928).

Equal contributions were made by the first two authors. The authors thank A. J. Smolka and H. Winkler for their generous

gifts of antisera; M. Blaeker, A. B. Leiter, and A. Kane for careful reading of the manuscript; S. Roffler-Tarlov, S. Ito, E. Solcia, and R. Fiocca for helpful discussions; and D. Gallant, S.M. Quinn, E.W. McBride, L. Lincicome, and V. Necchi for technical assistance.

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