In� ammopharmacology, Vol. 10, No. 4–6, pp. 351–364 (2002)Ó VSP 2002.Also available online - www.vsppub.com

Review

CCK2 receptors are necessary for the differentiation andproliferation of ECL cells in mouse and rat stomach

DUAN CHEN 1;2;¤, ROLF HÅKANSON3, JENS F. REHFELD4

and CHUN-MEI ZHAO 1;2

1 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology,Trondheim, Norway

2 Department of Laboratory Medicine, Norwegian University of Science and Technology, Trondheim,Norway

3 Institute of Physiological Sciences, University of Lund, Lund, Sweden4 Department of Clinical Biochemistry, Rigshospitalet of Copenhagen, Copenhagen, Denmark

Received 21 October 2002; accepted 6 November 2002

Abstract—ECL cells in the oxyntic mucosa of the mouse and rat stomach express CCK2 receptors,which enable them to respond to gastrin by the production and release of histamine. We havestudied CCK2 receptor-de�cient mice and rats subjected to pharmacologicalCCK2 receptor blockadein an attempt to demonstrate the importance of gastrin signaling for ECL-cell differentiation andproliferation. In CCK2 receptor knockout mice, the ECL cells were replaced by a novel endocrine-like cell type lacking histamine and histidine decarboxylase but retaining pancreastatin and vesiclemonoamine transporter type 2 (VMAT2). Ultrastructurally, they were characterized by the presenceof small dense-core granules and microvesicles and by the absence of the secretory vesicles that area hallmark feature of wild-type ECL cells. Upon pharmacological CCK2 receptor blockade, theECL cells became small and inactive, although unchanged in number. By immunocytochemistry(histamine, pancreastatin and VMAT2) and electron microscopy (secretory vesicles) they remainedrecognizable as ECL cells; their proliferation in response to hypergastrinemiawas prevented.

Key words: CCK2 receptors; ECL cells; differentiation; gastrin; knockout; proliferation.

1. BACKGROUND

The gastrointestinal tract in mammals is rich in endocrine cells that belong to oneof two categories: enterochromaf� n cells and enterochromaf� n-like cells. Theenterochromaf� n cells contain 5-hydroxytryptamine (5-HT) or dopamine, while

¤To whom correspondence should be addressed at Department of Surgery, St. Olavs Hospital(University Hospital in Trondheim), Olav Kyrres gate 17, 7006 Trondheim, Norway. E-mail:Duan.Chen@medisin.ntnu.no

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the enterochromaf� n-like cells do not store 5-HT but are able to take up anddecarboxylate exogenous dopa and 5-hydroxytryptophan, storing the resultingamine products in the secretory granules (Håkanson and Owman, 1969). Manydifferent subpopulations of enterochromaf� n-like cells have been identi� ed in thestomach of various animal species based on their histochemical and ultrastructuralfeatures (Solcia et al., 2000). Enterochromaf� n-like cells in the oxyntic mucosaof mouse and rat stomach include ECL cells, D cells, A-like cells and D1=P cells.The antrum harbors enterochromaf� n cells and enterochromaf� n-like cells, suchas G cells, A-like cells and D cells. The G cells produce gastrin (Pearse andBussolati, 1972), the D cells produce somatostatin (Canese and Bussolati, 1977),the A-like cells produce ghrelin (Dornonville de la Cour et al., 2001), while thepeptide hormones produced by the ECL cells, D1=P cells and enterochromaf� n cellshave not been identi� ed.

The ECL cells share many properties with other hormone-producing endocrinecells in that they express chromogranin A and harbour cytoplasmic granules/ves-icles, but they also have unique features, such as the ability to produce andsecrete histamine in response to gastrin and the neuropeptide PACAP (pituitaryadenylate cyclase-activating polypeptide) (Chen et al., 1999; Håkanson et al.,1994; Lindström et al., 1997; Zeng et al., 1998). The ECL cells are believed toexpress cholecystokinin type 2 (CCK2) receptors (previously referred to as CCK-B/gastrin receptors) (Bakke et al., 2001; Helander et al., 1997; Lindström et al.,1999; Nylander et al., 1993; Prinz et al., 1993). Activation of the CCK2 receptorsby gastrin will stimulate the synthesis and release of histamine, which in turnevokes acid secretion by activating histamine H2 receptors on parietal cells. Thisis the so-called gastrin-ECL cell-parietal cell axis which is a key physiologicalpathway controlling acid secretion (Håkanson and Sundler, 1991; Kidd et al., 1998;Lindström et al., 2001; Waldum et al., 1991). Hypergastrinemia evoked by gastrininfusion or treatment with acid secretion inhibitors induces ECL-cell hypertrophyand accelerates the self-replication rate of the ECL cells, resulting in ECL-cellhyperplasia (Håkanson et al., 1993; Ryberg et al., 1990). A recent study of gastrin-transgenic mice with elevated serum gastrin concentration has con� rmed the trophiceffect of gastrin on ECL cells (Wang et al., 2000). Nonetheless, ECL cells remain ingastrin-deprived animals, such as gastrin knockout mice (Friis-Hansen et al., 1998)and antrectomized rats (Chen et al., 1993), suggesting that lack of gastrin doesnot prevent basal ECL-cell replication and differentiation. Thus, studies of CCK2

receptor knockout mice or rats subjected to CCK2 receptor blockade by functionalmorphology may help clarify the importance of gastrin receptor activation for thedifferentiation and proliferation of ECL cells.

2. FUNCTIONAL MORPHOLOGY OF ECL CELLS

Resting ECL cells (in fasted animals) can be activated by gastrin. The change froman inactive to an active state is accompanied by a series of typical ultrastructural

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Figure 1. Schematic drawing illustrating the proposed exocytotic pathway (left part) of the secretoryorganelles and crinophagic pathway (right part) of the ECL cell (for details, see text). ¤ indicatescompounds that can be detected by immunostaining. CGA, chromogranin A; HDC, histidinedecarboxylase;VMAT2, vesicle monoamine transporter type 2; VAMP, vesicle-associatedmembraneprotein; SNAP-25, synaptosomal-associatedprotein of 25 kDa; CSP, cysteine string protein.

alterations. First, the process of exocytosis is associated with a drop in the numberof cell membrane-associated secretory vesicles and with the appearance of fairlymany microvesicles in the cell periphery (Chen et al., 1994, 1996b; Zhao et al.,1999b). Upon prolonged gastrin stimulation, there is an increase in the endoplasmicreticulum and Golgi area followed by an enlargement of the cell (Böttcher et al.,1989; Chen et al., 1996b). Functional morphology is the term used to describequantitatively these changes in ultrastructure and their relationship to functionalchanges. Based on a series of such studies of ECL cells, the life cycle of secretoryorganelles has been outlined and the intracellular localization of the differentsecretory products has been at least tentatively established (Fig. 1). We proposethat the secretory organelles originate from small, electron-lucent microvesicles thatbud off from the trans Golgi network. These microvesicles are probably loadedwith cargo, such as secretory peptide precursors (e.g. prohormone, chromogranin-Aand precursor processing enzymes) (Zhao et al., 1999b). As a result of proteincondensation (because of acidi� cation?), the electron-lucent microvesicles aretransformed into dense-cored granules. The granules are able to take up histaminefrom the cytosol by means of the vesicle monoamine transporter type 2 (VMAT2)

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in the granule/ vesicle membrane (Zhao et al., 1999a). Histamine is formed in thecytosol by the decarboxylation of histidine (histidine decarboxylase, HDC). Thecontinued accumulation of histamine into the granules leads to the transformationof the granules into large electron-lucent, dense-cored secretory vesicles (Chen etal., 1994, 1996a). These changes take place while the secretory organelles areon their way to the cell periphery, where they will dock onto the cell membrane.As a consequence of stimulation of the cell, primed secretory vesicles will fusewith the cell membrane to undergo exocytosis (Zhao et al., 1999b). This processinvolves several exocytotic proteins, e.g. vesicle-associated membrane proteins(VAMPs, also called synaptobrevins), synaptotagmins, synaptosomal-associatedprotein of 25 kDa (SNAP-25), syntaxin, Munc-18 and cysteine string protein (CSP)(Hohne-Zell et al., 1997; Zhao et al., 1997). In the process, small electron-lucentmicrovesicles appear in the periphery of the ECL cells. They are thought to be partof the regulated secretory pathway, arising from the process of membrane retrieval(endocytotic vesicles) (Chen et al., 1996a). In the case of exposure to long-termsustained stimulation with gastrin, some of the secretory vesicles will fuse notwith the cell membrane but with each other to form large vesicles, referred to asvacuoles (Chen et al., 1996a, b). The development of such vacuoles precedes thedevelopment of lipofuscin bodies that seem to arise from the oxidative conversionof native lipids and proteins to an electron-dense, polymerized, non-degradableproduct (Zhao et al., 1998, 2001). Both vacuoles and lipofuscin bodies are referred

Figure 2. A pair of photomicrographsshowing immuno� uorescent cells in a transverse section of theoxyntic mucosa of freely fed rat, double-immunostainedfor histamine (A) and histidinedecarboxylase(HDC) (B). Mucosal surface at the top. ECL cells are visualized in basal half of the mucosa in both(A) and (B), while some mast cells are shown in (A) (indicated by arrows). Bar D 50 ¹m.

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Figure 3. Electron micrographs showing: (A) a typical ECL cell with granules (indicated by smallarrows) and secretory vesicles (large arrows) and (B) typical vacuoles (asterisks) and lipofuscinbodies (arrows) in the cytoplasm of an ECL cell from rat treated with long-term omeprazole(hypergastrinemia). Bars D 400 nm.

to as crinophagic (and/or autophagic) organelles, which may incorporate or beincorporated by primary lysosomes to form part of the lysosomal compartment(Chen et al., 1999; Zhao et al., 2001).

ECL cells can be recognized by immunocytochemistry (histamine and HDC im-munostaining) (Fig. 2) and they can be identi� ed by their characteristic ultrastruc-ture as described above (Fig. 3). Hence, immunocytochemistry has been applied tostudy the changes in the density of ECL cells using antibodies to histamine (Håkan-son et al., 1986), HDC (Dartsch and Persson, 1998; Dartsch et al., 1999; Kubotaet al., 1984), chromogranin A (or its fragment pancreastatin) (Chen et al., 1994;Facer et al., 1985) or VMAT2 (De Giorgio et al., 1996; Rindi et al., 2000). How-ever, the method of immunostaining is not without pitfalls. For instance, histamineimmunostaining also reveals mast cells. In the mouse and rat stomach, mast cellsare few but they can be found at the surface of the mucosa and in the submucosaand muscle layer. The ECL cells, in contrast, are restricted to the basal half of thegastric glands. HDC immunostaining shows ECL cells but not mast cells. How-ever, it is important to realize that since the HDC immunoreactivity varies greatlydepending on the functional state of the ECL cells, HDC immunostaining does notalways demonstrate ECL cells successfully. When the cells are inactive, they willnot be detected by HDC antisera. When the cells are active, they will give a strongHDC signal. In contrast, the intensity of the histamine immunostaining varies only

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moderately despite functional changes in the ECL cells. Immunostaining with an-tisera to chromogranin A (or pancreastatin) or VMAT2 demonstrates not only ECLcells but also A-like cells and D cells (Norlén et al., 2001; Zhao et al., 1997).

3. ECL-CELL DIFFERENTIATION

Gastrin is an important stimulator of ECL-cell activation and proliferation in theadult rat and mouse (Håkanson et al., 1994). Interestingly, ECL cells in gastrinknockout mice remained in unchanged numbers and with recognizable ultrastruc-ture (Zhao, Friis-Hansen, Koh, Wang, Håkanson, and Chen, unpublished obser-vations). However, CCK2 receptor blockade promptly inactivated the ECL cells,which was re� ected in a reduced serum pancreastatin concentration and in a lowHDC activity in the oxyntic mucosa. Within 3 weeks of CCK2 receptor blockade,the ECL cells lost their typical irregular slender shape with long thin projections. In-stead, they became small and spherical, displaying reduced endoplasmic reticulumand Golgi complex. They displayed also a reduced number but an unchanged den-sity of granules and secretory vesicles, whereas both the number and the density ofmicrovesicles were reduced. Despite these changes, they wear still recognizable asECL cells (Chen et al., 2000). In contrast, the stomach of CCK2 receptor knockoutmice was devoid of proper ECL cells. They were replaced by a novel endocrine-like cell population (Chen et al., 2002). Analysis of the novel endocrine-like cellsin the stomach of CCK2 receptor knockout mice may throw light on how the CCK2

receptors control ECL-cell differentiation / ultrastructure.CCK2 receptor knockout mice were hypergastrinemic because of the impaired

acid secretion (Fig. 4) (Chen et al., 2002; Langhans et al., 1997; Nagata et al.,1996). The gastric HDC activity was measurable in wild-type mice but belowthe limit of detection in CCK2 receptor knockout mice (Chen et al., 2002). Dueto lack of HDC and histamine in the ECL cells, the cells in the mutant micecould no longer be visualized by histamine or HDC immunostaining. However,numerous endocrine-like cells were visualized in the oxyntic mucosa of CCK2

receptor knockout mice by immunostaining for pancreastatin or for VMAT2 (Fig. 5).Moreover, electron microscopy revealed that CCK2 receptor knockout mice lackedwild-type ECL cells in the oxyntic mucosa. Instead, numerous cells displaying anultrastructure distinct from that of the ECL cells were observed (Fig. 6). These novelcells were characterized immunocytochemically by the presence of pancreastatinand VMAT2 and ultrastructurally by the presence of small dense-core granulesand microvesicles (common features of most peptide hormone-producing cells).They differed from wild-type ECL cells by the absence of HDC, histamine andelectron-lucent secretory vesicles (hallmark features of wild-type ECL cells). Theultrastructure of the novel endocrine-like cells differed also from that of A-likecells, D cells and D1=P cells. Quantitative analysis of typical ECL cells inwild-type mice and of the endocrine-like cells in CCK2 receptor knockout micerevealed no difference with respect to cell pro� le size and volume densities of

ECL cells and CCK2 receptor 357

Figure 4. Plasma gastrin concentration in wild-type versus CCK2 receptor knockout mice. The bloodsamples were from freely fed animals and the original data from Chen et al., 2002. Means § SEM(n D 6), ¤¤P < 0:01.

nucleus, rough endoplasmic reticulum and Golgi complex (Chen et al., 2002).Comparison between the wild-type ECL cells and the novel endocrine-like cellsrevealed signi� cant differences in that secretory vesicles were virtually absent fromthe latter cells, while granules were small and condensed. The microvesicles werequite similar in the two cell types and occurred in similar numbers. It cannot beexcluded that the novel endocrine-like cells in the CCK2 receptor knockout micecorrespond to the ECL cells in wild-type mice and that they derive from the sameprecursor cell. The difference in ultrastructure between the ECL cells in gastrin-deprived animals and their replacement in the CCK2 receptor knockout mice canperhaps be explained if we assume that the CCK2 receptor is constitutively active.

4. PROLIFERATION OF ECL CELLS

ECL cells are capable of self-replication (Tielemans et al., 1990a). The turnovertime of ECL cells in mice and rats is somewhere between 20 and 100 days(Tielemans et al., 1992). Potent antisecretagogues, such as histamine H2 receptorantagonists and proton pump inhibitors (e.g. omeprazole), are widely used clinicallyto treat acid-related disorders. The consequent inhibition of gastric acid secretion

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Figure 5. (A) Histidine decarboxylase (HDC) activity and numbers of (B) histamine-, (C)pancreastatin- and (D) vesicle monoamine transporter type 2 (VMAT2)-immunostaining cells inthe oxyntic mucosa of wild-type versus CCK2 receptor knockout mice. Means § SEM (n D 6),¤¤¤P < 0:001; NS, not signi� cant. For the original data, see Chen et al., 2002.

Figure 6. Electron micrographs showing (A) well differentiatedECL cell from wild-type mouse and(B) endocrine-likecell (ECL-cell replacement) from CCK2 receptor knockout mouse. For details, seetext. Bars D 400 nm.

ECL cells and CCK2 receptor 359

Figure 7. Plasma gastrin concentration in rats treated with vehicle, YF 476, omeprazole or YF476 Comeprazole for 8 weeks. Means § SEM (n D 8), ¤¤¤P < 0:001; NS, not signi� cant. For the originaldata, see Chen et al., 2000.

abolishes the luminal acid feedback inhibition of the antral G cells and leadsto hypergastrinemia, which in turn stimulates the ECL cells, resulting � rst inhyperactivity and hypertrophy (due to an increased protein synthesis) and then ingeneral diffuse hyperplasia (due to an accelerated self-replication rate) (Håkansonand Sundler, 1990; Håkanson et al., 1993; Tielemans et al., 1990b). Indeed, gastriccarcinoids, i.e. ECL-cell tumours or ECLomas, are known to develop in the murinestomach after long-term treatment with drugs that cause sustained inhibition of acidsecretion (Havu, 1986; Havu et al., 1990).

CCK2 receptor antagonists, such as the benzodiazepine YF476, have made it pos-sible to examine the role of the CCK2 receptor in the proliferation of ECL cells(Chen et al., 2000). Treatment with YF476 raised the serum gastrin concentrationmuch like omeprazole (Fig. 7), probably because of acid inhibition. The numericaldensity of ECL cells was determined by immunocytochemistry. When stained forHDC, the density of visible ECL cells was increased by 8-weeks’ treatment withomeprazole and greatly reduced by YF476 alone or by the combination of omepra-zole and YF476 (Fig. 8A). The changes in the density of HDC-immunoreactivecells paralleled those in the HDC activity (Chen et al., 2000). When stained for his-tamine, the density of the ECL cells was increased by omeprazole, slightly reducedby YF476 and unchanged by the combination of omeprazole and YF476 (Fig. 8B).When stained for pancreastatin and VMAT2, omeprazole increased the density ofimmunoreactive cells, while YF476 alone and the combination of omeprazole andYF476 were without effect (Figs 8C, 8D). Thus, long-standing CCK2 receptorblockade reduced the apparent density of ECL-cell pro� les only marginally, if atall, and prevented the omeprazole-induced ECL-cell hyperplasia, supporting the

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Figure 8. Numbers of (A) histidine decarboxylase (HDC)-, (B) histamine-, (C) pancreastatin- and(D) vesicle monoamine transporter type 2 (VMAT2)-immunreactive cells in the oxyntic mucosa ofrats treated with vehicle, YF 476, omeprazole or YF476 C omeprazole for 8 weeks. Means § SEM(n D 8), ¤p < 0:05, ¤¤¤p < 0:001; NS, not signi� cant. For the original data, see Chen et al., 2000.

view that CCK2 receptors are responsible for gastrin-evoked proliferation of ECLcells but not for maintaining pre-existing ECL cells.

5. CONCLUDING REMARKS

ECL cells in the oxyntic mucosa of the mouse and rat stomach express CCK2

receptors. The importance of the CCK2 receptor for the ECL cells can be clari� edby comparing receptor knockout mice with rats under pharmacological CCK2

receptor blockade. Upon CCK2 receptor blockade (for 8 weeks), the ECL cellsare unchanged in number but small and inactive. Ultrastructurally, they remainrecognizable as ECL cells. In CCK2 receptor knockout mice, typical ECL cellsdisappear from the stomach only to be replaced by a novel endocrine-like cell type,lacking histamine and HDC and having an ultrastructure that differs from that of

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Figure 9. Schematic drawing illustrating that the differentiationof ECL cell and the novel endocrinecell (left part) and proliferation of ECL cells (right part) depend on the CCK2 receptors. It is notknown if the precursor cell is a stem cell.

wild-type ECL cells by the virtual lack of secretory vesicles. The number of suchcells equals the number of ECL cells in wild-type mice. Hence, the basal self-replication rate of these cells seems to be independent of gastrin and of its receptor.However, the CCK2 receptor is needed to ensure the proper differentiation of theECL cells and their ability to manufacture histamine (Fig. 9).

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