In normal physiology, the antral hormone gastrin stimulates the secretion of acid and the proliferation of cells of the gastric epithelium. Excess gastrin production is associated with states in which the normal inhibitory mechanisms are lost or suppressed (such as with the use of proton-pump inhibitor drugs) or in which there is a pathological source of gastrin secretion (such as a secretory ‘gastrinoma’ in the Zollinger-Ellison syndrome). The ‘classical’ gastrins are those peptides whose carboxy terminus is amidated and they can also be sulphated at their single tyrosine residue. The various forms of classical gastrins exhibit similar activity at the CCK2 receptor though they vary in half-life.
1.2.1 Biosynthesis and processing
The gastrin hormone is encoded by a single gene located on the long arm of chromosome 17(32). It is primarily synthesised in G-cells of pyloric glands in the gastric antrum though G-cells are also found in duodenal Brunner’s glands(33). The primary precursor – preprogastrin – is synthesised in the endoplasmic reticulum where the N-terminal sequence is cleaved to form progastrin for storage in exocytic vesicles (Figure 1-5). Here, progastrin is further modified by protease cleavage to generate COOH-terminal Gly-extended gastrins (G-Gly). These G-Gly peptides are then acted upon by peptidyl- -amidating mono-oxygenase (PAM) to form COOH- terminal amidated gastrins, the two major forms of which are G17 and G34 with 17 and 34 amino acid residues respectively(34). Gastric acidity inhibits gastrin release via the secretion of somatostatin by antral D-cells in a negative-feedback fashion. G- cells release gastrin in response to stimuli associated with food ingestion. Gastric
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nerves release gastrin-releasing peptide (GRP) and luminal amino acids and calcium ions act at luminal receptors on the G-cell.
Figure 1-5 Biosynthesis and processing of gastrin.
Gastrins are said to belong to a family of peptide hormones that also includes cholecystokinin (CCK) as they both possess the COOH-terminal pentapeptide amide, which confers their biological activity. These peptides act at the CCK1- and CCK2 receptors and whilst CCK exhibits a high affinity for both receptors, gastrin binds to CCK2R with an affinity approximately 100-times that of CCK1(35). After ingestion of a meal, the circulating concentration of gastrin exceeds that of CCK by 5-10 times and so gastrin is thought to be the most important physiological agonist of CCK2R(36). In normal physiological states, gastrin is expressed on gastric parietal and ECL-cells as well as in the pancreas and brain.
1.2.2 Cellular effects
The role of gastrin in the control of gastric acid secretion has been summarised in section 1.1.7.
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The other major role of amidated gastrins in normal physiology is in gastric epithelial cell proliferation – a phenomenon borne out through clinical observations and in numerous laboratory studies. As cellular proliferation is a key step in oncogenesis, this behaviour merits further discussion. Perhaps the best-characterised clinical effect of hypergastrinaemia is the gastric corpus hypertrophy with ECL-cell tumour formation and hyperacidity that is observed in patients with Zollinger-Ellison syndrome. In such patients, resection of the gastrin-secreting gastrinoma results in normalisation of gastric acid secretion and leads to reversal of parietal cell
hyperplasia(37,38).
The trophic effect of gastrin was first described in animal models almost 45 years ago when the augmenting effects of pentagastrin on protein synthesis and parietal cell mass were demonstrated in rats (39,40). This has been studied extensively in other animal models. In mice in which the genes encoding either gastrin or its receptor (CCK2R) are deleted, there are reduced gastric populations of parietal and ECL cells accompanied by hypochlorhydria(41) suggesting that the effect of gastrin is to stimulate gastric stem cell proliferation and to influence stem cell differentiation towards a parietal or ECL-cell fate(42). In contrast, transgenic mice engineered to produce gastrin in pancreatic -cells (INS-GAS) initially exhibit hyperproliferation of gastric epithelium, enhanced populations of parietal and ECL cells and
hyperacidity(34). Interestingly, the same animals later lose parietal cell mass and develop foveolar hyperplasia in a histologically similar fashion to that seen in the human disease of chronic atrophic gastritis. The molecular mechanisms responsible for the proliferative influence of gastrin have also been explored in vitro. MKN-45 cells constitutively expressing CCK2R exhibit diminished proliferation when treated with a CCK2R antagonist(43). The corollary of this is seen with AGS-B cells stably transfected to express CCK2R and treated with gastrin. These cells exhibited more rapid proliferation associated with upregulation of cyclin D1(44).
The thesis that classical gastrin acting via CCK2R is responsible for gastric epithelial proliferation is however complicated by several observations. First, proliferating cells in normal gastric epithelium are not seen to express CCK2R with the exception of
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ECL-cells and (to a lesser degree) parietal cells(45). Additionally, other cell-line studies have illustrated an inhibitory effect of gastrin on proliferation(46,47). Previous studies suggested a paracrine role for gastrin-induced ligands for the epidermal growth factor receptor (EGF-R) such as heparin-binding epidermal growth factor (HB-EGF)(48–50). Our own group examined this by first transfecting gastric cancer derived AGS cells with the CCK2R (AGS-GR). Exposure to gastrin inhibited the
proliferation of these cells but when the same cells were co-cultured with labelled AGS cells (AGS-GFP), exposure to gastrin induced proliferation of the latter, CCK2R deficient cells suggesting paracrine stimulation. The same study identified that the likely mechanism was via gastrin induced shedding of HB-EGF and that this was in- turn mediated by protein kinase C (PKC) dependent matrix metalloproteinase (MMP) activity(51). This mechanism is pertinent to the present study as H. pylori is known to induce hypergastrinaemia, MMP activity and the expression of growth factors
including HB-EGF(48,52–56). Recent work has identified mitogen activated protein kinase 1 interacting protein 1 (MP1) as an essential partner in gastrin-induced phosphorylation of ERK1 and ERK2 and that this is responsible for gastrin-induced proliferation via the mitogen-activated protein kinase (MAP) pathway(57).
These paracrine pathways also appear to be important when considering the effect of gastrin stimulation on cellular migration and invasion – also key steps in
oncogenesis and important when investigating preneoplastic epithelial remodelling. Using the same AGS-GR/AGS-GFP co-culture methodology described above, gastrin
stimulated cell migration both directly (for AGS-GR) and in a paracrine fashion (for
AGS-GFP). This phenomenon was found to be due to MAPK activity via HB-EGF(53). MAPK activity has also been implicated in the induction of MMP-9 expression and the gastrin-stimulated invasion of AGS-GR cells through basement membrane in
vitro(56).
The influence of gastrin on ECL-cell proliferation is one of direct stimulation. In rodents, it has been shown that ECL-cells are capable of self-replication(58) and that gastrin induces ECL cell proliferation in animals and in vitro(25,59). ECL-cell
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reaches a maximum plateau after 20 weeks(60). Pathological hypergastrinaemia in humans is considered as either a) primary - as seen in Zollinger-Ellison syndrome with excess production of gastrin by a secretory tumour or b) secondary – as seen in chronic autoimmune atrophic gastritis (AIG) with oxyntic atrophy, hypochlorhydria and loss of the negative feedback influence of gastric acid. The pathophysiology of the associated type 1 gastric neuroendocrine tumours is discussed in section 1.6.