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 The Esophageal
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Volume: The Esophagogastric Junction
Chapter: Esophageal columnar metaplasia (Barrett s esophagus)

What is the effect of epidermal growth factor on parietal cell function?

J. Del Valle (Ann Arbor)

Peptide growth factors play a central role in maintaining the integrity of the gastrointestinal tract. The past few decades have witnessed a steady increase in the number of proteins which regulate cell growth and proliferation [1]. These factors have been classified into families based on their similarities in structure and function. One such group is the epidermal growth factor (EGF) family. The members of this group include EGF, transforming growth factor a (TGFa), amphiregulin, heparin binding EGF (HB-EGF), pox virus growth factors, cripto and heregulin. This review will focus on the effect of EGF and TGFa on gastric parietal cell function.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGFa) are naturally-occurring homologous peptides which are present throughout the gastrointestinal tract [2, 3]. In addition to the well known effects of EGF on tissue proliferation, early studies using this growth factor showed that it could inhibit acid secretion [4]. The presence of EGF in submandibular glands and saliva, led to the hypothesis that it was a luminally-active acid-inhibitory agent. Contrary to this theory was the observation that the inhibitory effect of EGF on acid secretion in vivo was greater when administered by an intravenous rather than a luminal route [5]. Moreover, EGF-receptors were localized to the basolateral surface of parietal cells only, thus suggesting a blood-bourne or paracrine route of action [6]. However, an endocrine role for EGF as an acid inhibitor seemed unlikely since the measured plasma levels of EGF were substantially below the half maximal dose required for acid inhibition in rat [7]. These discordant data on the physiological role of EGF on acid secretion were rationalized by the discovery of TGFa. TGFa shares 30% amino acid homology with EGF, and is an agonist at the EGF-receptor [3, 8]. It can be deduced that TGFa is the natural ligand for the EGF-receptor on the parietal cell since concentrations of TGFa in gastric mucosa exceeds that of EGF by twentyfold [2]. Furthermore, TGFa and EGF-receptor mRNA are expressed abundantly in gastric parietal cells [9]. Both EGF and TGFa inhibit histamine-stimulated gastric acid secretion in vitro [5,10], thus, it is possible that TGFa acts in an autocrine regulatory loop.

In addition to the documented ability of these growth factors to inhibit gastric acid secretion directly via an EGF receptor located on parietal cells [7, 11, 12], Chew et al. have demonstrated that chronic exposure of parietal cells to EGF/TGFa can enhance secretagogue stimulated activity [13]. The mechanism by which these growth factors regulate parietal cell function has received much attention. We and others have attempted to characterize the mechanisms by which EGF/TGFa inhibit parietal cell function. The following is a brief overview of our studies examining the effect of EGF and TGFa on isolated parietal cell activity.

Effect of EGF and TGFa on histamine-stimulated parietal cell activity

We initially examined the effect of EGF and TGFa on basal- and histamine-stimulated parietal cell activity [14]. We utilized as our model, isolated enriched rabbit parietal cells prepared using well established methods.

Neither EGF nor TGFa (10-10-10-6M) altered basal parietal cell activity at the time intervals tested. Contrary to this, both growth factors dose dependently inhibited histamine (10-5M) stimulated parietal cell activity with similar IC5O'S (TGFa = 2.5 ± 0.32 x 10-9M; EGF = 1.75 ± 0.45 x10-9M) (Figure 1). EGF/TGFa mediated inhibition of parietal action was also time-dependent.

The EGF receptor is composed of a single polypeptide chain which contains a cytoplasmic domain that encodes an EGF-regulated tyrosine kinase which leads to phosphorylation of a large number of cellular proteins after activation [5]. The histamine H2 receptor has several potential phosphorylation sites at the carboxyl terminal portion [11], which in turn may serve to regulate receptor function once phosphorylated. Despite these interesting observations, neither EGF nor TGFa altered the affinity of the histamine H2 receptor for its ligand. Our data suggests that the inhibitory action of these growth factors involves a mechanism that is not coupled to the short term regulation of secretagogue receptor affinity.

Figure 1. Effect of EGF and TGFa on histamine (10-5M) stimulated aminopyrine accumulation.

Figure 2. Effect of EGF and TGFa on forskolin (10-5M) stimulated aminopyrine accumulation.

Next we examined the effects of EGF and TGFa on various levels of the second messenger system through which histamine exerts its stimulatory action (adenylate cyclase). First we investigated the effect of EGF and TGFa on forskolin-stimulated AP-uptake (Figure 2). Contrary to several previous reports, we observed that both growth factors inhibited the action of forskolin with similar potency, efficacy and time course of action [14, 15]. This observation suggests that the post receptor mechanism mediating the inhibitory action of EGF and TGFa on parietal cell function may be coupled to the enzyme which stimulates formation of cAMP in response to histamine exposure. This hypothesis is supported by the ability of both EGF and TGFa to inhibit forskolin and histamine mediated generation of cAMP (Figure 3). We next examined whether these growth factors could inhibit parietal cell function at a level distal to the synthesis of cAMP and observed that neither EGF nor TGFa significantly reversed DbcAMP mediated aminopyrine accumulation. Finally, we investigated whether the inhibitory effect of EGF and TGFa on histamine-stimulated parietal cell action was coupled to a guanine nucleotide binding protein [16] and observed that preincubation of parietal cells with pertussis toxin (PT) significantly reversed the inhibitory actions of both EGF and TGFa (Figure 4). These data suggest that both peptides have similar post receptor transduction events involving the activation of a PT-sensitive GTP binding protein.

Figure 3. Effect of EGF (E, 10-8M) and TGFa (T, 10-8M) on histamine (H, 10-5M) and forskolin (F, 10-5M) stimulated cAMP formation.

Figure 4. Effect of pertussin toxin (PT, 200 ng/ml) on EGF (E, 10-8M) and TGFa (T, 10-8M) mediated inhibition of histamine (10-5M) stimulated aminopyrine accumulation.

Effect of EGF and TGFa on carbachol-stimulated parietal cell activity

Although some have not demonstrated that EGF can inhibit carbachol-stimulated parietal cell activity [7], several studies have confirmed that the action of this secretagogue can be blocked by EGF [11, 13]. Despite this observation, the mechanism by which EGF inhibits carbachol-stimulated parietal cell activity remains unclear. We addressed this issue by examining the effect of EGF on carbachol stimulated isolated canine parietal cells [14]. We also explored whether EGF's action on parietal cell activity involved regulation of the protein kinase C (PKC) pathway.

Consistent with studies performed in other models such as rabbit parietal cells, we demonstrated that EGF could inhibit carbachol-stimulated parietal cell activity (Figure 5). EGF's inhibitory effect was dose and time dependent. Contrary to the effect of EGF on histamine stimulated parietal cell activity, the inhibitory action of EGF on carbachol was not sensitive to pertussis toxin.

We next examined whether EGF inhibited the ability of muscarinic receptors to activate the phosphoinositide [Ca2+]i signaling system. Consistent with previously published studies [11, 13], EGF did not inhibit the ability of carbachol to increase [Ca2+]i or IP3 generation in parietal cells. These observations suggest that EGF's action is independent and distal to the linkage of the cholinergic receptor to these early signal transduction events.

We sought to examine whether EGF's action on parietal cell activity involved a PKC-dependent mechanism. The rationale for this stems in part from the known inhibitory effect of PKC activation on parietal cells [16-23]. In addition to the ability of EGF to stimulate tyrosine kinase activity, elevate IP3 formation through activation of PLCg and interact with G-proteins, this growth factor has also been shown to activate PKC [24-26]. We explored whether EGF could activate PKC in parietal cells and whether this signaling pathway was involved in EGF's inhibitory action. The PKC inhibitors H-7 [27] (Figure 6) and staurosporine (Figure 7A) reversed EGF's inhibitory effect on carbachol stimulated parietal cell activation. Although H-7 has been considered a specific PKC inhibitor, several studies suggest that at the micromolar concentration range this compound can inhibit other kinases such as the cyclic AMP-dependent protein kinase (PKA). We addressed this possibility in part by examining the effect of the PKA inhibitor H-89 on EGF's inhibitory action
(Figure 7B). Of note, H-89 did not reverse EGF's effect on carbachol stimulated parietal cell activity. Taken together, our antagonist data support the hypothesis that EGF's inhibitory effect on parietal cells is in part mediated via PKC. The mechanism by which PKC inhibits parietal cell function is unclear and is the subject of further investigation.

Figure 5. Effect of EGF on carbachol (10-5M) stimulated aminopyrine accumulation. Pretreatment with pertussis toxin (PTX) did not reverse EGF's inhibitory effect.

Figure 6. Effect of H-7 on EGF-mediated inhibition of carbachol stimulated aminopyrine uptake.

Figure 7. Effect of staurosporine (staur) and H-89 on EGF's inhibitory action.

Although previous studies have demonstrated that EGF can activate PKC in several cell models [24, 26], the ability of this growth factor to regulate this kinase in parietal cells has not been shown. PKC represents a large family of isoenzymes [28] with varying levels of homology and tissue distribution. They are divided into two major groups, the
Ca2+-dependent or conventional PKC's (cPKC) and the Ca2+-independent or novel PKC's (nPKC's). We initially determined that the protein for the two Ca2+ dependent isoforms of PKC
a and b1 are expressed in parietal cells. We did not screen for the g isoform since it is expressed exclusively in brain and spinal cord. Our studies demonstrate that EGF is capable of promoting translocation of PKCa and to a lesser extent, PKCb1 into parietal cell membranes. Although PKC translocation often coincides with kinase activation we assessed whether actual PKC activity was also increased by EGF (Figure 8). Our results confirm that this growth factor can activate PKC in canine parietal cells.

Figure 8. Effect of EGF and TPA (10-6M) on PKC activity in canine gastric parietal cells.


We and others have demonstrated that short term treatment with EGF/TGFa inhibits secretogogue stimulated parietal cell activity in a direct manner. More recent studies [13, 29] have illustrated that long term exposure of parietal cells to EGF can lead to enhanced acid production. EGF receptor mediated stimulation of tyrosine kinase is important for both the inhibitory and stimulatory action of EGF [29, 30]. It also appears that EGF can induce expression of the gastric H+, K+-ATPase a subunit gene via interaction with a novel transcription factor [29].

EGF/TGFa mediated inhibition of parietal cell action involves multiple pathways. The inhibitory effect of these growth factors on histamine stimulated activity involves a pertussis toxin sensitive mechanism. EGF's inhibitory action on carbachol stimulated parietal cell activation does not involve a pertussis toxin dependent GTP binding protein, but does appear to involve activation of PKC. Ostrowski et al. have postulated that EGF inhibits parietal cell activity through yet another pathway by induction of ornithine decarboxylase (ODC) activity and through increased synthesis of polyamines [31]. In summary, EGF and TGFa influence gastric secretion via multiple complex cellular processes.



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Publication date: May 1998 OESO©2015