Epidermal growth factor modulates pepsinogen secretion in guinea pig gastric chief cells
Abstract
BACKGROUND & AIMS: Although epidermal growth factor (EGF) inhibits gastric acid secretion, the effects it exerts on gastric chief cells are unknown. The aim of this study was to investigate whether EGF modulates pepsinogen release and intracellular Ca2+ concentrations ([Ca2+]i) and whether the effect involves mitogen-activated protein (MAP) kinase, eicosanoid generation, and nitric oxide. METHODS: Chief cells were obtained by sequential digestion with collagenase and Ca2+ chelation. [Ca2+]i was measured in cells loaded with Fura-2 and NO generation by the NO coproduct citrulline. RESULTS: In situ hybridization, immunohistochemistry, and immunoblotting showed that EGF receptor and MAP kinases were constitutively expressed in chief cells. EGF caused a concentration-dependent stimulation of pepsinogen secretion and MAP kinase activity and determined a 2.5-7.0-fold increase in [Ca2+]i, inositol 1,4,5-tryphosphate, prostaglandin E2, and leukotriene B4. Tyrosine kinase inhibitors and cyclooxygenase and lipoxygenase inhibitors reduced pepsinogen secretion and eicosanoid generation induced by EGF. EGF increased citrulline generation and guanosine 3',5'-cyclic monophosphate accumulation sixfold; the effect was blocked by NG monomethyl-L-arginine, which is an NO synthase inhibitor. CONCLUSIONS: EGF stimulates pepsinogen secretion by activating eicosanoid generation, tyrosine kinases, MAP kinases, Ca2+, NO, and guanosine 3',5'-cyclic monophosphate. (Gastroenterology 1996 Oct;111(4):945-58)
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Role of 5-lipoxygenase pathway in the regulation of RAW 264.7 macrophage proliferation
2006, Biochemical PharmacologyCitation Excerpt :The interaction of growth factors with their cell surface receptors leads to multiple signalling events including the activation of several phospholipases, which in turn can lead to the release of AA and the production of AA metabolites. Thus, epidermal growth factor (EGF) induces LOX metabolism in A431 cells, HeLa cells and fibroblasts [8], and LTB4 synthesis in guinea pig gastric chief cells [9]. LOX metabolites are also involved in basic fibroblast growth factor-induced endothelial cell proliferation [10].
Arachidonic acid (AA) metabolites control cell proliferation, among other physiologic functions. RAW 264.7 macrophages can metabolise AA through the cyclooxygenase and lipoxygenase (LOX) pathways. We aimed to study the role of AA-metabolites derived from 5-LOX in the control of RAW 264.7 macrophage growth. Our results show that zileuton, a specific 5-LOX inhibitor, and nordihydroguaiaretic acid (NDGA), a non-specific LOX inhibitor, inhibit cell proliferation and [3H]-thymidine incorporation in a concentration-dependent fashion. Growth inhibition induced by NDGA can be explained by an apoptotic process, while zileuton does not seem to induce apoptosis. Moreover, these treatments delay the cell cycle, as analysed by flow cytometry. On the other hand, the leukotriene (LT) B4 receptor antagonist U-75302, the LTD4 receptor antagonists LY-171883 and MK-571, and the cysteinyl-LT receptor antagonist REV-5901 also inhibit cell proliferation and [3H]-thymidine incorporation in a concentration-dependent manner, and delay the RAW 264.7 cell cycle. However, these antagonists did not induce annexin V staining, caspase activation or DNA fragmentation. Furthermore, we demonstrated that exogenous addition of LTB4 or LTD4 revert the cell growth inhibition induced by zileuton or the leukotriene receptor antagonists mentioned above. Finally, we observed that LTB4 and LTD4, in the absence of growth factors, have pro-proliferative effects on macrophages, and we obtained preliminary evidences that this effect could be through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, our results show that the interaction between LTB4 and LTD4 with its respective receptor is involved in the control of RAW 264.7 macrophage growth.
Prostanoids and prostanoid receptors in signal transduction
2004, International Journal of Biochemistry and Cell BiologyProstanoids are arachidonic acid metabolites and are generally accepted to play pivotal functions in amongst others inflammation, platelet aggregation, and vasoconstriction/relaxation. Inhibition of their production with, for instance, aspirin has been used for over a century to combat a large variety of pathophysiological processes, with great clinical success. Hence, the cellular changes induced by prostanoids have been subject to an intensive research effort and especially prostanoid-dependent signal transduction has been extensively studied.
In this review, we discuss the impact of the five basic prostanoids, TxA2, PGF2α, PGE2, PGI2, and PGD2, via their receptors on cellular physiology. These inflammatory lipids may stimulate serpentine plasma membrane-localized receptors, which in turn affect major signaling pathways, such as the MAP kinase pathway and the protein kinase A pathway, finally resulting in altered cellular physiology. In addition, prostanoids may activate the PPARγ members of the steroid/thyroid family of nuclear hormone receptors, which act as transcription factors and may thus directly influence gene transcription. Finally, evidence exists that prostanoids act as second messengers downstream of mitogen receptor activation, mediating events, such as cytoskeletal changes, maybe via direct interaction with GTPase activating proteins. The final cellular reaction to prostaglandin stimulation will most likely depend on combined effects of the above-mentioned levels of interaction between prostaglandins and their cellular receptors.
The mucin biosynthesis stimulated by epidermal growth factor occurs in surface mucus cells, but not in gland mucus cells, of rat stomach
2000, Life SciencesAlthough epidermal growth factor (EGF) accelerates gastric mucin biosynthesis, information on whether its activation is limited to the specific mucus-producing cells is lacking. In this paper, we investigated the effects of EGF on mucin biosynthesis and the expression of its receptor in distinct layers of rat gastric mucosa, including the possible participation of nitric oxide (NO). EGF enhanced the incorporation of [3H]glucosamine and [14C]threonine into the mucin in the full-thickness tissues of the gastric mucosa. This stimulation disappeared on the removal treatment of the surface mucosal layer chiefly consisting of surface mucus cells. The EGF-induced increase in [3H]-labeled mucin in the full-thickness mucosa was not suppressed by either NG-nitro-l-arginine (10−5 M) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (10−5 M). The EGF-receptor-mRNA expression was high in the surface mucosal layer but low in the deep and muscle layers of the stomach. These results suggest that EGF-induced stimulation of mucin biosynthesis is limited to the surface mucus cells of the rat gastric mucosa and is independent of the NO pathway.
Pepsinogens: Physiology, pharmacology pathophysiology and exercise
2000, Pharmacological ResearchHuman gastric mucosa contains aspartic proteinases that can be separated electrophoretically on the basis of their physical properties into two major groups: Pepsinogen I (PGA, PGI); and Pepsinogen II (PGC, PGII). Pepsinogens consist of a single polypeptide chain with molecular weight of approximately 42"000 Da. Pepsinogens are mainly synthesized and secreted by the gastric chief cells of the human stomach before being converted into the proteolytic enzyme pepsin, which is crucial for the digestive processes in the stomach. Pepsinogen synthesis and secretion are regulated by positive and negative feed-back mechanisms. In the resting state pepsinogens are stored in granules, which inhibit further synthesis. After appropriate physiological or external chemical stimuli, pepsinogens are secreted in the stomach lumen where hydrochloric acid, secreted by the parietal cells, converts them into the corresponding active enzyme pepsins. The stimulus-secreting coupling mechanisms of pepsinogens appear to include at least two major pathways: one involving cAMP as a mediator, the other involving modification of intracellular Ca2+concentration. Physiological or external chemical stimuli acting through the intracellular metabolic adenyl cyclase are more effective in inducing ` de novo ' pepsinogen synthesis than those acting through intracellular Ca2+. The activation of protein kinase C (PK-C) would appear to be involved in regulatory processes. The measurement of pepsinogens A and C in the serum is considered to be one of the non-invasive biochemical markers for monitoring peptic secretion and obtaining information on the gastric mucosa status of healthy subjects. Recently, pepsinogen measurements have been used as an effective biochemical method for evaluating and monitoring patients with gastrointestinal diseases and for checking the effects of drug treatment. The level of PGA in the serum is always high in normal gastritis, while in atrophic gastritis it is always low. In both cases the PGC level in the serum is high. In most gastrointestinal pathologies the ratio between the PGA/PGC decreases. Various reports concerning hormone and/or enzyme modification as well as gastrointestinal distress in the case of long distance exercise have been reported. It has been suggested that the origin of the gastrointestinal distress experienced by long distance runners is a transient ischaemia of the gastric mucosa; it is also suggested that a hypobaric–hypoxic environment could contribute to induce gastric mucosa necrosis. Interrelation between gastrointestinal distress, hypobaric–hypoxic environment and modifications of PGA and PGC, gastrin and cortisol was evaluated in 13 athletes after a marathon performed at 4300 m. Gastrointestinal symptoms occurred in approximately 40% of the athletes. After the race the athletes showed a significant increase of gastrin and cortisol, while the ratio between PGA/PGC decreased. No relationship was observed between gastrointestinal symptoms and hormonal changes after the race. A control group of five subjects, who had been exposed to the same environmental conditions, showed no gastrointestinal or hormonal alteration. Conversely, control subjects presented a significant decrease of cortisol related to the circadian rhythm. The same incidence of gastrointestinal symptoms at high altitude and at sea level and the absence of pathological alteration of PGA and PGC in the serum of the athletes indicates that running a marathon and living for 6 days at 4300 m does not induce gastric mucosa necrosis. Cortisol and gastrin alteration observed in the athletes at this altitude would seem to be related to an activation of the mesopontine and forebrain structures involved in the behavioural and metabolic integration of the autonomic control and arousal and psychophysical-exercise stress. 2000 Academic Press@p$hr
A new primary culture system representative of the human gastric epithelium
1999, Experimental Cell ResearchThe gastric pit-gland unit is a highly dynamic and compartimentalized structure which assumes important key functions such as acid secretion, digestion of dietary proteins and triglycerides, protection, and epithelial restitution following injury. However, in vitro models representative of the intact gastric epithelium are still lacking. The current study was undertaken to investigate the possibility of generating such primary cultures from human fetal stomach. The use of Matrisperse, a nonenzymatic solution, allowed complete dissociation of the epithelial layer and the maintenance for at least 7 days of all gastric epithelial cell types in primary culture on plastic. Indirect immunofluorescence and Western blot analyses confirmed the purity of epithelial cultures, composed of 60% mucus-secreting cells, 25% zymogenic chief cells, 5% parietal cells, and a small proportion of mitotic precursors. Their functionality was demonstrated by the presence of zonulae occludens and adherens at cell to cell contacts, [3H]thymidine incorporation, Periodic acid Schiff staining, and expression of growth factor receptors (EGF/TGFα, IGF1, HGF, KGF), gastric H+/K+-ATPase, pepsinogen (Pg5), and human gastric lipase (HGL). Chief cells were able to produce and secrete both Pg5 and HGL and to respond to EGF treatment. In conclusion, we developed a new primary culture system of human gastric epithelium characterized for the first time by the absence of added matrix and the maintenance of functional chief cells. It represents an experimental breakthrough that will serve applications in investigating the actions of hormones, mesenchymal growth factors, and basement membrane proteins on human gastric functions in vitro.
Potential involvement of calcium, CaM kinase II, and MAP kinases in PCB- stimulated insulin release from RINm5F cells
1999, Toxicology and Applied PharmacologyPolychlorinated biphenyls (PCBs) are environmental contaminants that induce release of insulin in rat insulinoma cells, RINm5F (Fischer et al., Life Sci. (1996) 59, 2041–2049). In the present study the mechanisms of this effect were investigated using noncytotoxic concentrations (10 μg/ml) of a PCB mixture, Aroclor-1254, and the pure PCB congeners 2,2′,4,4′-tetrachlorobiphenyl and 2,2′,4,4′,5,5′-hexachlorobiphenyl. Treatment of RINm5F cells with each of these agents resulted in a rapid increase in intracellular free calcium. The presence of extracellular calcium was required for PCB-induced insulin release because removal of calcium from the medium attenuated the effect. In addition, pretreatment of RINm5F cells with the calcium channel blocker verapamil also blocked PCB-induced insulin release. To determine whether PCB-related insulin release could be associated with the enzyme, calcium/calmodulin-dependent kinase II (CaM kinase II), RINm5F cells were pretreated with the CaM kinase II inhibitor KN-93. PCB-induced insulin release was completely blocked by KN-93. Under similar treatment conditions, PCBs also induced the activity of mitogen-activated protein kinases (MAPK) 1 and 2. However, inhibition of MAPK activation by a specific inhibitor, PD-98059 (10.0 μM) did not prevent insulin release induced by PCBs. The results of the present investigation suggest a role for calcium and CaM kinase II in PCB-induced insulin release. Furthermore, the results suggest that insulin release by PCBs is independent of the activation of MAPKs.