Characterization of gastric mucosal prostaglandin E2 receptor

1. The binding characteristics of gastric mucosal prostaglandin (PG) E2 (PGE2) receptor were investigated using mucosal cell membranes from rat stomach. The binding was found to be dependent upon PGE2 and membrane protein concentration, the time of incubation and the pH of the mixture, being highest at pH 3.0. 2. Scatchard analysis of the binding data revealed a curvilinear plot with high affinity binding (Kd = 2 nM; Bmax = 0.106 pmol/mg protein) and low affinity binding (Kd = 319 nM; Bmax = 2.262 pmol/mg protein) sites. 3. Competitive displacement study indicated that the receptor was specific for PGs of the E series, as PGF2 alpha and 6-keto-PGF1 alpha failed to displace the PGE2. 4. The study is the first report to provide biochemical parameters of specific PGE receptors in rat gastric mucosa.     

Protective effect of human epidermal growth factor against the experimental gastric mucosal lesions in rats

The protective effect of human epidermal growth factor (hEGF) on the gastric mucosal lesions in rats was examined in relation to the immunoreactive concentration of plasma. Human EGF (30 micrograms/kg) was administered intravenously, intraperitoneally or subcutaneously. At different times following the administration of hEGF, rats received acidified ethanol solution to induce an experimental gastric mucosal lesion. Sum of lesion length in the gastric mucosa was used as a lesion index. Human EGF administered parenterally markedly decreased the gastric mucosal lesions in 10 min after administration of necrotizing solution, and 10 to 30 min delay was observed in the development of maximal protective action. Profiles of protective potency against the hEGF dose administered intraperitoneally or subcutaneously 30 min before administration of necrotizing solution revealed that the effective dose of hEGF (ED50) was about 5.2 and 2.6 micrograms/kg, for intraperitoneal and subcutaneous administrations, respectively.     

Effect of sialoadenectomy on ethanol-induced gastric mucosal damage in the rat: role of neutrophils

We have observed that removal of the salivary glands is associated with an increase in the susceptibility to gastric mucosal damage in the rat. In the present study, we have examined the effect of sialoadenectomy on ethanol-induced mucosal hemorrhagic damage and myeloperoxidase (MPO) activity. Hemorrhagic damage and MPO activity in response to intragastric 50% w/v ethanol were greater in sialoadenectomized rats when compared with sham-operated animals. Pretreatment with 16,16-dimethylprostaglandin E2 (0.3 micrograms/kg s.c.) reduced damage and MPO activity in both sialoadenectomized and sham control rats receiving 50% ethanol. The reduction in these parameters was greater in control than in sialoadenectomized rats. Pretreatment with epidermal growth factor (5 micrograms/kg s.c.) significantly reduced MPO activity but did not significantly affect the extent of damage. These data suggest that sialoadenectomy is associated with an increase in mucosal inflammation in animals given ethanol. However, in some situations tissue inflammation (as indicated by MPO activity) was reduced, while the proportion of gastric mucosa exhibiting hemorrhagic damage was not changed.     

Activation of gastric mucosal calcium channels by epidermal growth factor

• 1.1. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin.
• 2.2. The complex following labeling with [³H]PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active ⁴⁵Ca²⁺ uptake into intravesicular space as evidenced by La³⁺ displacement and osmolarity measurements. The ⁴⁵Ca²⁺ uptake was independent of sodium and potassium gradients indicating the electroneutral nature of the process.
• 3.3. The gastric mucosal channels on epidermal growth factor binding in the presence of ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa subunits of calcium channel.
• 4.4. The phosphorylated channels following reconstitution into vesicles displayed at 48% greater ⁴⁵Ca²⁺ uptake, thus indicating the tyrosine kinase involvement in EGF dependent activation of calcium channel.
• 5.5. The results point towards the importance of epidermal growth factor in the maintenance of gastric mucosal calcium homeostasis.

     

Aging alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha

Administration of pharmacological doses of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) in young rats stimulates gastric mucosal proliferation, but, in aged rats, the same treatment inhibits proliferation. This may be due to enhanced ligand-induced internalization of EGF receptor (EGFR). In support of this, we demonstrated that although a single injection of EGF (10 microg/kg) or TGF-alpha (5 microg/kg) in young (4-6 mo old) rats greatly increased membrane-associated EGFR tyrosine kinase activity, the same treatment slightly inhibited the enzyme activity in aged (24 mo old) rats. This treatment also produced a greater abundance of punctate cytoplasmic EGFR staining in gastric epithelium of aged rats, consistent with EGFR internalization. In vitro analyses demonstrated that exposure of isolated gastric mucosal cells from aged but not young rats to 100 pM TGF-alpha resulted in marked increases in intracellular EGFR tyrosine kinase activity and that induction of EGFR tyrosine kinase activity in mucosal membranes from aged rats occurred at doses 1,000-fold less than those required in young rats. Our data suggest that aging enhances sensitivity of the gastric mucosa to EGFR ligands. This may partly explain EGFR-mediated inhibition of gastric mucosal proliferation in aged rats.     

Possible mechanism of gastric mucosal protection by epidermal growth factor in rats

The mechanism of the protection by human epidermal growth factor (hEGF) against the gastric mucosal lesions induced by acidified ethanol was studied in rats. At different times following the subcutaneous administration of hEGF (30 micrograms/kg), intragastric acidified ethanol (EtOH: 0.125 M HC1 = 50:50 v/v%) was administered to induce an experimental gastric mucosal lesion. Mean length of the lesion in the gastric mucosa was used as a lesion index. Extravasation of intravenously injected Evans blue into the gastric wall and gastric contents was used as an indicator of vascular permeability. Pretreatment with hEGF decreased both the gastric mucosal lesions and the increase of vascular permeability caused by acidified ethanol with similar time profiles relative to pretreatment with hEGF. Maximal protective actions of hEGF occurred about 10 to 30 min after the observed peak plasma concentration of hEGF. Indomethacin and N-ethylmaleimide, but not iodoacetamide, blocked the protective action of hEGF, indicating that endogenous prostaglandins and/or sulfhydryls may participate in the protective action of hEGF. The content of endogenous nonprotein sulfhydryls in the gastric mucosa decreased markedly after acidified ethanol. However, pretreated hEGF did not restore the sulfhydryl contents. Thus, it seemed that endogenous prostaglandins, but not sulfhydryls, are the probable mediators for protection against gastric mucosal injury caused by acidified ethanol.     

Characterization of epidermal growth factor receptor in rat buccal mucosal cells

1. Receptor binding for epidermal growth factor (EGF) in rat buccal mucosa was characterized. Binding of [125I]EGF to rat buccal mucosa was time, temperature, cell number and [125I]EGF concentration dependent. 2. The [125I]EGF binding was reversible and specific. Unlabeled EGF competed for binding to buccal mucosal cells with an IC50 of 1.25 nM, whereas insulin failed to compete. 3. Scatchard analysis of the binding data revealed a curvilinear plot with dissociation constants of 3.39 nM and 2.14 microM, and binding capacities of 1.23 x 10(4) and 3.38 x 10(5) receptors per cell for high and low affinity sites, respectively. 4. Crosslinking of [125I]EGF to buccal mucosa followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major protein with Mw 170,000 which shares similar molecular weight with other known EGF receptors from different tissues and species. 5. The study is the first report to provide biochemical parameters of the specific EGF receptors in rat buccal mucosa.     

The effect of epidermal growth factor on prostaglandin synthesis of oestrogenized rat uterus is mediated by nitric oxide

We examined the possible relationship between cytokines, nitric oxide and prostaglandins (PGs) in the estrogenized rat uterus. Results indicate that epidermal growth factor (EGF) enhances the synthesis of prostaglandins in estrogenized rat uteri and induces the augmentation of nitric oxide (NO) production in this tissue by stimulating iNOS. While the effect of EGF is abolished by L-NMMA, an NO antagonist, the NS-398, a cyclooxygenase-II (COX-II) inhibitor, prevents the augmentation of prostanoids induced by EGF. These results suggest that there is an interaction among EGF, NO and PGs and that in this interrelationship are involved COX-II and iNOS. This mechanism might be important during implantation and labor.     

Effects of sialoadenectomy and epidermal growth factor administration on the duodenum of adult mice.

Sialoadenectomy (removal of the submandibular glands) of adult male and female mice caused significant or apparent decreases in duodenal weight, duodenal protein, DNA and RNA contents, and alkaline phosphatase activity. These decreased levels observed in sialoadenectomized mice were completely restored to or rather increased over the control levels by epidermal growth factor (EGF) administration. There was no appreciable difference in body weight between the control and sialoadenectomized mice. These results strongly suggest that salivary EGF secreted from the submandibular gland plays a role in the maintenance of growth and function(s) of the adult mouse duodenum.     

Inhibition of 15-hydroxy prostaglandin dehydrogenase and increase of prostaglandin E2: effect of sofalcone on rat gastric mucosa

The effect of sofalcone, an anti-ulcer agent, on gastric mucosal prostaglandin (PG) metabolism was studied. Gastric mucosal PGE2 was determined in rats in which PGE2 synthesis was inhibited by preadministration of indomethacin. Oral administration of sofalcone at doses of 200 and 400 mg/kg significantly inhibited the PG metabolizing enzyme, 15-hydroxy-PG-dehydrogenase (15-OH-PG-DH) activity and increased PGE2 contents in the rat gastric mucosa. The inhibition of 15-OH-PG-DH activity was accompanied by an increase of PGE2 contents up to 6 hours after the administration of sofalcone. These changes, however, were not observed 12 hours after its administration. Intraperitoneally administered sofalcone also inhibited 15-OH-PG-DH activity and increased PGE2 content. The inhibition of 15-OH-PG-DH activity by sofalcone was noncompetitive and uncompetitive against substrates NAD and PGE1, respectively. These results suggest that the increase of the gastric PGE2 level is mainly due to the inhibition of 15-OH-PG-DH activity, and this increase in PGE2 may be involved in the anti-ulcer effect of sofalcone.     

Epidermal growth factor and prostaglandin E(2) accelerate mucosal recovery from stress-induced gastric lesions via inhibition of apoptosis.

The repair of damaged gastric mucosa is a complex process involving prostaglandins (PG) and mucosal growth factors such as epidermal growth factor (EGF). Recently, we postulated that the increased occurrence of apoptosis in the gastric epithelium might be of pathophysiological importance in the development of stress lesions. The aim of the present study was to assess the effect of the pretreatment of rats, exposed to 3.5 h of water immersion and restraint stress (WRS), with EGF and PG (16,16 dmPGE(2)) on the number of stress lesions, recovery of gastric mucosa from stress and the expression of apoptosis related genes such as caspase-3 and antiapoptotic bcl-2. Rats were divided in following groups: (1) vehicle; (2) EGF 100 microg/kg i.p.; (3) 16,16 dm-PGE(2) (5 microg/kg i.g.) and caspase-1 inhibitor (ICE-I; 100 microg/kg i.p.). One hour later, the rats were exposed to 3.5 h of WRS and then sacrificed immediately (0 h) or at 6, 12, or 24 h after WRS. The number of acute gastric lesions was determined. Gastric epithelial apoptosis was assessed by TUNEL staining. In addition, mRNA expression of caspase-3, Bcl-2 and proinflammatory cytokines (IL-1 beta, TNFalpha) was assessed by RT-PCR. PGE(2) generation in gastric mucosa and luminal EGF were determined by RIA. Exposure to WRS resulted in the development of multiple acute stress erosions ( approximately 18) which almost completely healed during 24 h. The gastric blood flow was significantly reduced (approximately 70% of intact mucosa) immediately after WRS. The expression of mRNA for IL-1 beta and TNF alpha reached their peak at 12 h after stress exposure. The apoptosis rate was highest at 6 h after WRS and was accompanied by the highest caspase-3 expression. In rats pretreated with EGF or 16,16 dm-PGE(2), a significant decrease in caspase-3 mRNA and upregulation of bcl-2 mRNA as observed as compared to vehicle controls. Caspase-1 inhibitor significantly reduced the number of stress lesions. We conclude that EGF and PGE(2) accelerate healing of stress-induced lesions due to the attenuation of apoptosis via upregulation of bcl-2 in gastric mucosa. Inhibitors of apoptosis accelerate healing of stress lesions and may be potentially effective agents in the healing of damaged gastric mucosa.     

Role of salivary glands and epidermal growth factor (EGF) in gastric secretion and mucosal integrity in rats exposed to stress

EGF, produced mainly by salivary glands, inhibits gastric acid secretion, stimulates the proliferation of gastric mucosal cells and protects the mucosa against various ulcerogens, but its role in the pathogenesis of stress ulcerations is unknown. In this study, rats with intact or resected salivary glands were exposed to water immersion and restraint stress (WRS) without and with pretreatment with exogenous EGF or dimethyl PGE2 (dmPGE2) at doses which were shown previously to protect the mucosa against topical irritants. During 1.5-12 h of WRS, the formation of gastric ulcerations increased progressively with the duration of stress reaching peak after 6 h of stress and being significantly higher in rats with removed salivary glands than in intact animals. Gastric acid secretion and DNA synthesis in oxyntic mucosa declined with the duration of WRS, but after sialoadenectomy a significant increase in gastric acid secretion and a further decline in DNA synthesis were observed after WRS. EGF contents in the gastric lumen and the gastric mucosa were several times higher in rats subjected to stress than in control unstressed animals, indicating that stress causes an extensive release of EGF. Both exogenous EGF (17 nmol/kg/h) and dmPGE2 (143 nmol/kg) prevented, in part, the formation of gastric lesions, while inhibiting gastric acid secretion both in rats with intact or resected salivary glands. We conclude that water immersion and restraint stress is accompanied by an excessive release of EGF, which appears to attenuate gastric secretion, enhances the DNA synthesis and may limit the formation of stress-induced gastric ulcerations.     

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.     

Stimulatory effect of epidermal growth factor on prostaglandin E2 production in mouse fibrosarcoma cell line (HSDM1 C1)

Using HSDM1 C1 cell line derived from the mouse fibrosarcoma which synthesizes and secretes prostaglandin (PG) E2, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator of many tissues, and its effect on PGE2 production by cultured tumor cells were studied. HSDM1 C1 cell line possessed specific, high-affinity receptors for EGF: Kd (5.5 X 10(-10 M) and binding capacity (17,650 sites/cell). EGF significantly stimulated PGE2 production in HSDM1 C1 line cultured in serum-free medium for 24 h in a dose-dependent manner; a 2.5-fold increase over control was induced by as little as 0.1 ng/ml and the maximal effect (3.5-fold increase) by 1 ng/ml. Its stimulatory effect on PGE2 production was completely blocked by indomethacin, an inhibitor of PG biosynthesis. These data suggest that EGF may be involved in modulation of synthesis and/or secretion of PGE2, a potent bone-resorbing factor, by the tumors which may partly contribute to hypercalcemia in certain types of neoplasms.     

Effects of epidermal growth factor on serum zinc and plasma prostaglandin E2 levels of mice with pressure sores

This work was undertaken to study the effects of systemic application of EGF on the plasma PGE2 and serum zinc levels of mice with pressure sores. The pressure sores were made on the hind limb of the mice by bilateral sciatic nerve neurotomy. Mice were injected IP, 1 μg EGF daily for ten days, begining on the 21st day after operation. EGF administration increased the serum zinc and plasma PGE2 levels compared with controls. These results indicate that EGF can be effective on wound healing by elevating the serum zinc and plasma PGE2 concentrations together with other physiological roles in cellular events.     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine,and synergism with epidermal growth factor

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE₂ and PGFF_2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE₂, PGF_2α, PGD₂, and the synthetic analog dimethyl-PGE₂ markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE₂ and PGF_2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE₂ and PGF_2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE₂ and PGF_2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley-Liss, Inc.