C.Y. Wu-Wang

Decreased levels of salivary prostaglandin E2 and epidermal growth factor in recurrent aphthous stomatitis

Prostaglandin E2 and epidermal growth factor are two important cytoprotective compounds in saliva. This study investigated their salivary levels in controls and individuals with minor recurrent aphthous stomatitis. The development of recurrent aphthous stomatitis was divided into three stages: (1) early active stage (mucosal redness); (2) active stage (mucosal ulceration); (3) convalescent stage. Unstimulated mixed saliva was collected from each volunteer. Salivary prostaglandin E2 and epidermal growth factor concentrations were determined by radioimmunoassay. Their levels (mean +/- SEM) were significantly lower during the active stage of ulceration as compared to the control: (a) for prostaglandin E2, 200 +/- 55 versus 73 +/- 11 pg/mg salivary protein (p < 0.01), 447 +/- 123 versus 112 +/- 19 pg/ml saliva (p < 0.01), 215 +/- 30 versus 63 +/- 12 pg/min salivary flow (p < 0.01), control (n = 12) versus active stage (n = 15); (b) for epidermal growth factor, 1.09 +/- 0.17 versus 0.67 +/- 0.17 ng/mg salivary protein (p < 0.05); 2.51 +/- 0.53 versus 0.84 +/- 0.19 pg/ml saliva (p < 0.05), 1.24 +/- 0.26 versus 0.41 +/- 0.09 pg/min salivary flow (p < 0.05), control (n = 12) versus active stage (n = 12). Salivary prostaglandin E2 and epidermal growth factor showed stage-dependent alterations during the development of the stomatitis. The prostaglandin E2 concentration decreased significantly during the active stage of ulceration, and then increased significantly during the convalescent stage. However, the recovery of salivary epidermal growth factor after the ulceration was slower than that of the prostaglandin E2. It is suggested that the diminution of prostaglandin E2 and epidermal growth factor in the saliva may be associated with the ulcer development.

Characterization of the Epidermal Growth Factor Receptor in the Gastric Mucosa

The effects of epidermal growth factor (EGF), a potent mitogen involved in mucosal protection, are mediated by specific cellular receptors. Here, we present the characteristics and binding properties of EGF receptors in the gastric mucosa. The studies were conducted using cell membranes isolated by subcellular fractionation of rat stomach mucosal scrapings. Specific binding of [125I]-EGF to the membrane preparations was assessed at room temperature for various periods of time and at different pHs. The results showed that the binding was proportional to the incubation time up to 1 h and was not affected by a pH change between 4.0 and 7.0. Scatchard analysis of the binding data infer the presence of 2 binding sites, one of high affinity (Kd = 1.34 nM, Bmax = 34 fmol/mg protein) and the other of low affinity (Kd = 484 nM, Bmax = 2.29 pmol/mg protein). Cross-linking experiments using disuccinimidyl suberate to link the [125I]-EGF to gastric membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed that the major receptor for EGF was a protein of 170 kilodaltons. When the solubilized membranes were subjected to wheat germ agglutinin affinity chromatography, the purified material was found to act as substrate for EGF-stimulated phosphorylation. The major component which was labeled by the [gamma-32P]-ATP was also found to be a 170-kilodalton protein. The data are the first to provide evidence that the gastric mucosa possesses a functional EGF receptor and describe its binding characteristics.

Effect of cigarette smoking on salivary epidermal growth factor (EGF) and EGF receptor in human buccal mucosa

The mouth acts as a primary target for cigarette smoke which is associated with several oral diseases and cancer. The present study investigated the effect of cigarette smoking on salivary EGF and the buccal EGF receptor. Samples of whole saliva and buccal biopsy were obtained from 15 healthy volunteers (10 smokers and 5 non-smokers). The smokers smoked 20 or more cigarettes/day for more than 5 years. Salivary cotinine (a major metabolite of nicotine) was determined by radioimmunoassay (RIA). The salivary cotinine level was consistent with the self-reported smoking status (smokers, 106-530 ng/ml saliva; non-smokers, < 2 ng/ml saliva). As compared to the non-smokers, the salivary EGF concentration (determined by RIA) was 32% lower in those smokers whose salivary cotinine level was 250 ng/ml or higher (non-smokers, 2.21 +/- 0.16; smokers, 1.57 +/- 0.09 ng/ml saliva; mean +/- S.E.M., P < 0.01). There was no significant difference in 125I-labeled EGF binding to the buccal receptor between the two groups. However, EGF stimulated the autophosphorylation of a 170-kDa protein band in the sample of non-smokers, but not in the smokers. The immunoblot analysis using anti-EGF receptor antibody indicated that the smoking-related deficiency in EGF receptor autophosphorylation was due to the functional alteration of the receptor proteins. In conclusion, cigarette smoking reduces the salivary EGF level and impairs the function of buccal EGF receptor, which may be associated with the pathology of smoking-related oral disease.

Impairment by ethanol of prostaglandin production in rat salivary glands

Sublingual salivary acini and submandibular tissue were incubated in DMEM medium in the presence of various concentrations (0-5%) of ethanol and the content of the three major prostaglandins, PGE2, PGF2 alpha and 6-keto-PGF1 alpha, were determined by radioimmunoassay. In In the sublingual gland, ethanol caused a decrease in PGE2 and PGF2 alpha levels, but had no effect on 6-keto-PGF1 alpha, while all three prostaglandins were affected in the submandibular gland. At 2.5% ethanol, the production of PGF2 alpha and PGE2 in sublingual gland decreased by 10% and reached maximum inhibition at 5% ethanol, at which concentration there was a 20 and 30% decrease in their levels. In submandibular gland, 2.5% ethanol caused a 20% decrease in PGE2, 30% in PGF2 alpha and 50% in 6-keto-PGF1 alpha; 40% inhibition in PGE2, 57% in 6-keto-PGF1 alpha and 65% in PGF2 alpha occurred in the presence of 5% ethanol. These findings suggest that alcohol impairs the function of salivary glands by inhibiting prostaglandin production.

Identification of epidermal growth factor receptor in human buccal mucosa

EGF receptor was identified and its binding characteristics were determined. Buccal mucosa was obtained from 12 healthy volunteers (6 males and 6 females) and assayed individually for [125I]-EGF binding. The specific binding of [125I]-EGF to the receptor ranged from 2.85 to 6.12 fmol/mg protein. There was no significant difference in binding between male and female (4.31 +/- 0.61 versus 3.94 +/- 0.53 fmol/mg protein; mean +/- SEM). Individual tissue homogenates were pooled for Scatchard analysis and cross-linking experiments. Scatchard analysis produced curvilinear plots with a Kd of 0.71 nM and Bmax of 0.024 pmol/mg protein for the high-affinity binding sites, and Kd of 435 nM and Bmax of 9.92 pmol/mg protein for the low-affinity binding sites. To determine the molecular weight of the EGF receptor, the [125I]-EGF and receptor complex were cross-linked by DSS and subjected to SDS-PAGE. The autoradiogram of the gel revealed one major protein band of 160K and a minor band of 170 K, characteristics shared with the EGF receptors in other tissues. The study is thought to be the first to demonstrate the presence of the EGF receptor in human buccal tissue and to show its biochemical features.

Cigarette smoking reduces human salivary eicosanoids.

The effect of cigarette smoking on salivary eicosanoid levels was investigated in 10 smoker and 10 non-smoker volunteers. The smokers consumed an average of 20 cigarettes/day for the past 5 years or longer. The smoking status was validated by salivary cotinine level. Eicosanoids were extracted from saliva with ethanol, and the radioimmunoassay was performed to determine the concentrations of four major eicosanoids, i.e. prostaglandin E2 (PGE2), PGF2 alpha, 6-sulphidopeptide-containing leukotrienes (LTs) and 12-hydroxyeicosatetraenoic acid (12-HETE). The levels of PGE2, PGF2 alpha, and LTs were significantly lower in the saliva of smokers as compared to that of the non-smokers (1.74 +/- 0.32 vs 2.41 +/- 0.64, p = 0.006; 0.36 +/- 0.12 vs 0.54 +/- 0.18, p = 0.04; 2.24 +/- 0.96 vs 4.92 +/- 1.29, p = 0.006; mean +/- SD, ng/ml saliva). No significant differences were found in the levels of 12-HETE between the two groups. The results suggest that cigarette smoking reduces the concentrations of both the cyclooxygenase and 5-lipoxygenase products in saliva.

Chronic Ethanol Feeding Alters the Structure and Function of the Epidermal Growth Factor Receptor in Rat Stomach

We investigated the effect of chronic ethanol feeding on the EGF receptor in rat stomach. Adult male rats were fed either an isocaloric control or ethanol (EtOH)-containing liquid diet (36% total calories as EtOH) for 4 weeks EtOH significantly reduced the specific binding of 125I-EGF to the gastric mucosal membrane (control vs. EtOH, 2.07 +/- 0.2 vs. 0.94 +/- 0.16 fmol/mg protein; p < 0.01). Scatchard analysis suggested that the lower binding might be due to the reduction of EGF receptor number, and/or the affinity of the high-affinity binding site. Western blot analysis, using anti-EGF receptor antibody, revealed four immunoreactive protein bands (180, 150, 60, and 50 kDa) in the lectin-purified gastric membrane prepared from both groups. However, the intensities of these protein bands in the EtOH-fed animals were 90% lower compared to the controls. In the EGF-responsive protein kinase assay, 32P-ATP was incubated with lectin-purified samples in the absence or presence of 1 microM EGF. EGF stimulated autophosphorylation of the EGF receptor (180 kDa) in stomach from the control groups, but not the EtOH-fed animals. This EtOH-related alteration of the gastric EGF receptor may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.

Prostaglandin E2 receptor of rat submandibular salivary glands

The binding characteristics of the PGE2 receptor were investigated in membrane preparations from these glands. Specific [3H]PGE2 binding was linear as a function of the membrane protein concentration and reached steady state by 40 min of incubation at 37 degrees C under neutral pH. Scatchard analysis of the binding data produced a curvilinear plot with a Kd of 0.18 nM and Bmax of 1.02 fmol/mg protein for the high-affinity binding sites, and a Kd of 181 nM and Bmax of 5.72 pmol/mg protein for the low-affinity binding sites. A competitive displacement study indicated that the receptor was specific for prostaglandins of the E series. The study is the first to demonstrate the presence of the PGE2 receptor in rat submandibular gland and to provide its biochemical features.

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.

Effect of acute ethanol treatment on epidermal growth factor receptor in the rat stomach

The present study investigated the effect of acute ethanol (ETOH) treatment on the epidermal growth factor (EGF) receptor in rat gastric mucosa using two different experimental models. In the in vitro experiments, gastric mucosal cells were incubated with 0, 0.05, 0.1, 0.25, and 0.5% ethanol in Dulbeccos modified Eagles medium (DMEM) for 30 min and then used for the membrane preparation. The EGF receptor binding assay indicated that cells incubated in the presence of ethanol displayed a concentration-dependent increase (r = 0.85) in the 125I-EGF binding. The Western blot analysis using anti-EGF-receptor antibody revealed that ethanol in vitro caused reduction in the immunoreactivity of the major 170-kDa protein. There were also alterations in the minor protein bands (140, 120, and 50 kDa). In the in vivo experiments, rats that fasted overnight were given 1.0 ml of saline or ethanol (5, 10, or 15%) by gastric intubation 30 min before sacrifice. In comparison with the saline controls, ethanol treatment caused a decrease of the EGF receptor binding to the gastric mucosal membrane (saline: 5%: 10%; 15% ETOH, 1.46 +/- 0.18: 1.13 +/- 0.17: 1.27 +/- 0.19: 0.84 +/- 0.14, p < 0.02; mean +/- SEM, n = 9). Furthermore, the immunoblot analysis revealed concentration-dependent decrease in the intensity of the major 170-kDa protein with ethanol. The reduction in the EGF receptor binding and the impairment of the receptor protein might be due to the nonspecific damage of the gastric mucosal membrane by ethanol.