Relationship between epidermal growth factor in mouse submandibular glands, plasma, and bile: effects of catecholamines and fasting

The epidermal growth factor (EGF) concentration in bile is high (approximately 150 fold higher than that in plasma), but little is known about its physiological control. Acute administration of the alpha 1-adrenergic agonist phenylephrine (1.7 mg/kg, iv) to male mice produced a rapid increase in the EGF concentration in bile. We suggest that this EGF originates in submandibular glands and not in the liver. The bases for this are: 1) this increase was parallel to the increase in plasma, and the EGF content of the submandibular glands decreased after phenylephrine injection; and 2) the EGF concentrations in plasma and bile did not increase after phenylephrine administration to sialoadenalectomized mice. The concentration of EGF in bile is not only under pharmacological control, but is also regulated physiologically. Thus, the EGF concentrations in plasma, bile, and submandibular glands increased in fasted mice. All of these changes were reversed by refeeding. As 1) [125I]EGF binding to liver membranes decreased only after 2 days of fasting, but the level of circulating EGF was already increased in 1-day fasted mice, and 2) EGF secretion by submandibular glands from 1-day fasted mice incubated in vitro increased, we suggest that the increase in EGF concentrations in plasma and bile is the consequence of increased endocrine secretion by submandibular glands. Taken together, our results suggest that there is a flux of EGF from submandibular glands to bile in mice, which is under physiological control.     

Immunohistochemical localization of transforming growth factor alpha in the major salivary glands of male and female rats

The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF alpha stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF alpha antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF alpha in the rat salivary glands.     

Effects of gonadal steroid hormones on hypothalamic vasopressin mRNA level in male and female rats

Experiments were carried out in 10-11-week old gonadectomized male and female Sprague-Dawley rats. Dot-blot analysis and 3'-end digoxigenin-labeled 26 meroligonucleotide probe was used in detecting the mRNA level hypothalamic vasopressin (AVP). The basal hypothalamic AVP-mRNA level in the sham-operated intact males was 45% higher than that in the sham-operated intact females (P < 0.05). Plasma osmolality was also higher in the sham-operated intact males than in the sham-operated intact females (P < 0.05). The hypothalamic AVP-mRNA level in ovariectomized rats was 30% higher than that in sham-operated intact females (P < 0.05). Although the hypothalamic AVP mRNA level tended to be lower in castrated males than in sham-operated intact males, the difference was not statistically significant. The difference in plasma osmolality between gonadectomized males and females was statistically insignificant. In castrated males, hypothalamic AVP-mRNA level was decreased following sc injection of estradiol (P < 0.05), but testosterone, progesterone or a combination of estradiol and progesterone were without effect. In ovariectomized rats, sc injection of estradiol or a combination of estradiol and progesterone resulted in a decrease in hypothalamic AVP-mRNA level (P < 0.01), but progesterone or testosterone had no effect. The difference in plasma osmolality between gonadal steriod hormones-treated rats and vehicle-treated rats was not statistically significant. These findings indicate that gonadal steriod hormones can affect hypothalamic AVP-mRNA level in rats, through some central mechanism.     

Effects of growth hormone-releasing factor and(or) thyrotropin-releasing hormone on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers

The objective of this study was to determine the effect of long-term administration of a growth hormone (GH)-releasing factor analog (GRFa) and(or) thyrotropin-releasing hormone (TRH) on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers. Crossbred heifers (n = 48; 345.9 +/- 2.8 kg) were divided into four equal groups: control (vehicle), 1 microgram of GRFa (human GRF 1-29 analog).kg BW-1.d-1, 1 microgram of TRH.kg BW-1.d-1, or GRFa + TRH. Daily s.c. injections continued for 86 d. Blood samples were collected from half of the heifers after injection on d 1, 36, and 78. On d 89, all heifers were slaughtered. Treatments did not affect (P > .05) ADG but GRFa + TRH decreased (P < .05) ADFI relative to all other treatments. Feed conversion efficiency tended (P < .10) to be improved in the groups given GRFa alone or TRH alone. Treatment with GRFa and(or) TRH did not affect carcass weight, dressing percentage, conformation score, backfat thickness, or weights of liver, kidneys, pituitary, and ovaries. The GRFa + TRH treatment reduced (P < .05) fat score and increased (P < .05) longissimus muscle area relative to other treatments. The GRFa treatments reduced (P < .05) the weight and fat percentage of the mammary gland and increased (P < .05) heart weight. Treatment with TRH alone failed to stimulate GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) GH above controls on d 36, whereas GRFa + TRH increased (P < .05) GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) IGF-I only on d 1, whereas GRFa + TRH was without effect on all days. Across sampling days, treatments had little effect on blood concentrations of insulin, triiodothyronine, nonesterified fatty acids, urea nitrogen, and glucose. The GRFa alone and GRFa + TRH decreased (P < .05) and TRH alone increased (P < .05) thyroxine concentrations. In conclusion, with the dose and administration regimen used, GRFa and(or) TRH yielded small but positive improvements in animal performance.     

Effects of growth hormone, activin, and follistatin on the development of preantral follicle from immature female mice

The aim of this study was to investigate whether GH and insulin-like growth factor I (IGF-I) are involved in preantral folliculogenesis and, if so, to clarify the relationship between GH/IGF-I and activin/follistatin (FS) systems in immature female mice. Ovaries were obtained from 11-day-old mice, and preantral follicles, 100-105 microm in diameter, were mechanically isolated and selected for culture. Ten preantral follicles per well were cultured with different quantities and combinations of additives as follows: no additives (control), recombinant human FSH (rhFSH), IGF-I, recombinant human GH (rhGH), activin A, and recombinant human FS (rhFS). Mean diameters of the follicles were measured daily, and estradiol and immunoreactive inhibin levels in the cultured medium were assayed by RIA on day 4. rhGH showed stimulatory effects on the follicular diameter and the secretion of estradiol and immunoreactive inhibin. These effects were augmented by the presence of IGF-I and activin A. IGF-I alone did not show any stimulatory effect. The addition of rhFSH to activin A or to rhGH and activin A promoted preantral follicular growth and hormone production. On the other hand, GH- or activin-stimulated follicular growth was suppressed by rhFS in a dose-dependent manner. These results indicate that activin A and rhGH may play an important role in controlling earlier phases of follicular development during the infantile period, which is considered to be gonadotropin independent.     

Effects of chronic stimulation or antagonism of opiate receptors on GH secretion in male and female rats

The present study was undertaken to assess the role of endogenous opioid systems in the sexually dimorphic pattern of growth hormone (GH) secretion. To this end, male rats were treated chronically (6 to 12 h) with morphine and estrogen-exposed, ovariectomized female rats with morphine or naloxone. Chronic morphine exposure of male rats caused a 12-fold increase in basal GH levels and a modest rise in GH pulse frequency. These two changes resulted in a 3-fold increase in both mean GH concentration and total GH secretion over 6 h. In female rats, chronic morphine reduced GH pulse amplitudes but did not significantly affect other parameters of GH secretion. By contrast, chronic naloxone treatment of female rats reduced basal GH levels by 64% without affecting GH pulse amplitudes or pulse frequency. These data suggest that endogenous opioid systems are involved in the regulation of the basal GH secretion in both male and female rats.     

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.     

Effects of epidermal growth factor and insulin on migration and proliferation of primary cultured rabbit gastric epithelial cells

We assessed the influence of epidermal growth factor (EGF) and insulin on gastric epithelial restoration in vitro. Rabbit gastric epithelial cells were cultured and formed a complete monolayer cell sheet in 2 days. We created a wound (1.8 +/- 0.05 mm2) by denuding an area of cells, and EGF (0.1-30 ng/ml) and/or insulin (1 nM-1 microM) was added. The restoration process, which included cell migration and proliferation, was monitored by measuring the cell-free area every 12 h for 2 days. Proliferating cells were detected by sequential staining with bromodeoxyuridine (BrdU). Control cells showed complete repair in 36-48 h and restoration was accelerated dose-dependently by EGF or insulin. EGF plus insulin further accelerated restoration, which was then completed in 12-24 h. EGF and/or insulin increased the number of BrdU- positive cells. The results indicated that EGF and insulin additively accelerated gastric epithelial wound repair by stimulating both the migration and the proliferation of gastric epithelial cells (particularly the former).     

Effects of enalapril and nitrendipine on the excretion of epidermal growth factor and albumin in hypertensive NIDDM patients

OBJECTIVE: To compare the effect of the antihypertensive drugs nitrendipine and enalapril on the excretion of epidermal growth factor (EGF) and albumin in hypertensive non-insulin-dependent diabetes mellitus (NIDDM) subjects. RESEARCH DESIGN AND METHODS: After a 4-week washout period, mildly hypertensive (systolic blood pressure [sBP] > or = 140 mmHg and/or diastolic blood pressure [dBP] > or = 90 mmHg) NIDDM patients with albuminuria (15-200 micrograms/min) were randomized into an 8-month-long therapy with either nitrendipine (n = 11) or enalapril (n = 10). Blood pressure, EGF, and microalbumin excretion were measured at baseline and throughout the treatment period. RESULTS: A significant fall in sBP was noticed in the enalapril group and in dBP in the nitrendipine group. In the enalapril group, EGF excretion progressively increased from 188 to 214 nmol/mmol creatinine after 6 weeks and to 274 after 8 months of therapy (P = 0.03). There was a significant fall in albumin excretion while patients were on enalapril, but in the nitrendipine group, neither albuminuria nor EGF excretion changed significantly. There was no correlation of improved EGF excretion with a decrease in albuminuria or BP. CONCLUSIONS: The angiotensin-converting enzyme inhibitor enalapril has been effective in decreasing albumin and increasing EGF excretion. Measurement of urinary EGF may provide a new valuable index of renal function.     

Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor

We have previously demonstrated that insulin activates farnesyltransferase (FTase) and augments the amounts of farnesylated p21 (Goalstone, M. L., and Draznin, B. (1996) J. Biol. Chem. 271, 27585-27589). We postulated that this aspect of insulin action might explain the "priming effect" of insulin on the cellular response to other growth factors. In the present study, we show the specificity of the effect of insulin on FTase. Insulin, but not insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF), stimulated the phosphorylation of the alpha-subunit of FTase and the amounts of farnesylated p21. Even though all four growth factors utilized the Ras pathway to stimulate DNA synthesis, only insulin used this pathway to influence FTase. Insulin failed to stimulate FTase in cells expressing the chimeric insulin/IGF-1 receptor and in cells derived from the insulin receptor knock-out animals. Insulin potentiated the effects of IGF-1, EGF, and PDGF on DNA synthesis in cells expressing the wild type insulin receptor, but this potentiation was inhibited in the presence of the FTase inhibitor, alpha-hydroxyfarnesylphosphonic acid. We conclude that the effect of insulin on FTase is specific, requires the presence of an intact insulin receptor, and serves as a conduit for the "priming" influence of insulin on the nuclear effects of other growth factors.     

Effects of lactoferrin and its peptides on proliferation of rat intestinal epithelial cell line, IEC-18, in the presence of epidermal growth factor

The cell growth-stimulating activity of lactoferrin (LF) in combination with epidermal growth factor (EGF) was evaluated by using a rat intestinal epithelial cell line, IEC-18. LF was found to be more effective than EGF for inducing an increase in cell numbers when cultured for over 6 days using a medium containing 0.2% fetal calf serum (FCS), although the 3H-thymidine incorporation-stimulating activity of EGF was more potent than that of LF. A synergistic effect of LF and EGF was observed in both cell proliferation and DNA synthesis assays. The increase in cell numbers when stimulated with LF plus EGF corresponded to about 5 times that of the control. Iron was not required for manifestation of these effects of LF. On the other hand, iron-saturated transferrin (TF) had cell-growth-stimulating activity, but iron-free TF did not, either in the presence or absence of EGF. These results indicate that LF induces cell proliferation by a mechanism distinct from that of TF. A pepsin-generated hydrolysate of LF (LFH) had an activity similar to that of undigested LF, and a peptide with cell-growth-stimulating activity from bovine LFH was isolated by monitoring its effects in combination with EGF on DNA synthesis in IEC-18 cells. Sequence analysis indicated that the peptide has the structure Ala-Glu-Ile-Tyr-Gly-Thr-Lys-Glu-Ser-Pro-Gln-Thr-His-Tyr-Tyr, corresponding to residues 79-93 of bovine LF.     

Influence of recent modification of behavioral relationships of male and female OF1 mice on resistance to nitrogen or carbon monoxide hypoxia

A decrease of resistance to hypoxia is obtained in OF1 mice, 24 hours after an hyperactivity resulting from a grouping by 13 of male or female mice previously isolated. Besides, this decrease is statistically significant in an acute nitrogen hypoxia but not in an acute carbon monoxide poisoning. In both hypoxias, in mice of same age the males are less resistant than females.     

Effects of grafts of single anterior pituitary glands on the incidence and type of mammary neoplasm in neutron- or gamma-irradiated Fischer female rats

Three batches comprised of 48 young adult Fischer female rats each were subjected to total-body irradiation with 50 rads modified fission neutrons, or were given 600 rads 137Cs gamma-rays, or served as unirradiated controls. On the day following exposure, one-half of each batch was grafted with a single anterior pituitary gland beneath the left kidney capsule. The animals were observed for mammary neoplasia and all those that died during the experiment were autopsied. The experiment was terminated 538 +/- 13 days after irradiation when all neutron-irradiated, pituitary-grafted animals had one or more mammary tumors. Only 2 of the 23 untreated rats that survived until termination of the experiment developed mammary fibroadenomas, and none had mammary carcinomas. The incidence of fibroadenomas was increased, and a single carcinoma was found, in unirradiated rats with pituitary grafts. Irradiation alone caused an increase in the incidence of mammary fibroadenomas and the appearance of carcinomas. Fibroadenomas were markedly increased by the addition of pituitary grafts to irradiation. Carcinoma incidence was less markedly affected. The neutron dose of 50 rads was slightly more effective in inducing mammary neoplasms than the 600-rad dose of gamma-rays.     

Effect of castration on serum concentrations of gonadal hormones, insulin-like growth factor-I and its binding proteins in male pigs

Ten male pigs (Large White x Landrace), 7 months old, were randomly allocated to two experimental groups. Five of them were castrated and the other five served as controls. Sera were collected on the day of castration and 1, 5, 6 and 7 weeks after castration for hormone assay. There was a significant rise in the splenic and pancreatic weights in the castrates (P < 0.01). The weights of prostate, seminal vesicles and bulbourethral glands were significantly decreased (P < 0.01) in the castrates, which is attributed to a fall in testosterone levels (P < 0.001). The fall in oestradiol concentrations (P < 0.001) in castrates confirms that the testis is the major source of oestrogens in males. Although there was no significant change in the body weight, serum IGF-I levels were elevated in the castrates as compared to the controls after 5, 6 and 7 weeks (P < 0.001). IGFBP bands of 43 and 39 kda predominate in both control and experimental groups indicating that castration had no effect on the IGFBP pattern. It is suggested that the increase in IGF-I levels may be due to uncoupling of GH/IGF-I axis induced by the decrease in steroid concentrations due to castration.     

Acute effects of short-term feed deprivation and refeeding on circulating concentrations of metabolites, insulin-like growth factor I, insulin-like growth factor binding proteins, somatotropin, and thyroid hormones in adult geldings

Two studies were performed with Standardbred geldings 7 to 21 yr of age to determine the sequence of changes in blood plasma concentrations of some hormones and metabolites during feed deprivation for 48 h and for 12 h after refeeding. Plasma hormone and metabolite concentrations were determined with methods validated for horse plasma. Insulin-like growth factor binding proteins (IGFBP) were determined with radioligand analysis following SDS-PAGE electrophoresis. In both experiments, plasma concentrations of triiodothyronine and thyroxine decreased (P < .01) during feed deprivation and increased (P < .01) during refeeding. Plasma glucose and IGF-I either decreased or were not altered during feed deprivation. In contrast, plasma concentrations of NEFA and urea nitrogen increased (P < .01) during feed deprivation and decreased (P < .01) during the refeeding period. Plasma somatotropin (ST) increased (P < .01) approximately 80% at 24 to 36 h of feed deprivation, declined (P < .01) to control values at 48 h of feed deprivation, increased (P < .01) nearly three fold at 3 h after refeeding, and returned to control values by 6 h after refeeding. We identified five IGFBP, and their plasma concentrations were not significantly altered during feed deprivation or following refeeding. We conclude that metabolite availability during feed deprivation and following refeeding alters the secretion of thyroid hormones, ST, and possibly IGF-I, thereby maintaining homeostasis in horses.     

Effects of cyclosporine and dexamethasone on IgE antibody response in mice, and on passive cutaneous anaphylaxis in the rat

The suppressive effects of cyclosporine A (CsA) and dexamethasone (Dxt) on antigen-specific IgE responses to ovalbumin (OA) were studied in BALB/c mice. The effects upon other isotypes were also analyzed. The antiovalbumin IgE response did not change when low doses of CsA [8 mg/kg intraperitoneally (i.p.)] were administered; IgA also remained unchanged, while IgG and IgG1 decreased significantly. At higher CsA doses (16 mg/kg i.p. or orally), a decrease was noted for all the ispotypes assayed. Dxt administered orally at 0.3 mg/kg selectively inhibited IgE and IgA but did not influence IgG or IgG1 levels. Delayed hypersensitivity reactions to OA were not modified by CsA, but were depressed by Dxt. Although CsA had not effect on passive cutaneous anaphylaxis in the rat, Dxt significantly reduced this reaction when it was administered 6 h before challenge. These results suggest that Dxt has more specific antiallergic activity than CsA.     

Inverse effects of 20K and 22K human growth hormones on the growth of T-47D human breast cancer cells in culture and in nude mice

The major form of human growth hormone (22K hGH) stimulates the growth of T-47D human breast cancer cells in culture and in nude mice by binding to their receptors for growth hormone and prolactin. Another isoform of hGH having a smaller molecular mass (20K hGH) is known to show different binding affinities to these receptors. In this study, we have analyzed the effects of 20K hGH on the growth of T-47D cells in vitro and in vivo. 20K hGH (50 ng/ml) inhibited the proliferation and DNA synthesis of T-47D cells cultured in the presence and absence of 17 beta-estradiol (100 ng/ml), while 22K hGH (50 ng/ml) promoted the cellular growth. In estradiol-treated nude mice, 22K hGH (100 micrograms) remarkably promoted the growth of T-47D tumor, but 20K hGH again suppressed the tumor growth significantly. The results suggest the presence of different signal pathways for these two hGH isoforms and imply a possible clinical application for 20K hGH.