Effects of beta-naphthoflavone on insulin receptor binding and protein kinase activity in rat liver and placenta

Studies investigating the effects of beta-naphthoflavone (beta NF) on insulin receptor binding and its intrinsic protein kinase activity in rat liver and placenta were performed. Membranes were prepared from maternal liver and placenta on gestation day 11 and used for [125I]insulin radioreceptor assay. Scatchard analysis showed that association constants (Ka) for high affinity binding sites were similar for placental and liver membrane. The administration of beta NF, 15 mg/kg, 1 day before study did not alter the specific binding of insulin to liver membranes, whereas ligand binding to placental preparations was decreased 40% from control. Scatchard analysis of binding to placental membranes suggests that beta NF treatment was associated with a change in the number of high affinity binding sites. In further studies membrane receptors were solubilized and partially purified by wheat germ agglutinin affinity chromatography for protein kinase assay. Insulin stimulated the phosphorylation of the Mr 95,000 subunit of the receptor in lectin-purified membrane proteins from liver and placenta. In liver receptor preparations, beta NF treatment was associated with a nearly 3-fold increase in the insulin-stimulated phosphorylation of the 95-kD protein. In contrast, placental receptor preparations showed a 40% decrease in the extent of autophosphorylation following beta NF treatment. Insulin-stimulated phosphorylation of an exogenous substrate poly(Glu4, Tyr) also showed a divergent pattern of changes in liver and placental receptors following beta NF treatment. In studies during late gestation (day 18), beta NF treatment was also associated with an increase in liver receptor kinase activity, whereas placental receptors showed a decrease in autophosphorylation. Thus, acute treatment with beta NF during mid and late gestation was associated with significant alterations in insulin receptor protein kinase activity, and data suggest that fetal insulin receptors may respond in a different manner than maternal receptors to polyaromatic compounds like beta NF. The observed effects of beta NF on liver and placental receptor kinase activity may be related to alterations in insulin function in the regulation of pregnancy and fetoplacental growth.     

Smoking-related alterations in epidermal growth factor and insulin receptors in human placenta

Studies characterized insulin and EGF receptors in human placental tissue from smokers and nonsmokers. Specific binding of 125I-labeled insulin and EGF to placental membranes was not different for nonsmokers compared with smokers. EGF and insulin receptor kinases were further studied using a wheat germ agglutinin-purified preparation of solubilized placental membrane proteins. In extracts from the nonsmoker group, EGF stimulated the active phosphorylation of Mr 170,000 and 140,000 protein bands, which was half-maximal (EC50) at 5 x 10(-8) M. In extracts from the smokers group, however, phosphorylation of these two protein bands was barely detectable over a range of 0 to 10(-6) M EGF. Thus, EGF-stimulated phosphorylation of the 170,000 and 140,000 bands was markedly decreased in placental membranes from smokers. In contrast, insulin stimulated the phosphorylation of a 95,000 protein that was immunoprecipitated with anti-insulin receptor antiserum in membrane preparations from both nonsmokers and smokers. Dose-response curves for autophosphorylation indicate that EC50 values were 2.6 and 7.0 nM insulin for nonsmokers and smokers, respectively. Laser densitometry scan of the 95,000 band on autoradiograms further showed that maximal 32P incorporation was 30% greater in smokers compared with nonsmokers. Analysis of the insulin-dependent phosphorylation of an exogenous substrate, poly(Glu,Tyr) (4:1), showed a similar pattern of values for nonsmokers versus smokers. These results indicate that insulin receptor autophosphorylation and tyrosine kinase activity were normal or increased, whereas EGF-stimulated kinase activity was markedly decreased in placental membrane proteins from smokers. Western blot analysis using an antiserum to the EGF receptor showed the presence of immunoreactive bands of 126,000 and 150,000-170,000 in receptor preparations from nonsmokers, whereas only the 126,000 protein was detected in preparations from smokers. Thus, the smoking-related deficiency in EGF receptor autophosphorylation appeared to be due to the absence of a 150,000-170,000 receptor protein. In conclusion, maternal cigarette smoking is associated with selective alterations in two major receptor-mediated pathways thought to be involved in cell growth and differentiation in human placenta.     

Positional metabolism of benzo(a)pyrene in rat placenta and maternal liver. Comparison of induction effects

The biotransformation of [14C]benzo(a)pyrene (BP) was studied in vitro in the presence of microsomes prepared from isolated labyrinth and basal zone tissues of the rat placenta, as well as from maternal liver. Pregnant rats, day 14 of gestation, received beta-naphthoflavone (beta NF; 15 mg/kg, ip) or 3-methyl-cholanthrene (3MC; 30 mg/kg, ip). On day 15, placentae were dissected and microsomes were incubated with 17 microM [14C]BP and 2 mM NADPH. Metabolites formed in the incubation flasks were extracted and separated by HPLC utilizing a reverse phase column. Only trace BP metabolism occurred in basal zone microsomes from control, beta NF-, or 3MC-pretreated animals, as well as in labyrinth microsomes from control animals. In contrast, the preadministration of beta NF and 3MC increased labyrinth microsomal BP metabolism by 10- to 15-fold. Labyrinth and maternal liver microsomes from beta NF- and 3MC-treated animals actively converted BP to eight separate metabolites which co-chromatographed primarily with quinones and phenols. The overall formation of BP diol and phenolic metabolites by labyrinth microsomes was appreciably less than was observed for liver preparations. The very low activity of BP-4,5-oxide hydrolase in labyrinth microsomes compared to liver may in part explain the low level of formation of BP diols in placental microsomes. Labyrinth microsomes catalyzed the covalent binding of [3H]BP to calf thymus DNA, and this activity increased 5-fold following beta NF pretreatment. A comparison of induced tissues indicates that the amount of DNA binding in labyrinth microsomes is more extensive than would be expected by the level of total BP metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased human birth weights after in utero exposure to PCBs and PCDFs are associated with decreased placental EGF-stimulated receptor autophosphorylation capacity

Yucheng (oil disease) is a clinical and metabolic syndrome reported in Taiwanese who consumed rice oil contaminated with large amounts of various polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs), including the 2,3,4,7,8- and 1,2,3,4,7,8-PCDF congeners which are similar in structure and toxicity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. A well known characteristic of Yucheng is the marked decrease in birth weights, although the underlying mechanism of this effect is unclear. Placental epidermal growth factor (EGF) receptor binding and autophosphorylation studies were done using tissue samples taken from Yucheng and unexposed control patients. EGF-stimulated receptor autophosphorylation of the human placental EGF receptor in the Yucheng subjects was decreased more than 60% of control levels, 4-5 years after the exposure had occurred. The decrease in EGF receptor phosphorylation was significantly correlated with decrease in birth weights. Nonlinear regression analysis of the 125I-EGF receptor binding data revealed that there were two distinct EGF receptor binding isotherms representing the high affinity-low capacity (HALC) and the low affinity-high capacity (LAHC) binding sites. In contrast to the placental EGF-stimulated phosphorylation data described above, the binding kinetics of the EGF receptor were not significantly altered in the control [HALC site Kd = 0.10 +/- 0.02 (SE) nM, Bmax = 788 +/- 255 fmol/mg of protein; LAHC site Kd = 17.4 +/- 8.2 nM, Bmax = 62 +/- 32 pmol/mg) compared to the Yucheng subjects (HALC site Kd = 0.11 +/- 0.02 nM, Bmax = 784 +/- 305 fmol/mg; LAHC site Kd = 49.5 +/- 24.7 nM, Bmax = 147 +/- 80 pmol/mg). GC-MS analysis of placental specimens showed elevated levels of selected PCB and PCDF congeners in the Yucheng compared to control individuals. Total PCB levels were 0.5 +/- 0.2 ppb and 20.0 +/- 4.8 ppb for the control and Yucheng subjects, respectively. A significant dose-response relationship was observed between the placental EGF receptor phosphorylation levels and the PCB concentrations (total or concentrations of 2,2',4,4',5,5'-hexa- and 2,2'3,3'4,4',5-heptachlorobiphenyls). In contrast, no significant relationship was found between the EGF receptor phosphorylation activity and the 2,3,4,7,8- or 1,2,3,4,7,8-PCDF congeners, which were at nondetectable levels in the control and between 104 and 374 parts per trillion in the Yucheng subjects. In summary, our data reveal that decreased placental EGF receptor phosphorylation capacity is associated with decreased birth weight. Furthermore, PCB tissue concentrations might be a better predictor of effects than are PCDF concentrations.     

Prenatal cocaine use associated with down regulation of receptors in human placenta

Cocaine use during pregnancy has been associated with abruptio placentae and spontaneous abortions. These effects may be secondary to the vasoconstrictive effects of cocaine or to other alterations. Since it has been demonstrated that the use of opiates during pregnancy alters placental receptors, the effects of cocaine on placental receptors was studied. Women who used cocaine during pregnancy showed a significant lowering of the total number of beta-adrenergic receptor binding sites and delta-opiate receptor binding sites. The decreases in Bmax for each of these receptors was not associated with a decrease in the Kp. The potential causes for the receptor down regulation and effects are discussed.     

Benzo[a]pyrene and nicotine impair epidermal growth factor mediated cellular functions of buccal mucosa.

This study investigated the effect of two major ingredients in cigarette smoke, benzo[a]pyrene (BP) and nicotine, on epidermal growth factor (EGF) receptor binding and EGF-mediated cellular functions in rat buccal mucosa. Rat buccal tissue was incubated in DMEM in the absence (control) and presence of 10 microM BP or nicotine for 2.5 h at 25 degrees C. There were no significant differences in [125I]EGF binding to the buccal mucosal membranes between the control and treatment groups. Protein tyrosine kinase assay showed that EGF stimulated phosphorylation of a 170-kDa protein band in the controls, but not in the BP- and nicotine-treated samples. The basal [3H]thymidine incorporations were not significantly different between the groups. Nevertheless, addition of 5 nM EGF increased [3H]thymidine incorporation by 22% in the control, but not in the BP- or nicotine-treated group. The results demonstrate that BP and nicotine change the buccal mucosal functions associated with alteration of EGF receptor.     

Comparison of the Methods for Determining Cell-Surface and Intracellular Receptors for Epidermal Growth Factor in the Rat Liver

We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37°C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intra-cellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 µg/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.     

Antiproliferative and antiangiogenic effects of 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, in human umbilical vascular endothelial cells

There is increasing interest in the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and polycyclic aromatic hydrocarbons on cardiovascular diseases. Their chemical structures are similar, although polycyclic aromatic hydrocarbons contain no chlorine as does TCDD. The biochemical mechanism of their action is mainly mediated by the aryl hydrocarbon receptor. In addition, oxidative stress also plays a role in the biological and toxic effects of these chemicals. In this study, we used an aryl hydrocarbon receptor agonist, 3-methylcholanthrene (3-MC), to investigate its effect on the proliferation and angiogenesis of human umbilical vascular endothelial cells. 3-MC suppressed DNA synthesis of human umbilical vascular endothelial cells as determined by [(3)H]thymidine incorporation in a concentration-dependent fashion and arrested cells at the G0/G1 phase of the cell cycle. Interestingly, the inhibition of DNA synthesis by 3-MC was eliminated to a greater extent by aryl hydrocarbon receptor antagonists, alpha-NF (0.5 and 1 microM) and resveratrol (5 and 10 microM), than by the antioxidant, N-acetylcysteine (5 and 10 mM). Cell permeability, adhesion, and tube formation in human umbilical vascular endothelial cells exposed to 3-MC decreased in concentration-dependent manners. We also demonstrated that cell adhesion signaling (phosphorylated focal adhesion kinase (FAK)) decreased upon 3-MC treatment, suggesting that cell adhesion inhibited by 3-MC might be due to inhibition of cell adhesion signaling. Additionally, alpha-naphthoflavon (alpha-NF) ameliorated the effects of 3-MC on cell permeability, adhesion and tube formation, indicating the involvement of the aryl hydrocarbon receptor in angiogenesis. The results suggest that the adverse effects of 3-MC are mainly mediated by the aryl hydrocarbon receptor and not via increased oxidative stress.     

Heterologous regulation of EGF receptor function in cultured aortic smooth muscle cells

A10 cultured smooth muscle cells from rat embryonic thoracic aorta bound ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF), and responded to EGF by an increase in DNA synthesis. Scatchard analysis of binding data obtained at 4° C showed curvilinearity consistent with there being two affinity classes of binding site. The amount of ¹²⁵I-EGF that bound was decreased by treatment of the A10 cells at 37° C with [Arg⁸]vasopressin or with 5-hydroxytryptamine (5-HT). Scatchard analysis of binding (at 4° C after pretreatment at 37° C) revealed this effect to be due to a loss of the high-affinity component of ¹²⁵I-EGF binding, with no change in total receptor number. The presence of vasopressin or 5-HT raised the concentration of EGF required for the stimulation of DNA synthesis. Cultured A10 aortic smooth muscle cells therefore have receptors for EGF that mediate an increase in cell proliferation. EGF receptor function is modified by vasopressin and 5-HT, probably as a consequence of their effects on EGF receptor affinity.     

Regulation of epidermal growth factor binding in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) decreased the binding of epidermal growth factor (EGF) by the human keratinocyte cell line SCC-12F. This response was concentration dependent (half-maximal effective concentration, EC50 = 1.8 nM) and stereospecific. Scatchard analysis of EGF binding indicated that treatment with TCDD resulted in a loss of high-affinity (Kd = 0.28 nM) binding sites. This loss was accompanied by a concomitant inhibition of EGF-stimulated DNA synthesis. The kinetics for the decrease of EGF binding by TCDD and benzo[a]pyrene (BP) were compared. Inhibition of EGF binding by BP was maximal by 24 hr, with 90% recovery of EGF binding apparent by 48 hr. In contrast, TCDD treatment for 72 hr was required to produce maximal inhibition, and no recovery was evident up to 10 day after removal of TCDD from the growth medium. The data indicate that modulation of EGF binding by TCDD was mediated by the Ah receptor. Subsequent cellular responses, for example, inhibition of EGF-stimulated DNA synthesis, may be important in the expression of altered differentiation patterns observed in human epidermal keratinocytes exposed to TCDD.     

Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.     

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin and thyroid hormones act through different mechanisms in human keratinocytes

It has been proposed [J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41-53] that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361-1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T3) and T4 on human target cells. Keratinocytes were treated simultaneously with T3 and T4 in a 4:1 molar ratio (T3/T4; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P1-450 induction) were measured. T3/T4 (at concentrations up to 10 microM T3/2.5 microM T4) and T3 or T4 alone (0.1 to 10 microM) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T3/T4 was not due to metabolism of these hormones by the keratinocytes. T3 and T4 did not compete with [3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [125I]T3 or [125I]T4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.     

Effects of vinorelbine on epidermal growth factor-receptor binding of human breast cancer cell linesin vitro

Epidermal Growth Factor (EGF) is a mitogenic peptide that binds to surface membrane receptors (EGFR) of breast cancer cells. After binding, secondary transmitter molecules are activated by tyrosine phosphorylation of the intracellular receptor domaine. The activity of the EGF/EGFR system can be modulated by a variety of chemically unrelated compounds including cytostatic agents. The purpose of our present study was to determine the effects of vinorelbine, a novel semisynthetic vinca alkaloid on EGF receptor binding on human breast cancer cells. We have found that MDA-231 and MDA-468 cells bind substantially more [¹²⁵I]-EGF after preincubation with vinorelbine. This effect was concentration- and time-dependent reaching a maximum at 100 ng/ml and 24 h incubation. Subsequent experiments showed an increase in the rate of EGF binding as well as maximal binding capacity. Scatchard analysis of binding experiments under equilibrium conditions indicated that this was mainly due to an increase in the number of apparent EGF binding sites. Modulation of EGF receptor binding by vinorelbine was not detectable when isolated membranes were used indicating that intact cytoplasmatic mechanisms are required for the upregulation of EGF receptors.     

Decreased ligand binding to the hepatic glucocorticoid and epidermal growth factor receptors after 2,3,4,7,8-pentachlorodibenzofuran and 1,2,3,4,7,8-hexachlorodibenzofuran treatment of pregnant mice

2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) are environmental contaminants which mimic many of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Like TCDD, these polychlorinated dibenzofurans (PCDFs) induce hepatic benzo[a]pyrene hydroxylase activity (BPH) and possess high affinity for the Ah receptor. Another similarity of these PCDFs to TCDD is their ability to induce teratogenic effects such as cleft palate and hydronephrosis in mice. Recent studies have shown that TCDD modifies the equilibrium binding kinetics of the rat liver cytosolic glucocorticoid receptor (GRc) and the hepatic plasma membrane epidermal growth factor (EGF) receptor. To gain a better understanding of the action of halogenated hydrocarbons on these cytosolic and membrane-bound receptor systems during pregnancy, we investigated the biochemical effects of PeCDF and HCDF on the binding kinetics of maternal mouse liver GRc and EGF receptors and the induction of BPH activities. Pregnant C57BL/6N mice were treated once daily on gestation Days 10 through 13 with PeCDF (0-30 micrograms/kg) or HCDF (0-300 micrograms/kg). Hepatic [3H]dexamethasone and [125I]EGF equilibrium binding studies indicated that all doses of PeCDF tested (10, 20, and 30 micrograms/kg) significantly reduced the GRc and EGF receptor maximum binding capacities but did not affect the binding affinities of these receptors when compared to corn oil-treated control pregnant mice. Similar effects were observed for doses of HCDF greater than or equal to 100 micrograms/kg. These data suggest that the dibenzofuran-mediated decreases in GRc and EGF receptor binding capacities are similar to those caused by TCDD. Although the mechanism of action is not yet clear, our results indicate that halogenated aromatic compounds in addition to TCDD have profound effects on both steroid and growth factor receptor systems.     

Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons

BACKGROUND AND PURPOSE: Stimulation of astrocytes by the alpha(2)-adrenoceptor agonist dexmedetomidine, a neuroprotective drug, transactivates epidermal growth factor (EGF) receptors. The present study investigates signal pathways leading to release of an EGF receptor ligand and those activated during EGF receptor stimulation, and the response of neurons to dexmedetomidine and to astrocyte-conditioned medium. EXPERIMENTAL APPROACH: Phosphorylation of ERK(1/2) was determined by western blotting and immunocytochemistry, and phosphorylation of EGF receptors by immunoprecipitation and western blotting. mRNA expression of fos family was measured by RT-PCR. KEY RESULTS: Pertussis toxin (0.2 microg ml(-1)) an inhibitor of betagamma subunit dissociation from Galpha(i) protein, and GF 109203X (500 nM), a protein kinase C inhibitor, abolished ERK(1/2) phosphorylation. PP1 (10 microM), inhibiting Src kinase and GM 6001 (10 microM), an inhibitor of Zn-dependent metalloproteinase, abolished ERK(1/2) phosphorylation by dexmedetomidine (50 nM), but not that by EGF (10 ng ml(-1)), showing Src kinase and metalloproteinase activation during the first stage only; AG 1478 (1 microM), an inhibitor of the EGF receptor tyrosine kinase, abolished ERK(1/2) phosphorylation. Dexmedetomidine-induced EGF receptor phosphorylation was prevented by AG 1478, GM 6001, PP1 and GF 109203X and its induction of cfos and fosB by AG 1478 and by U0126 (10 microM), an inhibitor of ERK phosphorylation, indicating downstream effects of ERK(1/2) phosphorylation. EGF and conditioned medium from dexmedetomidine-treated astrocytes, but not dexmedetomidine itself, induced ERK phosphorylation in primary cultures of cerebellar neurons. CONCLUSIONS AND IMPLICATIONS: Dexmedetomidine-induced transactivation pathways were delineated. Its paracrine effect on neurons may account for its neuroprotective effects.     

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.     

Staphylococcus aureus alpha-toxin. 2. Reduction of epidermal growth factor receptor affinity in PC12 cells

Staphylococcus aureus alpha-toxin, at sub-cytotoxic concentrations, inhibits both the 125I-labeled epidermal growth factor (EGF) binding and autophosphorylation properties of EGF-receptors in PC12 cells. This inhibition occurred only in intact cells and is probably due to a decrease in the affinity of the receptor for EGF. Streptolysin S and parcelsin could mimic the alpha-toxin effect below cytotoxic concentrations, as measured by a 51Cr release assay. In contrast, other membrane perturbing toxins with different lipid specificity, such as tetanolysin and cobra direct lytic factor, inhibited [125I]EGF binding only at cytotoxic concentrations. Staphylococcal alpha-toxin also stimulated 3-fold the specific binding of a radioactive tumor-promoting phorbol ester (PDBu) to PC12 cells at concentrations similar to those required for the inhibition of [125I]EGF binding. Although the exact mechanism for the inhibition of EGF binding by alpha-toxin has not been established, our results suggest that protein kinase C may be involved in this time-dependent process.     

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.     

hCG和LHRH-A对人早孕和孕终末期胎盘绒毛分泌孕酮作用的体外研究

在妊娠的不同时期,胎盘中黄体生成素释放激素(LHRH)的含量不同。离体培养的绒毛组织实验也发现,相同剂量的外源性LHRH对妊娠中期和终末期胎盘绒毛组织分泌hCG和孕酮作用强度也不同。本实验室曾发现,LHRH类似物(LHRH-A)对体外培养孕早期(10～12周)和孕终末期(38～40周)绒毛组织分泌hCG均有兴奋作用,但对分泌孕酮的作用不同,即LHRH-A对早孕绒毛分泌孕酮有抑制作用,而对终末期分泌孕酮