Receptors for epidermal growth factor in the rat uterus.

Uterine membranes contain high affinity [dissociation constant (Kd) = 0.36 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I] iodo-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled insulin, fibroblast growth factor, or multiplication-stimulating activity. Incubation of [125I] iodo-EGF with uterine membranes, followed by chemical cross-linking with disuccinimidyl suberate and detergent extraction reveals a major species of specifically bound EGF (mol wt = 170,000) and a minor species (mol wt = 150,000) visualized by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the extracts. In detergent-solubilized preparations EGF also stimulates the phosphorylation of major (mol wt = 170,000) and minor (mol wt = 150,000) species of identical molecular weight. The increased phosphorylation produced by incubation of membrane extracts with EGF occurs largely at tyrosine residues, as indicated by phosphoamino acid analysis. These results indicate that the rat uterus contains high affinity EGF binding sites with the properties expected of EGF receptors.

Hormonal control of uterine growth: alterations in luminal epithelial deoxyribonucleic acid synthesis after intraluminal application of estrogen.

Administration of a single injection of estradiol (E2) causes a maximum increase in DNA synthesis in the uterine luminal epithelium approximately 24 h later. We previously reported that animals receiving a second injection of E2 15-18 h after the first show an apparent decrease in DNA synthesis measured in this cell type at 24 h. This apparent decrease in DNA synthesis is due to a shift in the time course of DNA synthesis rather than an absolute decrease in this parameter. In this report we demonstrate that this inhibitory effect of a second injection of E2 is also observed after intraluminal instillation of the hormone. This effect is dose dependent and appears to result from a shift in the time course of luminal epithelial DNA synthesis. The intraluminal instillation of E2 produces the same pattern of nuclear receptor localization as sc administration of the hormone. These results suggest that this inhibitory effect we previously described results primarily from a direct action of E2 on the luminal epithelium.

Stimulatory and inhibitory effects of estrogen and antiestrogen on uterine cell division.

Acute administration of 17 beta-estradiol or the antiestrogen nafoxidine to immature rats produces quantitatively similar responses in the uterine stroma and myometrium, although the responses to nafoxidine occur at slightly later times. Both the hormone and drug cause nuclear translocation of equivalent amounts of estrogen receptor in these two cell types, although receptor translocation is slower with nafoxidine, and nuclear receptor levels remain elevated for an extended time. Based upon labeling and mitotic indices, however, the response of the luminal epithelium to a maximum dose of nafoxidine is much lower than that produced by estradiol, although nafoxidine treatment causes massive hypertrophy of luminal epithelial cells and causes nuclear translocation of estrogen receptors in this cell type. If nafoxidine is administered 30 min after administration of estradiol, cell division in the luminal epithelium is inhibited. Furthermore, multiple injections of estradiol itself within a 24-h period decrease cell division in the luminal epithelium relative to controls receiving a single injection of the hormone. These results indicate that estradiol and the antiestrogen can have both inhibitory and stimulatory effects on the luminal epithelium and that cell division in different uterine cell types can be differentially affected.

Hormonal control of uterine growth: temporal relationships between estrogen administration and deoxyribonucleic acid synthesis.

Administration of a single injection of estradiol (E2) causes a maximum increase in DNA synthesis of all major uterine cell types approximately 24 h later. Animals given a second injection of E2 6-12 h after the first show an apparent increase in DNA synthesis in the luminal epithelium. Animals receiving a second injection of E2 15-18 h after the first show an apparent decrease in DNA synthesis at 24 h, which is most prominent in the luminal epithelium. This apparent decrease in DNA synthesis is most apparent if the second injection of E2 is given 18 h after the first, and is due to a shift of 10-12 h in the time course of DNA synthesis rather than to an absolute decrease in this parameter. This shift in the time course of uterine DNA synthesis is a dose-dependent phenomenon and displays a dose-response curve similar to that for the stimulation of DNA synthesis by a single injection of E2. A third injection of E2, 28 h after the initial hormone treatment, again causes a shift of 10-12 h in the time course of DNA synthesis relative to that in animals receiving two injections of hormone. These results suggest that nuclear levels of estrogen receptor, which initially increase after hormone administration, must decrease before the onset of uterine DNA synthesis.

Regulation of deoxyribonucleic acid polymerase activity in uterine luminal epithelium after multiple doses of estrogen.

Estradiol (E2) exerts both inhibitory and stimulatory effects on DNA synthesis in the rat uterine luminal epithelium (LE). This inhibitory effect is due to a shift in the time course of DNA synthesis, i.e. in animals receiving a single injection of E2, a peak of DNA synthesis occurs 24 h after treatment, but in animals receiving multiple injections of E2, DNA synthesis is suppressed until 10-12 h after hormone treatment ceases. In these previous studies LE DNA synthesis was assessed by measuring tritiated thymidine incorporation. In the present study, we sought to determine if the molecular basis for this decrease in DNA synthesis was due to a suppression of DNA polymerase activity in LE nuclei. Animals receiving a single injection of E2 exhibit a peak of nuclear DNA polymerase activity 20-24 h later. Animals receiving multiple injections of E2 (0, 12, 15, and 18 h) show more than a 50% decrease in DNA polymerase activity at 20-24 h, due to a shift in the maximum increase in enzyme activity to 32-36 h after the initial treatment. The observed differences between these groups are not due to different levels of DNase activity or different degrees of leakage of the nuclear enzyme. The observed enzyme activity is due to DNA polymerase-alpha, since it requires ATP as well as deoxyribonucleoside triphosphates, and is aphidicolin sensitive. These results indicate that the inhibitory effect of E2 on LE DNA synthesis is due at least in part to a suppression of nuclear DNA polymerase-alpha activity.

Hormonal control of uterine growth: the effect of hypothyroidism on estrogen-stimulated cell division.

The increase in mitotic indices of uterine luminal epithelium, stroma, and myometrium were determined as a function of time after the administration of a single dose of 17 beta-estradiol to euthyroid and hypothyroid rats. Hypothyroidism reduced the increase in the mitotic index 5-fold in the luminal epithelium, 6-fold in the stroma, and 9-fold in the myometrium. In addition to reducing mitotic indices, hypothyroidism also produced a shift of 12 h in the time course of estrogen-stimulated cell division of all uterine cell types relative to euthyroid animals. This shift in the time course of cell division was preceded by a shift in the time course of uterine DNA synthesis measured by tritiated thymidine incorporation. In contrast, hypothyroidism did not alter the magnitude or the time course of synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose after estrogenic stimulation. These results indicate that hypothyroidism decreases the ability of all major uterine cell types to undergo cell division in response to acute administration of estradiol, and also shifts the time course of the uterine growth response to the hormone.

A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody.

A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 micrograms/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.

Evidence for thyroid hormone receptors in uterine nuclei.

Nuclear preparations from the rat uterus contain specific, high affinity binding sites for T3, with the properties expected of a thyroid hormone receptor. The Kd value for the binding of T3 to these sites is 9.3 +/- 0.1 x 10(-10) M and the maximum number of binding sites is 0.11 +/- 0.02 pmoles T3 bound per mg of DNA. Competition experiments with Triac, D-T3, T4, and rT3 compared to L-T3 revealed relative affinities of 87%, 22%, 15%, and 1.6% respectively. T3 binding sites solubilized from uterine nuclear preparations by high salt extraction sediment at 3.6 S and are destroyed largely by trypsin treatment. Estradiol treatment of ovariectomized rats for 3 days did not alter the quantity of T3 binding sites expressed on the basis of DNA content. These results suggest that uterine nuclei contain a specific T3 receptor and raise the possibility that this hormone may have direct effects on the uterus.

IGF-I, growth hormone, and/or exercise effects on non-weight-bearing soleus of hypophysectomized rats.

The effects of insulin-like growth factor (IGF-I) or growth hormone (GH) with and without exercise on predominantly slow muscles of hypophysectomized hindlimb-suspended (HS) rats were determined. HS resulted in a 21, 23, and 30% decrease in soleus, adductor longus, and vastus intermedius masses, respectively, compared with ambulatory rats. Compared with values in HS rats, IGF-I increased the vastus intermedius mass and GH or exercise alone increased both the soleus and vastus intermedius masses. There was a strong interactive effect between GH, but not IGF-I, and exercise in all three muscles of HS rats. The soleus fiber type distribution of HS rats was not affected by any treatment. HS resulted in a 24, 18 (P > 0.05), 32, and 20% (P > 0.05) decrease in the size of soleus fibers containing type I, IIa, I + IIa, and IIa + IIx myosin heavy chains, respectively, compared with ambulatory hypophysectomized rats. Hormone or exercise alone had no effect on fiber size in HS rats. However, all fiber sizes (except for type IIa + IIx in IGF-I with exercise rats) were larger in HS rats treated with GH or IGF-I and exercise than those in HS rats. These data indicate an interactive effect of both GH and IGF-I with exercise in maintaining fiber size of chronically non-weight-bearing predominantly slow muscles. Furthermore, the results suggest that the myosin heavy-chain phenotype in rats deficient in all pituitary factors is unresponsive to short-term administration of either GH or IGF-I or to exercise or HS.

Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment.

The purpose of this study was to determine the effects of functional overload (FO) combined with growth hormone/insulin-like growth factor I (GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receiving saline vehicle served as controls (Con group). Muscle wet weight was 32% greater in the FO than in the Con group: 162 +/- 8 vs. 123 +/- 16 mg. Muscle weight in the FO + GH/IGF-I group (196 +/- 14 mg) was 59 and 21% larger than in the Con and FO groups, respectively. Mean soleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 +/- 445 microm2) was increased compared with the Con (2,044 +/- 108 microm2) and FO (2,267 +/- 301 microm2) groups. The difference in fiber size between the FO and Con groups was not significant. Mean myonuclear number increased in FO (187 +/- 15 myonuclei/mm) and FO + GH/IGF-I (217 +/- 23 myonuclei/mm) rats compared with Con (155 +/- 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups. These results demonstrate that the larger mean muscle weight and fiber cross-sectional area occurred when FO was combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume. Thus there appears to be some mechanism(s) that maintains the myonuclear domain when a fiber hypertrophies.

Passive immunization with an antibody to the beta-subunit of ovine luteinizing hormone as a method of early abortion--a feasibility study in monkeys (Macaca radiata).

Antiserum to the beta-subunit of ovine luteinizing hormone (oLH-beta) raised in monkeys (Macaca radiata) has been tested by a variety of criteria both in vivo and in vitro to establish its ability to neutralize oLH, hLH, and human chorionic gonadotropin (hCG). Passive administration of this antiserum caused inhibition of ovulation and termination of pregnancy in recipient monkeys as indicated by premature vaginal bleeding and a significant reduction in serum progesterone and estrogen levels. The results suggest that antiserum raised in monkeys against oLH-beta can neutralize monkey LH as well as monkey CG.

Stimulation of myofibrillar protein synthesis in hindlimb suspended rats by resistance exercise and growth hormone.

The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 degrees), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of 3H-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks approximately 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p < or = 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p < or = 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH+exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p < or = 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

Interactions between estrogen and EGF in uterine growth and function.

The rat uterus contains specific, high-affinity EGF receptors which possess a tyrosine kinase activity. As demonstrated autoradiographically, these receptors are present in the epithelial, stromal and myometrial cells of the uterus. Estrogen treatment in vivo produces a 2-3-fold increase in EGF receptor levels in the immature rat, the immature mouse and the ovariectomized adult rat; furthermore, EGF receptor levels vary throughout the estrus cycle in concert with levels of occupied nuclear estrogen receptor. This estrogen-induced increase in EGF receptor is preceded by an increase in the level of EGF receptor mRNA as judged by Northern blot analysis. In general, there is a good correlation between estrogen-induced DNA synthesis and EGF receptor levels in the uterus, although in certain situations EGF receptor levels are elevated without a subsequent increase in DNA synthesis. These observations suggest that an increase in tissue EGF receptor levels is important in estrogen-induced uterine growth, but that this increase in receptor levels alone is not sufficient to stimulate DNA synthesis. In addition to its possible role in tissue growth, we have shown very recently that EGF causes contraction of myometrial smooth muscle in a completely in vitro organ bath system. The qualitative nature of this contractile response is distinct from that produced by other classical uterotonic agents. The physiological significance of this uterine response to EGF remains to be elucidated.

Regulation of epidermal growth factor receptor levels by thyroid hormone.

The binding of iodinated epidermal growth factor (125I-EGF) to membrane preparations from the livers of euthyroid and hypothyroid rats was examined to determine if thyroid hormones regulate EGF receptors. Binding in membrane preparations from hypothyroid livers was only 30-40% compared to that of euthyroid controls. Treatment of hypothyroid animals with a single dose of L-T3 96 h prior to sacrifice restored EGF binding nearly to the control levels. Scatchard analysis of the binding data indicated that the decrease in EGF binding in hypothyroid membranes was due to a decrease in the number of the receptors. Affinity labeling/cross-linking of 125I-EGF to hepatic membranes with disuccinimidyl suberate followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a prominent band of Mr = 170,000, which was decreased in hypothyroid samples to the same extent as EGF binding. EGF-stimulated receptor kinase activity in detergent extracts of hepatic membranes also revealed a major phosphorylated protein species of Mr = 170,000, which was also decreased in samples from hypothyroid animals to approximately the same degree as EGF binding. Binding, affinity labeling/cross-linking, and phosphorylation measurements thus indicate that the levels of hepatic EGF receptor are regulated by thyroid status. These results raise the possibility that some in vivo affects of thyroid hormones may be mediated by alterations in EGF receptor levels.

Evidence in intact cells for an involvement of GTP in the activation of adenylate cyclase.

Treatment of cultured normal rat kidney cells with virazole or mycophenolic acid which are inhibitors of IMP dehydrogenase decreases by 50 to 70% the ability of prostaglandin E1 or isoproterenol to elevate cAMP levels. Inhibition is maximal by 2 h. The response to cholera toxin is not significantly decreased. Basal cAMP is not affected. Under these conditions, GTP is decreased by 80%, ATP is only 10 to 15% decreased, and UTP and CTP are slightly increased. Normal GTP levels and the responses to prostaglandin E1 and isoproterenol are restored if guanosine, but not inosine, is added with the inhibitor. The response to isoproterenol is recovered within 5 min after removal of mycophenolic acid. Desensitization to prostaglandin E1 or isoproterenol stimulation occurs under conditions where GTP is 80% decreased. These results in intact cells provide direct evidence for a role for GTP in the activation of adenylate cyclase and support previous conclusions from studies with cell homogenates.

Regulation of epidermal growth factor receptor by estrogen.

Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 125I-epidermal growth factor (EGF) binding to uterine membranes with no change in the affinity of membrane receptors for EGF. E2 treatment also increases the EGF receptor visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after affinity labeling of uterine membranes and the EGF-stimulated receptor autophosphorylation activity. In addition, E2 administration stimulates EGF-dependent tyrosine kinase activity in an assay system using exogenous angiotensin II as substrate. Following hormone treatment, EGF receptor levels increase between 6 and 12 h, remain elevated at 18 h, and decline between 24 and 36 h. This stimulation of EGF receptor levels by E2 is specific, since the non-estrogenic hormones progesterone, dexamethasone, and dihydrotestosterone fail to elevate receptor levels. E2-stimulated increases in EGF receptor levels are also blocked by cycloheximide and actinomycin D, suggesting that the observed effect represents de novo synthesis of the EGF receptor and may be mediated by a transcriptional mechanism. These results demonstrate that estrogen can regulate acutely the levels of EGF receptor in vivo and raise the possibility that events coupled to this receptor may play a role in estrogen-stimulated growth.

Identification of the major sites of phosphorylation in IGF binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth factor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues and neighboring amino acids potentially involved in defining a protein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by > 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17 kDa fragment contained serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of amino acid substitutions rather than lack of phosphorylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.