Biomedical subjects

D M Baldwin

Publications and source records attributed to D M Baldwin.

At least 19 recordsLinked to original sources

PubMed1996

An enzyme immunoassay for serum and urinary levonorgestrel in human and non-human primates.

A microtiter plate enzyme immunoassay (EIA) is reported for the measurement of levonorgestrel (LNG) in serum and urine samples of human and non-human primates, and the results are compared to data obtained by radioimmunoassay (RIA). Rabbit polyclonal antibodies were raised against the bovine serum albumin conjugate of the 3-O-carboxymethyl oxime (CMO) derivative of LNG. The enzyme label was produced by the conjugation of horseradish peroxidase to LNG at the 3-position by the same CMO bridge used for the immunogen. The assay requires 2.5 hours to perform using 2.2-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt as the chromogenic substrate. Serum (100 microliters) is extracted with petroleum ether prior to assay, whereas urine samples (25 microliters) are diluted and measured directly. The sensitivity of the assay is 0.25 pg/well with a 50% displacement of label at 7.5-9.5 pg and a linear response through 250 pg/well. Minimum levels of 8.7 and 10.0 pg/ml can be detected in serum and urine samples, respectively. Changes in serum LNG concentrations were measured in women and non-human primates following LNG implantation or injection. In the non-human primate study, serum LNG concentrations began to rise rapidly following i.m. injection of LNG, with peak levels occurring on days 3 to 5, then decreasing to approximately 25-35% of peak levels for the duration of the study. Circulating concentrations of 1.86 +/- 0.18 ng/ml LNG were reached in women the first week post-insertion of Norplant implants and decreased by 50% at 7-10 days, 75% after 14-21 days, followed by a steady decrease during the next 60-70 days to constant low levels that exhibited a high individual variation. Correlation coefficients of EIA and RIA results were 0.988 for human serum, 0.926 for human urine, and 0.972 for non-human primate serum.

Adolescent↗

PubMed1996

Immediate postpartum insertion of the norplant contraceptive device.

OBJECTIVE: To determine the safety and efficacy of Norplant (Wyeth-Ayerst Laboratories, Philadelphia, PA) insertion immediately postpartum. DESIGN: Prospective study of 14 women receiving Norplant immediately postpartum compared with controls (n = 6) having a bilateral tubal ligation. Subjects were followed for 3 months postpartum, and data were analyzed by analysis of variance and chi2. SETTING: Academic Health Sciences Center. PATIENTS: Female subjects 18 to 35 years old who had an uncomplicated term pregnancy, normal spontaneous vaginal delivery, and did not breast-feed. INTERVENTION: A brief interview, physical exam, and blood and urine samples were evaluated during a 12-week postpartum period. MAIN OUTCOME MEASURES: Major complaints, serum chemistry panels, hematologic and coagulative measures, serum E2, P, levonorgestrel, PRL, LH, FSH, and urinary estrone-3 conjugates and pregnanediol-3-glucuronide concentrations. RESULTS: Serum levonorgestrel peaked at approximately 2,000 pg/mL (6,400 pmol/L) during the 1st week after Norplant insertion, declining to approximately 250 pg/mL (800 pmol/L) by the 8th week. Significant differences between Norplant and control groups included bleeding irregularities, headaches, alopecia, and abdominal discomfort. Serum electrolytes, metabolic markers, and blood components were within normal limits. Serum E2, P, and urinary steriod biomarkers indicated that steroid secretion was suppressed severely in the Norplant group compared with controls who exhibited normal postpartum ovarian activity. CONCLUSION: Norplant inserted immediately postpartum appears to be a safe and effective method of contraception. However, the long-term hypoestrogenic state and contraceptive efficacy beyond the 3-month postpartum period as observed in this study are concerns that need further clinical evaluation.

Adolescent↗

PubMed1993

Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids.

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Testosterone suppresses 8-bromo-adenosine 3',5'-monophosphate and gonadotropin-releasing hormone-stimulated luteinizing hormone subunit synthesis.

The major objective of this study was to determine the effects of testosterone (T) on 8-bromo-cAMP (8-br-cAMP)- and GnRH-stimulated LH subunit polypeptide synthesis and glycosylation in cultured male anterior pituitary cells. The anterior pituitaries from 1-week castrate male rats were enzymatically dispersed and incubated for 48 h in steroid-free medium, followed by a 48-h incubation with or without 10 nM T. The cells were then incubated for 12 h in medium containing [35S]methionine ([35S]Met) and [3H]glucosamine ([3H]Gln) with or without 1 mM 8-br-cAMP or 1 nM GnRH, with or without 10 nM T. Incorporation of radiolabeled precursors into LH subunits was determined by specific immunoprecipitation of the LH dimer with subsequent analysis of the individual LH alpha- and beta-subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LH content was quantified by RIA (iLH). Both 8-br-cAMP and GnRH stimulated iLH release. T suppressed basal and GnRH-induced iLH secretion, whereas it enhanced iLH release stimulated by 8-br-cAMP. Both 8-br-cAMP and GnRH stimulated total (cell plus medium) [35S]Met and [3H]Gln incorporation into LH alpha and LH beta, and these responses were suppressed by T. Basal [35S]Met incorporation into the LH subunits was inhibited by T, whereas T had no effect on basal levels of [3H]Gln incorporation. Neither T nor GnRH altered [3H]Gln cell uptake or incorporation into total proteins, whereas 8-br-cAMP increased these responses. There were no treatment effects on [35S]Met cell uptake or incorporation into total proteins. These results suggest that 8-br-cAMP, similar to GnRH, stimulates both polypeptide synthesis and glycosylation of the LH alpha- and beta-subunits and that T suppresses these responses to 8-br-cAMP and GnRH in a similar fashion. These data indicate that cAMP is involved in mediating the actions of GnRH on LH subunit biosynthesis and that the inhibition of LH subunit polypeptide synthesis and glycosylation by T involves postreceptor events that are regulated by cAMP-dependent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Hepatic metabolism of 7,12-dimethylbenz(a)anthracene in male, female, and ovariectomized Sprague-Dawley rats.

Dimethylbenz(a)anthracene (DMBA) is a potent inducer of mammary tumors in intact female Sprague-Dawley rats, but not in males or ovariectomized females (OVX). Qualitative and quantitative aspects of hepatic metabolism of DMBA were examined in these three groups of rats, using the nonrecirculating perfused liver, to determine whether the production of proximate carcinogenic metabolites of DMBA by the liver differed among these groups in the same manner as does sensitivity to tumor induction. DMBA was infused into the liver at a constant rate for 60 min. Rates of appearance of DMBA and its metabolites were measured in perfusate and bile during the infusion period and the first 60 min thereafter. The maximum rate of appearance of total metabolites in the perfusate, seen at the end of the infusion period, was highest in the intact female [2.6 +/- 0.3 nmol/(g x min)], slightly lower in the OVX [2.3 +/- 0.2 nmol/(g x min)] and significantly lower in the male [1.0 +/- 0.1 nmol/(g x min)]. The rates of appearance of metabolites in the bile showed the same order as those seen in the perfusate. The major metabolites extracted from the perfusate in all three groups were dihydrodiols, hydroxymethyl metabolites, and several unidentified metabolites. The 3,4-dihydrodiol, a proximate carcinogenic metabolite, appeared in the perfusate at higher rates in the intact female and OVX than in the male. Hydrolysis of bile samples showed that glucuronidation was a major pathway in the excretion of DMBA metabolites in bile. High performance liquid chromatographic analysis indicated that hydrolysis of DMBA glucuronides yielded the 7- and 12-hydroxymethyl metabolites and an unidentified metabolite designated X. The major hydrolysis product in the male was 12-hydroxymethyl while X was found to be the major product in the intact female and OVX. Under the conditions of this study, there were differences in the metabolic activation of DMBA by male and female rat liver. Ovariectomy, followed by DMBA perfusion 7 days later, did not result in significant changes in DMBA metabolism relative to the intact female, except for a decreased rate of excretion of metabolites in bile.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Comparative metabolism of 7,12-dimethylbenz[a]anthracene by the perfused liver and liver microsomal preparations from Sprague-Dawley rats.

The metabolism of DMBA by microsomes and various cell cultures has been widely studied. However, the biotransformation of this compound by intact organs has not been well characterized. In order to compare the metabolism of DMBA in the whole liver with that in subcellular preparations, we used an in situ single-pass rat liver perfusion system and rat liver microsomes. [14C]DMBA was infused into the livers of Sprague-Dawley rats during the first 60 min of a 120 min perfusion. HPLC analysis of extracts of perfusate samples indicated that DMBA was rapidly oxidized in this system to a series of metabolites. The major products were polar metabolites including the trans-5,6- and the trans-10,11-dihydrodiols (46%), the trans-3,4-dihydrodiol (5%) and the 7-OHM-12-MBA and the 12-OHM-7-MBA metabolites (12%) of DMBA. Microsomes prepared from livers of corn oil treated rats were incubated with [14C]DMBA for 60 min, then extracted. In the microsomal system the major DMBA metabolites were the trans-8,9-dihydrodiol (6%), the 7- and 12-hydroxymethyl (20%), and the 3- and 4-hydroxy (11%) of DMBA with the more polar metabolites and the trans-3,4-dihydrodiol present at lower levels (12 and 3% respectively). This is the first report of DMBA metabolism in a whole liver preparation and the results are clearly different from those obtained in subcellular preparations in our laboratory and in cell culture systems elsewhere. These results have important implications for understanding DMBA biotransformation in vivo.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol.

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Direct effects of free and conjugated steroids on GnRH stimulated LH release in cultured equine anterior pituitary cells.

Enzymatically dispersed anterior pituitary cells from donor mares were cultured for 48 h in alpha-modified Eagles' medium containing 10% steroid-free horse serum. The cells were then incubated for 24 h in fresh medium oestrogen followed by a 4-h incubation with or without GnRH. Media and cell extracts were analyzed for LH by radioimmunoassay. In the first series of experiments, pituitary cells from Day-3 dioestrous mares were preincubated with ethanol (control) or different concentrations of E2 (10(-11) to 10(-7) M) for 24 h prior to a 4-h incubation without (basal) or with 1.0 nM GnRH. E2 increased (P less than 0.001) GnRH-stimulated LH release with a maximal response obtained at 10(-10) M E2 with an ED50 of 3 x 10(-11) M E2. Also E2 enhanced greatly the responsiveness of the pituitary to varying concentrations of GnRH by decreasing the minimum effective dose and ED50 by 93% and 78%, respectively, and by increasing the maximum response to GnRH by 250%. E2 had no effect on basal LH secretion. In a second experiment, the effects of E2, E1 and E1S on basal and GnRH stimulated LH release were evaluated. Although E1 increased GnRH-stimulated LH release, the maximally effective dose of E1 to enhance LH release and the ED50 for E1 were 100 times greater than those found for E2. E1S had no oestrogenic activity even at concentrations as high as 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Meeting production: the economics of contracting mental illness.

This article presents a case study which illustrates the impact that economic forces can have on organizational structure and clinical practice. Specifically, the stresses and double-binds which staff experience as a result of having to manipulate different sources of funding in order to 'meet production' are examined. Ethnographic fieldwork conducted at a day treatment center connected with a Community Mental Health Center revealed that contracts are made and quotas are set each year to 'produce' different kinds of services. The two main types of services, habilitative and socialization, are not funded equally. Funding agencies pay the most for habilitative services because these are meant to be directed toward the most symptomatic clients requiring the highest level of care. However, who is billed habilitative at any one time may or may not have anything to do with who is actually symptomatic and receiving a higher level of care. The reasons for this are explored, with special attention paid to how staff must adjust to constantly changing funding requirements. Despite the obstacles, staff attempt to provide clients with the level of care that they need, regardless of whether or not this is reflected in production quotas. Taken together, these two elements--the efforts of staff to conform to funding agency requirements plus their attempts to provide clients with the level of care that they need--require that staff engage in a constant and very wearisome juggling act. It is concluded that as a consequence of the demands of meeting production, both staff morale and the treatment of clients is adversely affected.

Capital Financing↗

PubMed1988

Regulation of luteinizing hormone subunit biosynthesis in cultured male anterior pituitary cells: effects of gonadotropin-releasing hormone and testosterone.

The purpose of this study was to evaluate the direct effects of testosterone (T) on LH subunit apoprotein synthesis, glycosylation, and release by the male pituitary. Cells from 1-week castrate rats were cultured for 48 h in steroid-free medium, followed by 48 h in medium with or without 10 nM T. The cells were then incubated for 2, 4, 6, 8, or 12 h in medium containing [35S]methionine (35S-Met) or [3H]glucosamine (3H-Gln), with or without 1 nM GnRH (Exp 1) or in medium containing precursors with or without 10 nM T and/or 1 nM GnRH (Exp 2). Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Exp 1, precursor incorporation into total protein (TP) and LH subunits increased linearly over time for at least 8 h. GnRH did not affect precursor incorporation into total protein or 35S-Met labeling of LH subunits, but stimulated a linear time-dependent accumulation of 3H-Gln into total (cells plus media) LH subunits and release of radioimmunoassayable LH into the medium. Based on these results, the effects of T on LH subunit biosynthesis (with or without GnRH) were studied during an 8-h incubation. In Exp 2, GnRH enhanced total 3H-Gln (but not 35S-Met) incorporation into both LH subunits. GnRH stimulated the release of 35S-Met LH alpha and 3H-Gln LH subunits and increased the relative glycosylation of secreted LH subunits without altering the relative glycosylation of intracellular LH subunits. T inhibited radioimmunoassayable LH release and incorporation of both precursors into total and secreted LH subunits (with or without GnRH). However, only the relative glycosylation of secreted LH alpha was reduced by T (with or without GnRH). These data indicate that T acts directly at the pituitary to inhibit LH subunit apoprotein synthesis and selectively inhibit LH alpha glycosylation. Further, these data support the hypothesis that changes in LH glycosylation may be one of the ways by which GnRH and T regulate LH release.

Animals↗

PubMed1987

Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species.

The present study describes the development and characterization of a monoclonal antibody (518B7) generated against bovine LH (bLH). Although 518B7 was extremely specific for LH, very low species specificity was observed. A RIA using this antibody and radioiodinated equine LH (eLH) showed good sensitivity for all mammalian LH preparations tested, with the exception of human LH (15%, relative to the eLH reference standard). Activities of most mammalian LH's ranged between approximately 50-200%. Much less activity was detected with reptilian LH (less than 1.5%). Amphibian and avian LH fractions were essentially inactive. The reactivities of LH alpha and beta subunits from a variety of mammals clearly showed that the antibody reacts with the beta subunit. Sensitive RIAs were also developed utilizing 125I-bovine and 125I-rat LH. Interestingly, all hormone preparations which showed sufficient reactivity for statistical analysis within the dose ranges used in the present study (0.01-1000 ng/tube) produced a displacement curve parallel to the reference standard. We have also validated the use of 518B7 in detecting LH in serum. Parallel dilution curves relative to purified LH reference standards were observed with equine and bovine serum samples and equine pituitary extract. High (average 94%) recoveries were also seen with bovine serum with known amounts of exogenously added bLH. Similar patterns of LH secretion were detected with a RIA based upon 125I-bLH and 518B7 and a previously described polyclonal antibody-based RIA in bovine serum samples during estrus. Thus, a monoclonal antibody for LH has been produced which can be used to develop sensitive and specific RIAs in many different mammalian species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Biological and immunological properties of zebra pituitary gonadotropins: comparison with horse and donkey gonadotropins.

Previous studies from this laboratory have described the properties of purified luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from horse and donkey anterior pituitary glands. The present study afforded the opportunity to further characterize these previously purified hormone preparations and to compare them with enriched gonadotropin fractions from zebra pituitary glands. Although a single LH and FSH fraction was usually obtained for each pool of pituitaries, two separate zebra LH and two donkey FSH preparations were generated. Purified hormone preparations from the horse were designated eLH and eFSH. Preparations zLH-A, zLH-B, and zFSH were obtained from zebra pituitaries, and fractions dLH, dFSH-A, and dFSH-B were prepared from donkey pituitary glands. These preparations were analyzed by LH and FSH radioimmunoassays (RIAs), radioreceptor assays (RRAs), LH bioassay, and chromatofocusing. Clear immunological differences were observed between equid gonadotropins. Homologous RIAs for eLH and eFSH did not cross-react similarly, or in a parallel fashion, with gonadotropins from the donkey and zebra. In contrast, RIAs capable of assessing LH or FSH in a wide number of species showed all equid gonadotropin preparations to have considerable activity and to produce parallel dilution curves. Relative to eLH (1.00), zLH-A was found to have higher LH bioactivity:LH RIA (2.50), LH RRA:LH RIA (1.42), and LH bioactivity: LH RRA (2.21) activity ratios. The dLH and zLH-B fractions only differed from eLH in LH RRA:LH RIA activity (0.69 and 0.62, respectively). Only LH from the horse possessed clear intrinsic FSH-receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Evidence for cAMP as a mediator of gonadotropin secretion from female pituitaries.

Sodium flufenamate, which inhibited gonadotropin-releasing hormone (GnRH)-stimulated increases in adenosine 3',5'-cyclic monophosphate (cAMP), was used to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion. Quartered pituitaries from diestrous II female rats were perifused at 37 degrees C, and sequential effluent fractions were collected every 10 min. Administration of GnRH resulted in a characteristic biphasic response for both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas 5 microM cycloheximide inhibited the secondary augmented responses (phase II) of both hormones. Infusions of 0.1 mM flufenamate inhibited GnRH-stimulated gonadotropin secretion in a manner similar to that of cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate resulted in the restoration of LH and FSH secretion. The dibutyryl cAMP-restored response appeared to be protein synthesis dependent and specific for cAMP. These results suggest that although the cyclic nucleotide is not involved in the acute release of LH and FSH, it does appear to play a pivotal but indirect role in phase II release of the hormones, by effects involving the stimulation of de novo protein synthesis.

Adenosine Monophosphate↗

PubMed1987

Evidence for cAMP as a mediator of gonadotropin secretion from male pituitaries.

The purpose of this study was to use sodium flufenamate, a compound that inhibits gonadotropin-releasing hormone (GnRH)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in the pituitary, to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion from male pituitaries. Quartered male pituitaries were perifused at 37 degrees C and sequential effluent fractions collected every 10 min. Infusions of GnRH resulted in a twofold increase in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Cycloheximide, 5 microM, completely inhibited the GnRH-stimulated LH and FSH secretion. Infusions of 0.1 mM flufenamate had similar effects on gonadotropin secretion as cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate restored the secretory responses of both hormones. The flufenamate-inhibited GnRH stimulated LH and FSH release, which was restored by DBcAMP and appeared to be protein synthesis dependent and specific for cAMP. These results suggest an indirect role for cAMP as a mediator of gonadotropin secretion from male pituitaries. However, in contrast to female pituitaries, the secretion of these hormones from male pituitaries is completely dependent on cAMP and de novo protein synthesis.

Animals↗

PubMed1987

The effects of gonadotropin-releasing hormone and estradiol on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

This study investigated the effects of physiological concentrations of GnRH and estradiol (E2) on LH biosynthesis and release using cultured anterior pituitary cells. Pituitaries from female rats were enzymatically dispersed and cultured for 48 h in steroid-free alpha-Modified Eagle's Medium, followed by a 24-h culture in medium with or without E2. The cells were then incubated for a 4-h (Exp 1 and 2) or 8-h (Exp 3) period in medium containing radiolabeled precursors with or without GnRH. Radioactive precursor incorporation into LH was determined by immunoprecipitation, while immunoreactive LH (iLH) content was quantified by RIA. In the first experiment, all concentrations of E2 (10(-11)-10(-8) M) enhanced iLH release in response to 1 nM GnRH, confirming previous reports. GnRH increased [3H]glucosamine (3H-Gln) incorporation into LH, but had no effect on [35S]methionine (35S-Met) incorporation. The higher concentrations of E2 enhanced GnRH-stimulated 3H-Gln LH production. In the second experiment, the effects of GnRH (10(-9) M) and E2 (5 X 10(-10) M) on the incorporation of [3H]galactose, [3H]mannose, [3H]fucose, or [35S]sulfate into LH were investigated. Although all precursors were incorporated into LH, no specific effect of GnRH and/or E2 on incorporation of any of the precursors into LH was noted. In Exp 3, pituitary cells were cultured with or without 0.5 nM E2 followed by an 8-h incubation with varying physiological concentrations of GnRH (10(-11)-10(-9) M) and radiolabeled precursors (3H-Gln and 35S-Met). GnRH stimulated iLH release in a dose-dependent manner, and this response was enhanced by E2. GnRH also increased the incorporation of both 3H-Gln and 35S-Met into LH, but the dose of GnRH required for this response was dependent upon the estrogen environment. In the absence of E2, only 10(-9) M GnRH increased 3H-Gln LH and 35S-Met LH production, whereas in cells exposed to E2, all concentrations of GnRH (10(-11)-10(-9) M) increased 3H-Gln LH and 35S-Met LH production. In all experiments, the specific activity of radiolabeled LH released under basal conditions was greatly reduced by stimulation with GnRH. These results suggest that GnRH regulates both LH glycosylation and LH polypeptide synthesis and that E2 lowers the physiological concentration of GnRH necessary to stimulate this biosynthetic response. Moreover, estrogen's enhancement of GnRH-stimulated LH release appears to be due to its action on mechanisms regulating the release of previously synthesized stored hormone as well as the release of newly synthesized LH.

Animals↗

PubMed1987

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells.

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

Animals↗

PubMed1986

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats.

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.

Animals↗

PubMed1984

Teratogenicity of nitrofen (2,4-dichloro-4'-nitrodiphenyl ether) and its effects on thyroid function in the rat.

Nitrofen is a herbicide with potent teratogenic activity in rodent species. Previous studies have indicated that this agent has a stereochemical structure similar to thyroid hormone, and that exposure of adult mice results in depression of thryoxine (T4) levels. The present study was undertaken to determine if teratogenic exposure to nitrofen alters pituitary-thyroid function in nonpregnant, pregnant, and fetal rats, and if these potential alterations could be related to induction of birth defects. In adult thyroparathyroidectomized (TPTX) female rats, nitrofen exposure for 2 weeks resulted in a significant suppression of thyrotropin-stimulating hormone (TSH) levels. When a single dose of nitrofen was administered to euthyroid female rats, a trend toward reduction (p = 0.058) in the release of TSH after a thyrotropin-releasing hormone (TRH) challenge was observed 4 and 5 hr after exposure. Pregnant euthryoid rats given a single dose of nitrofen on Day 11 of gestation had significantly depressed TSH and T4 levels, and fetal T4 levels were markedly depressed at term. Administration of T4 on Day 2 through 22 of pregnancy plus nitrofen on Day 9 through 11 to TPTX dams resulted in a 70% reduction in the frequency of malformed fetuses, especially in regard to the frequency of heart anomalies, compared to nitrofen exposure alone. Competitive displacement studies in radioimmunoassays for T4 and T3 indicated that a nitrofen metabolite (4-hydroxy-2,5-dichloro-4'-aminodiphenyl ether) competed with [125I]T3 for antibody binding, while the parent compound and six isolated metabolites failed to compete with [125I]T4 for antibody binding. These results have been interpreted to indicate that nitrofen teratogenicity is mediated at least in part by alterations in maternal and/or fetal thyroid hormone status, and may be due to a premature and pharmacologic exposure to the embryo to a nitrofen-derived, T3-active metabolite.

Animals↗