Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig

Corpora allata of adult female Euborellia annulipes, incubated in medium containing ³H-methionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 ± 1.6% of the total juvenile hormone III synthesized was released and 96.5% ± 0.3 in medium supplemented with 60 μM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 ± 0.004 mm), hormone production was only 0.59 ± 0.13 fmol hr⁻/corpus allatum; production increased to 1.52 ± 0.25 fmol hr⁻¹/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 ± 0.13 fmol hr⁻¹/corpus allatum. The addition of 60 μM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of l-methionine in the medium also enhanced hormone biosynthesis. Maximal hormone production was 32.8 ± 10.9 fmol hr⁻¹/corpus allatum, at an l-methionine concentration of 51 μM.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.     

Binding capacity of luteinizing hormone-releasing hormone and its analogues for pituitary receptor sites.

Competition for luteinizing hormone-releasing hormone (LH-RH) receptor sites by the inhibitory analog [D-Phe², D-Trp³, D-Phe⁶]-LH-RH and by the superactive stimulatory analog [D-Trp⁶]-LH-RH was observed in adenohypophysial homogenates incubated at 4°C. Competition for LH-RH binding sites was less evident with adenohypophysial plasma membranes. The binding affinities of these analogues to LH-RH pituitary receptors can explain at least in part their respective action in blocking ovulation and in inducing a greater release of luteinizing hormone and follicle stimulating hormone than the parent hormone.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

[125I] gonadotropin binding to the ovary of an Indian major carp,Catla catla,at different stages of reproductive cycle

At different stages of the annual reproductive cycle ofCatla catla, a major Indian carp, specific binding of gonadotropic hormone to the plasma membrane receptors was demonstrated. Maximum specific binding of [¹²⁵I]Catla gonadotropic hormone was obtained at 30‡C and pH 7.5 during 2 h of incubation.Catla gonadotropic hormone binding was saturable with high affinity. Competitive inhibition experiment showed that binding site was specifically occupied by piscine gonadotropic hormone,Catla gonadotropic hormone and murrel gonadotropic hormone, human chorionic gonadotropin was a weak competitor while bovine thyroid stimulating hormone, bovine prolactin and ovine follicle stimulating hormone had no effect. Scatchard analysis ofCatla gonadotropic hormone binding to the plasma membrane preparation from the carp oocytes of different reproductive stages showed that the range of dissociation constant(K _d ) varied from 0.78 to 0.97 x 10^-10 M. However, maximum binding capacity (B-max) varied remarkably between the different stages of reproductive cycle, it was 6.11 ± 0.36 fmol/mg protein in the preparatory stage which increased to about three-fold in prespawning stage of reproductive cycle (17.0 ± 0.29 fmol/mg protein) and spawning (18.7 ± 0.17 fmol/mg protein) and lowest in postspawning stage of reproductive cycle (5.28 ± 0.28 fmol/mg protein). Fluctuation in the number of gonadotropic hormone binding site at different stages of annual reproductive cycle was found to be coincided well with the pattern of ovarian steroidogenesis in response toCatla gonadotropic hormone as determined by the formation of progesterone from pregnenolone.     

Effect of bovine growth hormone on rat liver plasma membranes as studied by circular dichroism and fluorescence using the extrinsic probe 7,12-dimethylbenzanthracene

Conformational changes produced by in vitro bovine growth hormone addition to plasma membranes of hypophysectomized rat liver proteins and lipids have been studied by circular dichroism as well as intrinsic and extrinsic fluorescence. 7,12-Dimethylbenzanthracene has been used as a fluorescent probe of changes in membrane structure. The exposure of membranes to bovine growth hormone produced a change in membrane negative ellipticity. Dimethylbenzanthracene at concentrations similar to those employed in fluorescence studies had no effect on the membrane circular dichroism spectrum. Its presence did, however, prevent a response to growth hormone. There was a decrease in peak fluorescence intensity and a peak shift when bovine growth hormone (0.5 · 10^?12 M) was added to liver membranes. The addition of dimethylbenzanthracene (1.6 · 10^?6 M) to membranes resulted in a decrease in the intensity of the protein fluorescence peak at 335 nm and the appearance of two peaks at 430 and 407 nm, assignable to the probe. The addition of bovine growth hormone (0.5 · 10^?12 M) produced a decrease in fluorescence at 335 nm and also in the peaks at 407 and 430 nm. These data are consistent with the conclusion that bovine growth hormone produces a conformational change in rat liver plasma membrane proteins and lipids.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormones and labelled antibody binding techniques

The parathyrin receptor in renal cortex has been investigated by studying the binding of ¹²⁵I-labelled parathyrin, or of unlabelled parathyrin detected with ¹²⁵I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled ¹²⁵I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was $\text{2.3 · 10}{}^8\text{M}{}^{\mathrm{?1}}$ and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Critical Sensitivity of the Ovary of <Emphasis Type="Italic">Aedes aegypti</Emphasis> Adults to Sterilization by Juvenile Hormone Mimics

STERILIZATION of adult insects with juvenile hormone mimics has been reported several times^1–5, but only once for adult mosquitoes and no details were given². I report here on the sterilizing effect of three juvenile hormone mimics on female Aedes aegypti. They were (i) mixed geometric isomers of methyl 10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate (CJH2), one of two substances with high juvenile hormone activity isolated from Cecropia oil⁶; (ii) mixed geometric isomers of farnesyl methyl ether (FME) and (iii) 2 cis/trans, 6 trans farnesenic acid ethyl ester (FAE). All three compounds were first tested on fifth instar Rhodnius prolixus using Wiggles-worth's method⁷. The doses to give a score of ten were as follows: CJH2 1.3µg; FME 1.4 µg; FAE 31.7 µg.     

Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly Sarcophaga bullata.

Ecdysterone has been shown to stimulate hydrocarbon biosynthesis in Sarcophaga bullata at pupariation. When post-feeding larva were treated with ³H-acetate 10·5 hr after hormone administration, a 1·3 times greater quantity of ³H-acetate was incorporated into hydrocarbon in the ecdysterone injected insects than controls. A similar experiment with a 24 hr delay of ³H-acetate administration following hormone treatment resulted in 3·3 times greater incorporation into hydrocarbon of treated animals.Isolated integuments synthesize hydrocarbon from acetate better than internal tissues, and the integuments of ecdysterone-treated insects incorporate acetate into hydrocarbon 9·8 times better than integuments of control insects. This indicates that cuticular hydrocarbon biosynthesis not only occurs in the integument, but that a locus of regulation is present in the integument.     

Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina,in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development.As well as morphogenetic effects, a proper concentration (3 × 10^?5 M to 3 × 10^?7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10^?9 M seemed to be rather toxic to both development and metabolic activity.     

Effects of growth hormone on ATPase and fluorescence of isolated liver membranes utilizing the fluorescent substrate, 1,N6-etheno-adenosine triphosphate

The influence of bovine growth hormone on Mg²⁺-ATPase (EC 3.6.1.4) in isolated liver plasma membranes of hypophysectomized rats has been investigated in vitro by means of spectrofluorescence measurements in parallel with Mg²⁺-ATPase assays, using 1, N⁶-etheno-ATP as substrate and fluorescence probe.Bovine growth hormone, at concentrations of 10^?14m and above, enhanced significantly Mg²⁺-ATPase activity in the presence of GTP at concentrations from 10^?6m to 10^?10m. Moreover, bovine growth hormone decreased fluorescence intensity of membrane protein at the peak at 330 nm and of 1, N⁶-etheno-ATP at its peak at 395 nm as well. The greatest decrease in fluorescence intensity of 1, N⁶-etheno-ATP was observed in the presence of 5 mm MgCl₂ and 10^?8m GTP, consistent with the stimulating effect of bovine growth hormone on Mg²⁺-ATPase activity. In addition, bovine growth hormone caused a small decrease in fluorescence intensity of 1, N⁶-etheno-ADP, but not the corresponding fluorescent analogs of AMP, cyclic AMP, and adenosine. The decrease in fluorescence intensity of 1, N⁶-etheno-ATP by bovine growth hormone was completely eliminated by addition of ATP, ADP, and cyclic AMP at concentrations five times that of 1, N⁶-etheno-ATP. Neither AMP nor adenosine exerted any effects.Bovine growth hormone also increased the fluorescence polarization of 1, N⁶-etheno-ATP from 0.177 ± 0.006 to 0.212 ± 0.010 at 300 nm under the same conditions employed for the Mg²⁺-ATPase assay.These observations suggest that bovine growth hormone produced changes in tertiary structure of membrane proteins in general and probably Mg²⁺-ATPase in particular with consequent enhanced enzyme activity.     

Labelled antibody membrane assay for parathyroid hormone a new approach to the measurement of receptor bound hormone.

A new method is described for the measurement of hormone bound to membrane receptors. Antibodies specific for the C-terminal and N-terminal regions of parathyroid hormone were labelled with ¹²⁵I and incubated with renal membranes which had been previously incubated with unlabelled hormone. The uptake of hormone demonstrated pH and time dependence and was a saturable process. Treatment of the membranes with acid or heating to 100°C, or inactivation of the hormone with hydrogen peroxide, completely abolished detectable hormone uptake to the membranes.     

Water Soluble Receptors for Human Growth Hormone from Rabbit Liver

Abstract

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes from pregnant female rabbits in 1 mM Tris buffer (pH 7.5 or 9.0) at 4°C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (K_a 2.2 × 10⁹ M^-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (K_a ～1 × 10⁸ M^-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Properties of human growth hormone binding sites solubilized from female rabbit kidney

Human growth hormone binding sites from female rabbit kidney microsomes were solubilized by treatment with the nonionic detergent Triton X-100. The binding of ¹²⁵I-labelled human growth hormone to the solubilized sites retains many of the properties observed in the particulate fraction, such as saturability, reversibility, high affinity and structural specificity. The association and the dissociation process are time- and temperature-dependent. The association rate constant, k₁, is 1.6·10⁷ mol^?1·l·min^?1 at 25°C, and the dissociation rate constant, k_?1, is 2.8·10^?4 min^?1 at 25°C. Solubilization causes an increase in affinity as well as in binding capacity. Scatchard plots from saturation curves suggest the presence of a single class of binding site with a dissociation equilibrium constant, K_d, of 1.3·10^?11 M and a binding capacity of 133 fmol/mg of protein. Similar results were obtained from competition experiments. Specificity studies revealed the lactogenic characteristics of the solubilized sites. The Stokes radii of the free binding sites and of the ¹²⁵I-labelled human growth hormone-binding site complex, determined on a Sepharose CL-6B column, are 57 and 53 Å, respectively.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Pituitary receptor binding activity of active, inactive, superactive and inhibitory analogs of gonadotropin-releasing hormone.

We have previously described the binding of biologically active ¹²⁵I gonadotropin-releasing hormone to the 10,800 × g membrane fraction prepared from 7-day castrate adult female rat anterior pituitary glands. Specific binding with two equilibrium association constants (10⁹ liters per mole and 10⁵ liters per mole) was found and an equilibrium competitive binding radio-receptor assay established. In order to further characterize the gonadotropin-releasing hormone receptor, 20 synthetic analogs with known bioactivity were tested in the radioreceptor assay. In vivo biological activity correlated with high affinity receptor binding but not with low affinity binding. Inhibitory analogs with no in vivo biological activity and weak antagonistic properties did not bind, while in vivo active or superactive analogs bound to high affinity receptors. These findings suggest that the high affinity gonadotropin-releasing hormone receptor binds only biologically active gonadotropin-releasing hormone like peptides and that this binding may be the initial step in gonadotropin-releasing hormone actions at the pituitary level.