Thyroxine transport in choroid plexus

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon increasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of 125I-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated 125I-thyroxine, but not [125I]triiodothyronine, for many hours. The accumulation of 125I-thyroxine in choroid plexus preceded that in brain. The amount of 125I-thyroxine in non-brain tissues and the [125I]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on partitioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthyretin newly synthesized and secreted by choroid plexus.     

Magnesium-dependent non-specific binding of [125I]prolactin to myelinated tracts in the rat central nervous system

An in vitro autoradiographic assay was used in identifying a magnesium-dependent, non-specific binding of [125I] prolactin to myelinated fiber tracts in the rat brain. Frozen tissue sections were incubated for 18 h at 4 degrees C in media which included [125I]prolactin alone or with a 500 fold excess of unlabelled prolactin. Magnesium in the incubation medium caused a non-specific binding of radiolabelled prolactin to the myelinated fiber tracts in the brain. In contrast, calcium did not facilitate prolactin non-specific binding to myelin. Hence, calcium should optimize the detection of specific prolactin binding sites in the brain by in vitro autoradiographic or radioreceptor assays.     

Studies with anti-growth hormone receptor antibodies.

Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.     

Lack of interaction between hormonal proteins and synthetic carrier ampholytes (Ampholine)

Using ¹⁴C-labeled Ampholine, radioiodinated and unlabeled human growth hormone, and ovine prolactin, no significant degree of interaction could be detected by polyacrylamide gel electrophoresis and gel chromatography. Protein binding to excess Ampholine was negligible. Ampholine binding to excess protein was below the detection limit of 0.2 moles of Ampholine per mole of protein. Commercial Ampholine contains large molecular weight components that cannot be separated by gel chromatography from proteins in the 20–30,000-molecular weight range.     

Measurement of specific radioactivities in labelled hormones by self-displacement analysis.

A graphical method is described that allows the determination of specific radioactivities of radioactively labelled hormones. This method combines the self-displacement technique, plotting bound/free ratios versus mass of unlabelled hormone or total radioactivity of labelled preparation added to the receptor preparation, and the maximal binding capacity of the labelled hormone. The procedure presented herein provides a more realistic specific radioactivity for use in all binding experiments. Application of the method is demonstrated for 125I-labelled ovine prolactin, and data are presented for 125I-labelled human choriogonadotropin and [3H]testosterone.     

Development of transport mechanisms for sulphate and iodide in immature choroid plexus

The time-course of development of sulphate and iodide transport mechanisms in choroid plexus was studied by measuring uptake of [³⁵S]sulphate and [¹²⁵I]iodide from an incubating medium by isolated choroid plexuses of foetal and newborn rabbits and cats. Sulphate uptake by choroid plexus was poorly developed in rabbit foetuses just before term, but highly developed in newborn animals. Iodide uptake was already well developed in the most immature foetuses studied.     

Human growth hormone-variant demonstrates a receptor binding profile distinct from that of normal pituitary growth hormone

We have recently established that the human growth hormone-variant (hGH-V) gene is functional in vivo by documenting its expression in the placenta. We have subsequently generated transformed murine cell lines stably expressing the genes for normal pituitary growth hormone (hGH-N), hGH-V, and each of two chimeric genes generated by exon 3 exchanges, hGH-NV3 and hGH-VN3. In the present study, we utilize these cell lines as sources of hormone to characterize and compare the receptor binding profiles of hGH-N with hGH-V. hGH-V was found to displace 125I-ovine prolactin bound to rat liver microsomes (lactogen binding) and to displace 125I-hGH bound to rabbit liver microsomes (somatogen binding). Therefore, hGH-V would be predicted to display both somatogenic and lactogenic bioactivity, a dual specificity previously thought to be unique to hGH-N. The concentrations of hormone necessary to displace 50% (IC50) of the 125I-hGH from somatogen receptors and 125I-ovine prolactin from lactogen receptors was expressed as a ratio, IC50 somatogen: IC50 lactogen, for each hormone tested. A 7.4-fold difference in this ratio was observed for hGH-N compared to hGH-V, suggesting significantly greater selectivity by hGH-V in binding to the somatogen receptor. The intermediate binding ratios of the hGH-NV3 and hGH-VN3 chimeric proteins confirmed the distinct receptor binding profiles of the two parent hormones and served to identify three amino acids of potential importance in defining their respective receptor binding specificities.     

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.     

Presence of prolactin receptors in eel liver and carp kidney and growth hormone receptor in eel liver.

1. 125I-labelled ovine prolactin and bovine growth hormone were used to test for the presence of prolactin and growth hormone receptors in membrane prepared from tissues of the white eel Anguilla japonica, the carp Ctenopharynogodon idellus and the ricefield eel Monopterus albus. 2. High levels of specific 125I-labelled ovine prolactin binding were found in white eel liver membranes and carp kidney membranes. 3. High levels of specific 125I-labelled bovine growth hormone binding were detected in white eel liver membranes. 4. Tissues of the ricefield eel did not bind 125I-labelled ovine prolactin or bovine growth hormone. 5. The results suggest the presence of prolactin receptors in white eel liver and carp kidney membranes and growth hormone receptors in white eel liver membranes.     

Endogenous Prolactin Maintains its own Binding Sites in the Pigeon Crop Sac Mucosa

Abstract

The specific binding of lactoperoxidase-labelled ¹²⁵I-labelled ovine prolactin was determined in a membrane particulate of the pigeon crop-sac mucosal epithelium. Binding was found to be dependent upon the particulate preparation used, its protein concentration and the length of the incubation at 5°C. Scatchard analysis of the binding to crop-sacs from saline or prolactin-injected (1.9 μg per pigeon) revealed that prolactin stimulated 7-fold its own receptors by increasing the number of binding sites per mg protein: saline - 392±75 fmol/mg protein and prolactin 2736±602 fmol/mg protein (p<0.01). This increase did not affect the affinity constant (Ka): saline - 5.28±0.75x10⁸ l/mol and prolactin-3.28±0.40x10⁸ l/mol (N.S.), in keeping with the stimulatory effect of prolactin in the rat liver and mammary gland. This study further demonstrates the physiological role of endogenous prolactin in maintaining its own binding-sites in the pigeon crop-sac, since the administration of 0.8 ml anti-serum to prolactin resulted in a 63% reduction in the specific binding of the labelled hormone in vitro. These results confirm the prolactin binding to the pigeon crop-sac mucosa, quantify the stimulation of this binding by prolactin itself, and demonstrate the role of the endogenous hormone in the maintenance of these receptors.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Prolactin-Brain Interactions and Reproductive Function

SYNOPSIS. There is mounting evidence that prolactin influencesreproductive function in several vertebrate species via directaction on the central nervous system. In most instances, however,the basic properties of these prolactin-induced alterationsin brain function, the neurochemical mechanisms underlying theseeffects, and the identity of the prolactinlike molecules thatmight normally promote these changes have yet to be adequatelycharacterized. Several properties of the prolactin-brain relationshipthat have emerged from our work in the ring dove (Streptopeliarisoria) are described in this paper and discussed in relationto similar work in other species. Receptor binding studies usingradiolabelled mammalian prolactin preparations consistentlyindicate the existence of saturable, high affinity binding sitesfor prolactin in the brains of several vertebrate species. Theapparent concentration of these sites in the diencephalon iscorroborated by recent mapping studies in rabbits, ring doves,and three song birds in which in vitro autoradiography and densitometrywas employed. Nevertheless, direct comparisons among the threesongbird species suggest some differences in the precise distributionand/or concentration of prolactin binding sites within the preoptic-hypothalamiccontinuumthat may relate to species differences in the display of prolactin-related incubation behavior and parental activities. Althoughdefinitive evidence is currently lacking, it would appear thatblood-borne prolactin gains access to binding sites in the centralnervous system and that a receptor-mediated, blood-to-cerebrospinalfluid transport process in the choroid plexus may be a majorconduit for such uptake. Recent findings from several vertebratespecies also suggest that the brain may synthesize one or moreprolactin-like molecules that could conceivably interact withthese binding sites. Direct actions of prolactin and relatedhormones on brain mechanisms underlying reproductive processesare suggested by the profound dose-dependent suppression ofgonadotropin secretion and gonadal activity in ring doves byintracerebroventricular administration of prolactin or growthhormone at doses that are below those required for significantperipheral actions. A major challenge for future investigationwill be to identify the brain site(s) at which these and othereffects of prolactin are exerted and to determine how blood-borneprolactin of pituitary origin, prolactin-like molecules of brainorigin, and growth hormone-like molecules interact with eachother and with other neurochemical systems to promote thesetypes of changes under normal physiological conditions.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Modulation of prolactin binding sites in vitro by membrane fluidizers II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in ¹²⁵I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.     

Modulation of insulin receptors and catecholamines in rat brain in hyperthyroidism and hypothyroidism

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat brain using Triton X-100 extracts from total membranes, synaptosomes and choroid plexus in experimental hypothyroidism and hyperthyroidism. Insulin receptor activity was assessed by binding to [125I]insulin and catecholamines by high performance liquid chromatography. In choroid plexus thyronines effects are well pronounced and there is modulation vis a vis plasma hormone concentrations. Triiodothyronine levels increase in brain in all experimental groups. This suggests that rat may serve as a useful model for thyronine homeostasis in brain and there may be involvement of very complex regulatory mechanisms in glucose tolerance.     

A quantitative electron microscope autoradiographic study on I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 °C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 ° or 38 °C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 ° than at 38 °C. Co-incubation of ¹²⁵I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with ¹²⁵I-hCG.The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 °C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 °C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of ¹²⁵I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.