Hormone Uptake Research Articles

A new method for measurements of plasma protein steroid-binding kinetics in human plasma at 37 C

The equilibrium kinetics in vivo of free and protein-bound steroid hormone were examined at 37 C. Human plasma was pumped through a 1 m PVC catheter and tritiated steroid hormone continuously added at the inlet. The plasma was collected and rerun through a new catheter 24 h later. A significantly (P<0.05) higher uptake of steroid hormone was observed in the first parts of the catheter during the first passage when compared to the second passage. The results indicate that non-equilibrium conditions existed for testosterone (>10 sec), estradiol (4 sec), estrone (>2 sec), and estriol (>2 sec) while no delay was observed for progesterone. The results indicate that the counter-current transfer between the testicular and ovarian vessels, respectively, may create a physiologically important, temporarily increased concentration of available hormones in the arterial blood supply to the organs.

The free hormone hypothesis: a physiologically based mathematical model.

The free hormone hypothesis states that the biological activity of a given hormone is affected by its unbound (free) rather than protein-bound concentration in the plasma. The fundamental mathematical and physiological principles relating to this hypothesis are reviewed, along with experimental data that shed light on its validity. It is shown that whether or not this hypothesis is likely to be valid for any given hormone will depend largely on which step in the tissue uptake process (plasma flow, dissociation from plasma binding proteins, influx, or intracellular elimination) is rate-limiting to the net tissue uptake of that hormone. It is further shown that the free hormone hypothesis could hold even if tissue uptake of hormone occurred by a mechanism that acted directly on one or more circulating protein-bound pools of hormone. Indeed, many of the data previously interpreted as being inconsistent with the free hormone hypothesis are in fact readily consistent with it when its predictions are fully understood. Nevertheless, the free hormone hypothesis is not likely to be valid for all hormones with respect to all tissues. It is likely to be valid with respect to all tissues for the thyroid hormones, for cortisol, and for the hydroxylated metabolites of vitamin D. For many of the other steroid hormones, however, it is likely to be valid with respect to some tissues, but not with respect to others (in particular, the liver). And for some of the steroid hormones (in particular, progesterone) it may not hold at all.

Effects of vitamin B6 deficiency and repletion on the uptake of steroid hormones into uterus slices and isolated liver cells of rats.

1. In vitro, pyridoxal phosphate extracts steroid-hormone receptors from tight nuclear binding (Cidlowski & Thanassi, 1981); in vitamin B6-deficient rats there is increased and prolonged nuclear accumulation of oestradiol in the uterus and testosterone in the prostate, associated with enhanced biological responsiveness of these target tissues to steroid hormone action (Symes et al. 1984; Bowden et al. 1986). 2. Slices of uterus from vitamin B6-deficient rats accumulated more [3H]oestradiol than did tissue from repleted animals. Acute repletion with vitamin B6 (0.5-1 h before killing) further increased the uptake of the steroid. 3. Isolated hepatocytes from vitamin B6-deficient rats accumulated more [3H]dexamethasone than did cells from repleted animals. Pre-incubation of the hepatocytes with pyridoxal phosphate resulted in a further increase in the uptake of the steroid. 4. The results suggest that in addition to the putative role of pyridoxal phosphate in releasing steroid-hormone-receptor complexes from tight nuclear binding (Cidlowski & Thanassi, 1981), vitamin B6 deficiency may also increase the concentration of steroid-hormone receptors or enzymes and other steroid-binding proteins in target tissues.

In-vivo uptake of human growth hormone in male rat liver.

125I-Labelled human GH (hGH) was injected i.v. to male rats and its subcellular distribution in the hepatocyte was examined using fractionation techniques. Uptake into liver homogenates was maximal by 15 min after injection and represented 24% of the injected radioactivity; it was markedly inhibited by coinjection of native hGH. 125I-Labelled hGH taken up by the liver underwent a time-dependent translocation process. The peak of specific labelling of plasma membranes occurred at 3 min whereas later on the radioactivity was concentrated in low-density structures present in Golgi-endosome fractions. To characterize the ligand-associated structures better, endosome-enriched fractions were prepared from a microsomal fraction by isopycnic centrifugation in a sucrose gradient and a Nycodenz gradient. The radioactivity was in one peak with a median density of 1.096 g/cm3 in the Nycodenz gradient fractions. The peak of radioactivity was distinct from that of galactosyltransferase activity which appeared at a median density of 1.114 g/cm3. The labelled material eluted from the various subcellular fractions appeared as intact hGH. Upon in-vivo interaction with male rat hepatocytes, 125I-Labelled hGH was internalized with a sequential association with plasma membranes and endocytic structures distinct from Golgi elements.

The effects of environmental stress on steroid receptor levels and steroid-induced morphogenesis in Achlya ambisexualis

Incubation of Achlya ambisexualis at elevated temperature (heat shock) or in the presence of sodium arsenite resulted in an inhibition of steroid hormone-induced responsiveness. The effect of heat shock was time- and temperature-dependent and more severe than the effect of sodium arsenite. Incubation at 37 °C for 30 min completely abolished the steroid-induced response and full recovery was not observed until 6 h after a return to the normal growth temperature of 22 °C. Heat shock and arsenite treatment had no effect on the cellular uptake of the steroid hormone, but heat shock resulted in a time- and temperature-dependent loss in the cellular level of steroid receptors. In contrast, arsenite treatment had little effect on the concentration of steroid receptors. However, both heat shock and arsenite treatment produced a long-term (4 h) and transient (1 h) inhibition of total protein synthesis, respectively. The recovery of steroid-induced responsiveness following heat shock was observed after both protein synthesis and steroid hormone receptor levels had returned to normal values.

Thyrotropin releasing hormone uptake into serum and cerebrospinal fluid following intravenous or subcutaneous administration.

Intravenous thyrotropin releasing hormone (TRH) was administered to 6 amyotrophic lateral sclerosis (ALS) patients at the dose rate of 10 mg/kg. Blood samples were obtained prior to and at 10, 20, 40, 60, and 120 min during the TRH infusions. Lumbar punctures were performed at 90 min following the start of infusion. The mean serum TRH concentration rose from 0.03 +/- 0.02 (SD) to 17 +/- 2 ng/ml by 60 min and remained constant to 120 min. The mean CSF TRH concentration rose 10-fold from 0.02 +/- 0.01 to 0.2 +/- 0.02 ng/ml at 90 min and increased further to 0.5 +/- 0.2 ng/ml at 120 min. Subcutaneous TRH was administered to 4 ALS patients at 2.5 mg/kg and to 5 ALS patients at 5.0 mg/kg. The mean serum TRH concentration increased to 1.4 +/- 0.6 ng/ml (2.5 mg/kg) and 3.2 +/- 1.1 ng/ml (5.0 mg/kg) by 60 min. The mean CSF TRH concentration at 60 min increased to 0.3 +/- 0.08 ng/ml following 2.5 mg/kg TRH and 0.8 +/- 0.04 ng/ml following 5.0 mg/kg TRH. TRH entry into the CSF is comparable following subcutaneous or intravenous administration.

The influence of thyroid hormones on colony growth of peripheral CFU-GM from normal and leukemic subjects.

In order to clarify the alterations of granuloblastic cells in chronic and acute myeloid leukemia, the colony growth behavior of cultured CFU-GM from the peripheral blood of normal and leukemic subjects was examined in basal conditions and after adding to the medium T3 or T4 and/or thioproline and/or flurbiprofen. These drugs had in previous investigations proved their ability to modify cellular receptors and the uptake of thyroid hormones. The study was carried out in semisolid (double agar layer) and liquid medium, utilizing the techniques described previously. Both thyroid hormones enhanced the colony growth from normal peripheral blood CFU-GM and the response was more evident with T4 than T3. The effect on leukemic CFU-GM (from CML and AML) was less clear, probably due to the presence in leukemic cells of a defect of cellular uptake and to the utilization of T3 and T4. Indeed, on addition to the culture medium of thioproline, which modifies membrane permeability, and of fluorbiprofen, which inhibits PGE synthesis, the colony number and growth from leukemic CFU increased considerably in accordance with the results of our previous studies on these substances showing that they are able to modify cellular receptors for thyroid and several other hormones.

Inhibition of Uptake of Thyroid Hormone into Rat Hepatocytes by Preincubation withN-Bromoacetyl 3,3′,5-Triiodothy r onine

To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.

The biological expression of natural progesterone

Progesterone tablets were orally administered to women with no recent luteal activity. Three hypoestrogenic subjects who received, respectively, a single dose of 150 mg, 300mg and 300 mg/d/7d, showed absorption and a progressive increase in circulating values in a dose-dependent manner. Five out of nine oligomenorrheic patients experienced withdrawal uterine bleeding with 150 mg/12 h/5d. Postmenopausal endometrium estrogenised by mestranol ( n = 5) showed secretory histological changes after 300 mg/d/7d of oral progesterone. Six women with luteal insufficiency received 300 mg/d after control studies, and this dosage was effective in supplementing the circulating natural hormone so that it reached the level of that of fertile women. However, the endometrial progesterone uptake of these women showed no parallel increase. This study supports the clinical usage of oral progesterone and suggests evaluation of the dynamics of hormone uptake by the reproductive tissues.

Insulin effect on thyroid hormone uptake in rat skeletal muscle

Previous work has indicated that thyroid hormone entry into cells includes an energy-dependent, saturable process. In this study we investigated the effect of insulin on T 3 uptake in rat skeletal muscle. Intact soleus muscles were preincubated for 30 minuted at 37°C, pH 7.4, in modified Krebs-Ringer bicarbonate buffer in the presence or absence of insulin and then for 60 minutes after adding 50 pmol/L [ 125I] T 3. The results showed a stimulatory effect of insulin that was half maximal at 33 nmol/L and maximal at 100 nmol/L. Addition of 10 μmol/L T 3 to the incubation medium completely blocked the effect, showing that the action of insulin is exerted only on the specific component of T 3 uptake. The substitution of extracellular sodium with an equimolar amount of lithium also prevented the insulin effect. [ 125I] T 4 uptake was unaffected by insulin. These results indicate that T 3 uptake in skeletal muscle is sensitive to insulin, that insulin stimulates the specific component of T 3 uptake in a dose-dependent manner, and that its action requires extracellular sodium. In contrast, T 4 uptake is insensitive to insulin action in accordance with previous results that indicated passive diffusion as its major pathway for cell entry.

Impaired transport of thyroid hormones into livers of obese (ob/ob) mice

Obese (ob/ob) mice exhibit impaired hepatic thyroid hormone action that is mediated, at least in part, by a reduced nuclear 3,5,3'-triiodothyronine (T3) receptor occupancy. The possibility that lowered occupancy in obese mice may be caused by decreased transport of T3 across the hepatic plasma membrane was examined by measuring the unidirectional influx of [125I]T3 into livers of 8- to 10-wk-old obese and lean mice using a tissue-sampling portal vein-injection technique. Influx of [125I]thyroxine (T4), a substrate for T4 5'-deiodinase, was also measured. Unidirectional clearance of T3 and T4 was 64 and 80% lower, respectively, in obese mice than in lean mice. Hepatic T3 and T4 uptake was nonsaturable in both lean and obese mice, suggesting that transport occurs by lipid-mediated free diffusion. Clearance of another lipid-soluble hormone, hydrocortisone, was also lower in obese mice than in lean mice. Decreased membrane permeability to the above hormones in obese mice may result from reported changes in membrane lipid composition. In conclusion, decreased hepatic thyroid hormone uptake may contribute to impaired thyroid hormone action and T3 production in livers of obese mice.

Active Transport of Iodothyronines into Human Cultured Fibroblasts*

Thyroid hormone uptake into human cultured fibroblasts was studied using 2-min incubations with labeled iodothyronines. The results indicate the presence of an active T4 uptake process with two saturable sites with apparent Km values of 1.9 and 141 nM, respectively, and an active T3 uptake process with two saturable sites with Km values of 29 and 650 nM. The uptake of both hormones was energy dependent, i.e. inhibited by KCN or by incubation of the cells in the absence of glucose. By analogy with similar findings in rat hepatocytes we postulate that the high affinity systems represent active transport of thyroid hormone into the cell. Preincubation of the cells with 2 mM ouabain resulted in a decrease in the uptake of both T3 and T4, suggesting that a sodium gradient is necessary for transport. Similar to that in rat hepatocytes, uptake of T3 was inhibited by high concentrations of T4, and uptake of T4 was inhibited by high concentrations of T3. These data indicate that regulation of thyroid hormone uptake at the level of the plasma membrane may be operative in humans.

Role of sodium in thyroid hormone uptake by rat skeletal muscle.

Whether Na+ movement through the plasma membrane plays a role in thyroid hormone uptake was investigated in intact rat soleus muscles. After preincubation for 120 min at 37 degrees C in modified Krebs-Ringer bicarbonate containing 140 or 5 mM Na+ plus choline or lithium to maintain osmolarity, muscles were incubated with 50 pM [125I]triiodo-L-thyronine (T3) or [125I]L-thyroxine (T4) for 60 min. T3 uptake was decreased when extracellular Na+ was replaced by either choline or lithium, the amount of decrease corresponding to the specific (or saturable) uptake component. Monensin, an ionophore that stimulates Na+ entry, increased T3 uptake at 140 mM Na+ but not at 5 mM Na+. Amiloride, a Na+/H+ exchange inhibitor, had no effect on T3 uptake under basal conditions or when Na+ was replaced by choline, but reversed the action of lithium. Ouabain, an inhibitor of Na+/K+ ATPase, reduced specific T3 uptake. T4 uptake was unaffected by low extracellular Na+. These results are consistent with a major role of Na+ movement in T3 uptake by skeletal muscle, but not in T4 uptake, and suggest an involvement of membrane pumps in this process.

Uptake of thyroid hormones (L-T3 and L-T4) by isolated rat adipocytes

3,5,3′-triiodo-L-thyronine is taken up by isolated rat adipocytes under physiological conditions by a saturable sigmoidal process, while L-thyroxine uptake follows Michaelian kinetics. Comparative studies performed with intact adipocytes and derived liposomes suggest that thyroid hormones are taken up into cells via carrier-mediated transport.

Proton pumps and chemiosmotic coupling as a generalized mechanism for neurotransmitter and hormone transport.

Neuroendocrine secretory vesicles contain within their membranes a highly specialized H1-translocating ATPase responsible for the generation and maintenance of an electrochemical proton gradient, delta pH inside acidic, and delta psi inside positive. Coupled with a high internal buffering capacity and extremely low permeability of the membrane to protons, this proton pump can generate and maintain an intravesicular pH of 5.5, independent of the external pH, and transmembrane electrical potential of 60 mV. The chemiosmotic gradient has important implications for several functions of the secretory vesicles: (1) maintaining oxidizable substances (such as biogenic amines) in the unoxidized form; (2) stimulating (or inhibiting) peptide processing enzymes; (3) permitting precipitation of intravesicular protein complexes, thereby increasing the amount that can be stored within the vesicle; and (4) serving as the driving force for the uptake of certain hormones and neurotransmitters such as acetylcholine, biogenic amines, and ATP. By using the putative biogenic amine transporter as an example, it can be demonstrated that based purely upon the existence of a transporter in equilibrium with the electrochemical proton gradients, an amine concentration approaching 135,000 to 1 can be achieved. The bioenergetics of amine transport do not predict the molecular mechanism of amine translocation. By using kinetic analyses of amine accumulation under a variety of situations, however, initial information concerning the salient aspects of amine transport is being obtained.

Cellular distribution and hormonal regulation of h-SBP in human hepatoma cells

The cellular distribution of human Sex Steroid Binding Plasma Protein (h-SBP) was studied in human cells and tissues by indirect immunofluorescence, h-SBP was detected in the cytoplasm of hepatocytes, of prostate and epididymis epithelial cells and in endometrium. Sexual and non-sexual skin, intestine epithelium, striated muscle and some rodent organs were not labelled. The intracellular localization of h-SBP indicate that h-SBP could be taken up from the extracellular compartment or synthesized in situ in sex steroid target organs, where it may play a role in hormone uptake. The hormonal regulation of h-SBP secretion by a human hepatoma cell line, H5A, showed that tri-iodothyronine was more potent than estradiol or tamoxifen, which acted as estrogen agonist, in increasing secreted h-SBP and the combined effect of both thyroid and estrogen hormones resulted in an additive stimulation of h-SBP secretion. As shown by Northern blot analysis, oligonucleotides synthesized from the known sequence of h-SBP hybridized with a RNA of ∼2 kb which was more represented in H5A cells than in normal human liver, and was increased 2–3 times after hormonal stimulation of the cells. The presence of a poly(A +)RNA coding for h-SBP in the human liver indicated the hepatic synthesis of this protein.

Active transport of L-tri-iodothyronine through the red cell plasma membrane—true or false?

The membrane transport of L-tri-iodothyronine has been studied in human erythrocyte ghosts. Initial uptake showed saturability with a Km of 33.8 nmol/l, suggesting carrier-mediated transport. Ouabain partly inhibited this uptake. ATP, on the other hand, caused a significant increase in the Vmax indicating an active transport of L-tri-iodothyronine across the erythrocyte plasma membrane. Ghosts from patients with hyperthyroidism, hypothyroidism or thyroid hormone resistance did not differ significantly from controls in their uptake of the hormone.

5-HT 2 receptor-stimulated inositol phosphate formation in rat aortic myocytes

Serotonin (5-HT) stimulated inositol phosphate production in primary cultures of rat aortic myocytes via a 5-HT 2 receptor. Agonists active at 5-HT 2 receptors in other systems were also active here but the response to some agonists was potentiated by the hormone uptake blocker, cocaine HCl. Two 5-HT 2 selective antagonists, ketanserin and spiperone, inhibited the serotonin-induced response while compounds selective for other 5-HT receptor subtypes did not.

Carrier-mediated transport of thyroid hormone into rat hepatocytes is rate-limiting in total cellular uptake and metabolism.

We investigated if carrier-mediated transport into rat hepatocytes is rate-limiting in total cellular uptake and metabolism of thyroid hormone. Rat hepatocytes in primary monolayer culture were incubated under equilibrium conditions with tracer T4, T3 or rT3 in the absence or presence of inhibitors of thyroid hormone uptake, i.e., ouabain and ER-22, a monoclonal antibody against the rat hepatocyte plasma membrane. The results for all three iodothyronines show that inhibition of clearance from the medium during incubation is paralleled by a similar decrease in iodide production. This indicates that the decrease in metabolism of thyroid hormone is directly related to the inhibition of cellular uptake. These findings underline the potential importance of the plasma membrane in the regulation of thyroid hormone metabolism and, therefore, determination of expression of thyroid hormone activity.

Reduced affinity of intestinal receptors for 1,25-dihydroxycholecalciferol in phosphorus-deficient chicks.

The binding of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) to its intestinal receptor was studied in chicks fed a phosphorus (P)-deficient diet. The equilibrium association constant (Ka) was determined by Scatchard analysis. Association (Kass) and dissociation (Kdis) rate constants were determined in experiments on hormone uptake and release respectively. The Ka, determined at 4, 12 and 19 degrees C, decreased progressively during P deficiency, due to the decrease in Kass, but Kdis was not affected. During prolonged P deficiency the concentration of binding sites (Nmax) also decreased. Duodenal calcium-binding protein (CaBP) increased during 10 days of P deficiency and then decreased. The long-term decrease in receptor affinity and Nmax may account for the observed reduction in receptor occupancy and the decrease in the high level of intestinal CaBP stimulated during early P deficiency. The resulting decrease in Ca absorption may minimize the hypercalcaemia induced by the deficiency.