Clearance and metabolism of 125I-labeled tonin in the rat

125I-Labeled rat tonin was injected iv into male Wistar rats weighing 303 +/- 17 g (mean +/- SE), and the disappearance from the circulation, plasma interaction, tissue uptake, and metabolism of radioactivity was studied. After injection, 125I-labeled tonin was immediately bound by alpha 1-macroglobulin, the major circulating protease inhibitor of rats, and the complex was cleared in a biexponential manner: t1/2 of the fast component = 1.8 +2- 0.1 min; t1/2 of the slow component = 84 +/- 10 min; MCR = 2.1 +/- 0.2 ml/min (mean +/- SE, n = 8). The complex was taken up intact by tissues, the major ones being the liver, spleen, adrenal, thyroid, and kidney. Analysis of tissue extracts by gel filtration on Sephadex G-100 showed that the kidney was the major site of metabolism, and radioactivity similar in size to 125I appeared rapidly in urine. Therefore tonin circulates wholly as a complex with alpha 1-macroglobulin (the rat equivalent of human alpha 2-macroglobulin), which once formed is quickly eliminated from the bloodstream by tissue uptake and metabolized.     

Release of thyroid hormone from circulating thyroglobulin in the rat

Under normal conditions, a small amount of thyroglobulin (Tg) exists in peripheral blood. However, the fate of circulating Tg is unclear. In the present study, in vivo labelled rat Tg was injected iv into rats whose thyroids had been blocked with KI to determine whether circulating Tg released thyroid hormone by hydrolysis in extrathyroidal tissues. Radiolabelled Tg was obtained from thyroid of rats injected with 125I 24h before sacrifice, and subsequently purified by ammonium sulphate precipitation. The plasma samples were obtained from tail veins or by cardiac punctures at various times following injection of [125I]rat Tg. The radioactive samples were separated into iodoprotein, iodoaminoacid and iodide fractions using columns of anion and cation exchange resins. The per cent radioactivity of the iodoprotein, iodoaminoacid an iodide fractions, respectively, was 91.2, 3.8 and 5.2 at 15 min and 66.9, 17.4 and 15.4 at 20 h after injection. In the iodoaminoacid fractions, the presence of T4, T3, MIT and DIT was defined by further fractionation using a Sephadex G-25 column. At 20 h after injection, more than 75% of the radioactivity of the iodoaminoacid fraction was found to be incorporated in T4 and T3. It is concluded that circulating Tg is hydrolyzed in extrathyroidal tissues and that thyroid hormone is released into the circulation, but the amounts of T4 and T3 released are not physiologically significant.     

Renal handling of high-density lipoproteins by isolated perfused kidneys

Recent studies show that the normal and diseased kidney is an important organ in the catabolism of high-density lipoproteins (HDL). However, little is known about the renal handling of HDL. To investigate this aspect, kidneys were isolated from normal rats and rats made nephrotic with puromycin aminonucleoside. They were perfused in a chamber at 37 degrees C with a modified Krebs-Hensleit Bicarbonate Buffer containing 1%, 3%, 6%, and 10% Bovine Serum Albumin (BSA). Presence of 10% BSA in the perfusate prevented glomerular filtration and urine formation. Thus, the filtering and the nonfiltering kidney perfusion models distinguish the renal parenchymal function independently of luminal events that follow filtration. 125I-labeled rat HDL was injected into the perfusate and radioactivity in perfusate, urine, and kidney was examined. At the end of perfusion (30 minutes or four hours), each kidney was flushed with 125I-HDL-free perfusate and kidney radioactivity was measured. At four hours, 1.9% +/- 0.5% of injected radioactivity was present in urine from kidneys perfused with 6% BSA. Kidneys with intact glomerular filtration sequestered significantly more radioactivity (1.1% +/- 0.2% of injected radioactivity) than nonfiltering kidneys (0.7% +/- 0.2%); P less than 0.05. Radioactivity in filtering kidneys was significantly higher than in nonfiltering kidneys (33.9 +/- 7.8 v 15.6 +/- 2.6 cpm/mg kidney tissue protein, respectively; P less than 0.001). Nephrotic kidneys (filtering and nonfiltering) sequestered two to four times more 125I-HDL than normal kidneys. These data support the hypothesis that prior to urinary excretion, partial reabsorption of filtered HDL (or subfractions) occurs in the normal kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial characterization and sexual dimorphism of serum growth hormone-binding protein in adult rats

The in vitro binding of 125I-bovine growth hormone (bGH) to adult rat serum was studied using Ultrogel AcA34 filtration. When analytical chromatography on a 1.6 x 100 cm column was performed, four peaks of radioactivity were revealed: the first two peaks with Mr +/- 220,000 and +/- 110,000 corresponded to bound 125I-bGH (abolished by excess of unlabeled bGH), the third corresponded to free 125I-bGH and the fourth to free Na125I (Vt). On a short (1 x 40 cm) column, bound 125I-bGH eluted as a single peak between the void volume (Vo) and the peak of free 125I-bGH. Serum 125I-bGH binding was specific, saturable, and time-dependent. Specific serum 125I-bGH binding (bound/total radioactivity x 100), calculated as the difference of binding in the absence and the presence of an excess unlabeled bGH, was higher in female than in male rats (26 +/- 2% vs. 11 +/- 1%, respectively; mean +/- SE; n = 6; P less than 0.01). Scatchard analysis revealed a binding affinity of 2 x 10(8) M-1 for both sexes, and a binding capacity of 6.4 x 10(-8) mol/liter for the female rats and 1.6 x 10(-8) mol/liter for the male rats (mean of three serum pools of three animals each). Specific binding of 125I-bGH to serum correlated significantly with 125I-bGH binding to liver homogenates (r = 0.83; n = 12; P less than 0.01). These results suggest the presence of a specific GH-binding protein in rat serum and provide further evidence for a close relationship between serum GH-binding protein and hepatic GH receptors.     

Kinetic study of immunoreactive human thyroglobulin

Thyroglobulin (Tg) can be detected in the circulation of normal subjects. Serum Tg is increased in patients with various thyroidal disorders including Graves' disease; however, little is known about Tg metabolism. Therefore, a kinetic study of human Tg was carried out in 13 normal men, 19-28 yr old, and 6 untreated hyperthyroid patients with Graves' disease, 3 men (22 to 25 yr old), and 3 women (21 to 63 yr old). Ten milligrams of Tg were injected as a bolus dose. Blood samples were collected before and 10 min, and 2, 4, 6, 8, 12 h and every 12 h up to 72 h after injection. Concentrations of serum Tg were measured by an RIA method developed in our laboratory. Anti-Tg antibody was not detected in any subject. Various indices of this kinetic study were calculated using single compartmental analysis. In 13 normal subjects, the mean serum concentrations of Tg were 17 +/- 12.6 (SD) ng/ml; mean half-life was 29.6 +/- 2.8 h; distribution volume was 11,210 +/- 3,076 ml/60 kg body weight; fractional decay was 2.40 +/- 0.22%/h; MCR was 268.9 +/- 87.8 ml/h X 60 kg; and release rate was 100.3 +/- 50.2 micrograms/day X 60 kg. Serum concentrations of Tg were increased in four of the six untreated hyperthyroid patients with Graves' disease. Their Tg half-lives and MCR were within the normal range. In the two patients who had normal serum concentrations of Tg, the Tg half-lives were shorter and MCR were greater than in normal subjects. The release rates of Tg were increased in all six of these patients. In summary, in hyperthyroid patients, Tg release is significantly greater than normal, whereas Tg metabolism is similar to that in normal subjects.     

The mouse fibroblast growth hormone receptor: ligand processing and receptor modulation and turnover

The recent observation that adipose conversion of mouse 3T3 fibroblasts is stimulated by physiological concentrations of human GH (hGH) and rat GH in vitro suggested that this cell line may be suitable for the study of GH-receptor interactions. The aim of this study was to examine the binding and subsequent processing of [125I]iodo-hGH by BALB/c 3T3 mouse fibroblasts. Binding of [125I]iodo-hGH to 3T3 fibroblasts was time and temperature dependent. Apparent steady state binding was achieved after 1 and 2 h at 37 and 30 C, respectively. At 37, 30, and 20 C specifically bound [125I]iodo-hGH became increasingly resistant to removal by acid treatment (0.15 M NaCl/0.05 M glycine, pH 2.5). In contrast at 4 C or at higher temperatures in the presence of metabolic inhibitors, a greater proportion of specifically bound hGH was removed by acid treatment. Inclusion of 0.2 mM chloroquine in the incubation medium resulted in significantly more accumulation of trichloroacetic acid (TCA)-precipitable radioactivity compared to control cells without affecting the shift of radioactivity from the acid-elutable to the acid-inaccessible compartment. After removal of [125I]iodo-hGH from the medium there was a rapid loss of radioactivity (t 1/2 = 36.5 +/- 7.2 min, SE, n = 3) from the cell monolayer with a concomitant appearance in the medium of TCA-soluble radioactive species. Chloroquine reduced the rate of efflux of radioactivity from the monolayer (t 1/2 = 4.5 +/- 0.6 h, n = 3) and the appearance of TCA-soluble material in the medium. The half-time of GH receptor loss after inhibition of protein synthesis with cycloheximide (0.1 mM) was 1.25 +/- 0.14 h, n = 3). In contrast half-time of net receptor synthesis calculated from the recovery of specific [125I]iodo-hGH binding capacity after ligand-induced down-regulation was 10.2 +/- 1.5 h, n = 3). These data reveal that after binding of [125I]iodo-hGH to specific cell surface receptors there is rapid irreversible binding of GH to its receptor with a resultant reduction in receptor concentration. Degradation of [125I]iodo-hGH occurs intracellularly and involves processes which are inhibited by lysosomotropic agents. On the basis of these studies we conclude that the binding and subsequent processing of GH by 3T3 fibroblasts is qualitatively similar to that described for other polypeptide hormones and growth factors in this and other cell lines.     

Low-molecular-weight iodoproteins in the congenital goiters of cog/cog mice.

Previously we described sedimentation and immunologic abnormalities of thyroglobulin (Tg) in a strain of mice with inherited congenital goiter and hypothyroidism (cog/cog). The goals of the present study were to determine the extent to which thyroid gland stimulation by TSH accounts for the abnormal properties of cog/cog Tg and to characterize further the abnormally small iodoproteins found in cog/cog mice. Cog/cog and control +/cog and BALB/c mice were fed with either normal or thyroid-hormone-containing diets and were injected with Na125I. Sucrose density gradient centrifugation of labeled thyroid extracts from cog/cog mice on normal diet showed that 82% of the iodine was in iodoproteins smaller than Tg, with sedimentation rates of 3-8S. No 12S and 19S peaks, characteristic of normal Tg, were present, but distinct and stable 12S and 19S peaks emerged after recentrifugation of the 12S and 19S areas. In contrast, in cog/cog mice treated with T4, a smaller (55%) amount of 3-8S iodoproteins and distinct 12S and 19S peaks were present. In both groups of mice, the labeled 3-8S iodoproteins were composed of three fractions: 15% precipitated by antirat Tg serum, 38% precipitated by antimouse albumin serum, and 47% not precipitated by either serum. The 3-8S iodoproteins contained labeled MIT and DIT and no T4. On sodium dodecyl sulfate polyacrylamide gel electrophoresis the 3-8S iodoprotein fraction that reacted with anti-Tg serum contained a distinct electrophoretic band at 49K. The 3-8S nonreactive iodoproteins resolved into several bands of lower molecular weight. We conclude that the 3-8S iodoproteins in cog/cog mice are heterogeneous and that TSH stimulation contributes to the production of these low-molecular-weight iodoproteins.     

Retroendocytosis of insulin in rat adipocytes

A variety of ligands internalized by receptor-mediated endocytosis follow a short circuit pathway that does not lead to degradation but results in rapid exocytosis of intact ligand, a process termed retroendocytosis. We studied the time course of [125I]iodoinsulin processing and retroendocytosis after internalization in isolated rat adipocytes. After steady state binding and internalization, surface receptor-bound insulin was removed by exposing cells to a low pH at low temperatures. The cells containing internalized [125I]iodoinsulin were reincubated in fresh medium; subsequently, the radioactivity remaining within the cells and released into the medium were analyzed at various times by trichloroacetic acid (TCA) precipitation, Sephadex G-50 gel filtration, and reverse phase HPLC. Cell-associated radioactivity progressively decreased after reincubation in 37 C buffer, with 50% released in 9 min and 85% by 45 min. In the media, TCA-precipitable material appeared quickly, with a t1/2 of 2 min, and plateaued by 10 min. TCA-soluble material was released continually throughout the 45-min period. The release of both TCA-precipitable and TCA-soluble material was temperature and energy dependent. Sephadex G-50 chromatography demonstrated the loss of insulin from the intracellular pool and its appearance in the medium with a time course similar to that of TCA-precipitable material. Reverse phase HPLC demonstrated that the intracellular and medium radioactivity eluting in peak II (insulin peak) on Sephadex G-50 was composed of both intact insulin and intermediates. In conclusion, these studies demonstrated that after the internalization of insulin, rat adipocytes release not only small mol wt degradation products of insulin, but also insulin intermediates and intact insulin. The rate of retroendocytosis reported here is almost identical to the rate of insulin receptor recycling in rat adipocytes. Therefore, retroendocytosis may serve as an excellent in vitro reflection of the extent and rate of insulin receptor recycling.     

Metabolism of 125 I-luteinizing hormone-releasing hormone

With the use of prepared iodine-125-monoiodo-LH-RH, the disappearance rates of LH-RH (luteinizing hormone-releasing hormone) in man and in the rat were investigated, as well as the distribution of LH-RH in the rat. After 10 microcuries of iodine-125-LH-RH were injected as an iv bolus into 3 male volunteers, blood samples from the opposite arm were obtained through an indwelling venous catheter at 2, 5, 8, 11, 15, 20, 30, 45, and 60 minutes postinjection and at 6 and 24 hours postinjection. To describe the disappearance curve a 3-term exponential equation was necessary and sufficient. The ranges of the t1/2s for the first, second, and third components were 2-4 minutes, 30-55 minutes, and starting at more than 10 hours. The initial distribution space was 3-4.7 1 or 37-47 ml/kg body weight. Similar procedures in the rat (45) resulted in an equation with 2 exponentials, with the ranges of the t1/2s for the first and second components being 5-10 minutes and 150-600 minutes. In both man and rat the distribution volume approximated estimated plasma volume. Following the injection of iodine-125-LH-RH in the rat, pituitary radioactivity increased, as expected, reaching a maximum tissue/serum ratio of 1.5 at 90 minutes. 2 iodinated oligomers of LH-RH were found to have different disappearance rates and distributions in the rat than did iodine-125-LHrh. the disappearance rate of iodine-125-LH-RH in this study is consistent with values for other small peptide hormones.     

Heterogeneous acinar localization of the asialoglycoprotein internalization system in rat hepatocytes

Desialylated glycoprotein is rapidly cleared from plasma by a receptor-mediated endocytic mechanism located on hepatocytes. We studied the hepatic acinar distribution of this asialoglycoprotein transport system with the ligand 125I-asialoorosomucoid using rat liver perfused in either antegrade or retrograde direction in combination with quantitative light microscopic autoradiography. Grain distribution along the acinus appeared dependent on the perfusion direction. A rather shallow zone 1 to zone 3 gradient was observed if livers were perfused in the normal direction. However, a statistically significantly steeper zone 3 to zone 1 gradient was detected in retrograde perfusions. Kinetic analysis of perfusate clearance profiles yielded a hepatic clearance of 21.6 +/- 1.3 ml per min in antegradely perfused liver. Hepatic extraction was calculated to be 60.1 +/- 7.4%. Biliary secretion of radioactivity amounted to 1.89 +/- 0.18% of the dose within 1 hr after injection and consisted of intact material (1.39 +/- 0.25%) and radioactive low-molecular-weight degradation products (0.52 +/- 0.08%), of which more than 90% could be accounted for by 125I-. Apart from a minor difference regarding biliary secretion of an unidentified glycopeptide (less than 0.1% of the injected dose), transport data for the retrogradely perfused livers were identical to those obtained with livers perfused in antegrade direction, emphasizing the functional equivalence of both groups of livers. The autoradiographic data indicate that zone 3 hepatocytes take up 125I-asialoorosomucoid more avidly than zone 1 cells. The kinetic and biochemical data indicate that further processing in the hepatocytes is virtually similar in the two zones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyposialylated thyroglobulin in a patient with congenital goiter and hypothyroidism.

A large family (14 children) with congenital goiter whose parents are first cousins was studied. Thyroid tissue was obtained, after 125I in vivo labeling, from one of the siblings (JBM). Gel filtration of thyroid proteins indicated that thyroglobulin (Tg) eluted as a single symmetrical peak in the same position as authentic 19S Tg. Gel electrophoresis in a 7.5% sodium dodecyl sulfate-polyacrylamide gel revealed a major band with the same mobility and immunoreactivity as normal 19S Tg. Hydrolysis of the patient's Tg indicated that most of the radioactivity was mono- and diiodotyrosines. The yield of T4 from JBM Tg (26 pmol/mg protein) was 5-fold less than normal thyroid tissue (140 pmol/mg protein) and approximately half of that in thyroid tissue from endemic goiter (51 pmol/mg). Total T3 released from JBM Tg was similar to the other two tissues. When the carbohydrate content of normal and patient Tg was analyzed, there was no differences in glucosamine, galactose or mannose content. However, unlike normal and endemic-goiter Tg, that had a mean sialic acid content of 7.3 and 5.6 micrograms/mg protein, respectively, the sialic acid concentration of the patients Tg was only 0.3 microgram/mg. Sialyltransferase activity was readily demonstrated in homogenate from normal thyroid or endemic goiter, but no sialyltransferase activity was detectable in a homogenate of JBM-thyroid tissue. We conclude that the finding of severely hyposialylated Tg is linked to a defect in iodotyrosine coupling seen in this patient with a possibly abnormal migration of Tg into the follicular lumen.     

Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.     

Insulin degradation into monocytes from normal subjects: a high performance liquid chromatographic analysis

In this work the fate of A14-125 I-insulin inside human cells has been investigated by the complementary use of gel permeation and reversed-phase high performance liquid chromatography to obtain a better resolution of the cell processed radioactive material resulting from the internalization of labeled insulin. Mononuclear leukocytes from 12 normals were incubated with pure A14-125 I insulin at 37 C and internalized radioactivity was characterized after 2, 15 and 60 min. Nearly 14% of intracellular radioactivity was associated to materials with a molecular weight of approximately 300,000. The remaining 86% had a molecular weight lower than 20,000. High molecular weight material showed an elution profile very similar to that obtained from purified human placental insulin receptor and was partially precipitable with antireceptor antibody. The reversed phase high performance liquid chromatography analysis of the low molecular weight material showed two main peaks corresponding to 125 I and A14-125 I-insulin and three intermediate peaks, a, b, c, accounting for about 8% of the recovered radioactivity. By increasing the incubation time of A14-125 I-insulin with monocytes a decrease of insulin peak (2 min: 38 +/- 18%; 15 min: 25 +/- 11%; 60 min: 6 +/- 4%) and a corresponding increase of iodide peak was observed. Immunoprecipitability with anti-insulin antibody was 0% for iodide and a peaks, 60% for peak b, 78% for peak c and 90% for A14-insulin peak. Our results show that intracellular insulin degradation procedes rapidly and in a time-dependent manner and that this process produces insulin derivatives which partially retain the immunological properties of intact A14-125 I insulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intralysosomal hydrolysis of thyroglobulin: specific and cAMP-mediated activation by TSH

Pig thyroid slices, pre-labelled with [125I]iodide, were incubated with or without thyrotropin (TSH), dibutyryl cyclic AMP (dbcAMP) or forskolin. After lysosome isolation, intralysosomal thyroglobulin (Tg) hydrolysis was determined by the increment in trichloroacetic acid (TCA)-soluble radioactivity. TSH stimulated the intralysosomal Tg hydrolysis. This stimulation was time and concentration dependent and was mimicked by forskolin or dbcAMP. When endocytosis and protein synthesis were blocked by inhibitors (nocodazole and puromycin) the stimulatory effect was still maintained. We conclude that TSH increases quickly and specifically, via a cAMP-mediated process, intralysosomal Tg hydrolysis, independent of its effects on endocytosis of Tg and lysosomal protease synthesis.     

Corticotropin-releasing hormone inhibition of growth hormone-releasing hormone-induced growth hormone release in man

Recent studies in the rat have shown that intracerebroventricular administration of CRH inhibited spontaneous pulsatile GH secretion and prevented GH-releasing hormone (GHRH)-induced GH release. We have studied the effect of CRH on GHRH-induced GH release in man. In the first study, CRH was injected iv at three different doses (100, 50, or 25 micrograms) at 0800 h together with 50 micrograms GHRH in six men and six women. In a second study, 100 micrograms CRH were given iv at 0800 h, 1 h before the administration of 50 micrograms GHRH in five men and five women. Each subject demonstrated a normal GH response after the administration of 50 micrograms GHRH plus saline. All doses of CRH administered simultaneously with GHRH significantly inhibited GHRH-induced GH release in women [peak value +/- SE after GHRH plus saline, 28.9 +/- 2.9 micrograms/L; after GHRH plus 100 micrograms CRH, 9.9 +/- 0.7 micrograms/L (P less than 0.001); after GHRH plus 50 micrograms CRH, 8.7 +/- 0.8 micrograms/L (P less than 0.001); after GHRH plus 25 microgram CRH, 9.5 +/- 1.6 microgram/L (P less than 0.001]). In contrast, in men, while a dose of 100 micrograms CRH was capable of suppressing GHRH-induced GH secretion (peak value +/- SE, 8.1 +/- 0.6 vs. 20 +/- 2.9 micrograms/L; P less than 0.001), no inhibition was observed after 50- and 25-micrograms doses. When 100 micrograms CRH were injected 1 h before the administration of 50 micrograms GHRH, it strongly inhibited GHRH-induced GH secretion in both men (peak value +/- SE, 6.2 +/- 2.8 vs. 24.6 +/- 5.9 micrograms/L; P less than 0.02) and women (peak value +/- SE, 14.2 +/- 4.5 vs. 37.8 +/- 6.7 micrograms/L; P less than 0.005), and this inhibition lasted up to 2 h post-CRH administration. These results demonstrate that CRH is capable of inhibiting GHRH-induced GH release in both men and women. Furthermore, the findings suggest that a sexual dimorphism in the neuroregulation of GH secretion may be present in man. In view of the inhibitory action of CRH on GH secretion, simultaneous administration of CRH and GHRH for testing should be avoided in clinical practice.     

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.     

Half-life of plasma growth hormone in young and old conscious female rats

The kinetics of disappearance of plasma GH was studied in young (3-4 months) and old (24-27 months) Sprague-Dawley female rats. Conscious, free moving animals carrying indwelling atrial and carotid cannulas received a single injection of 125I-rGH via the carotid cannula. Sequential blood samples were removed at intervals during the following hour, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. Both TR and IPR displayed biexponential kinetics in vivo which did not differ significantly, for each variable, between young and old animals. The volumes of distribution of GH were also similar in both age-groups. The IPR/TR ratio, an estimate of GH inactivation within the plasma space, showed a decreasing sigmoid-shaped kinetics in vivo with a time of semi-inactivation (ti1/2) of 23.8 +/- 1.2 and 29.0 +/- 1.0 min (mean +/- SE) for young and old rats, respectively (P less than 0.02). The estrous status did not significantly affect ti1/2 values in vivo. The in vitro t1/2 was estimated by incubating plasma from the young and old animals at 37 degrees C with 125I-rGH for several hours. The IPR/TR ratio displayed a linear kinetics in vitro with t1/2 values of 23.7 +/- 1.7 and 25.8 +/- 1.9 h (NS) for young and old animals, respectively. The above results show that GH catabolism decreases slightly with age in the female rat, although it is unlikely that this change has a significant effect on plasma levels of GH. The data also suggest that GH is physiologically inactivated in the extravascular space.     

Degradation of porcine relaxin by glutathione-insulin transhydrogenase and a neutral peptidase

The susceptibility of porcine relaxin and 125I-polytyrosyl-porcine relaxin to degradation by 3 purified enzymes involved in the degradation of insulin and proinsulin was examined. Rat liver glutathione-insulin transhydrogenase (GIT), which cleaves disulfide bonds in insulin, catalyzed a time- and concentration-dependent increase in trichloroacetic acid (TCA)-soluble radioactivity of relaxin. The Sephadex G-50 profile of the reaction products revealed conversion to the A- and B-chains. Relaxin competitively inhibited the degradation of insulin by GIT; however, kinetic analysis revealed insulin to be preferred over relaxin as a substrate. Rat liver cytosol neutral thiol peptidase (NTP) catalyzed a time- and concentration-dependent increase in the TCA solubility of relaxin and a shift in the Sephadex G-50 radioactivity profile to low molecular weight products. Kinetic analysis revealed that insulin and B-chain are preferred over relaxin as substrates for NTP. A third enzyme, rat kidney neutral metalloendopeptidase, which degrades proinsulin and insulin C-peptide but not insulin, also did not degrade porcine relaxin.     

Binding of the 35-s of 35-s-propylthiouracil by follicular thyroglobulin in vivo and in vitro.

Radioactivity was found bound to follicular thyroglobulin after administration of 35-S-propylthiouracil (PTU) to rats. Denaturation of the thyroglobulin using various procedures could not separate the 35-S from the protein; it was concluded that the 35-S is bound to thyroglobulin covalently. Fractionation of saline-soluble thyroid proteins was performed by ultracentrifugation on sucrose gradients. The PTU-sulphur/thyroglobulin (S/Tg) molar ratio was calculated in all fractions. One hour after the injection of PTU the S/Tg molar ratio was the same for 19S thyroglobulin from rats on stock diet and 18S thyroglobulin from rats on low iodine diet. Injection of KI to the animals before administration of 35-S-PTU significantly reduced the ratio. The highest S/Tg ratio 1.10 was noted at 19S thyroglobulin, 17 h after a single injection of PTU. Daily injection of PTU for six days increased the S/Tg ratio to 3.3. Inverse relationship between dose of PTU and S/Tg ratio was noted at one hour. In animals injected with large dose of 35-S-PTU and sacrificed several hours later the S/Tg was higher at the 12S subunits than at the 19S protein. The amount of PTU bound to 3-8S subunits was minimal. Sulphite liberated 64 percent of the 35-S bound to thyroglobulin which appeared as four compounds on thin layer chromatography plates. The main 35-S compound liberated by sulphite was sulphate.     

Distribution of follitropin and deglycosylated follitropin in the rat: a quantitative and radioautographic study

125I-labeled ovine follitropin (125I-oFSH) and deglycosylated follitropin (125I-DG-oFSH) were injected into rats and the tissue uptake was quantified and correlated with radioautographic data. Trichloroacetic acid (TCA)-precipitable radioactivity and gel filtration analysis of blood samples indicated no degradation of follitropin or analogue with time. Clearance of follitropin from the circulation was accelerated after its deglycosylation. Disappearance of both molecules from the blood was associated with uptake and/or loss of radioactivity from liver, kidney, ovary and spleen. The more rapid removal of deglycosylated follitropin from blood was associated with higher renal levels of accumulated radioactivity than native follitropin. This was associated with its localization within the cortex, specifically the proximal convoluted tubules of the nephron. Binding of 125I-labeled follitropin and analogue to granulosa cells was specific and time-dependent. 125-I-DG-oFSH demonstrated greater avidity of binding to rat granulosa cells with time than 125I-oFSH. This was associated with slower dissociation kinetics and/or metabolism for 125I-DG-oFSH. The absence of localization of either 125I-follitropin or analogue in hepatic tissue suggests that hepatic mechanisms may not significantly contribute to the clearance of these molecules. Implications of these findings in regard to the metabolism of oFSH and its antagonist are discussed.