Excess Unlabeled Hormone Research Articles

Differential effects of TR ligands on hormone dissociation rates: Evidence for multiple ligand entry/exit pathways

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself. This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here, we show that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation ( T 1/2 < 2 h) of radiolabeled T 3 vs. T 3 ( T 1/2 ≈ 5–7 h). We cannot discern relationships between this effect and ligand size, activity or affinity for TRβ. One ligand, GC-24, binds the TR LBC and (weakly) to the TRβ-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T 3 dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T 3 release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T 3 release and that this effect enhances hormone dissociation.

Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor

In porcine Leydig cells in primary culture, 95% of the internalization of [ 125I]porcine lutropin ([ 125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [ 125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [ 125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars.

Photoaffinity labeling and characterization of a juvenile hormone binding protein in the membranes of follicle cells of Locusta migratoria

Membrane preparations from vitellogenic follicles of Locusta migratoria incubated with tritium-labeled photoaffinity analogs of juvenile hormones and methoprene in the presence or absence of excess unlabeled hormone or methoprene were irradiated, subsequently solubilized, the peptides separated by SDS-PAGE and the radioactive proteins visualized by fluorography. Both [ 3H] epoxyhomofarnesyl diazoacetate ([ 3H]EHDA), the photoaffinity analog of JH II, and [ 3H] epoxyfarnesyl diazoacetate ([ 3H]EFDA), the photoaffinity analog of JH III, covalently modified a 35 kDa peptide, and this labeling was displaced by an excess of unlabeled JH II and JH III respectively. No JH 1-displaceable labeling by [ 3H] epoxybishomofarnesyl diazoacetate ([ 3H]EBDA), the photoaffinity analog of JH I, was detected. [ 3H] methoprene diazoacetate ([ 3H]MDK), the photoaffinity analog of methoprene, exhibited specific binding to a 17 kDa protein. Using (10R)-[ 3H]JH III as a ligand, membrane preparations were demonstrated to possess specific and saturable binding: Scatchard analysis revealed a single binding site with a Kp of 3.68 ± 0.46 nM and a total binding capacity ( B max ) of 0.375 ± 0.07 pmol per mg protein. The ability of the various JHs to bind reflects previous findings concerning their biological activity on follicle cells in vitro, suggesting that the binding site is a membrane receptor for JH III.

Presence of growth hormone-binding proteins in cattle plasma and milk

The presence of GH-binding proteins (GHBPs) in the plasma of adult cattle was investigated using Sephadex G-200 filtration, Western ligand blotting and Western blotting. The changes in the concentration of GHBP in the plasma of dairy half-sister heifers during the first year of life as well as the presence of GHBP in milk were also investigated. When analytical chromatography (on a 1.6 x 100 cm column) was performed, five peaks of recombinant bovine GH (rbGH)-associated radioactivity were revealed in cattle plasma; the first peak, which appeared near the void volume, was presumed to represent aggregates, the second (M(r) 290 kDa) and the third peaks (M(r) 75 kDa) corresponded to specific rbGH-GHBP complexes; the last two peaks representing free 125I-labelled rbGH and Na[125I]. Western ligand blotting revealed multiple GHBPs. Three major bands were observed at approximately 190, 58 and 31 kDa; an excess of unlabelled hormone blocked the binding of 125I-labelled rbGH. Minor non-specific binding bands were also detected in cattle plasma with molecular weights between 40 and 136 kDa. One monoclonal antibody (8H7) produced against synthetic peptide (amino acids 54-63 of the extracellular domain of the bovine GH receptor) specifically interacted with 190 and 58 kDa bands while the 31 kDa band was not recognized. Finally, Western ligand blots were performed to evaluate the changes in plasma GHBP during the first year of life in 55 dairy half-sister heifers and to identify GHBP in milk. In plasma, the intensity of the 31 kDa band varied greatly between animals while the other specific bands remained stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin d nuclear binding to neurons of the septal, substriatal and amygdaloid area in the siberian hamster ( Phodopus sungorus) brain

Autoradiographic experiments were performed on brains of Siberian hamsters ( Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alzheimer's disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, K d, approximately 10 −10 m) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 × 10 6 cells/250 μl) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

Characterization of hepatic growth hormone binding in domestic poultry

Specific binding of 125I-recombinant chicken growth hormone (r-cGH) to avian hepatic crude membrane preparations was characterized in terms of time, temperature, and pH dependence of binding as well as by demonstration of specificity and dissociability of the binding phenomenon. Crude membranes (100,000g pellet) were prepared from livers of female, 4-week-old broiler-strain chickens and 24-week-old random-bred turkeys. Optimal conditions for binding in a 25 m M Tris-HCl, 10 m M CaCl 2 0.5% BSA buffer system were determined to be 24 hr at 30°, pH 7.0, for chicken membrane preparations and 36 hr at 30°, pH 7.2, for turkey membrane preparations. Total specific binding was proportional to the concentration of membrane protein, and remained linear over the range of 200 to 1400 μg per tube (500–3500 μg/ml) for both chicken and turkey systems. Binding of 125I-r-cGH was reduced by addition of unlabeled, pituitary-derived cGH (p-cGH) (IC 50 = 0.42 ng/tube/600 μg membrane protein for turkey; 3.8 ng/tube/600 μg membrane protein for chicken). Bovine GH (bGH) was an even more effective competitor than cGH, such that at approximately the IC 50 for p-cGH, bGH displaced 83.5% of 125I-r-cGH. Bovine prolactin also competed with 125I-r-cGH; however, cross-reactivity was only 1% that of bGH. Rat TSH exhibited negligible and LH and FSH essentially no cross-reactivity in the avian system. Binding was reversible as indicated by dissociation of 75% of bound 125I-r-cGH from turkey preparations and complete dissociation from chicken preparations by 12 hr following addition of excess unlabeled hormone.

Thyroid hormone transport in a human glioma cell line

The uptake of 3,5,3'-triiodothyronine (T 3) and thyroxine (T 4) was studied in human glioma cells (Hs 683) and compared with that in several other neural cell lines. At 25 ° C or 37° C, total cell uptake rose rapidly and reached equilibrium within 60 min. The glioma cells had the highest uptake: 47.6 fmol of l-T 3 and 43.4 fmol of l-T 4 per 10 6 cells at 37 °C. These were inhibited 77% and 72%, respectively, by excess unlabeled hormone. Uptake in the nuclei reached equilibrium between 90 and 120 min and was also highest in glioma cells: 1.46 fmol of l-T 3 and 0.49 fmol of l-T 4 per 10 6 cells. When expressed as percent of total cell uptake, however, glioma cells had the lowest values (3.1% for l-T 3 and 1.1% for l-T 4). Also in contrast to other cell lines, glioma cells transported l-T 4 almost as effectively as l-T 3. d-T 3 and d-T 4 total cell uptake was 86% and 96% lower than that of the respective l-isomers, and the nuclear uptake as a fraction of the cell uptake was similar. Kinetic analysis of the initial rate of cell uptake gave V max values for d-T 3 and d-T 4 that were 97% and 98% lower than for the l-isomers. Antimycin and monodansylcadaverine decreased the V max as well as the equilibrium cell and nuclear uptake of the l-isomers. The apparent nuclear affinity constant for l-T 4 in intact cells was inhibited 90% in the presence of antimycin, whereas no effect was observed in isolated nuclei. This suggests that nuclear availability of l-T 4 is decreased when plasma membrane transport is blocked, resulting in an apparently lower free T 4 concentration in the cytosol. Address for correspondence: Dr. J. Robbins, Clinical Endocrinology Branch, Bldg 10, Rm 8N315, National Institutes of Health, Bethesda, MD, 20892 U.S.A.

A correlative study between glucocorticoid receptor levels in human mononuclear leukocytes and biochemical data in Cushing's disease.

We determined glucocorticoid receptors in human mononuclear leukocytes in 9 patients with Cushing's disease, in order to correlate them with laboratory data. Receptors were measured by a whole-cell assay method, after incubation with [3H]-dexamethasone in the presence or absence of excess unlabelled hormone. In Cushing's disease, there were 4425 +/- 364 sites/cell (N = 9), similar to in the controls: 4473 +/- 476 (N = 10); average Kd was 2.42 +/- 0.52 nmol/l (N = 3) similar to in the controls: 2.0 +/- 0.20 nmol/l (N = 3). In Cushing's patients we found significant negative correlations between basal glucocorticoid receptors and: 1) morning blood cortisol (r = -0.67, P less than 0.05), and 2) 17-ketogenic steroids after 2 mg of dexamethasone (r = -0.85, P less than 0.01). No correlations were observed with afternoon blood cortisol, free urinary cortisol, basal and post-8-mg dexamethasone 17-ketogenic steroids, TRH-TSH area, urinary calcium, plasma glucose, or systolic blood pressure. In Cushing's disease, a subtle receptor down-regulation may exist, as suggested by the inverse relationship between glucocorticoid receptors and morning blood cortisol. Secondly, the relationship between basal receptors and 17-ketogenic steroids after 2 mg of dexamethasone suggests that glucocorticoid receptors in human mononuclear leukocytes could reflect the sensitivity of the nervous system-pituitary-adrenal axis to dexamethasone inhibition.

In vivo autoradiographic analysis of prolactin binding in brain and choroid plexus of the domestic ring dove

The binding of intravenously administered prolactin to choroid plexus and brain tissue was determined radioautographically in the ring dove, a species that exhibits prolactin-induced alterations in brain function. An intense autoradiographic reaction was detected over the epithelial cells of the choroid plexus 5 min after the intravenous injection of 125I-ovine prolactin. A significant reaction was also observed over the infundibulum but no significant uptake of prolactin occurred in other brain areas. The binding of radiolabelled prolactin to infundibulum appeared to be non-specific, since excess unlabelled hormone did not reduce silver grain density. In contrast, 125I-ovine prolactin binding in choroid plexus was significantly reduced by excess unlabelled ovine prolactin or human growth hormone, but not by ovine luteinizing hormone. Specific binding to choroid plexus was also detected in vitro. The lack of significant brain uptake of prolactin in vivo is discussed in relation to recent in vitro evidence for specific binding sites for prolactin in several dove brain regions. Similarities between the binding results obtained in this avian species and those reported previously in mammals suggest that the two vertebrate groups exhibit similar patterns of prolactin interaction with neural target tissues.

Specific insulin binding in bovine chromaffin cells; Demonstration of preferential binding to adrenalin-storing cells

Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125I-insulin was carried out at 15°C for 3 hrs in the absence or presence of excess unlabelled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. We conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells.

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits.

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.

Radioautographic localization of 125I-atrial natriuretic factor binding sites in the brain.

Rats were injected through the carotid artery (cephalad direction) with 18.9 mu Ci of either 125I-Arg 101-Tyr 126 atrial natriuretic factor alone or together with an excess of unlabeled hormone. At 2 min after injection, all rats were fixed in vivo by perfusion and serial sections of the whole brain were processed for light microscope radioautography. The radioautographic reaction produced by 125I-atrial natriuretic factor alone was localized in all circumventricular organs (except the subcommissural organ), the smooth muscle cells and endothelial cells of arteries, arterioles, veins, venules, the endothelial cells of intraparenchymal capillaries and the epithelial cells of the choroid plexus. In rats which received 125I-atrial natriuretic factor plus an excess of unlabeled hormone, the radioautographic reaction was reduced by 70-90%. Binding sites are thus localized in regions of the brain, some of them involved in the central monitoring of blood pressure and osmolarity. In addition, the presence of binding sites in the cerebral vasculature and in the epithelium of the choroid plexus suggests that circulating ANF may play a role in the control of cerebral blood flow and in the production of vertebrospinal fluid.

Theoretical basis for a new in vivo radioreceptor assay for polypeptide hormones

To date the in vivo identification and quantitation of specific hormone receptors has been difficult, time consuming, and lacking in sensitivity. We present the theory underlying a new in vivo radioreceptor assay for polypeptide hormones based on receptor theory derived from in vitro investigations and in vivo kinetic and autoradiographic studies. The assay was developed from a tissue model and a theory of hormone distribution. Measuring the labeled hormone distributed between the plasma and interstitial space in the presence or absence of excess unlabeled hormone permits the accurate determination of hormone specifically bound to receptors. This approach eliminates the problem of nonspecific binding due to free tracer, hormone degradation products, or labeled non-hormone molecules. A receptor compartment and specific binding of hormone are calculated from only four measured parameters: total tissue labeled hormone, tissue albumin, plasma labeled hormone, and plasma albumin. The method is applicable to most tissues and hormones under a variety of conditions and permits simultaneous comparison of multiple tissues in the same animal under identical conditions.

Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor

When young rats (less than 14 days old) were treated once a day for 2 days with 100 μg/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 μg/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.

Characteristics of somatotropic growth hormone binding to homologous liv plasma membranes

The binding of 125I-labeled oGH, rGH and hGH to homologous liver membranes is compared to define the characteristics of somatotropic hormone-receptor interactions. The effect of pH and cations and the time-course of binding of these different GHs was similar when binding was determined in liver membranes from the homologous species. The time-course of binding was characterized by stable binding by 90–120 min of incubation at 22°C with relatively high nonspecific binding that continued to increase with time. This resulted in a peak of specific binding for each hormone. Addition of excess unlabeled hormone at 30 min of the binding incubation resulted in partial displacement of the bound GH. The binding of rGH was somewhat dependent upon the presence of divalent cations (Mg 2+, Ca 2+) in the incubation medium. In the absence of added divalent cation, a small but appreciable amount of binding was detected at pH 7–8. With 10 mM calcium, binding increased several-fold with a definite peak of rGH binding at pH 4–5. Both oGH and hGH exhibited a broad peak of binding at pH 5–7. Monovalent cations (Na +, K +) were either ineffective in enhancing GH binding or produced a slight enhancement at higher concentrations (100 mM). Competitive displacement curves confirm the somatotropic nature of the GH-receptor interaction. Human Pl was ineffective in competing for GH binding. We conclude that ‘somatotropic’ growth hormone binding to its homologous liver receptor exhibits phylogenetically consistent and distinguishing characteristics of binding. Comparison of the characteristics of hGH binding to human, female rodent and bovine liver membranes suggests the presence of three separate receptors for hGH in human, female rodent and nonhuman mammalian liver.

Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease

Rat liver nuclei were digested with micrococcal nuclease following incubation with 125I-angiotensin II (AII) or with 125I-AII and excess unlabeled hormone. Chromatin enriched in 125I was solubilized after 3 min and was applied to a BIO-GEL A-5 M column. Labeled hormone was 40 – 60% displaceable by unlabeled hormone, in nucleoprotein eluting with a V/Vo near 1.9, indicating that these solubilized chromatin fragments contained specific receptors for AII. Furthermore, a discrete AII binding nucleoprotein particle was resolved on DNP gel electrophoresis. Additionally, binding to specific AII nuclear receptors appeared to bring about changes in chromatin structure consistent with the induction of transcriptional activity.

Aldosterone binding in isolated tubules. III. Autoradiography along the rat nephron.

Aldosterone binding was investigated along the nephron of adrenalectomized Wistar rats. Autoradiographs of microdissected tubular segments were performed on dry film after incubation with [3H]aldosterone [( 3H]A) at 2 X 10(-10), 2 X 10(-9), and 2 X 10(-8) M in the presence or absence of an excess unlabeled hormone. Competition studies were done with a 10 times excess A, dexamethasone (D), and estradiol in the presence of 2 X 10(-8) [3H]A. Specific nuclear labeling predominated in the cortical collecting tubule (CCT) and A specificity of the binding was assessed by the much better displacing potency of unlabeled A as compared with D. Labeling in the distal tubule (DCT) resembled that of CCT, but D was nearly as good a competitor as A. In the loop and medullary collecting tubule, labeling was lower, with mixed steroid specificity. No specific nuclear labeling was detectable in the proximal tubule. We conclude that the CCT and DCT present the highest binding capacity for aldosterone. Competition studies revealed that the CCT is probably the more specific mineralocorticoid target segment. In other segments beyond the pars recta (loop of Henle, medullary collecting tubule), aldosterone probably binds to both mineralocorticoid and glucocorticoid binding sites. No major difference appeared between the profile of aldosterone binding along the rat tubule and the one previously described in the rabbit.

Mouse liver specific uptake of iodinated growth hormones: evidence for the presence of somatogenic and lactogenic sites.

The distribution of human growth hormone labelled with 125I (125I-hGH) was studied in normal adult female and male mice. The radioactivity was basically concentrated by the liver and kidney reading a maximum 15 minutes after the labelled hormone injection. Only the liver showed a significant reduction of radioactivity uptake when 125I-hGH was injected together with an excess of unlabelled hormone. This reduction was dose-dependent and the amount of unlabelled hormone that prevented 50% of the liver uptake (ED50) of 125I-hGH was close to 3 micrograms for both female and male mice. Similar results were obtained in studies where bovine growth hormone labelled with 125I (125I-bGH) was injected, except that the maximum uptake value was significantly lower than that observed when 125I-hGH was used. This observation could be attributed to the difference that exists between the biological properties of both hormones since hGH has growth-promoting and lactogenic effects in rodents, whereas bGH exhibits exclusively somatotropic activity. In order to examine the nature of the radioactive material which localized in the liver soluble extracts were prepared using Triton X-100 and analyzed on Sepharose CL-6B. The majority of the radioactivity appeared as an homogeneous peak with KD = 0.31 which could be attributed to a molecular species of Stokes radius of approximately 64A. This magnitude is consistent with the effective molecular size reported for various hormone-receptor complexes.

Dynamic 131I-labelled insulin distribution in rabbits as seen by in-vivo scintigraphic studies.

The kinetics of 131I-labelled insulin distribution in heart, liver, kidneys and urinary bladder of rabbits were studied in vivo by gamma-camera techniques combined with plasma measurements (glucose concentrations and chromatographic separation of insulin and its degradation products). The distribution space of radioiodinated insulin differed from the vascular bed delineated by radioiodinated serum albumin. During a 20-min gamma-camera recording, radioactive degradation products only appeared in the plasma after 10-12 min. Previous administration of a 10 000-fold excess of unlabelled insulin and 5 ml glucose (20%) did not modify the evolution of 131I-labelled insulin cardiac invasion and the subsequent linear decrease of radioactivity. Conversely, wash-out of radioactivity from the liver and kidney was accelerated after preadministration of this excess of unlabelled hormone, binding in these organs accounting for this acceleration. Urinary bladder filling was imaged later than cardiac, hepatic or renal labelling and was only accelerated by polyuria induced by glucose injection, independent of preadministration of unlabelled hormone.