The use of [125I]polyvinyl pyrrolidone K. 60 in the quantitative assessment of the uptake of macromolecular substances by the intestine of the young rat

1. A method has been developed which allows the quantitative estimation of the uptake of labelled polyvinyl pyrrolidone (PVP) of mean mol. wt. 160,000 (K. 60) by the wall of the small intestine of young rats.2. Four hours after feeding a standard dose of [(125)I]PVP by stomach tube, the small intestine was thoroughly washed out, and the radioactivity of the intestinal wall measured. Under these conditions, the small intestine of animals less than 18 days old took up more than 50% of the radioactivity which had left the stomach. There was no increase in PVP uptake if the duration of absorption exceeded 4 hr. The PVP was taken up by the epithelial cells of the villus, and its intracellular localization has been demonstrated by fluorescence microscopy and can be related to vacuolation in the cells.3. In animals between 18 and 20 days old the uptake of PVP declined progressively, until, in animals more than 20 days old, less than 5% of the radioactivity was taken up by the intestinal wall.4. There is good agreement between the reported age of termination of antibody absorption in young rats and the age at which PVP uptake ceased in the present experiments. It is suggested that the loss of ability of the intestine to take up substances of high mol. wt. may be the factor which limits the duration of the period of antibody absorption in this species.     

The absorption of polyvinyl pyrrolidone by the new-born pig intestine

1. The intestinal absorption of [(131)I]polyvinyl pyrrolidone of mean mol. wt. 160,000 (K. 60) and 40,000 (K. 30) after oral administration has been measured in unsuckled conscious pigs less than 20 hr old. Absorption was assessed by the measurement of the concentration of [(131)I]PVP in venous blood during the 6 hr after feeding and also by the distribution at the end of the experiment of [(131)I]PVP between homogenates of the alimentary tract and homogenates of the rest of the animal.2. The concentration of [(131)I]PVP in the peripheral blood after feeding was dependent upon the mol. wt. of the polymer, when comparable amounts had been absorbed from the intestine. PVP K. 60 attained higher blood concentrations than PVP K. 30 and the blood concentrations of PVP K. 60 were close to the values to be expected if all the material which had left the intestine had remained in the blood. The lower blood concentrations found when PVP K. 30 was fed were associated with the disappearance of labelled solute from the gut and were thus the consequence of the relatively rapid escape of labelled solute from the plasma after absorption had taken place.3. The ability of the intestine to absorb [(131)I]PVP K. 60 declined progressively after birth but did not terminate abruptly unless the animal was fed colostrum. In unsuckled animals the rate and extent of absorption at 3 hr was much greater than at 20 hr after birth, but some absorption was still present at least 65 hr after birth.4. The transfer of PVP K. 60 to the peripheral blood was dependent upon factors in sow colostrum, since significant absorption did not occur when PVP was fed in water or simple salt solutions.5. The factors which accelerated absorption were present in colostrum from the goat, cow and ewe as well as that from the sow; they remained in the whey, but, in contrast to the factors which accelerate absorption in the calf, were largely inactivated by boiling. Similarly, neither phosphate, lactate, pyruvate, nor lower volatile fatty acid salts, which were effective in the calf, accelerated absorption in the pig.6. The absorption of [(131)I]PVP K. 30 was found to be much less dependent upon the composition of the solvent than the absorption of [(131)I]PVP K. 60, although absorption was most rapid when PVP K. 30 was fed in colostrum.     

Structural changes in the small intestine associated with the uptake of polyvinyl pyrrolidone by the young ferret, rabbit, guinea-pig, cat and chicken

1. The entry of [(125)I]polyvinyl pyrrolidone (PVP) of mean mol. wt. 160,000 (K. 60) into the epithelial cells of the small intestine has been measured in new-born animals of five species.2. The distribution along the intestine of cells capable of taking up [(125)I]PVP and the decrease and eventual cessation of uptake (closure) with increasing age have been investigated, and have been related to changes in the histological appearance of the small intestine.3. The small intestine of the ferret took up PVP readily until 33-34 days after birth. From 34 to 37 days of age PVP uptake declined sharply and disappeared completely by 40-45 days.4. In the ferret, unlike other species studied, some PVP was taken up by the duodenum. This continued for the first 4 weeks after birth. Thereafter PVP uptake gradually became confined to the terminal ileum.5. In the guinea-pig, PVP uptake was limited to the first 48 hr after birth. During this period the site of uptake was progressively restricted to the terminal ileum.6. In the rabbit, PVP could be taken up in the distal two-thirds of the small intestine for at least 20 days after birth. A decline in uptake occurred between 20 and 22 days after birth in most animals.7. Wide individual variations were seen in the kitten, but PVP uptake was seen in some animals up to 14 days after birth.8. Newly hatched chicks and chicks tested 48 hr after hatching did not take up PVP.9. Histological examination of the small intestine with the light microscope demonstrated that in all species PVP uptake was associated with the presence of vacuoles in the epithelial cells of the villus.10. In the young guinea-pig, large PAS-positive granules were seen in the macrophages of the lamina propria. These appeared to migrate through the epithelium into the intestinal lumen. The significance of this finding and its relation to macromolecular uptake remain unclear.     

The effects of corticosterone and cortisone on the uptake of polyvinyl pyrrolidone and the transmission of immunoglobulin G by the small intestine in young rats.

1. The distribution of polyvinyl pyrrolidone along the intestinal lumen and in the intestinal wall, following oral administration to normal and corticosterone treated rats, was found to be extremely variable. Valid comparisons between the two groups of animals could not be made using this technique. 2. Three, 4 and 5 days after corticosterone treatment there was no significant change in the uptake of 125I-labelled polyvinyl pyrrolidone from standard doses injected into ligated segments of the distal small intestine; nor did the treatment induce precocious replacement of the absorptive cells in this region. Cortisone induced precocious cell replacement, a process which took up to 4 days to complete, and also led to a marked reduction in the uptake of 125I-labelled polyvinyl pyrrolidone from ligated segments of the distal intestine. 3. Three days after treatment with corticosterone (5 mg I.P. at 12 days) there was a marked reduction of labelled immunoglobulin G transport into the blood. Four and 5 days after treatment there was some recovery of the immunoglobulin G transport function. Three days after treatment with cortisone (5 mg I.P. at 12 days) there was closure of the gut to labelled immunoglobulin G. 4. The relevance of these results to antibody transmission and the termination of immunoglobulin transport is discussed.     

Measurement of the clearance function of macrophages with 125I-labelled polyvinyl pyrrolidone

The rate constant of the exponential decay in blood radioactivity between 18 and 48 hr after intravenous injection of 30–80 μg of ¹²⁵I-labelled polyvinyl pyrrolidone (PVP) in mice has the characteristics of a test of the clearance function of macrophages. It is reduced by carbon, PVP or hydrocortisone, and increased by oestrogen. Clearance is independent of dose over this range, and probably measures resting unstimulated phagocytic function; this is perhaps different from the function measured by existing techniques. Preliminary evidence suggests that it is applicable to man.     

Transport of [125I] IgG and [125I] ovalbumin between lymphocyte-like and macrophage-like rat spleen cells: chromatin binding

Spleen cells from rats were incubated for 10 min in Eagle's medium containing [125I] IgG or [125I] ovalbumin. Lymphocyte-like and macrophage-like cells were separated by centrifugation in Ficoll-metrizoate solution (d = 1.095). Control spleen cells were incubated in Eagle's medium without [125I] IgG or [125I] ovalbumin and both subpopulations were separated analogically. Lymphocyte-like cells labelled with [125I] IgG or [125I] ovalbumin were incubated for 10 min with control macrophage-like cells and labelled macrophage-like cells with control lymphocyte-like cells. After the incubation time all subpopulations were separated again in Ficoll-metrizoate solution. Radioactivity of [125I] IgG or [125I] ovalbumin was found in cytoplasmic fraction, sap protein fraction (nucleoplasm) and chromatin of control lymphocyte-like and macrophage-like cells. It may be concluded that transport of these potential antigenic compounds occurs directly in lymphocyte-like and macrophage-like cells as well as transport between these two subpopulations.     

Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain

Autoradiograms of [125I]-endothelin (ET) binding in the rat brain demonstrated that the receptors for endothelin are localized mainly in the brainstem, basal ganglia, and cerebellum. Among the many other nuclei in these regions, there also appeared nuclei which are considered to play important roles in the central nervous regulation of the cardiovascular system: they include the nuclei of the anteroventral third ventricle area, the supraoptic nucleus, and the subfornical organ, for example. From these findings, we suggest that ET-1 or its analogous peptide(s) may act as a neuropeptide regulating central nervous functions, including cardiovascular functions.     

Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules

Binding of [¹²⁵I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensititive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16–27×10^–18 mol mm^–1) were found in the thick ascending limb of the Henle's loop and in the distal convoluted tubule and lower binding levels (2–5×10^–18 mol mm^–) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henle's loop. In the medullary thick ascending limb, Scatchard analysis of specific [¹²⁵I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation.Abbreviations PCT proximal convoluted tubule - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT medullary collecting tubule     

Autoradiographic localization of [125I]charybdotoxin binding sites in rat brain.

Charybdotoxin, a 37 amino acid peptide isolated from scorpion venom, is a potent inhibitor of potassium channel function. [125I]charybdotoxin was originally believed to be a selective ligand for the Ca(2+)-sensitive channel in many tissues, but it appears to bind only to a voltage-sensitive potassium channel in brain. We found high densities of [125I]charybdotoxin binding in lateral olfactory tract, interpeduncular nucleus and a variety of mesencephalic nuclei. Moderate levels were found in the cerebral cortex, medial thalamus, hypothalamus and selected thalamic nuclei. These results indicate that [125I]charybdotoxin identifies a potassium channel or channels with a unique distribution in the brain.     

The effect of protein restricted diets on the clearance of 125I-labelled polyvinyl pyrrolidone in mice

Ajax mice pair-fed isocaloric diets in which protein was restricted to 4% had impaired overall phagocytosis (K_PVP)—the clearance of ¹²⁵I-labelled polyvinyl pyrrolidone (PVP). This effect was produced and reversed within 3 days and occurred without effect on growth. In other mouse strains when the K_PVP was reduced, α_PVP, perhaps the function of individual macrophages, was increased with protein deprivation. Both these effects showed inter-strain variation.     

Quantitative studies with [125I]IgM anti-Lea

One of two volunteers with anti-Le^a in their plasma developed rises in titre following the intramuscular injection of Le^a glycoprotein.

With partially purified [¹²⁵I]IgM anti-Le^a, it was found that the maximum number of antibody molecules that would bind to red cells of phenotype Le(a+b-) ranged from 4500 to 7300 molecules per cell for different donors.

Lewis substance in serum samples was assayed by estimating its ability to inhibit the subsequent uptake of [¹²⁵I]anti-Le^a on to red cells. Somewhat unexpectedly, no clear correlation was found between the red cell and serum concentrations of Le^a antigen of particular donors. The discrepancy between the amount of Le^a on the red cells and in the serum may be due to the presence in the serum of antigen-bearing molecules (either glycoprotein or glycolipid) which fail to bind to red cells.

IgM anti-Le^a obtained from a single donor had an equilibrium constant of 8.4 × 10⁹ l/mol. but no antibody activity was detectable in subunit preparations. It is concluded that IgM anti-Le^a binds multivalently to each red cell.

     

Specific [125I]Bolton-Hunter substance P binding sites in human and rat skin

[125I]Bolton-Hunter substance P [( 125I]BH-SP) binding sites in rat and human skin were investigated, using quantitative receptor autoradiographic and emulsion autoradiographic methods. [125I]BH-SP binding sites were discretely localized in skin areas anatomically corresponding to dermal papillae, sweat glands, and hair follicles. The highest density of the binding sites was in the dermal papilla of the finger, followed by the sweat gland. [125I]BH-SP binding to the dermal papillae of the human finger pad skin and rat paw pad skin was displaced by unlabeled SP, with a high affinity, and Kd values were calculated to be 744 pM and 297 pM, respectively. The existence of [125I]BH-SP binding sites supports the idea of the neurotransmitter role of substance P in skin dermal papilla.     

Autoradiographic visualization of the binding sites for [125I]endothelin in rat and human brain

Putative receptors for endothelin were localised autoradiographically in frozen sections of rat and human brain using [125I]endothelin as a ligand. In the rat brain the highest densities were in the granular layer of the cerebellum, choroid plexus, hippocampus, and habenular nucleus. Similar brain high densities were found in the human cerebellum and hippocampus. The non-vascular pattern of distribution suggests that endothelin may have a function as a modulator of neuronal function in addition to its possible involvement in the regulation of vascular tone.     

Lack of alpha-adrenoreceptor binding of [125I]BE2254 to rat basilar artery membranes

Rat basilar arteries do not contain classical alpha- or beta-adrenoreceptors as assessed by electrophysiological techniques even though these arteries are innervated by catecholamine-containing perivascular nerves. These arteries were therefore examined for their ability to selectively bind an alpha-adrenoceptor radioligand, [125I]BE2254 (2/beta/4-hydroxyphenyl)-ethylaminomethyl)-tetralone). For comparison, rat tail arteries were also studied as these are known to contain functional alpha-adrenoreceptors. It was found that basilar artery membranes had only one-third of the specific binding of tail artery membranes and this finding collaborates the electrophysiological data.     

Autoradiographic mapping of [mono[125I]iodo-Tyr10, MetO17]vasoactive intestinal peptide binding sites in the rat brain

The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.     

Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain

Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.     

The effect of amino acid restricted diets on the clearance of 125I-labelled polyvinyl pyrrolidone in mice.

Impaired overall phagocytic function, as measured by clearance of radio-labelled polyvinyl pyrrolidone from the blood (KPVP), has been demonstrated in mice whose dietary intake of the essential amino acids phenylalanine and tryptophan was reduced to less than a quarter of that in a standard diet which supported optimal growth. This was associated with loss in weight of body, liver, spleen and thymus and an increase in the phagocytic index, alphaPVP.