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PubMed1979

Time course of increased collateral arterial and venous endothelial cell turnover after renal artery stenosis in the rat.

We induced left renal artery stenosis in rats and studied collateral arterial formation by angiography, histology, and radioautography with tritiated thymidine. Endothelial cell turnover was estimated by radioautography with tritiated thymidine in the periureteric blood supply of 10 normal and 38 collateral-forming kidneys 1 to 100 days after stenosis. Periureteric arterial endothelial cell labeling showed a highly significant (P less than 0.005) increase, apparent within 1 day and gradually falling as the vessels grew, until a baseline was reached in 35 days. A smaller but statistically significant increase in the labeling index also was found in endothelial cells of the renal vein during the first week (P less than 0.01), and had a similar time course. A marked increase in epithelial cell labeling in the ureters draining the stenotic kidneys also was evident (P less than 0.005). Thus, collateral vessel development is characterized by active DNA synthesis in the cellular elements which is maximal during the first week. A humoral factor is implicated in the vascular response by the parallel proliferation of venous and uretic cellular elements that are unlikely to experience the biophysical forces, such as increased blood flow or tangential wall force, which might stimulate proliferation in ther arterial vessels.

Animals↗

PubMed1981

Specific binding and internalization of blood-borne [125I]-iodoinsulin by neurons of the rat area postrema.

The specificity and cellular location of binding sites for blood-borne [125I]iodoinsulin in the area postrema and adjacent paravagal region of the rat brain was determined by means of quantitative light and electron microscope radioautography. In both regions coinjected unlabeled insulin inhibited the binding of [125I]iodoinsulin in a dose-dependent manner, and insulin analogs blocked [125I]iodoinsulin binding in rank order of their known in vitro bioactivity. Electron microscope radioautography of this region showed that insulin specifically bound to neuronal dendrites and cell bodies and that some of the specifically bound radioactivity was internalized and concentrated in a variety of subcellular vacuoles. These observations demonstrate that certain neurons of the area postrema are endowed with the receptive capacity common to all known insulin-sensitive cells. Anatomic considerations suggest that insulin target neurons in the area postrema could mediate insulin feedback on parasympathetic function by relaying information regarding circulating insulin status to central autonomic circuits governing vagal activity. This study represents the first demonstration of a receptor-mediated uptake and concentration of a blood-borne polypeptide hormone by central neurons.

Animals↗

PubMed1981

In vivo demonstration of receptors in rat liver to the amino-terminal region of parathyroid hormone.

The specific binding of the amino-terminal region of parathyroid hormone (PTH) to liver has been assessed by in vivo radioautography employing a biological active 125I-labeled synthetic bPTH analog ([Nle-8,18, Tyr-34]bPTH-(1-34) amide) as a probe. Two minutes after intrajugular injection of the labeled analog into rats, free hormone was separated from that bound to cells by intracardiac perfusion with buffer (30 sec), followed by perfusion-fixation with glutaraldehyde. By light and electron microscope radioautography, the distribution of silver grains over the liver sections revealed localization over the periphery of hepatocytes as well as over endothelial cells. In simultaneous control experiments, the unlabeled synthetic active fragment bPTH-(1-34) and unlabeled intact native bPTH-(1-84) significantly inhibited binding of the labeled analog to liver hepatocytes but not to endothelial cells. Greater uptake of the 125I-labeled bPTH analog was found in rat liver (28% of the injected dose) than in kidney, bone, or other organs examined. In parallel experiments using purified plasmalemma fractions from hepatocytes, both bPTH-(1-34) and intact bPTH-(1-84) demonstrated a dose-dependent activation of adenylate cyclase which was less than that of glucagon but greater than that of epinephrine. The combined results support the concept of the liver as a target organ for the amino-terminal biologically active region of PTH.

Adenylyl Cyclases↗

PubMed1989

Increased binding of epidermal growth factor at preimplantation sites in mouse uteri.

The presence of receptors for epidermal growth factor (EGF) in nonpregnant uteri and the elevation of EGF levels in blood during early pregnancy suggest that EGF and its receptor may play important roles in the early stages of pregnancy. We determined the distribution of EGF receptors in uteri of nonpregnant and pregnant mice during the late preimplantation period (days 4.5-5.0 of pregnancy) using radioautograph and quantitative binding techniques. Radioautography of [125I]EGF binding to cornua from nonpregnant mice showed low levels of specific binding evenly distributed throughout the cornua. In contrast, radioautographs of cornua from pregnant mice showed bands of elevated binding encircling the lumen at sites of preimplantation. Results from radioautography were supported by quantitative analysis of [125I]EGF binding to uterine homogenates from nonpregnant and pregnant mice. Binding of [125I]EGF to uterine membranes was highly specific and time dependent. The average level of specific EGF binding calculated from Scatchard plots of nonpregnant uteri (27 +/- 13 fmol/mg protein) was significantly (P less than 0.05) lower than that in pregnant superovulated uteri (106 +/- 67 fmol/mg protein). Furthermore, specific binding of EGF was significantly (P less than 0.05) higher in preimplantation sites than in the intervening nonimplantation regions from the same uteri (42 +/- 6 vs. 29 +/- 4 fmol/mg protein, respectively). Differences in EGF binding appear to be due to changes in the number of EGF receptors, since half-displacement values (1 nM) were similar in all samples. These results demonstrate that alterations of EGF receptor levels occur at sites where implantation will occur in mouse uteri and support the concept that the transforming growth factor-alpha/EGF receptor and its ligands are involved in implantation of concepti.

Animals↗

PubMed1998

Radioactive fucose as a tool for studying glycoprotein secretion.

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose. [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose.

Animals↗

PubMed1998

Histologic distribution of insulin and glucagon receptors.

Insulin and glucagon are the hormonal polypeptides secreted by the B and A cells of the endocrine pancreas, respectively. Their major physiologic effects are regulation of carbohydrate metabolism, but they have opposite effects. Insulin and glucagon have various physiologic roles, in addition to the regulation of carbohydrate metabolism. The physiologic effects of insulin and glucagon on the cell are initiated by the binding of each hormone to receptors on the target cells. Morphologic studies may be useful for relating biochemical, physiologic, and pharmacologic information on the receptors to an anatomic background. Receptor radioautography techniques using radioligands to label specific insulin and glucagon receptors have been successfully applied to many tissues and organs. In this review, current knowledge of the histologic distribution of insulin and glucagon receptors is presented with a brief description of receptor radioautography techniques.

Animals↗

PubMed1980

Biosynthesis of proinsulin and other islet cell proteins in pancreatic beta cells of the rat: a radioautographic evaluation of glucose effects in vitro.

Rat pancreatic islets were incubated in vitro with L-[4,5-3H]leucine or with L-[2,3-3H]tryptophan in Krebs-Ringer bicarbonate buffer, containing 0, 5, or 20 mM glucose. Incorporation of labeled amino acids in islet cells was evaluated quantitatively by a validated radioautographic procedure. Incorporation of labeled leucine into [3H]proinsulin and [3H]insulin was measured by immunoprecipitation and into other islet proteins by trichloroacetic acid precipitation. Incorporation of labeled amino acids in pancreatic beta cells was patchy and not uniform. Up to 20 to 35% of beta cells, mainly in central regions of the islets, showed poor or no incorporation of label. Peripheral nonbeta, endocrine islet cells and mesenchymal islet cells were all uniformly labeled. Incorporation of either amino acid into nonbeta endocrine and mesenchymal cells was not much affected by the absence of glucose in the incubation buffer. In contrast, incorporation of the amino acids into beta cells was strikingly affected. Incorporation of [3H]leucine into proinsulin and insulin at 0 mM glucose measured by specific immunoprecipitation and by silver grain densities over beta cells was 15 to 20 times less than at 20 mM glucose. Incorporation of [3H]tryptophan, an amino acid absent in proinsulin, into nonhormonal, sedentary beta cell proteins studied by radioautography, similarly, was strikingly affected in the absence of glucose. Thus, radioautography revealed a great sensitivity of both hormonal and nonhormonal protein biosynthesis in the beta cell to the concentration of glucose in the medium.

Amino Acids↗

PubMed1986

Radioautographic localization and regulation of the insulin receptors in rat testis.

Luteinizing hormone (LH) is involved in maintenance of the insulin receptor in rat Leydig cells. In the cryptorchid model, spontaneous LH elevation increases insulin binding in Leydig cell membrane; this result is confirmed by the number of insulin receptors determined by radioautography. Administration of testosterone or LH-RH to control or cryptorchid rats lower insulin binding in testis membranes indicating the key role of LH. Isolated Leydig cells respond in vitro to LH by increasing their insulin binding. Localization and specificity of this binding has been studied by radioautography and we have demonstrated the preferential localization of insulin receptor in Leydig cells vs tubular cells. We conclude that insulin plays a synergistic role with LH on steroidogenesis via a specific membrane receptor which number appears modulated by pituitary LH.

Animals↗

PubMed1993

Evidence that estrogen binding sites are present in bone cells and mediate medullary bone formation in Japanese quail.

Studies were carried out in Japanese quail to characterize the binding of estrogen to bone cells and to determine whether induction of medullary bone by estrogen is mediated by estrogen receptors. First, attempts were made to identify specific, high affinity nuclear binding sites for estrogen with a nuclear exchange assay in oviduct and femora from laying female quail and in liver, kidney, and femora from adult male quail treated with estradiol valerate (2 mg per bird). High-affinity nuclear estrogen binding sites were detected in each of the tissues except femora. Second, estrogen binding sites in femora from male quail were localized by radioautography after treatment with 100 microCi of [3H]-17 beta-estradiol ([3H]-E). Estrogen binding sites were present at 1 and 3 h after administration of [3H]-E, and binding of the radiolabeled hormone was prevented by the simultaneous administration of an excess of radioinert estrogen. Third, estradiol valerate (.4 mg) was given as a bolus to adult male Japanese quail. Five days later, the quail had hypercalcemia due to accumulation of phosvitin in serum, had an extensive network of medullary bone at the femur midshaft, and had reduced cortical bone area. The nonsteroidal, anti-estrogen tamoxifen (2 mg/day) prevented estrogen-induced hypercalcemia, medullary bone formation, and reduction of cortical bone area. Fourth, the estrogen target cells in femora from adult male quail that were induced to differentiate to osteoblasts by estrogen treatment were located by [3H]-proline radioautography as early as 12 h following administration of the hormone. These results are interpreted as evidence that induction of medullary bone is a process mediated by estrogen receptor.

Animals↗

PubMed2002

[Study on low density lipoprotein receptor of adult Schistosoma japonicum].

OBJECTIVE: To provide a theoretical basis for the study of vaccines against Schistosoma japonicum, the receptor for human LDL in the tegument of adult Schistosoma japonicum was investigated. METHODS: Proteins existed in adult Schistosoma japonicum membrane were extracted by Triton X-100 and purified through reverse-phase high performance liquid chromatography (HPLC), and the main protein peaks were then collected separately. 125I-LDL of human plasma as the ligand, through the radioautography and radioligand binding assay, the protein which can bind human serum 125I-LDL specifically was identified. The molecular weight and IEF were detected by SDS-PAGE. RESULTS: According to the radioautography and radioligand binding assay, a protein with retention time of 10.5 min was proved to be able to bind human serum 125I-LDL specifically. SDS-PAGE revealed that the molecular weight of the purified protein is 60-65 kDa, and its IEF is 6.7. CONCLUSION: LDL binding protein may exist on the surface of both male and female adult Schistosoma japonicum with the function of obtaining cholesterol from host circulating system.

Animals↗

PubMed1976

Uptake of circulating iron by the duodenum of normal mice and mice with altered iron stores, including sex-linked anemia: high resolution radioautographic study.

Using electron microscopy radioautography, the deposition of intravenously administered iron in the duodenal epithelium was studied in normal mice, iron-overloaded and iron-deficient mice, and in mice with X-linked anemia (gene symbol sla) 4 and 24 hours after injection of 59Fe. The resolution of radioautography with 59Fe was determined with a line source and the distance from the hot line within which half of the grains fell (HD value) was 1650 A. In normal, iron-overloaded, and sla mice, radioiron was localized in the undifferentiated crypt cells at 4 hours and in the absorptive cells of the luminal half of the villi, at 24 hours. At both times, the vast majority of the grains was seen over the areas rich in free ribosomes and rough endoplasmic reticulum. In iron-deficient mice, grains were not found at either time. The amount of iron incorporated in the crypt cells was related to the size of the body iron stores. It is postulated that the amount of iron incorporated in the crypt cells is the result of interaction between uptake and return to the blood of the circulating iron. Once the crypt cells have differentiated into absorptive cells, the uptake and recirculation of iron from and to the blood would cease, leaving an amount of "message" iron which determines the absorptive cell's subsequent capacity for iron transfer to the plasma. In sla mice, in spite of tissue iron deficiency, the amount of iron deposited was similar to that of normal mice and markedly increased after treatment of the anemia. The accumulation of iron in the crypt cells may result from the decreased return to the blood of the incorporated circulating iron, due to the postulated deficient iron carrier mechanism in sla, or may be a consequence of increased avidity of the absorptive cells for circulating iron. In either event, the result would be that the absorptive cell receives an inappropriate message with resulting inappropriate absorption of iron.

Anemia, Hypochromic↗

PubMed1989

Radioautographic study on the localization of an anti-allergic agent, tranilast, in the rat liver.

In order to demonstrate the localization associated with metabolism of an anti-allergic agent, Tranilast, in the liver, light microscopic radioautography of the liver was performed. Rats were administrated orally with 3H-Tranilast, and were sacrificed at 15 minutes to 24 hours after the administration. The livers were taken out and fixed, embedded and processed for light microscopic radioautography. 3H-Tranilast was absorbed rapidly, and the radioactivity in the liver increased and decreased within several hours. The number of radioautographic silver grains reached a maximum 3 hours after the administration. From 1 to 6 hours after the administration, the silver grains decreased from the portal area toward the central area. Seventy to 80% of all silver grains on the hepatocytes were retained in the cytoplasms of the hepatocytes at any experimental period. From these results, it was concluded that the localization of radioautographic silver grains was associated with Tranilast uptake of hepatocytes in each hepatic lobular compartment and that the metabolic process from uptake to excretion of Tranilast took part in the hepatocytes in each hepatic lobular compartment.

Administration, Oral↗

PubMed1989

[True pemphigus: electron microscopic and radioautographic research on the skin].

Skin biopsy specimens of 19 patients with pemphigus vulgaris have been examined by electron microscopy and radioautography. Ultrastructural changes in the epidermocytes (impaired nucleolar system, lysis of the cytoplasmic organelles, disappearance of desmosomes) reflect impaired protein biosynthesis. In vitro radioautography with 3H uridine and 3H thymidine has shown inadequate RNA and DNA synthesis in the epidermal cells. These findings are regarded in the light of the plastic insufficiency concept: the plastic deficiency syndrome.

Adult↗

PubMed1988

[Ultrastructural localization of mRNA coding for oxytocin by in situ hybridization. Study by high resolution autoradiography using a tritiated oligonucleotide probe].

In situ hybridization (ISH) using a 25 mer tritiated oligonucleotide probe has been performed to study at the electron microscopic level the subcellular localization of the oxytocin mRNA in the rat hypothalamic magnocellular neurons. After high resolution radioautography, silver grains appeared to be localized over the cytoplasm of some magnocellular neurons of the supra-optic nucleus and frequently overlapped the ergastoplasmic "cisternae" of the Nissl bodies. These results demonstrate the possible application of ISH at a subcellular level using high resolution radioautography and a tritiated probe.

Animals↗

PubMed1986

[Determination of the DNA-synthesizing cell count by the immunofluorescence method].

In DNA-synthesizing cells DNA is partially single-stranded. Anti-thymidine antibodies, while specifically reacting with this DNA, form a complex which may be revealed using indirect immunofluorescent technique. A comparative determination of DNA-synthesizing cell number in tumor tissue (larynx squamous cell carcinoma) was performed using immunofluorescent technique and radioautography. The former method showed the labeling index (LI) to vary from 1.2 to 9.9%, while the latter showed it to vary from 1.0 to 8.2%. The correlation ratio between the LI values obtained by the two techniques was 0.79. To eliminate a possible reaction of anti-thymidine antibodies with cellular RNA, specimens were preincubated in solutions with RNAase. No more than 6 hours were required to stain specimens using this LI estimation technique. This investigation allows to reveal DNA synthesizing cells not only in the periphery of a histological section, as does routinely radioautography, but also in its centre.

Animals↗

PubMed1987

[Electron microscopic-radioautographic study of bacterial invasion in severely burned patients].

Wound pieces taken from 3-6 sites at a time during surgical treatment of the wound in 10 patients with thermal burns involving 25-50% of the body surface area, were examined by electron-microscopic radioautography and bacteriologically. The burn wounds were found to contain not only live (RNA-synthesizing) bacteria but also replicating (DNA-synthesizing) bacteria, and to be heterogeneous in terms of bacterial seeding (the largest numbers being present in the scab and the smallest, in the granulation tissue), but not of bacterial species; these were represented in all layers by mixed populations (Pseudomonas aeruginosa, pathogenic Staphylococcus, Proteus), although the deep layers contained more pathogenic and more virulent subpopulations than the more superficial ones. Septic patients had considerably more organisms in all layers of their wounds than nonseptic patients, and contained proliferating organisms in the granulation tissue. The main steps in the penetration of individual microbial species into the circulation were traced out by means of electron-microscopic radioautography.

Acute Disease↗

PubMed1985

Proteins rapidly transported to the synapses of a single identified neuron of Aplysia californica.

The cell body of R2, a giant cholinergic neuron of Aplysia californica, resides in the abdominal ganglion, whereas its synapses are on thousands of unicellular mucus glands located in the skin. Due to the great spatial separation between the site of macromolecular synthesis and the presynaptic terminals, rapid axonal transport can be used to segregate synaptic proteins from those to be used elsewhere in the cell. The proteins of R2 were labeled by incubating the abdominal ganglion in [35S]methionine for 5 hr in a chamber separated from the rest of the isolated central nervous system. After 50 hr, 28 radiolabeled proteins were reproducibly found by one- and two-dimensional polyacrylamide gel electrophoresis to be transported to the distal regions of peripheral nerves P6, P7, and P8 that innervate the parapodia and middle body wall. We are sure that R2 is the source of these proteins since radioautography of sections taken throughout the nervous system, complemented by cobalt tracings, showed that R2 is the only neuron in the abdominal ganglion with axons in these nerves. Nine of the 28 transported proteins are glycoproteins since they were also labeled after injecting R2's cell body with [3H]-L-fucose. There is evidence that the proteins and glycoproteins are destined for R2's presynaptic terminals. For example, in experiments in which the body wall and parapodium remained attached to the nerves, the proteins were transported to the skin region that contains the glands. Moreover, analyses of the distribution of the rapidly transported proteins by qualitative radioautography and by extrusion of axoplasm indicated that none are constituents of the axolemma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1979

[Rhythm of protein concentration and incorporation of 3H-lysine into hepatocytes in vitro].

"In vitro" incubated slices of the rat liver, by the method of interferometry combined with radioautography, have demonstrated hour's rhythmic fluctuations in protein concentration and 3H-lysine incorporation into proteins. The biochemical method has demonstrated fluctuations in protein synthesis rate in the same cells with an average period of about one hour. The possibility to apply the interferometric method in combination with radioautography for revealing quantitative changes of protein in slices of organic cultures is demonstrated.

Animals↗