Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.
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ABSTRACT:
SUBMITTER: Livesey G
PROVIDER: S-EPMC1163305 | biostudies-other | 1981 Sep
REPOSITORIES: biostudies-other
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Project description:When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.
| S-EPMC1152318 | biostudies-other
Project description:The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.
| S-EPMC1163306 | biostudies-other
Project description:Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.
| S-EPMC1186580 | biostudies-other
Project description:In rat yolk sacs incubated in vitro, the rates of degradation of endogenous [3H]leucine-labelled proteins and of pinocytically ingested 125I-labelled bovine serum albumin were both decreased in the presence of either ammonium, methylammonium or ethylammonium ions (0-20 mM) or much lower concentrations of chloroquine (0-500 microM). These effects were also accompanied by an inhibition of pinocytosis, as measured by the rate of uptake of 125I-labelled polyvinylpyrrolidone, and by a fall in the [ATP]/[ADP] ratio within the tissue. Re-incubation in inhibitor-free medium of yolk sacs previously exposed to a weak base restored pinocytic and proteolytic capacities, except for tissues exposed to chloroquine at concentrations above 0.1 mM (these appeared to be cytotoxic); an attendent rise in [ATP]/[ADP] ratios to near normal values was also observed. Weak bases, at concentrations that fully arrested the breakdown of 125I-labelled albumin, failed to inhibit by more than 45% the degradation of [3H]leucine-labelled endogenous proteins. Since 125I-labelled bovine serum albumin has been shown to be degraded entirely intralysosomally by yolk sacs, this suggests either that the hydrolysis of endogenous proteins is shared between lysosomes and some other site or that, unlike 125I-labelled albumin, some endogenous proteins can be degraded within lysosomes at abnormally high pH.
| S-EPMC1161975 | biostudies-other
Project description:1. The effects of leupeptin and other microbial proteinase inhibitors were measured in rat yolk sacs on the uptake and degradation of formaldehyde-denatured 125I-labelled bovine serum albumin as well as on the degradation of 3H-labelled endogenous protein. 2. Leupeptin, at concentrations between 1 and 100 micrograms/ml, inhibits the degradation of added albumin without affecting pinocytic uptake. Accordingly large amounts of undegraded albumin accumulate within the tissue. 3. Removal of leupeptin produces a rapid recovery of the capacity to degrade albumin. 4. Endogenous protein degradation is rapidly inhibited by leupeptin, but to a far lesser extent than the breakdown of albumin. However, the inhibition is only slightly reversed on removal of leupeptin. 5. Degradation of both albumin and endogenous protein in intact yolk sacs is inhibited by the microbial proteinase inhibitors in the order: leupeptin greater than antipain greater than chymostatin; elastatinal, pepstatin and bestatin are ineffective. 6. Similar results are found when albumin is incubated in yolk-sac homogenates at pH 4 with the inhibitors. 7. The marked inhibitory effects of leupeptin, antipain and chymostatin suggest that cathepsin B and possibly cathepsin L participate in the degradation of 125I-labelled albumin in yolk sacs. By comparison, the smaller inhibitory effects of the proteinase inhibitors on endogenous protein breakdown imply a minor role of lysosomal cathepsins in this process.
| S-EPMC1162965 | biostudies-other
Project description:Isolated soleus and extensor digitorum longus muscles from small (40 or 70 g) rats developed a central and substantial (13-57%) loss of glycogen and alpha-glucan phosphorylase activity after incubation for up to 2 h in vitro. The central 'core' of the muscles showed a marked decrease in the rate of protein synthesis. It is suggested that during brief periods of incubation the central core of isolated rat muscles becomes hypoxic, and that consequently the viability of such muscles must be in question.
| S-EPMC1152972 | biostudies-other
Project description:Though phospho-receptor-interacting protein 3 (RIP3 or RIPK3) antibodies are used in western blot, immunostaining of murine phospho-RIPK3 is challenging. Here, we verify and describe a detailed protocol for immunofluorescent detection of phospho-RIPK3 in L929 cells and mouse yolk sacs. We also describe in detail the model construction methods, sample preparation steps, and staining procedures for immunohistochemical labeling of RIPK3 activation in mouse ceca and small intestines by utilizing a specific commercially available antibody. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021) and Wang et al. (2020).
| S-EPMC9254451 | biostudies-literature
Project description:The yolk sac is phylogenetically the oldest of the extraembryonic membranes. The human embryo retains a yolk sac, which goes through primary and secondary phases of development, but its importance is controversial. Although it is known to synthesize proteins, its transport functions are widely considered vestigial. Here, we report RNA-sequencing (RNA-seq) data for the human and murine yolk sacs and compare those data with data for the chicken. We also relate the human RNA-seq data to proteomic data for the coelomic fluid bathing the yolk sac. Conservation of transcriptomes across the species indicates that the human secondary yolk sac likely performs key functions early in development, particularly uptake and processing of macro- and micronutrients, many of which are found in coelomic fluid. More generally, our findings shed light on evolutionary mechanisms that give rise to complex structures such as the placenta. We identify genetic modules that are conserved across mammals and birds, suggesting these modules are part of the core amniote genetic repertoire and are the building blocks for both oviparous and viviparous reproductive modes. We propose that although a choriovitelline placenta is never established physically in the human, the placental villi, the exocoelomic cavity, and the secondary yolk sac function together as a physiological equivalent.
| S-EPMC5474779 | biostudies-literature
Project description:When added to the serum-free medium in which 17.5-day rat yolk sacs were incubated, formaldehyde-denatured 125I-labelled bovine serum albumin was rapidly degraded. More than 80% of the radiolabelled digestion products appearing in the incubation medium consisted of [125I]iodo-L-tyrosine; larger digestion products were found only in association with the yolk-sac tissue. In the early stages of an incubation, low-molecular-weight digestion products began to appear in the incubation medium only after they could be detected within the tissue, and progressive association of trichloroacetic acid-insoluble radioactivity with the tissue preceded both these events. None of the observed proteolysis could be attributed to proteinases released into the incubation medium. Tissue-associated acid-insoluble radioactivity showed a lysosomal distribution on sub-cellular fractionation, and cell-free homogenates of yolk sacs degraded albumin only at acid pH values. Progressively decreasing the rat of pinosome formation (either by progressively lowering the incubation temperature or by the use of increasing concentrations of the metabolic inhibitor rotenone) caused a corresponding decrease in the rate of degradation of albumin. These findings indicate that, in vitro, formaldehyde-denatured 125I-labelled bovine serum albumin is digested by rat yolk sacs exclusively intracellularly, within lysosomes.
| S-EPMC1161833 | biostudies-other
Project description:The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 ?M or 20 ?M over a 26-h period (GD10-GD11) and 50 ?M over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual tissues following leupeptin treatment. Analysis of the thiol proteome showed few alterations to specific pathways mapped to the Kyoto Encyclopedia of Genes and Genomes Pathway database, but did reveal significant increases in concentrations of proteins associated with glycolysis/gluconeogenesis in the VYS and decreased concentrations proteins associated with ribosome biogenesis and function in the EMB. A subset of proteins elevated by >2-23-fold in the VYS were identified as serum (blood) proteins and represent the maternal-side proteins captured by the VYS and which are not degraded in the lysosomes as a result of leupeptin's inhibitory action. The observed constellation of proteins decreased in the EMB by leupeptin represent proteins from several adaptive pathways that are commonly altered in responses to amino acid starvation. These studies show clear differential responses to protease inhibition in VYS and EMB during organogenesis and suggest the possibility for additional roles of redox regulation, cellular adaptations and metabolic insufficiency caused by protease inhibition.
| S-EPMC4679479 | biostudies-literature