Evidence for phosphorylation of actin by the insulin receptor-associated protein kinase from human placenta

A purified preparation of rabbit muscle actin (43-kDa protein) is phosphorylated at tyrosine residues when incubated with solubilized insulin receptor from human placenta. Phosphorylation of the 95-kDa receptor subunit and of 43-kDa protein is stimulated by insulin and vanadate, respectively; however, the mode of action of the two agents is distinguishable.     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.     

Characteristics of the solubilized insulin receptor of human placenta

A soluble human insulin receptor can be obtained in high yield from placenta membranes, using the detergent Ammonyx-LO. In crude soluble preparations, the placenta receptor exhibits complex insulin binding kinetics (two binding plateaus, half-saturated at about 40 pM and 700 pM insulin) and an apparent chromatographic heterogeneity (Sepharose 6B) with two insulin binding components having apparent Stokes radii of 72Å and 38Å. However, subsequent to purification by affinity chromatography on insulin-Sepharose, the placenta receptor exhibits a simple insulin binding isotherm, without evidence for binding cooperativity (K_D about 830 pM), and upon chromatography behaves as a single component with a Stokes radius of 38Å_A. When combined with a previously described non-receptor glyco-protein preparation isolated from liver membranes, the affinity-purified plancenta receptor undergoes an increase in its Stokes radius from 38Å to 72Å. Because of the physicochemical similarities between the placenta receptor and the insulin receptor previously isolated from liver cell membranes, it is concluded that the placenta receptor is representative of the insulin receptor present in other traditional target tissues for insulin. The study underscores a possible role for insulin in placental physiology and provides for the large scale isolation of the human receptor from a readily available source.     

Evidence for a clathrin-mediated recycling of albumin in human term placenta

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 microM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-beta-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.     

Evidence for modulation by ATP of amino acid transport in human placenta

The effect of ATP on placental amino acid transport was studied by measuring the uptake of alpha-(methylamino)-isobutyrate in brush border microvillous plasma membrane vesicles prepared from human full-term placental syncytiotrophoblasts which were incubated with or without ATP. The presence of a Na+ gradient from the outside to the inside of the vesicles prepared after incubation with ATP resulted in a higher initial rate and an increased transport of alpha-(methylamino)-isobutyrate, while Na+ gradient-independent alpha-(methylamino)-isobutyrate uptake was not different in either type of membrane vesicle. The increase in transport activity was not inhibited by cycloheximide. Kinetic analysis showed that ATP enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in the affinity (Km) of the carrier for the substrate, suggesting an increase in carrier number in placental syncytiotrophoblasts incubated with ATP.     

Differential expression of the receptors for epidermal growth factor and insulin in the developing human placenta

The detailed cellular distribution of epidermal growth factor (EGF) receptors and insulin receptors during the development of the human placenta was examined. We show that EGF receptors are expressed by villous cytotrophoblast cells in first trimester human placentae. However, where these cells proliferate to form extravillous cytotrophoblast cell columns, there is a dramatic decrease in EGF receptor expression. There is no such differential expression of insulin receptors on this cell population. In contrast, both EGF-and insulin-receptors are present throughout gestation on the microvillous membrane of the terminally differentiated and non-proliferative syncytiotrophoblast although, at term, EGF-but not insulin-receptors are also found on the basolateral membrane of this epithelium. We further show that EGF receptors isolated from first trimester and term human placentae have functional tyrosine kinase activities but differ in their extent of glycosylation. These results suggest that EGF receptors probably play several distinct functional roles in these epithelial cells depending on their proliferative capacity and differentiation status.     

Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta

Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.     

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.     

Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract.

Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.     

Towards a rational modeling for the human placenta

An attempt is made to provide a general framework sufficient of handling analytically typical questions concerned with a physiological functioning of a human placenta. The principles of mechanics and thermodynamics, as they apply to a continuum theory of mixtures, are the essential tools in constructing the model. In general terms, it is assumed that each point of the placenta space is simultaneously occupied by placenta tissue, fetal blood, and maternal blood. These three constituents are allowed to interact mechanically, chemically, and thermally. One of the basic features of the model is its capability of providing quantitative information on the deformation of the placenta tissue and the pressure distribution of the fetal and maternal blood. Effects of inertia, deformation, and changes of shape and weight of the placenta during gestation are also taken into account.     

Impermeability of the rat placenta to insulin during organogenesis

The cause of the embryopathy associated with diabetes mellitus is uncertain. To examine whether exogenously administered insulin may be teratogenic, tracer amounts of radiolabelled insulin were infused for two hours during organogenesis (day 12 1/2 of gestation) into three groups of pregnant rats: control (n = 8), diabetic (n = 5), and hyperinsulinemic (n = 4). For maternal plasma, no differences were found among the three study groups in the percentage of the protein-precipitable (insulin-containing) radioactivity. Tissue radioactivities were expressed relative to the two-hour maternal plasma sample. Maternal kidney samples had the highest total and protein precipitable counts followed in descending order by the maternal plasma, maternal liver, placenta, and embryo. No differences in radioactivities were noted among the three study groups for specific tissues studied. Protein-precipitable radioactivities in the embryo were more than 100-fold less than the maternal plasma values. In 11 of the 17 litters, the acid-insoluble fractions of the embryos were not distinguishable from background counts, and none of the remaining six were greater than twice background. These studies demonstrate that during the period of organogenesis, the rat embryo is protected from maternal insulin by the placenta, and hence, maternal insulin is an unlikely teratogen.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Evidence for nuclear processing of progesterone receptors in rat placenta

The distribution of the progesterone receptor (Rp) in cytosolic and nuclear compartments of placenta has been studied in intact and ovariectomized (Ovx) rats on the 14th day of pregnancy. Removal of estradiol (E) and progesterone (P) by Ovx caused a 50% decrease in progesterone receptors from cytosolic and nuclear compartments. Estradiol replacement restored binding to intact levels. Progesterone, given 19 h after E, induced an additional 3-fold increment in the number of cytosolic and nuclear binding sites 1 h later. Four hours after progesterone the number of receptor sites in the placenta fell 60%, signifying processing. This was followed 4 h later by reversal of processing mechanisms leading to full recovery of nuclear and cytoplasmic binding sites. Actinomycin D (0.6 mg/ani) was found to have no influence on these events. On the other hand cycloheximide (0.5 mg/ani) completely prevented processing of binding sites when administered at the same time as progesterone or 2 h before, but did not influence the unmasking of nuclear sites which occurred 1 h after a progesterone challenge. The cycloheximide block to processing was partial when given 2 or 3 h after progesterone (61 and 43% complete, respectively). The full complement of receptors was processed when cycloheximide treatment was delayed 3.75 h after progesterone administration. These findings have led to the view that processing represents rapid and reversible changes in binding properties of the receptor rather than a gain or loss of receptor protein per se. The findings of this study suggest that a hypothetical substance, "processin", whose production is blocked by cycloheximide binds to the receptor and in some undefined manner inhibits ligand-receptor interaction within 4 h after an in vivo progesterone challenge. Nuclear accumulation of receptor induced by progesterone was not accompanied by cytoplasmic depletion of receptor nor was the apparent loss of processed nuclear receptor due to recycling of receptor to cytoplasm. We propose that nuclear receptors continually recycle within the nucleus in masked and unmasked states regulated by delicate interplay between progesterone and processin.