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Biomedical subjects

B I Posner

Publications and source records attributed to B I Posner.

At least 91 records · Page 5Linked to original sources
PubMed1988

Increase in specific binding of insulin-like growth factor (IGF) II to type 1 IGF receptors on erythrocytes of hypopituitary children receiving growth hormone therapy.

Specific receptor binding for insulin-like growth factors (IGFs) is measurable in young erythrocytes. Cells of similar age, Fraction A, can be reproducibly obtained by dextran gradient centrifugation from 5-10 ml of blood. We now report IGF-II specific binding to Fraction A erythrocytes from normal children and children with growth hormone deficiency. Normal controls (Group 1) were 5 male volunteers (14.7 +/- .6 years, mean +/- SEM) and 10 children with constitutional short stature (11.4 +/- 1.6 years) who had normal 6-hour daytime growth hormone profiles and plasma IGF-I values. Twelve growth hormone deficient children (Group 2), aged 13.7 +/- 1.1 years, had samples taken after 2 months without growth hormone therapy and again following 2 months with growth hormone (0.1 U/kg 3 times per week) therapy. The percent of total erythrocytes in Fraction A did not differ in the two groups of children. Group 1 had IGF-II specific binding of 10.2 +/- 0.6% (per 3 X 10(9) cells). IGF-II specific binding was less in Group 2 at 6.6 +/- 0.8% (p less than 0.002). With growth hormone therapy, IGF-II specific binding increased to 10.4 +/- 1.0% (p less than 0.02), a value not different from that seen in Group 1. Corresponding plasma IGF-II and IGF-I values showed a positive correlation with IGF-II specific binding (r = 0.54 and r = 0.56 respectively, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1987

Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver.

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.

Animals↗
PubMed1987

Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase.

The actions of insulin, vanadate (V) and hydrogen peroxide (H2O2) on IGF-II binding and insulin receptor tyrosine kinase activity were studied in rat adipocytes. Incubating adipocytes with a combination of V plus H2O2 resulted in a potent synergistic effect on both the increase in IGF-II binding and the activation of the insulin receptor kinase. Catalase, which removes H2O2, abolished this synergism if added at the time of mixing of V plus H2O2 but not if added 10 min. later, suggesting that the formation of peroxide(s) of vanadate generated a potent insulin mimicker. The data support a critical role for the insulin receptor kinase in insulin action. The novel insulin-mimetic compound, a presumed peroxide of vanadate, could prove useful for investigating insulin action and may be valuable for treating insulin resistance.

Adipose Tissue↗
PubMed1987

Stimulation of insulin-like growth factor II receptor binding and insulin receptor kinase activity in rat adipocytes. Effects of vanadate and H2O2.

Autophosphorylation of the insulin receptor on tyrosine residues and activation of the endogenous insulin receptor kinase is postulated to be a critical step in the mechanism of action of insulin. To investigate this hypothesis, the insulin-mimicking effects of vanadate (sodium orthovanadate) and H2O2 (hydrogen peroxide) alone and in combination were examined in freshly isolated rat adipocytes. Vanadate and H2O2 stimulated the translocation of insulin-like growth factor II (IGF-II) receptors to the plasma membrane of rat adipocytes in a manner analogous to insulin. IGF-II binding was increased by maximally effective doses of vanadate (1 mM), H2O2 (1 mM), and insulin (10 ng/ml) to 172 +/- 10, 138 +/- 12, and 289 +/- 16% of control, respectively. Previously (Kadota, S., Fantus, I. G., Hersh, B., and Posner, B. I. (1986) Biochem. Biophys. Res. Commun. 138, 174-178), we showed that the combination of these concentrations of vanadate plus insulin was not more potent than insulin alone. In this study, similar results were found with H2O2 plus insulin. In contrast, the combination of vanadate plus H2O2 was synergistic, effecting an increase of IGF-II binding to 488 +/- 23% of control. Amiloride inhibited the effects of vanadate, H2O2, and insulin. Adipocyte insulin receptors purified by wheat germ agglutinin chromatography were assayed for tyrosine kinase activity using the synthetic substrate poly(Glu,Tyr) (4:1). Basal activity (no in vitro insulin) was stimulated by exposure of intact cells to vanadate, H2O2, insulin, and vanadate + H2O2 to 147.7 +/- 4.3, 178.2 +/- 43.4, 495.0 +/- 67.1, and 913.2 +/- 92.0% of control, respectively. The stimulation of tyrosine kinase activity by these agents was accounted for by the insulin receptor as the augmented activity was completely immunoprecipitated with insulin receptor antibody. In these studies, the increase in IGF-II binding correlated significantly with the activation of the insulin receptor-tyrosine kinase (r = 0.927, p less than 0.001). These data support the hypothesis that activation of the insulin receptor kinase is linked to insulin action.

Adipose Tissue↗
PubMed1987

Insulin-like growth factor (IGF) binding in hypophysectomized rat liver microsomes: alteration by a soluble binding moiety.

In this study, we compared the binding of IGF-I and IGF-II to liver microsomes of normal and hypophysectomized (Hypox) rats. The binding of [125I]-IGF-II, measured by centrifugation of membrane-bound ligand, was lower in hypox than in normal rats (15 +/- 2 vs 26 +/- 1%, p less than 0.001) but binding was increased (46 +/- 1.5 vs 31 +/- 1%, p less than 0.001) when bound and free hormones were separated using polyethyleneglycol (PEG) precipitation. This was due to the presence of soluble binding activity which dissociated from membranes to compete for IGF binding. When soluble binding activity was first removed from microsomal membranes by a washing procedure no difference was found in [125I]-IGF-II binding to microsomes of Hypox and normal animals (33 +/- 1 s 32 +/- 1%). However, in the microsomal washing supernatant from Hypox (containing soluble binding activity) IGF-II binding was much higher than in that from normals (17 +/- 2 vs 6 +/- 0.5%, p less than 0.001). The binding of [125I]-IGF-I was lower than that of [125I]-IGF-II but was comparably changed. By contrast, [125I]-insulin binding was similar in Hypox and normal rats and was not influenced by PEG precipitation or prewashing of the membranes. Inhibition dose-response curves showed a paradoxical increase in [125I]-IGF-II binding to unwashed microsomes of Hypox rats in the range of 0.5-5 ng/ml cold IGF-II. In normal animals [125I]-IGF-II binding to microscomes was progressively inhibited by IGF-II in a range of 0.5-500 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

A receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid.

PRL interacts with areas of the central nervous system which reside behind the blood-brain barrier. While vascular PRL does not cross this barrier, it is readily accessible to the cerebrospinal fluid (CSF) from which it may gain access to the PRL-responsive areas of the brain. Studies were undertaken to characterize the mechanism responsible for the translocation of PRL from blood to CSF. Rats were given external jugular vein injections of [125-I]iodo-PRL in the presence or absence of an excess of unlabeled ovine PRL (oPRL), human GH, bovine GH, or porcine insulin. CSF and choroid plexus were removed 60 min later. CSF samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide slab gels and resultant autoradiographs were analyzed with quantitative microdensitometry. The data revealed that unlabeled lactogenic hormones, viz. oPRL and human GH, caused a statistically significant inhibition of [125I]iodo-PRL transport from blood to CSF. In contrast, nonlactogenic hormones, viz bovine GH and insulin, had no effect on [125I]iodo-PRL transport into the CSF. An identical pattern of competition was observed in the binding of hormone to the choroid plexus. Furthermore, vascular injections of [125I]iodo-PRL administered with a range of concentrations of unlabeled oPRL revealed a dose-response inhibition in the transport of [125I]iodo-PRL from blood to CSF. The study demonstrates that PRL enters the CSF by a specific, PRL receptor-mediated transport mechanism. The data is consistent with the hypothesis that the transport mechanism resides at the choroid plexus. The existence of this transport mechanism reflects the importance of the cerebroventricular system in PRL-brain interactions.

Animals↗
PubMed1987

Diabetes insipidus: a postpartum complication.

Sheehan's syndrome and diabetes insipidus were diagnosed in a 31-year-old woman seven months after postpartum bleeding with a short duration of hypotension. The diagnosis of diabetes insipidus was established by the inability to concentrate urine during water deprivation and the marked increase in urinary osmolality after administration of 1-Desamino-8-D-arginine-vasopressin (DDAVP). Obstetricians should be aware of diabetes insipidus as a postpartum complication.

Adult↗
PubMed1986

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells.

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.

Animals↗
PubMed1986

Vanadate stimulation of IGF binding to rat adipocytes.

Preincubation of adipocytes with insulin (10 ng/ml) stimulated binding of IGF-II to maximal levels of 160% above controls. Vanadate also augmented IGF-II binding with an increase of 126% above controls at a concentration of 1 mM. Coincubation of vanadate (1 mM) with a maximal stimulatory dose of insulin (10 ng/ml) produced no additive effect. However, at submaximal doses of insulin (0.1 ng/ml) the effect of vanadate was additive. Amiloride, a potent inhibitor of the insulin receptor kinase, inhibited the effects of both vanadate and insulin. The data are consistent with an effect of vanadate via a similar sequence of steps to that of insulin; perhaps involving activation of the insulin receptor kinase.

Adipose Tissue↗
PubMed1986

Epidermal growth factor receptor kinase translocation and activation in vivo.

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.

Animals↗
PubMed1986

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures.

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.

Animals↗
PubMed1986

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin.

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The ontogeny of specific prolactin binding sites in the rat choroid plexus.

The development of prolactin receptors in the choroid plexus of the rat was examined using the in vivo autoradiographic approach employing the principle of competitive binding. Experimental animals were injected with [125I]prolactin alone (total binding) while control animals received [125I]prolactin and a 500-fold excess of unlabelled prolactin (non-specific binding). Newborns as well as animals 10, 14 and 18 days postnatal were studied. Three minutes following hormone injection animals received an intracardiac perfusion with fixative and tissues were prepared for quantitative light microscopic autoradiography. The choroid plexus first demonstrated specific binding of prolactin, i.e. a statistically significant difference in the autoradiographic reactions between experimental and control animals, at 14 days postnatal. The lactogen specificity of these binding sites was further defined by the ability of [125I]prolactin to be displaced by unlabelled human growth hormone, which is lactogenic in rats, and not by unlabelled insulin, which is structurally dissimilar to prolactin. Morphometric analyses were performed on electron micrographs of choroid plexus from 10- and 14-day postnatal rats. The volume densities of constituents known to be enriched in polypeptide hormone receptors were measured and compared. Small cytoplasmic vesicles and tubules were statistically significantly more abundant in 10-day-old rats than in 14-day-old animals. It is conjectured that these vesicles and tubules contain an intracellular pool of prolactin receptors whose decrease at 14 days parallels the expression of specific binding sites at the cell surface.

Adrenal Cortex↗
PubMed1986

Tunicamycin sensitivity of prolactin, insulin and epidermal growth factor receptors in rat liver plasmalemma.

We have used the glycosylation inhibitor tunicamycin to assess the stability of the receptors for prolactin, insulin and epidermal growth factor (EGF) in rat liver cell membrane. Direct binding studies on liver plasmalemma fractions which were isolated from tunicamycin-treated rats revealed a rapid loss of prolactin receptors (t1/2 approximately 35 min) with a more prolonged half-life for insulin (10 h) and EGF receptors (8 h). The rates of receptor loss were similar to the respective half-lives of the receptors as documented by others using cultured cells. The respective ligands for each receptor were lost more rapidly from liver, i.e. prolactin, t1/2 approximately 10 min, insulin, t1/2 approximately 5 min and EGF, t1/2 approximately 17 min. Previous studies have shown ligand loss in vivo to be receptor mediated. Thus, receptors and their ligands do not turn over synchronously in vivo. These studies also point to a major role for N-linked oligosaccharide side chains in the functional insertion of prolactin, insulin and EGF receptors into the hepatocyte cell surface in vivo.

Animals↗