Rat Placental Membrane Research Articles

Mechanism of Nucleoside Uptake in Rat Placenta and Induction of Placental CNT2 in Experimental Diabetes

The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [(3)H]adenosine and [(3)H]zidovudine from maternal blood was much higher than that of [(14)C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [(3)H]adenosine with K(m) values of 6.1 and 26 µM, respectively. [(3)H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [(3)H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 µM NBMPR on [(3)H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)-induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZ-induced diabetic rats. Consistently, Na(+)-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus.

Contribution of Residues A54 and L55 of the Human Insulin-like Growth Factor-II (IGF-II) A Domain to Type 2 IGF Receptor Binding Specificity

The underlying specificity of the interaction between insulin-like growth factor-II (IGF-II) and mammalian Type 2 insulin-like growth factor/cation-independent mannose 6 phosphate receptor (IGF2R) is not understood. We have mutated residues A54 and L55 of IGF-II in the second A domain helix to arginine (found in the corresponding positions of IGF-I) and measured IGF2R binding. There is a 4- and 3.3-fold difference in dissociation constants for A54R IGF-II and L55R IGF-II, respectively, and a 6.6-fold difference for A54R L55R IGF-II compared with IGF-II as measured by BIAcore analysis using purified rat IGF2R. This is also confirmed using cross-linking and soluble rat placental membrane receptor binding assays. Binding to the type 1 IGF receptor (IGF1R) and IGF binding protein-2 (IGFBP-2) is not altered. We can, therefore, conclude that residues at positions 54 and 55 in IGF-II are important for and equally contribute to IGF2R binding.

Neutral amino acid transport in placental plasma membrane vesicles in the late pregnant rat: Evidence for a B 0-like transport system

Rat placental plasma membrane vesicles have been used to study both alanine and leucine transport at late gestation. The results presented are consistent with the presence of more than two separate transport systems for neutral amino acids in the rat placenta. One system is clearly Na +-independent and transports alanine ( K M ≅ 2 mM; V max ≅ 360 pmol ala/mg prot × 5 s), leucine ( K M ≅ 0.07 mM; V max ≅ 100 pmol leu/mg prot × 5 s), serine, cysteine and 2-amino-2 norbornane carboxylic acid (BCH) showing similar properties to the L system, present in many cell types. The other systems are Na +-dependent and transport alanine (a, K M ≅ 5 mM; V max ≅ 3761 pmol ala/mg prot × 5 s; b, K M ≅ 0.07 mM; V max ≅ 376 pmol ala/mg prot × 5 s), cysteine, serine and leucine ( K M ≅ 0.2 mM; V max ≅ 112 pmol leu/mg prot × 5 s) with different kinetic behaviour referring affinity and capacity. While one of them is sensitive to inhibition by methylaminoisobutyric acid (MeAIB), the other is a B 0-like system similar to that characterized in bovine brush-border enterocyte membrane vesicles.

Analysis of rat placental plasma membrane proteins by two-dimensional gel electrophoresis

The placenta plays an essential role in fetal growth and the maintenance of pregnancy and its functions are strictly controlled in a stage-specific manner. To gain a insight into placental functions and their regulation, we analyzed the plasma membrane proteins of rat placenta by two-dimensional polyacrylamide gel electrophoresis ( 2D E ). Plasma membrane fractions of the placenta obtained on days 12, 14, 16, 18 and 20 of pregnancy were purified by Percoll gradient centrifugation, and subjected to 2D E analysis. After the proteins on the 2D E gels had been visualized by silver staining, the patterns on the gels at different stages of pregnancy were compared using image analysis software. Proteins within an isoelectric point (p I) range of 4.0 to 7.0 and a molecular weight ( M w) range of 20–100 kDa were analyzed in detail, and about 800 proteins on average were recognized on each gel. Of these, the expression of 150 proteins was found to change dramatically according to the stage of pregnancy. According to their expression patterns, these proteins were categorized into two groups, Group I and Group II. The proteins belonging to Group I showed a higher intensity of expression on day 12 and disappeared on day 20. They included 119 plasma membrane proteins and were divided into five subgroups. Group II, which consisted of three subgroups, included 31 proteins showing a low or negligible expression on day 12 and higher expression on day 20. Most of the other membrane proteins (about 600) were expressed constantly during pregnancy. On the basis of our data, we constructed a database for plasma membrane proteins of the rat placenta.

Effect of chronic cocaine administration on amino acid uptake in rat placental membrane vesicles

This study evaluated the effects of chronic exposure to cocaine during pregnancy on amino acid uptake in placental membrane vesicles. Pregnant rats received 62 mg/kg of cocaine hydrochloride by intraperitoneal (IP) injection as a divided daily dose on gestation days 8–19 inclusive. Fetal body weights were significantly decreased by 19% in the cocaine group, while placental weights were unchanged. Placental apical membrane vesicles were prepared from control and cocaine-treated animals, and marker enzyme enrichments for alkaline phosphatase and [ 3H]-dihydroalprenolol binding did not differ between cocaine and control groups. Rates of uptake (10 sec) of selected radiolabeled amino acids were measured utilizing a rapid filtration technique. Na +-dependent apical membrane [ 3H]-glutamine transport (50 μM) was reduced by 95% (p < 0.05) in cocaine-treated compared to control placentas. Uptake of 50 μM [ 3H]-methyl aminoisobutyric acid (MeAIB) into apical membranes was also decreased by 43% (p < 0.05) in cocaine membranes. Na +-independent [ 3H]-arginine transport (10 μM), however, did not differ between control or cocaine-treated groups. In summary, chronic cocaine administration selectively inhibited the transport of glutamine and MeAIB into apical membrane vesicles, but had minimal effect on arginine transport. We postulate that this diminution in uptake may contribute to the fetal growth retardation noted in our model.

Insulin‐Like Growth Factor‐II is a Substrate for Dipeptidylpeptidase I (Cathepsin C)

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP‐I) caused the release of trichloroacetic‐acid‐soluble radioactivity from rat 125I‐insulin‐like growth factor‐II (IGF‐II). This activity could be blocked by dipeptide inhibitors of DAP‐I, and was enhanced by chloride. Treatment of unlabeled rat IGF‐II with DAP‐I converted approximately 50% of the IGF‐II to a species with a slightly shorter elution time on reverse‐phase HPLC, whereas treatment of human IGF‐II caused complete conversion to the species with the shorter elution time. Rat IGF‐II purified from the rat BRL 3A cell line is a mixture of two molecules beginning with Ala‐Tyr‐Arg‐Pro‐Ser‐ and Tyr‐Arg‐Pro‐Ser‐ [Marquardt, H., Todaro, G. J., Henderson, L. E. &amp; Oroszlan, S. (1981) J. Biol. Chem. 256, 6859–6865] while human IGF‐II begins with Ala‐Tyr‐Arg‐Pro‐Ser‐. Determination of the N‐terminal amino acid sequence of human IGF‐II before and after digestion with DAP‐I showed that DAP‐I cleaved Ala‐Tyr, terminating at Arg‐Pro‐; the rat IGF‐II species beginning with Tyr‐Arg‐Pro‐Ser‐ was resistant to digestion. In order to compare DAP‐I‐treated IGF‐II with native IGF‐II for binding to IGF receptors and IGF‐binding proteins and in a bioassay, rat and human IGF‐II were treated with DAP‐I and the digested and undigested species were isolated by reverse‐phase HPLC. The IGF‐II/mannose 6‐phosphate receptor was purified from rat placental membranes, the IGF‐I receptor was solubilized from human placental membranes and IGF‐binding proteins were partially purified from adult and three‐day‐old rat sera by sequential gel filtration on Sephadex G‐200 (pH 8.0) and Sephadex G‐50 (acid pH). The dose/response curves of the two IGF‐II species were indistinguishable in radioreceptor assays utilizing the IGF‐II/mannose 6‐phosphate receptor and the IGF‐I receptor and in IGF competitive binding assays utilizing partially purified IGF‐binding proteins. The DAP‐I‐digested and native IGF‐II species were also equipotent in stimulating [3H]thymidine incorporation into DNA in the human osteosarcoma cell line, MG‐63. We conclude that DAP‐I cleaves an N‐terminal dipeptide from IGF‐II and that this does not result in a change in the biological activity of the molecule.

Insulin-like growth factor-II is a substrate for dipeptidylpeptidase I (cathepsin C). Biological properties of the product.

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP-I) caused the release of trichloroacetic-acid-soluble radioactivity from rat 125I-insulin-like growth factor-II (IGF-II). This activity could be blocked by dipeptide inhibitors of DAP-I, and was enhanced by chloride. Treatment of unlabeled rat IGF-II with DAP-I converted approximately 50% of the IGF-II to a species with a slightly shorter elution time on reverse-phase HPLC, whereas treatment of human IGF-II caused complete conversion to the species with the shorter elution time. Rat IGF-II purified from the rat BRL 3A cell line is a mixture of two molecules beginning with Ala-Tyr-Arg-Pro-Ser- and Tyr-Arg-Pro-Ser- [Marquardt, H., Todaro, G. J., Henderson, L. E. & Oroszlan, S. (1981) J. Biol. Chem. 256, 6859-6865] while human IGF-II begins with Ala-Tyr-Arg-Pro-Ser-. Determination of the N-terminal amino acid sequence of human IGF-II before and after digestion with DAP-I showed that DAP-I cleaved Ala-Tyr, terminating at Arg-Pro-; the rat IGF-II species beginning with Tyr-Arg-Pro-Ser- was resistant to digestion. In order to compare DAP-I-treated IGF-II with native IGF-II for binding to IGF receptors and IGF-binding proteins and in a bioassay, rat and human IGF-II were treated with DAP-I and the digested and undigested species were isolated by reverse-phase HPLC. The IGF-II/mannose 6-phosphate receptor was purified from rat placental membranes, the IGF-I receptor was solubilized from human placental membranes and IGF-binding proteins were partially purified from adult and three-day-old rat sera by sequential gel filtration on Sephadex G-200 (pH 8.0) and Sephadex G-50 (acid pH). The dose/response curves of the two IGF-II species were indistinguishable in radioreceptor assays utilizing the IGF-II/mannose 6-phosphate receptor and the IGF-I receptor and in IGF competitive binding assays utilizing partially purified IGF-binding proteins. The DAP-I-digested and native IGF-II species were also equipotent in stimulating [3H]thymidine incorporation into DNA in the human osteosarcoma cell line, MG-63. We conclude that DAP-I cleaves an N-terminal dipeptide from IGF-II and that this does not result in a change in the biological activity of the molecule.

Membrane-bound and water-soluble nonclassical class I MHC antigens on rat placenta

We have used mouse monoclonal antibodies to different determinants on rat class I major histocompatibility complex (MHC) antigens in order to identify water-soluble and membrane-bound nonclassical (i. e., non-RT1.A) class I MHC antigens on the spongiotrophoblast and labyrinthine trophoblast of rat placenta. Initial immunohistological studies with monoclonal antibodies reacting with a determinant restricted to classical (RT1.A) rat class I antigens confirmed the presence of these antigens on spongiothrophoblast, but not on labyrinthine trophoblast. Staining with another monoclonal antibody, which reacts with both classical and at least some nonclassical rat class I antigens, gave strong staining of both the labyrinthine and spongiotrophoblast. To distinguish membrane-bound from water-soluble class I molecules, quantitative absorption analyses were carried out using both placental cell membranes and ultracentrifuged aqueous extracts of placenta. The aqueous placental extract had no absorptive capacity for the RT1.A-specific antibodies, but it had very strong absorptive capacity for the more broadly reactive antibody. This strongly suggests the presence of large quantities of a soluble nonclassical class I MHC antigen in rat placenta. The placental cell membranes had four to fivefold greater absorptive capacity for the broadly reactive antibody when compared to the antibodies to classical class I antigens, a result that was consistent with the presence of membrane-bound nonclassical class I antigens on rat placenta. The membrane-bound nonclassical class I antigen was purified from detergent extracts of DA rat placental membranes using monoclonal antibody affinity and lentil lectin affinity chromatography. The putative nonclassical class I antigen had a heavy chain of Mr 43,000, which is 2000 smaller than the classical (RT1.A) class I antigen. N-terminal amino acid sequence analysis demonstrated that the nonclassical placental antigen differed at three amino acid residues from the classical RT1.A class I molecule and also from the Q10-like class I molecule of the DA strain. It differed also from the pAR 1.5 cDNA sequence, the only full-length rat class I DNA sequence available so far.

Serum Form of the Rat Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor Is Truncated in the Carboxyl-terminal Domain

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.

Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor.

Cloning and sequencing of the human type II insulin-like growth factor (IGF) receptor cDNA revealed an 80% deduced amino acid sequence homology with the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor, suggesting identity of the two receptors (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). We have performed biochemical experiments that support this proposal. Rat liver type II IGF receptor, purified by the conventional method of IGF-II affinity chromatography, bound quantitatively to a beta-galactosidase affinity column and was eluted with Man-6-P. Bovine liver Man-6-P receptor, prepared by the conventional method of affinity chromatography on phosphomannan-Sepharose, bound IGF-II with high affinity (Kd = 1 nM). Affinity cross-linking of 125I-IGF-II to the Man-6-P receptor and analysis by sodium dodecyl sulfate-gel electrophoresis showed that beta-galactosidase, but not Man-6-P, inhibited the formation of the 250-kDa 125I-IGF-II-receptor complex. The inhibition by beta-galactosidase was prevented by coincubation with Man-6-P. 125I-IGF-II did not bind to the 46-kDa cation-dependent Man-6-P receptor. For immunologic studies we purified type II IGF receptors and Man-6-P receptors in parallel from rat placental membranes using either IGF-II- or beta-galactosidase affinity chromatography. A panel of five antisera that previously had been raised against either type II IGF receptor or Man-6-P receptor behaved identically toward type II IGF receptor versus Man-6-P receptor in ligand blocking and immunoprecipitation assays. Our data support the conclusion that the type II IGF receptor and the cation-independent Man-6-P receptor are the same protein and that the IGF-II and Man-6-P-binding sites are distinct.

Type II insulin-like growth factor receptor is present in rat serum.

We previously identified in fetal rat serum a component capable of specifically binding radiolabeled insulin-like growth factor type II (IGF-II) that is considerably larger than both the fetal (40 kDa) and the adult (150 kDa) carrier proteins. We now present immunologic and affinity crosslinking data to show that this binding species is the type II IGF receptor. Rat serum was gel-filtered on a Sephadex G-200 column (0.05 M NH4HCO3, pH 8), and 125I-labeled IGF-II (125I-IGF-II) binding was measured in individual column fractions. 125I-IGF-II binding activity was found in the void volume of the column in addition to the carrier protein regions. Competitive binding studies using 125I-IGF-II and binding activity from the Sephadex G-200 void volume showed the characteristics of the type II receptor: IGF-II was more potent than IGF-I, and insulin did not compete. Moreover, a specific anti-type II IGF receptor antibody (no. 3637) completely blocked 125I-IGF-II binding. 125I-IGF-I did not bind to the void volume pool, demonstrating the absence of the type I IGF receptor in rat serum. Affinity crosslinking of 125I-IGF-II to the Sephadex G-200 void volume material demonstrated a specific band at 210 kDa without reduction and at 240 kDa after reduction of disulfide bonds. The serum type II IGF receptor size was confirmed by immunoblotting the void volume material with antiserum 3637, which revealed a band slightly smaller (approximately 10 kDa) than the type II IGF receptor from rat placental membranes. Immunoquantitation by immunoblotting using pure type II IGF receptor from rat placental membranes as standard showed a developmental pattern. In fetal rat serum (19-days gestation) and in sera from 3-, 10-, and 20-day-old rats, the concentrations of receptor protein were similar (1-5 micrograms/ml). The level of the type II IGF receptor in serum declined dramatically between age 20 and 40 days, but the receptor was still measurable at age 12 mo. We conclude that the type II IGF receptor is present in rat serum and is developmentally regulated.

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 IC 50 of 65 n M, while IGF-I was much less potent with an IC 50 of 2.12 m M. 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 K d 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 M r 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 α-and β-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.

Purification and Characterization of a Unique High Molecular Weight Form of Insulin-Like Growth Factor II

A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: Comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18–54, SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10–20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10–25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, there data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.

Serum somatomedin levels in adults with chronic renal failure: the importance of measuring insulin-like growth factor I (IGF-I) and IGF-II in acid-chromatographed uremic serum.

Somatomedin levels measured by radioreceptor assay (RRA) or RIA on acid-ethanol-extracted or unextracted serum from patients with uremia are quite variable relative to normal values. We have investigated whether compounds accumulating in uremic serum were interfering with these RRA and RIA measurements. Insulin-like growth factor I (IGF-I) and IGF-II were separated from carrier proteins by either Sephadex G-50 acid chromatography or acid-ethanol extraction. IGF-I was measured by RIA, and IGF-II was determined by rat placental membrane RRA. IGF-I levels in the acid-ethanol extracts of serum from eight uremic adults were only 50% of the levels found in seven normal subjects, and IGF-II levels in these uremic patients were 350% of normal values. However, these significant differences were not found when comparable serum samples were acid chromatographed rather than acid-ethanol extracted; instead, IGF-I levels were 343 +/- 168 (mean +/- SD) ng/ml in uremic patients and 325 +/- 54 ng/ml in normal subjects, while IGF-II levels were 780 +/- 215 ng/ml in uremic patients and 588 +/- 46 ng/ml in normal subjects. To determine whether compounds that interfere with somatomedin assays were present in the acid-ethanol extracts of uremic serum, we acid chromatographed these extracts and found that a compound eluting in the carrier protein fraction of the uremic extract artifactually decreased IGF-I levels measured by RIA and increased IGF-II levels measured by RRA. We also found that unsaturated carrier protein binding of IGF-I and IGF-II was significantly increased in the carrier protein fraction of uremic acid-ethanol extracts at the dilutions used for somatomedin assays. We conclude that acid chromatography of unextracted or acid-ethanol-extracted uremic serum is necessary to remove a compound, probably unsaturated carrier protein, which interferes with RIA of IGF-I and RRA of IGF-II. Once this compound is removed, serum IGF-I levels (RIA) in uremic patients are not different from normal, and serum IGF-II levels (RRA) are not lower than normal.

An Autocrine/Paracrine Role forInsulin-Like Growth Factors inthe Regulation ofHuman Placental Growth*

Tissue derived from preterm (9-19 weeks gestation) and term (38-41 weeks gestation) human placentae were examined for their ability to synthesize and secrete insulin-like growth factors (IGFs) in organ culture. IGF-I was measured by a specific RIA, and IGF-II by a rat placental membrane radioreceptor assay. First, explants of placental tissue were maintained in organ culture. These explants secreted immunoreactive IGF-I (IR-IGF-I). There were no differences in the IR-IGF-I content of media conditioned by term and preterm placentae under these conditions. The similarity of this material to authentic human IGF-I was supported by parallel displacement in a specific RIA and coelution during Sephadex G-50 gel filtration. Second, monolayer cultures of fibroblasts from normal human preterm placentae (15-19 weeks gestation) were established. Confluent monolayers of these fibroblasts secreted IR-IGF-I (3-10 pg/10(5) cells X 40 h). IR-IGF-I secretion was reversibly inhibited by 5.3 microM cycloheximide, suggesting that the IR-IGF-I was the result of de novo protein synthesis. IR-IGF-I secretion was stimulated 5-fold by platelet-derived growth factor (0.6 U/ml). The response of monolayers of placental fibroblasts to IGF-I also was tested. IGF-I stimulated alpha-[3H]aminoisobutyric acid transport in these fibroblasts, with half-maximal stimulation occurring at 2-3 ng/ml. Stimulation of alpha-[3H]aminoisobutyric acid uptake by IGF-I correlated with specific binding of [125I]iodo-IGF-I. Half-maximal inhibition of [125I]iodo-IGF-I binding occurred at 2-3 ng/ml IGF-I. Placental tissue also secreted IGF-II-like activity, as measured by radioreceptor assay. Media conditioned by placental explants contained 15-20 ng/mg protein X 48 h, and media conditioned by placental fibroblasts contained 3-7 ng/10(5) cells X 40 h IGF-II determined by radioreceptor assay. These data support the hypothesis that the human placenta produces IGFs (IGF-II and/or IGF-I) that act locally to regulate placental growth.

Ethanol and Fetal Nutrition Effect of Chronic Ethanol exposure on rat Placental Growth and Membrane Associated folic Acid Receptor Binding Activity

Rat placental composition and specific folate receptor activity were measured at 20 days gestation in dams exposed to chronic high doses of ethanol (6%, vol/vol) throughout gestation and in isocalorically pair-fed controls. Ethanol-exposed fetuses were smaller (ethanol = 3.28 +/- 0.08 vs. control = 4.01 +/- 0.10 g, p less than 0.001), but their placentae were larger (experimental = 0.534 +/- 0.02 vs. control = 0.399 +/- 0.01 g, p less than 0.001). The increased weight appears to be secondary to hyperplasia as total DNA was increased while the wet/dry, RNA/DNA, and protein/DNA ratios were not different. Despite larger placentae, specific folate receptor activity was significantly reduced in the ethanol-exposed tissue, whether expressed relative to membrane protein, placental weight, or total placental binding. These results confirm that ethanol exposure is placentotoxic and suggest an additional mechanism by which ethanol may lead to intrauterine growth retardation; namely, decreased folate receptor activity.

Ethanol and Fetal Nutrition Effect of Chronic Ethanol exposure on rat Placental Growth and Membrane Associated folic Acid Receptor Binding Activity

SummaryRat placental composition and specific folate receptor activity were measured at 20 days gestation in dams exposed to chronic high doses of ethanol (6%, vol/vol) throughout gestation and in isocalorically pair fed controls. Ethanol exposed fetuses were smaller (ethanol = 3.28 ± 0.08 vs. control = 4.01 ± 0.10 g, p &lt; 0.001), but their placentae were larger (experimental = 0.534 ± 0.02 vs. control = 0.399 ± 0.01 g, p &lt; 0.001). The increased weight appears to be secondary to hyperplasia as total DNA was increased while the wet/dry, RNA/DNA, and protein/ DNA ratios were not different. Despite larger placentae, specific folate receptor activity was significantly reduced in the ethanol exposed tissue, whether expressed relative to membrane protein, placental weight, or total placental binding. These results confirm that ethanol exposure is placen totoxic and suggest an additional mechanism by which ethanol may lead to intrauterine growth retardation; namely, decreased folate receptor activity.

Insulin-like growth factors I and II of nonmammalian sera

We have compared the insulin-like growth factors (IGFs) of a number of mammalian and nonmammalian vertebrate sera. Sera were subjected to acid gel filtration through G-75 Sephadex and separate binding protein and free IGF fractions were pooled. The IGF pools were subjected to flatbed isoelectric focusing and the concentration of IGF I in the fractions was detected with an immunoassay using 125I-hIGF I and the somatomedin C antibody of Underwood and Van Wyk. IGF II in the fractions was detected with either the 125I-IGF rat placental membrane RRA or the 125I-IGF II serum binding protein assay. One or more basic peaks of IGF I immunoactivity were detected in all the mammalian sera studied (human, guinea pig, rat, bovine, kangaroo, and opossum) and all the nonmammalian species studied (chicken, turtle, toad, and trout). Neutral and slightly acidic peaks of IGGF II receptor reactivity were observed in human, cow, and guinea pig serum. No distinct peaks of IGF II were observed in adult rat, opossum, or kangaroo serum. Distinct peaks oof IGF II activity in the neutral to slightly acidic range were clearly recognized in chicken and turtle serum, no such peaks were recognized in toad and trout serum. All mammalian sera studied contained acid stable, binding proteins with high affinity, saturable binding of 125I-IGF II. None of the nonmammalian sera studied contained demonstrable specific 125I-IGF II binding. These observations document the presence of separate IGF I and IGF II peptides in chickens and turtles but only IGF I in amphibians and fish. These observations indicate that the gene duplication giving rise to two separate IGFs arose before reptilian evolution. The acquisition of a specific IGF binding protein is limited to mammals.

Insulin-like growth factor receptors in rat placental membranes.

The nature and relative quantity of insulin-like growth factor (IGF) receptors in rat placental microsomal membranes was investigated by competitive binding studies and covalent cross-linking followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding studies revealed that 100 micrograms membrane protein specifically bound 26.5% of [125I]iodo-IGF-II, 19.6% of [125I]iodomultiplication-stimulating activity III-2 [( 125I]iodo-MSA-III-2), and 11.5% [125I]iodo-IGF-I. IGF-II was equipotent with MSA-III-2, and both were approximately twice as potent as IGF-I in competing with [125I]iodo-IGF-I for binding. In contrast, IGF-I competed for the binding of [125I]iodo-MSA and [125I]iodo-IGF-II with only 5-20% the potency of unlabeled MSA and IGF-II. Insulin competed weakly with [125I] iodo-IGF-I for binding (achieving half-maximal displacement at 20 micrograms/ml), but did not compete with [125I]iodo-MSA for binding. This evidence suggested that while IGF-I binds to both IGF-I and IGF-II receptors, the majority of IGF-I binding is due to an interaction with IGF-II receptors. Studies using [125I]iodo-IGF-I covalently cross-linked to placental membrane receptors followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that molecular species characteristic of the subunits of IGF-I receptors are present in rat placenta. It is concluded that rat placenta, like human placenta, contains receptors for both types of IGFs. Unlike human placenta, the majority of the receptors are of the IGF-II type.