A single receptor binds both insulin-like growth factor II and mannose-6-phosphate.

Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins.

Heterotrimeric G proteins and the single-transmembrane domain IGF-II/M6P receptor: functional interaction and relevance to cell signaling.

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor's structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.

Insulin-Like Growth Factor-II/Cation-Independent Mannose 6-Phosphate Receptor in Neurodegenerative Diseases.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional single transmembrane glycoprotein. Recent studies have advanced our understanding of the structure, ligand-binding properties, and trafficking of the IGF-II/M6P receptor. This receptor has been implicated in a variety of important cellular processes including growth and development, clearance of IGF-II, proteolytic activation of enzymes, and growth factor precursors, in addition to its well-known role in the delivery of lysosomal enzymes. The IGF-II/M6P receptor, distributed widely in the central nervous system, has additional roles in mediating neurotransmitter release and memory enhancement/consolidation, possibly through activating IGF-II-related intracellular signaling pathways. Recent studies suggest that overexpression of the IGF-II/M6P receptor may have an important role in regulating the levels of transcripts and proteins involved in the development of Alzheimer's disease (AD)-the prevalent cause of dementia affecting the elderly population in our society. It is reported that IGF-II/M6P receptor overexpression can increase the levels/processing of amyloid precursor protein leading to the generation of ß-amyloid peptide, which is associated with degeneration of neurons and subsequent development of AD pathology. Given the significance of the receptor in mediating the transport and functioning of the lysosomal enzymes, it is being considered for therapeutic delivery of enzymes to the lysosomes to treat lysosomal storage disorders. Notwithstanding these results, additional studies are required to validate and fully characterize the function of the IGF-II/M6P receptor in the normal brain and its involvement in various neurodegenerative disorders including AD. It is also critical to understand the interaction between the IGF-II/M6P receptor and lysosomal enzymes in neurodegenerative processes, which may shed some light on developing approaches to detect and prevent neurodegeneration through the dysfunction of the receptor and the endosomal-lysosomal system.

Altered ligand binding by insulin-like growth factor II/mannose 6-phosphate receptors bearing missense mutations in human cancers.

The M6P/IGF2R gene, encoding the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor (IGF2R), is frequently inactivated during carcinogenesis. M6P/IGF2R is postulated to be a tumor suppressor gene due to its ability to bind and degrade the mitogen IGF-II, promote activation of the growth inhibitor transforming growth factor beta, and regulate the targeting of lysosomal enzymes. In this study, we determined the effects of four M6P/IGF2R missense mutations associated with loss of heterozygosity in hepatocellular and breast cancers on the ligand binding properties of full-length membrane-bound receptors. Site-directed mutagenesis was used to prepare COOH-terminal, c-myc epitope-tagged human IGF2R cDNA expression constructs bearing point mutations that lead to the substitutions I1572T, G1464E, G1449V, and Q1445H, all of which are located in the receptor's extracytoplasmic domain. Ligand binding was measured in plasma membranes from 293T cells expressing full-length receptors. No binding of 125I-IGF-II to I1572T mutant receptors was observed. Binding to G1449V mutant receptors was decreased by 50% relative to wild-type (WT). However, IGF-II binding to the G1464E and Q1445H mutant receptors was equivalent to WT when plasma membranes were assayed immediately after preparation. The phosphomannosylated pseudoglycoprotein pentamannose 6-phosphate-BSA (PMP-BSA) was synthesized as a ligand for the M6P binding site. Binding of 125I-PMP-BSA was equivalent to WT for the I1572T, G1464E, and Q1445H mutations, but there was a 60% reduction in PMP-BSA binding to the G1449V mutant receptor. Thus, several missense mutations in M6P/IGF2R disrupt the ligand binding functions of the intact IGF2R, lending further support to the hypothesis that the M6P/IGF2R is a tumor suppressor gene.

Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro.

A quantitative assay employing binding of [3H] diisopropylfluorophosphate ([3H]DFP) and SDS-polyacrylamide gel electrophoresis was used to measure serine hydrolases in cell-free extracts from rat splenic lymphocytes. After labeling with [3H]DFP at pH7, six major serine hydrolases are detected on 10% gels, having molecular weights of 78, 55, 34, 30, 28, and 17 (.10(-3)). When labeled at pH4, only four activities are measured, with Mr of 79, 55, 33 and 17(.10(-3)). Incubation of splenic lymphocytes for 8 h in vitro with 1 microM dexamethasone followed by [3H]-DFP labeling at pH 7 produces a 91% increase in the 17000 [3H]DFP. Hormone treatment for 8 h with subsequent labeling at pH 4 results in a 15% increase in the largest (78000) species, as well as 73% increase in the 17000 enzyme, compared with lysates from cells incubated without steroid. These effects are not observed after only 4 h of glucocorticoid exposure. Dexamethasone treatment for 8 h does not produce a decrease in any of these serine hydrolases, nor is there an apparent induction of new enzymes (i.e., having a molecular weight different from the preexisting species). Studies examining the effect of protease inhibitors on the [3H]DFP capacity of these proteins, show that the 17000 enzyme is sensitive to the protease inhibitor, pepstatin A, as well as the sulfhydryl reagents dithiothreitol and N-ethylmaleimide. These results suggest that this dexamethasone-responsive enzyme is a protease which requires a free thiol group for optimal activity. These findings are discussed with regard to the mechanism of glucocorticoid action in lymphocytes.

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools.

In the presence of tracer concentrations of extracellular leucine (5 microM), treatment of rat splenic lymphocyte suspensions in vitro with 1 microM dexamethasone for 2.5-4 h caused a 30-35% inhibition of [3H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5mM, this inhibition was progressively reduced to 0-12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [3H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibiton of [3H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [3H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [3H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [3H]leucine by the cells regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 microM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino aicd incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.

Conditions for the measurement of nuclear estrogen receptor at low temperature.

This study was undertaken to determine optimum conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycerol, 0.5 M KCl, 10 mM pyridoxal 5'-phosphate, and 0.5 M NaSCN on the dissociation of estradiol from the receptor at 0 degree C. The half-time (t 1/2) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate (t 1/2 = 35 h). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital buffer) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the t 1/2 decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The t 1/2 of estradiol dissociation from the receptor at 0 degree C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptor by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30 degree C for 1 h and 0 degree C for 24 h. We verified that unoccupied receptor was measured reliably in KCl extract during incubation at 0 degrees C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in the same extract.

Biochemical studies of RT6 alloantigens in BB/Wor and normal rats. Evidence for intact unexpressed RT6a structural gene in diabetes-prone BB rats.

Lymphocytes bearing the T-lymphocyte differentiation antigen RT6 play an important immunoregulatory role in the development of autoimmune diabetes in BB rats. Immunofluorescence studies suggest that diabetes-prone (DP)- but not diabetes-resistant (DR)-BB rat lymphocytes fail to express RT6 antigen during ontogeny. Two alloantigenic forms of the molecule exist, i.e., RT6.1 and RT6.2; both are linked to cell membranes by a phosphatidylinositol (PI) linkage. In these studies, PI-phospholipase C (PLC) treatment of lymphocytes from BB and normal rats followed by immunoabsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of released proteins with anti-RT6 allotype-specific monoclonal antibodies was performed. RT6.1 in several nondiabetic rat strains was found to consist of a family of nonglycosylated and variably glycosylated molecules: an N-Glycanase-resistant 24,000- to 26,000-Mr peptide and four N-Glycanase-sensitive peptides of 29,000, 31,000, 33,000, and 34,000 Mr. In contrast, RT6.2 was found to be a 24,000- to 26,000-Mr nonglycosylated polypeptide. The electrophoretic pattern of RT6.1 was observed to be the same when the antigen was extracted from W3/25+ (CD4+) versus W3/25- T lymphocytes or from resting versus mitogen-activated cells. A pattern of bands characteristic of the RT6.1 antigen found in normal rat strains was detected after PLC treatment or detergent solubilization of lymphocytes obtained from DR rats. In contrast, no evidence of either RT6 species was found after PLC or detergent treatment of comparable numbers of T lymphocytes from DP-BB rats. Interestingly, T lymphocytes from Wistar-Furth (RT6.2+) x DP (RT6-) F1 crosses were observed to coexpress both RT6.2 and RT6.1 molecules, with the electrophoretic pattern of RT6.1 being similar to that obtained in DR and other rat strains. This study provides biochemical evidence that DP rats may have an intact RT6a structural gene.

Restenosis after coronary angioplasty: a multivariate statistical model to relate lesion and procedure variables to restenosis. The M-HEART Investigators.

The Multi-Hospital Eastern Atlantic Restenosis Trial group obtained follow-up angiography in 510 patients with 598 successfully dilated coronary lesions who were enrolled in a controlled trial of the effects of a single dose of 1 g of methylprednisolone on restenosis after coronary angioplasty. The overall restenosis rate was 39.6%. The strongest univariate relations to the restenosis rate were found for lesion location (saphenous vein graft, 68%; left anterior descending artery, 45%; left circumflex artery and right coronary artery, 32%; p = 0.002); lesion length (less than or equal to 4.6 mm, 33%; greater than 4.6 mm, 45%; p = 0.001); percent stenosis before angioplasty (less than or equal to 73%, 25%; greater than 73%, 43%; p = 0.005), percent stenosis after angioplasty (less than or equal to 21%, 33%; greater than 21%, 46%; p = 0.017) and arterial diameter (less than 2.9 mm, 44%; greater than or equal to 2.9 mm, 34%; p = 0.036). Two multivariate models to predict restenosis probability were developed with use of stepwise logistic regression. The preprocedural model, which included only variables whose values were known before angioplasty, entered lesion length, vein graft location, left anterior descending artery location, percent stenosis before angioplasty, eccentric lesion and arterial diameter. The postprocedural model, which also included variables whose values were known after angioplasty was performed, was similar to the preangioplasty model except that it also entered postangioplasty percent stenosis and "optimal" balloon sizing but did not enter eccentric lesion. These data indicate that the probability of restenosis after angioplasty is determined predominantly by the characteristics of the lesion being dilated. They are consistent with the known intimal proliferative mechanism of restenosis, offer a means of identifying lesions at unusually high or low risk of restenosis, and of predicting the likelihood that a particular lesion will restenose after angioplasty and provide a rationale for stratification by restenosis probability in the design of future studies of restenosis.

Clinical and angiographic determinants of primary coronary angioplasty success. M-HEART Investigators.

Clinical and anatomic determinants of the initial success of percutaneous transluminal coronary angioplasty were prospectively evaluated in 826 patients enrolled in the Multi-Hospital Eastern Atlantic Restenosis Trial (M-HEART). The 639 men and 187 women ranged in age from 31 to 85 years. Successful angioplasty (residual stenosis less than 50% and no major complications) was achieved in 886 (88.6%) of 1,000 lesions. Success rates were uniform among the eight individual centers. Outcome was not influenced by gender, age or other clinical features, including severity and duration of angina, prior myocardial infarction, rest pain, transient ST segment elevation, history of smoking or diabetes. In contrast, procedural outcome was significantly associated with lesion-specific angiographic factors. Stenoses 60% to 74%, 75% to 89%, 90% to 99% and 100% were associated with success rates of 96%, 90%, 84% and 69%, respectively (p less than 0.001). Angioplasty was less successful in calcified than in noncalcified lesions (82% versus 90%, p less than 0.01), in thrombotic than in nonthrombotic lesions (82% versus 90%, p less than 0.05) and in lesions in the right coronary artery than in other vessels (84% versus 90%, p less than 0.01). Outcome was not related to other anatomic variables, including lesion location (proximal versus distal), vessel size, eccentricity, stenosis length or translesional gradient. By multivariate logistic regression, preangioplasty percent stenosis, right coronary artery location and lesion calcification were demonstrated to be significant independent predictors of angioplasty success. Alternative clinical and angiographic variables did not contribute to this regression model.(ABSTRACT TRUNCATED AT 250 WORDS)

An insulin-like growth factor II (IGF-II) affinity-enhancing domain localized within extracytoplasmic repeat 13 of the IGF-II/mannose 6-phosphate receptor.

Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high-affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has approximately 50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.

Truncated forms of the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor encompassing the IGF-II binding site: characterization of a point mutation that abolishes IGF-II binding.

Complete understanding of the functional significance of insulin-like growth factor II (IGF-II) binding by the IGF-II/mannose-6-phosphate (Man-6-P) receptor requires mapping and ultimately mutational analysis of the receptor's IGF-II binding domain. Recent advances have localized the IGF-II binding site to extracytoplasmic repeats 10-11. To improve resolution of the binding site map, a nested set of epitope-tagged, truncated forms of the human IGF-II/Man-6-P receptor were transiently expressed in COS-7 cells. The IGF-II binding properties of truncated receptors immunoprecipitated from cell lysates and conditioned media were determined by affinity cross-linking. From the largest truncated receptor, encompassing extracytoplasmic repeats 8-11 (M(r) 68 K), through the smallest, comprised primarily of repeat 11 (M(r) 23 K), all were able to bind and cross-link to IGF-II. As a group, the truncated receptors had similar affinities for IGF-II, but with relative binding affinities 5-to 10-fold lower than those of full-length receptors. A point mutation substituting threonine for isoleucine at residue 1572, located in the NH2-terminal half of repeat 11, completely abolished IGF-II binding. We conclude that repeat 11 of the IGF-II/Man-6-P receptor's extracytoplasmic domain contains the minimal elements required for binding and cross-linking to IGF-II, and that lle1572 and other residues within the NH2-terminal half of repeat 11 are particularly important for IGF-II interaction.

Overexpression of the Insulin-Like Growth Factor II Receptor Increases ß-Amyloid Production and Affects Cell Viability.

Amyloid ß (Aß) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimer's disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from the trans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of ß-site APP-cleaving enzyme 1 levels and an increase of ß- and γ-secretase enzyme activities, leading to enhanced Aß production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by ß-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability.

Mannose-6-phosphate enhances cross-linking efficiency between insulin-like growth factor-II (IGF-II) and IGF-II/mannose-6-phosphate receptors in membranes.

Endogenous and exogenous phosphomannosyl ligands inhibit binding of insulin-like growth factor-II (IGF-II) to the IGF-II/mannose-6-phosphate receptor (IGF-II/Man-6-P receptor). In the present study, the mechanism of this antagonism was examined using a [125I]IGF-II cross-linking assay with disuccinimidyl suberate in cell membranes. Treatment with 5 mM Man-6-P enhanced [125I]IGF-II cross-linking to the receptor. The magnitude of the Man-6-P enhancement differed depending on the source of the membranes, ranging from a 30% increase in JEG-3 human choriocarcinoma up to a 560% increase in B16-F1 mouse melanoma. Man-6-P stimulated [125I]IGF-II-receptor cross-linking in H-35 hepatoma membranes by about 80%, even at concentrations of labeled IGF-II (greater than or equal to 10 nM) that nearly saturated the receptors. Thus, in addition to its effect on IGF-II-binding affinity, Man-6-P caused a 1.5- to 2-fold increase in cross-linking efficiency within the IGF-II-receptor complex. Furthermore, Man-6-P enhanced [125I]IGF-II cross-linking to the H-35 receptor by a constant (approximately 80%) increment 1) when the cross-linking reaction was conducted in buffers of different pH over the range 6.8-8.0, or 2) using cross-linking agents differing in spacer arm length from 6.4-16.1 A. Washing membranes before assay with either Man-6-P (pH 7.4) or 0.5 M NaCl (pH 4.5) reduced the subsequent Man-6-P enhancement of [125I]IGF-II-receptor cross-linking, suggesting that this phenomenon was actually due to displacement of inhibitory phosphomannosyl ligands bound endogenously to the Man-6-P sites of the receptor. In support of this hypothesis, Man-6-P produced a minimal (8-14%) enhancement of [125I]IGF-II-receptor cross-linking in membranes from I-cell fibroblasts lacking such phosphomannosyl ligands. Thus, phosphomannosyl ligands bound to the IGF-II/Man-6-P receptor decrease both IGF-II-binding affinity and IGF-II-receptor cross-linking efficiency. Membrane-associated receptors appear to exist in experimentally and perhaps functionally distinct populations, depending on occupancy of the Man-6-P-binding sites.

Characterization of type I and type II insulin-like growth factor receptors in an intestinal epithelial cell line.

Insulin-like growth factors (IGFs) and insulin stimulate DNA and protein synthesis in IEC-6 cells (an intestinal epithelial cell line) grown in a chemically defined medium. IGF-I stimulates proliferation of IEC-6 cells at a lower concentration (ED50 = 1.6 nM) than either insulin or IGF-II. To gain insight into the mechanisms by which IGFs stimulate IEC-6 cell growth, we have examined the characteristics of specific IGF receptors on IEC-6 cells. Binding of 125I-IGF-I and 125I-IGF-II to IEC-6 monolayers was analyzed by incubation with various concentrations (0.2 nM to 0.5 microM) of radiolabeled IGFs for 16 h at 3 C. Scatchard plots of 125I-IGF-I binding were linear, suggesting a single class of binding sites with KD = 3.1 +/- 0.35 nM and Bmax = 50.7 +/- 6 fmol/10(6) cells. IGF-II was potent in displacing 125I-IGF-I (KI = 8.1 +/- 0.85 nM), but insulin had little effect. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three bands with Mr of 270,000, 245,000, and 133,000, and the major band was the 133,000 species. Labeling of the 133,000 and 270,000 bands was greater than or equal to 80% inhibited by 10(-7) M unlabeled IGF-I, less potently inhibited by IGF-II and not at all by insulin. These results suggest that the 133,000 band represents the alpha-subunit of the type I IGF receptor. Scatchard plots of 125I-IGF-II binding to IEC-6 cell monolayers were curvilinear, suggesting two classes of binding sites: high affinity, low capacity sites, KD = 0.87 +/- 0.08 nM and Bmax = 28 +/- 2.5 fmol/10(6) cells; low affinity, high capacity sites, KD = 60 = +/- 8.8 nM and Bmax = 1780 +/- 230 fmol/10(6) cells. Neither IGF-I nor insulin was effective in inhibiting 125I-IGF-II binding. Affinity cross-linking with 125I-IGF-II labeled predominantly a 245,000 band, suggesting that this species is the type II receptor. A band with Mr 131,000 was barely detectable with 125I-insulin. These results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and few insulin receptors, suggesting that the mitogenic effect of IGFs is mediated through the type I IGF receptor.

Progesterone-induced estrogen receptor-regulatory factor in hamster uterine nuclei: preliminary characterization in a cell-free system.

In vitro studies have demonstrated a progesterone-induced activity associated with the uterine nuclear fraction which resulted in the loss of nuclear estrogen receptor. Uterine nuclear suspension or nuclear KCl (0.5 M) extract from control and progesterone-treated (30 min or 2h) hamsters were incubated at 37 C for 0, 15, or 30 min in Tris-glycerol buffer. Preparations from progesterone-treated hamsters showed an accelerated reduction of total estrogen receptor which was primarily due to preferential loss of occupied receptor. This progesterone-dependent stimulation of estrogen receptor loss was absent when nuclear extract was prepared in phosphate buffer rather than Tris buffer. In addition, sodium molybdate and sodium metavanadate (both at 10 mM) inhibited this activity in nuclear extract. These observations support the hypothesis that progesterone modulation of estrogen action may be accomplished by induction (or activation) of an estrogen receptor-regulatory factor (Re-RF), and this factor may in turn act to eliminate the occupied form of estrogen receptor from the nucleus, perhaps through a hypothetical dephosphorylation-inactivation mechanism.

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.

Rationale for use of glucocorticoids in modification of restenosis after percutaneous transluminal coronary angioplasty.

Recurrence of stenosis occurs in 15% to 45% of patients who have undergone successful percutaneous transluminal coronary angioplasty. Restenosis is believed to represent an accelerated form of atherosclerosis. It is recognized that the human coronary artery response to balloon dilatation reflects complex interactions among mediators of tissue injury and inflammation. Prominent among these are monocytes/macrophages, neutrophils, platelets, smooth muscle cells and endothelial cells. Certain changes suggest the potential for immunologically mediated inflammatory responses. It is therefore considered that restenosis, at least in part, reflects perturbations of cellular mediators of tissue injury leading to accelerated atherosclerosis, and that modulation of these processes by glucocorticoids might be therapeutically beneficial.

Angiographic morphology of restenosis after percutaneous transluminal coronary angioplasty.

Restenosis after successful percutaneous transluminal coronary angioplasty (PTCA) occurs frequently. To better define the restenosis process, a quantitative analysis was performed of coronary angiographic morphologic characteristics at restenosis, before and immediately after PTCA. In 22 patients cine frames showing stenosis at its most severe narrowing were traced and quantitatively analyzed. Immediately after PTCA, stenosis diameter (0.7 +/- 0.3 to 1.9 +/- 0.6 mm, mean +/- standard deviation, p less than 0.05) was increased; percent stenosis (77 +/- 11 to 34 +/- 16%, p less than 0.05), neck index (1.2 +/- 1.4 to 0.5 +/- 0.6, p less than 0.05) and irregularity (9 of 22 patients) were decreased. At follow-up, quantitative coronary morphologic values in most cases were similar to those before PTCA. There were individual changes, which occurred in an unpredictable and highly variable fashion, so that average values were not changed. The eccentricity ratio was not significantly changed by angioplasty or at restenosis. Thus, although successful PTCA results in specific changes in angiographic coronary stenotic morphology, these are reversed by the restenosis process.

Effects of acute coronary occlusion on hemodynamics in an adjacent coronary artery in dogs.

The effects of acute occlusion of 1 coronary artery on flow responses in another were studied in 24 open-chest dogs. Left circumflex (LC) flow was measured with and without LC stenoses before and during reactive hyperemia. In 19 dogs the left anterior descending artery (LAD) was occluded and measurements were repeated after 1 hour (group 1). Four dogs had measurements before and after 1 hour without LAD occlusion (group 2). In group 2 no systemic, left ventricular (LV) or coronary hemodynamic changes were observed after 1 hour. In group 1, an hour after LAD occlusion, heart rate and aortic pressure had not changed but stroke volume decreased slightly (-8 +/- 7%, mean +/- SD, p = not significant) and LV end-diastolic pressure had increased (2 +/- 3 mm Hg, p less than 0.05). Basal LC flow was not changed by less than 90% LC stenosis. Ninety percent LC stenosis decreased LC flow both before and after LAD occlusion. During reactive hyperemia without LC stenosis, LC flow decreased after LAD occlusion in 15 of 19 dogs (from 154 +/- 80 to 141 +/- 75 ml/min, p less than 0.05). With 60 and 80% LC stenoses, LC flow during reactive hyperemia decreased before LAD occlusion (110 +/- 62 and 74 +/- 40 ml/min, respectively), but decreased further (both p less than 0.05) after LAD occlusion (98 +/- 54 and 63 +/- 43 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)