Biomedical subjects

R S Bar

Publications and source records attributed to R S Bar.

At least 37 records · Page 2Linked to original sources

PubMed1992

Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.

Amino Acid Sequence↗

PubMed1992

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Adipose Tissue↗

PubMed1991

Interactions of cultured endothelial cells with TGF-beta, bFGF, PDGF and IGF-I.

Endothelial cells in culture synthesize the growth factors transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and, perhaps, insulin like growth factor I (IGF-I). We have previously demonstrated that IGF-I and PDGF have both high affinity receptors and stimulate glucose and AIB uptake in the microvessel cells under study and that IGF-I, but not PDGF, has similar high affinity receptors in cultured large vessel endothelial cells. In the present study, cultured bovine endothelial cells were exposed to these four growth factors to determine a) their effects on the acute metabolic processes of neutral amino acid (AIB) and glucose uptake and b) their interactions at the endothelial cell surface. In microvessel endothelial cells, each growth factor stimulated AIB and glucose uptake 2-4 fold whereas in large vessel endothelial cells only bFGF stimulated glucose uptake. Each growth factor had specific high affinity binding to the microvessel cells that was not influenced by the presence of the other growth factors. In large vessel endothelial cells, similar high affinity binding was present only for IGF-I and to a lesser degree TGF-beta. When cells were exposed to a given growth factor for 18 hours, homologous receptor downregulation was observed, with a maximal 60-95% decrease in surface binding. These findings suggest several potential levels of interaction of the growth factors TGF-beta, bFGF, PDGF and IGF-I in cultured vascular endothelial cells.

Animals↗

PubMed1990

Insulin differentially alters transcapillary movement of intravascular IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins in the rat heart.

Insulin-like growth factor binding-proteins 1 and 2 (IGFBP-1, IGFBP-2) and endothelial cell IGF binding proteins (ECBP) were individually perfused through isolated beating rat hearts in the absence and presence of insulin. Insulin caused an increased movement of IGFBP-1 from the vascular space to tissues of the heart. Subendothelial content of IGFBP-1 was 110%, 126% (p less than .01) and 132% (p less than 0.05) of control hearts when perfused with 1, 10 and 100 ng/ml insulin, respectively. . In contrast, insulin treatment was associated with a decrease in ECBP content in cardiac tissue, being 83%, 62% (p less than 0.005) and 73% (p less than 0.05) of control when perfused with 1, 10 and 100 ng/ml insulin. The efflux of IGFBP-2 from the intravascular space was unaffected by insulin. The subendothelial tissue distribution of the transported binding proteins was not changed by insulin perfusion, with IGFBP-1 and IGFBP-2 localizing predominantly in cardiac muscle and ECBP having greater affinity for connective tissue elements. We conclude that in the perfused rat heart, insulin can differentially alter transcapillary movement of IGFBP-1, IGFBP-2 and endothelial cell IGF-binding proteins. Such insulin-facilitated changes could potentially mediate nutrient-dependent transport of IGF-I and IGF-II to peripheral tissues.

Animals↗

PubMed1990

Transcapillary permeability and subendothelial distribution of endothelial and amniotic fluid insulin-like growth factor binding proteins in the rat heart.

Insulin-like growth factor (IGF) binding proteins (IGFBP) were purified from conditioned media of cultured bovine endothelial cells (ECBP) and from human amniotic fluid (IGFBP-1), and then labeled by radioiodination. 125I-ECBP and 125I-IGFBP-1 were perfused through isolated beating rat hearts for 1 and 5 min, and the hearts fixed and analyzed for 125I-BP content and distribution. One to 4% of the perfused 125I-ECBP and 125I-IGFBP-1 crossed the capillary boundary. The ECBPs predominantly localized as intact 125I-BP in connective tissue elements of the heart with less 125I-BP in cardiac muscle. The ratio of 125I-ECBP in connective tissue: muscle (normalized to percent vol of these compartments) was greater than or equal to 10:1. In contrast, the IGFBP-1 had a greater affinity for cardiac muscle with ratios of 125I-IGFBP-1 in connective tissue:muscle of approximately 1:2. When 125I-IGF-I, in the absence of any BPs, was perfused through the hearts approximately 3-5% left the microcirculation and was found in subendothelial tissues. 125I-IGF-I localized primarily to cardiac muscle with a distribution of connective tissue:cardiac muscle of about 1:3. The findings in the isolated perfused heart were confirmed in intact animals. After 125I-IGFBP-1 was injected into anesthetized rats and allowed to circulate for 5 min, substantial radioactivity was associated with the heart. As in the isolated heart, the IGFBP-1 preferentially localized to cardiac muscle with a connective tissue:cardiac muscle ratio of 1:3. We conclude that IGFBPs produced by endothelial cells and the IGFBP-1 contained in amniotic fluid can cross the capillary boundaries of the rat heart, and that the ECBPs preferentially localize in connective tissue elements of the myocardium, whereas IGFBP-1 predominantly localizes in cardiac muscle.

Amniotic Fluid↗

PubMed1990

Intrinsic bioactivity of insulin-like growth factor-binding proteins from vascular endothelial cells.

Conditioned medium from cultured vascular endothelial cells contains material capable of stimulating acute metabolic processes in endothelial cells. The bioactivity of the conditioned medium is not caused by the copurification of known growth factors produced by the cells, in particular platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor (IGF)-I/II. We now demonstrate that the bioactivity is directly due to an IGF-binding protein(s) (ECBP) and, further, that the bioactive domain of the binding protein differs from the IGF-binding domain. Binding proteins (BPs) from cultured pulmonary artery endothelial cells were purified by sequential passage over sizing, multiplication-stimulating activity affinity, and hydrophobic columns. BP fractions were separated into those with and those without biological activity. The bioactive binding protein(s) was cross-linked with disuccinimidyl suberate to IGF-I or the recombinant IGF analog [1-27,Gly4,38-70]IGF-I (Analog). The IGF-I Analog, by itself, had minimal interaction with the type I IGF receptor in cultured microvessel endothelial cells and no intrinsic bioactivity, but did bind with high affinity to ECBP. All free BP and free IGF-I/Analog were removed from the cross-linked mixture by passage over gel filtration and IGF affinity columns. The cross-linked BP-IGF-I complex did not bind to the type I receptor of cultured endothelial cells, but did stimulate glucose and alpha-aminoisobutyric acid uptake in endothelial cells (approximately 2-fold increase); the magnitude of the response was nearly equal to the effect of ECBP or IGF-I alone. The BP-Analog complex also stimulated glucose and alpha-aminoisobutyric acid uptake, with the magnitude of the response approaching the effect of ECBP alone. The BP-Analog complex also did not react with type I IGF receptors on the cultured endothelial cells. We conclude 1) IGF-BP produced by endothelial cells possess intrinsic biological activity; 2) bioactivity of the BP(s) is retained when the IGF-binding domain of the BP is occupied by IGF-I or an inactive IGF-I analog; and 3) IGF-I bound to the bioactive BP does not react with its receptor and possesses minimal, if any, bioactivity in vitro.

Aminoisobutyric Acids↗

PubMed1990

Tissue localization of perfused endothelial cell IGF binding protein is markedly altered by association with IGF-I.

Perfused endothelial cell IGF binding proteins (ECBP) have been previously demonstrated to leave the microcirculation of the rat heart and distribute primarily in connective tissue elements of the heart. In the present study, ECBP have been crosslinked to IGF-I and the biologically inactive [1-27,gly4,38-70]-hlGF-I, an analog of IGF-I lacking the type I IGF receptor domain. The crosslinked ECBPs were perfused through the isolated rat heart and their tissue distributions determined. Both [ECBP-Analog] and [ECBP-IGF-I] left the microcirculation of the heart. [ECBP-Analog] preferentially localized in connective tissue elements with a muscle:connective tissue ratio of approximately 1:6, similar to the tissue distribution of perfused ECBP. In contrast, the [ECBP-IGF-I] complexes localized in cardiac muscle with a muscle to connective tissue ratio of approximately 3:1, virtually identical to the tissue distribution of IGF-I when the IGF-I is perfused through the heart in the absence of any binding proteins. We conclude that 1) ECBP in the presence of IGF will cross capillary boundaries and 2) the tissue distribution of [ECBP-IGF-I] is dictated by the IGF-I molecule.

Animals↗

PubMed1989

The effects of platelet-derived growth factor in cultured microvessel endothelial cells.

The effects of platelet-derived growth factor (PDGF) on thymidine incorporation into DNA and glucose and neutral amino acid uptake were studied in endothelial cells cultured from macrovessels (bovine aorta and pulmonary artery) and microvessels (bovine fat and mouse brain). Similar to previous studies, PDGF did not bind to macrovessel cells, nor did it influence their metabolic function. In contrast, PDGF bound specifically to the two types of microvessel cell culture and in these cells also stimulated the uptake of glucose and neutral amino acids as well as the incorporation of thymidine into DNA. Stimulatory effects of PDGF occurred at concentrations of 2 ng/ml, with maximal stimulation up to 5-fold of the control value for amino isobutyric acid and glucose uptake and up to 8- to 10-fold for thymidine incorporation. The maximal effects of PDGF were additive to those of insulin-like growth factor, I, a known stimulator of all three metabolic processes in microvessel endothelial cells. The binding of PDGF to the endothelial cells was, in general, equivalent to PDGF binding to human foreskin fibroblasts, both in the magnitude of tracer binding and in the affinity of binding. Similar effects were found with recombinant and platelet-derived PDGF. We conclude that these two cultured microvessel endothelial cells not only produce PDGF-like material, but are capable of binding and responding to PDGF.

Adipose Tissue↗

PubMed1989

Insulin-like growth factor-binding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities.

Insulin-like growth factor (IGF)-binding proteins are produced by several cell types, including vascular endothelial cells. The production of IGF-binding proteins by endothelial cells is of particular interest, since these cells are directly bathed by the circulating IGFs and form the initial barrier to the passage of circulating IGFs from the bloodstream to subendothelial tissues. We have purified IGF-binding proteins from medium conditioned by cultured bovine endothelial cells by sequential passage over Bio-Gel P-60, multiplication-stimulating activity affinity, anion exchange (DEAE cellulose) and/or hydrophobic (phenyl-Sepharose) chromatography. Two peaks of IGF-binding activity were eluted from the phenyl-Sepharose column. After cross-linking, each peak contained two to five protein bands on gels that specifically bound IGF-I and -II with mol wt ranging from about 28-44K. Material in peak 1 bound IGF-I congruent to IGF-II and had no affinity for insulin and proinsulin. Peak 2 IGF-binding proteins bound IGF-II with substantially higher affinity than IGF-I and did not recognize insulin or proinsulin. Peak 1 material from phenyl-Sepharose chromatography was a potent stimulator of both glucose transport and aminoisobutyric acid (AIB) uptake in microvessel endothelial cells, with maximal stimulation of both processes being 300-400% of control values. In contrast, peak 2 material either had no intrinsic bioactivity or was slightly inhibitory or stimulatory, depending on the concentration of peak 2 material that was added. The bioactivity in peak 1 was not due to copurification of other endothelial proteins capable of stimulating glucose and AIB uptake, such as IGF-I/-II, platelet-derived growth factor, and basic fibroblast growth factor, since bioactivity was retained after acid treatment, antibody neutralization, and selective affinity chromatography to deplete these other factors. When peak 1 material was added to IGF-I the bioeffects (glucose and AIB uptake) of IGF and binding proteins were additive, and in some experiments the binding proteins potentiated the effect of IGF on endothelial cells, suggesting that the binding protein-IGF complex may retain the bioactivity of both the binding protein(s) and the IGF.

Animals↗

PubMed1989

Medical treatment of hypercalcemia.

Calcium homeostasis and the symptoms, etiology, and general medical management of hypercalcemia are reviewed. Hypercalcemia, an elevation in total serum calcium concentration, may produce neurologic, gastrointestinal, renal, and cardiovascular disturbances; it may also cause calcification in extraskeletal tissue. Hyperparathyroidism and malignancy cause more than 90% of cases of this potentially fatal disorder. When correction of the underlying cause of hypercalcemia is impossible, calcium-lowering therapy is necessary. Acute management of hypercalcemia often involves rehydration, mobilization of the patient, and furosemide-induced diuresis. These measures may be followed by intravenous administration of etidronate, plicamycin, or calcitonin. Agents used in the long-term management of hypercalcemia include oral phosphates, oral etidronate, intermittent intravenous plicamycin, and, in selected patients, corticosteroids and nonsteroidal anti-inflammatory agents. Investigational calcium-lowering agents include gallium nitrate, ethiofos, dichloromethylene diphosphonate, and aminohydroxyproline diphosphonate. Many agents are available for the treatment of hypercalcemia. Therapy can be individualized by integrating knowledge of the physiologic causes of hypercalcemia, the mechanism of action of calcium-lowering drugs, and the patient's other disease states. Further studies are needed to define the role of investigational calcium-lowering agents.

Adrenal Cortex Hormones↗

PubMed1988

Insulin, insulin-like growth factors, and vascular endothelium.

Endothelial cells form the intimal lining of the entire vascular system. The vascular endothelium is continuously and directly bathed by components of the bloodstream and represents the initial fixed anatomical surface with which these components come in contact. In the past decade, the methodologies for studying endothelial cell functions have markedly advanced, enabling direct and detailed study of the vascular endothelium. From such studies, it is now apparent that the vascular endothelium represents an extraordinarily complex network of cells demonstrating a multitude of distinct anatomic, metabolic, and immunologic properties critical to such processes as angiogenesis, atherosclerosis, thrombosis, neoplasia, and a variety of metabolic disorders including homocystinuria and diabetes mellitus. This report will focus on the interactions of insulin and the insulin-like growth factors (IGFs) with vascular endothelium, based on studies with cultured endothelial cells, isolated microvessels, and perfused organ systems. Data will be presented relevant to the following concepts: (1) endothelial cells, in culture and in vivo, have specific receptors for insulin, IGF-I, and IGF-II; (2) insulin, IGF-I, and IGF-II have both distinct and overlapping functions in cultured endothelial cells; (3) cultured endothelial cells process receptor-bound insulin, IGF-I, and IGF-II, by distinct processes; (4) in vivo, capillary endothelial receptors are integrally involved in the transport of intact insulin to subendothelial sites of insulin action; and (5) vascular endothelium has specialized cellular features that are likely to contribute to the unique interactions of endothelial cells with insulin and the IGFs.

Animals↗

PubMed1988

IGF receptors in myocardial capillary endothelium: potential regulation of IGF-I transport to cardiac muscle.

Beating rat hearts were perfused with 125I-IGF-II alone or 125I-IGF-II and unlabeled IGF-II or insulin, then prepared for radioautography. Maximal 125I-IGF-II grain counts over capillaries were decreased in a dose-dependent manner by unlabeled IGF-II but were unaffected by coperfusion with insulin. To determine a potential role for capillary receptors in the transfer of circulating IGF to cardiac muscle, the effects of sequential loss of capillary IGF binding sites was determined. For IGF-I, loss of capillary binding sites by trypsin perfusion was accompanied by proportional decreases in the subsequent appearance of IGF-I in cardiac muscle. In contrast, similar decrements of capillary IGF-II binding did not affect muscle levels of IGF-II. We conclude that capillary endothelium of the intact heart possesses distinct IGF-I and IGF-II binding sites, with the capillary IGF-I binding sites being of potential importance in the transfer of vascular IGF-I to subendothelial cardiac muscle.

Animals↗

PubMed1988

Actions of insulin and insulinlike growth factors I and II in cultured microvessel endothelial cells from bovine adipose tissue.

Endothelial cells were cultured from bovine adipose microvessels, pulmonary arteries, and aortas. The effects of insulin, IGF-I, and IGF-II (MSA) on glucose uptake, neutral amino acid (AIB) uptake, and thymidine incorporation into DNA by the endothelial cells were determined. Each hormone markedly stimulated all three processes in the microvessel endothelial cells but had no effect on the larger-vessel endothelial cells. In the microvessel cells the monoclonal antiinsulin receptor antibody, Ab 47-9, was observed to specifically inhibit insulin binding in the bovine microvessel cells without having intrinsic activity on the three biologic processes that were stimulated by insulin and the IGFs. When insulin binding was first inhibited by Ab 47-9, dose-response curves for insulin were markedly shifted to the right for glucose uptake, AIB uptake, and thymidine incorporation into DNA. Similar antibody treatment had no effect on dose-response curves of IGF stimulation of any of the three processes. These data further extend the biologic actions of insulin and the IGFs in cultured microvessel endothelial cells. They also suggest that certain functions mutually stimulated by both insulin and the IGFs, ie, glucose uptake, AIB uptake, and thymidine incorporation into DNA, are substantially mediated through homologous receptors.

Adipose Tissue↗

PubMed1988

Vascular transport of insulin to rat cardiac muscle. Central role of the capillary endothelium.

Using intact, beating hearts, we have assessed the interaction of insulin with capillary endothelium and the subsequent appearance of insulin in cardiac muscle. Rat hearts were perfused with 125I-insulin (10(-10) M) alone or in combination with unlabeled insulin (10(-9)-10(-5) M). 125I grains (shown to represent greater than 90% intact insulin) over both capillary endothelium and cardiac muscle decreased in a dose-dependent manner when hearts were co-perfused with labeled insulin and increasing concentrations of unlabeled insulin. Perfusion of 125I-desoctapeptide (DOP) insulin, a low affinity insulin analogue, with unlabeled insulin (10(-9)-10(-5) M) had no effect on the appearance of 125I-DOP insulin over microvessel endothelium and muscle. When capillary receptors were first destroyed by trypsin treatment or blocked by anti-receptor antibodies, the appearance of 125I-insulin in cardiac muscle decreased proportional to the inhibition of insulin binding to the capillary receptors. We conclude that insulin binding to capillary endothelial receptors is a central step in the transport of intravascular insulin to rat cardiac muscle.

Animals↗

PubMed1987

Production of IGF-binding proteins by vascular endothelial cells.

Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.

Animals↗

PubMed1987

Stimulation of proteoglycans by IGF I and II in microvessel and large vessel endothelial cells.

Endothelial cells were cultured from bovine capillaries and pulmonary arteries, and the effect of insulinlike growth factor (IGF) I and II (multiplication-stimulating activity) and insulin on the synthesis of proteoglycans was determined. IGF I and II stimulated 35SO4 incorporation into proteoglycans in a dose-dependent manner in both microvessel and pulmonary artery endothelial cells with maximum threefold increases. In pulmonary artery cells, the IGFs caused a general stimulation of all classes of glycosaminoglycan-containing proteoglycans. In microvessel endothelial cells, the IGFs appeared to preferentially increase heparan sulfate-containing proteoglycans. Insulin, at concentrations up to 10(-6) M, had no effect on the synthesis of proteoglycans in either microvessel or pulmonary arterial endothelial cells. Thus, the IGFs stimulate the synthesis of proteoglycans in both microvessel and large vessel endothelial cells, a property that is not mimicked by insulin. Because vascular endothelial cells are bathed by IGFs in vivo, such IGF-mediated functions are likely to be significant in both the normal physiology of vascular endothelium and in disease states such as diabetes mellitus.

Animals↗

PubMed1986

Cultured capillary endothelial cells from bovine adipose tissue: a model for insulin binding and action in microvascular endothelium.

Capillary endothelial cells were cultured from bovine adipose tissue. The endothelial nature of the cultures was documented by characteristic morphology, uniform presence of factor VIII antigen, and uptake of the endothelial cell marker Dil-Ac-LDL. The capillary cell cultures had specific, high affinity binding sites for insulin, demonstrating time and temperature dependence of binding, pH optimum, analog specificity, and inhibition of insulin binding by anti-insulin receptor antibodies. In both subconfluent and confluent cultures, insulin stimulated thymidine incorporation into DNA; significant stimulatory effects of insulin were observed at insulin concentrations of 1 ng/ml with maximal 8- to 10-fold increases at hormone concentrations of 1,000 to 10,000 ng/ml. Because of the ease of routine preparation, cell purity, presence of high affinity insulin binding sites, and insulin-sensitive metabolic responses, we suggest that the bovine capillary endothelial cultures could serve as a model cell system for the detailed study of insulin interactions with capillary endothelial cells.

Adipose Tissue↗

PubMed1986

Processing of insulin-like growth factors I and II by capillary and large vessel endothelial cells.

Cultured endothelial cells from bovine capillaries and pulmonary arteries were incubated with highly purified [125I]insulin-like growth factor I ([125I]IGF-I), [125I]IGF-II, or [125I]insulin for periods up to 120 min, the cells were washed, and the cell-bound radioactivity was allowed to dissociate from the cells into fresh incubation medium. For insulin, 85-95% of the 125I dissociated from cells in 15 min, with 75% dissociating by 5 min. The 125I material released into the medium during the first 5 min of dissociation was entirely intact insulin, while the material released in the next 10 min was 80% intact insulin. For the [125I]IGFs, several differences were observed. First, after 5 min of dissociation, only 30-45% of the cell-bound 125I was released into the medium. For IGF-I, this rapidly dissociating material was entirely intact peptide, whereas for IGF-II, up to 55% of the dissociated radioactivity was degraded peptide. Second, during the next 10 min of dissociation, an additional 20% of the 125I was released from the cells. For IGF-I, this was 85% intact peptide; however, for IGF-II, this dissociated fraction contained as little as 25% intact peptide. Third, for both IGFs, after 15 min of dissociation, 40-65% of the initial cell-associated 125I remained within the endothelial cells; after the 15-min dissociation period, 95% of the remaining internalized 125I was intact IGF-I, whereas for IGF-II, the internalized 125I was 90% intact IGF-II after short periods of association (less than 15 min) and progressively decreased to 65% intact peptide after 60 min of association. We conclude that in addition to having separate surface receptors for insulin, IGF-I, and IGF-II, endothelial cells also process each hormone by distinct pathways.

Animals↗