Characterization and Regulation of Insulin Receptors in Rat Brain

An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.     

Structural differences between insulin receptors in the brain and peripheral target tissues

Insulin receptors in various brain regions (olfactory tubercle, hippocampus, and hypothalamus) were photoaffinity labeled using the photoreactive analogue of insulin B2(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). A protein with an apparent Mr of 400,000 was specifically labeled with 125I-NAPA-DP-insulin in all three brain regions. When radiolabeled proteins were reduced with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a protein with an apparent Mr of 115,000 and to a much lesser extent in a protein with an apparent Mr of 83,000. The size of these receptor proteins, based on their electrophoretic mobilities, was consistently smaller than insulin receptor proteins in adipocytes. The covalent labeling of insulin receptors in brain by 125I-NAPA-DP-insulin was not blocked by anti-insulin receptor antiserum. Additionally, in contrast to effects observed in peripheral target tissues, this antisera did not inhibit the binding of 125I-insulin to brain membranes. Neuraminidase treatment resulted in an increase in the electrophoretic mobilities of insulin receptor subunits in adipocytes, but, had no effect on receptor subunits in brain. Solubilized insulin receptors from adipocytes were retained by wheat germ agglutinin columns and specifically eluted with N-acetylglucosamine. In contrast, solubilized insulin receptors from brain did not bind to these columns. The results from this study indicate that structural differences, including molecular weight, antigenicity, and carbohydrate composition exist between insulin receptors in brain and peripheral target tissues.     

beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Characterization of lactoferrin receptor in brain endothelial capillary cells and mouse brain

We present, herein, the evidence for lactoferrin (Lf) binding sites in brain endothelial capillary cells (BCECs) and mouse brain. The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells. Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs (high affinity: dissociation constant (K (d)) = 6.77 nM, binding site density (B (max)) = 10.3 fmol bound/mug protein; low affinity: K (d) = 4815 nM, B (max) = 1190 fmol bound/mug protein) and membrane preparations of mouse brain (high affinity: K (d) = 10.61 nM, B (max) = 410 fmol bound/mug protein; low affinity: K (d) = 2228 nM, B (max) = 51641 fmol bound/mug protein). The distribution study indicated the effective uptake of (125)I-Lf in brain after intravenous administration. The present study provides experimental evidence for the application of Lf as a novel ligand for brain targeting.     

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.     

Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an A1 adenosine receptor agonist

Adenosine, via interaction with A1 adenosine receptors, increases insulin sensitivity and inhibits lipolysis in adipocytes. To investigate regulation of this system, adipocytes were incubated for up to 72 h with the nonmetabolizable adenosine receptor agonist, N6-phenylisopropyl adenosine (PIA). Adenosine receptors were measured by the binding of 125I-hydroxyphenylisopropyl adenosine to membranes. PIA down-regulated adenosine receptors, decreasing the number of binding sites with no change in affinity. Adipocytes were incubated for 48 h without or with 100 nM PIA to down-regulate the A1 receptors by approximately 60%. The cells were washed, and lipolysis and glucose transport were assessed. The ability of PIA to inhibit lipolysis was markedly attenuated in the down-regulated cells. Furthermore, the EC50 of insulin was increased approximately 3-fold in the PIA-treated cells. 125I-Insulin binding to the PIA-treated cells was unchanged, demonstrating that the decreased insulin sensitivity is not due to decreased insulin receptor binding. Pertussis toxin catalyzed ADP-ribosylation of a 41-kDa protein thought to be the alpha-subunit of Gi. This 41-kDa protein was decreased in membranes from cells treated with PIA, with a maximal 50% loss. This suggests that Gi is down-regulated and that loss of both the A1 adenosine receptor and Gi are involved in the metabolic changes observed after PIA treatment.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.     

Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor alpha-subunit. Up-regulation of neuronal insulin receptors in diabetes

Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.     

Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [³H]-cis-methyldioxolane ([³H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitivel the binding of receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [³H]-cGMP synthesis in N1E-115 neuroblastoma cells—both acting as m receptor antagonist. All five OPs inhibited [³H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [³H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [¹²⁵I]-α-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.     

Insulin: Its binding to specific receptors and its stimulation of DNA synthesis and 2′,3′-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain

The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (K _d=7.77×10^–9 M; number of receptor sites,B _max=350 fmol/mg protein) while the other type was of low affinity type (K _d=5.75×10^–8 M;B _max=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [¹²⁵I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2,3-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase.

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.     

Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B(1) and B(2) receptors was detected with real-time PCR and B(2) receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of (125)I-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 +/- 27 pM. Competition studies with bradykinin B(1) and B(2) receptor antagonists showed that binding was competed by a B(1) antagonist, and when internalization was inhibited with hypertonic buffer, by both B(1) and B(2) antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased (125)I-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding experiments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged. Binding was competed similarly by the B(1) antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in (125)I-[Tyr(8)]-bradykinin binding is probably due to increased B(1) receptor affinity with no change in receptor capacity. In conclusion, bradykinin B(1) and B(2) receptor mRNA was shown in human endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B(1) receptor affinity, without changing capacity, and thus increase bradykinin binding.     

Glucose starvation alters insulin but not IGF-I binding to Chinese hamster ovary (CHO) cells

The pattern of cellular protein glycosylation can be altered in CHO cells by glucose starvation. When wild type CHO cells are deprived of glucose, 125I-insulin binding increases from a B/F of 0.033 +/- 0.004 to 0.063 +/- 0.011, due to an increase in receptor affinity. The already elevated insulin binding to mutant B4-2-1 CHO cells, whose genetic defect causes abnormal glycosylation mimicking the pattern seen in the glucose starved normal cells, is not affected by glucose starvation. In neither cell line is 125I-IGF-I binding affected by glucose starvation. These data support the hypothesis that abnormal glycosylation can alter insulin binding to its receptor. Furthermore, there is a striking difference in the susceptibility of IGF-I and insulin receptors to alterations in glycosylation.