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 M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM 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 M_r 145 000 and an M_r 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.     

Substrate binding by crude membranes and solubilized membrane extracts fromPseudomonas natriegens

A component formed in the membranes ofPseudomonas natriegens during induction to utilization of mannitol orl-arabinose was found to bind the specific inducing substrate. Chloramphenicol inhibited the synthesis of the component. Binding was optimal at 25–30 C in the presence of Na⁺ and K⁺. Trypsin, KCN, and N-ethylmaleimide destroyed or suppressed the capacity to bind, but DNase and RNase had little effect. Specific binding was demonstrated by equilibrium dialysis with labeled substrate on one side of the dialysis cell and membranes or alkaline extracts of membranes on the other. Membranes from mannitol-grown cells broke the equilibrium established for C¹⁴-labeled mannitol, whereas membranes from cells grown in sea water nutrient broth did not. The substrate was unchanged after binding. Simple alkaline extraction followed by centrifugation at 105,000 ×g for 60 min gave significant purification of the binding component. Specific requirement of Na⁺ and K⁺ for substrate binding by alkaline extracts of membranes could not be demonstrated. The binding capacity of the solubilized component in alkaline extracts was destroyed by heating, but was stable to storage at 0 C for several days.     

Insulin-like effects of polyamines spermine binding to fat cells and fat cell membranes

Two naturally occurring polyamines, spermine and spermidine, mimic the action of insulin on lipid and glucose metabolism in adipocytes. To evaluate the role of cell membranes in the action of polyamines, studies of [¹⁴C] spermine binding using an oil separation method were conducted in isolated rat adipocytes and adipose cell membranes. Spermine binding and dissociation in fat cells and fat cell membranes were rapid and complete within 3–6 min. Following a 30-min incubation of [¹⁴C] spermine with fat cell membranes, over 90% of bound [¹⁴C] spermine was dissociable while under similar conditions only 25% of bound [¹⁴C] spermine was dissociable in cells. The non-dissociable fractions in cells likely represented intracellular accumulation. Binding and stimulation of glucose oxidation were demonstrated at similar concentrations. Bound spermine was displaced by spermine, spermidine and 1,8-diaminooctane with greater efficacy than putrescine (a polyamine devoid of insulin-like properties) or insulin. Similarly, polyamines did not complete with insulin for binding to isolated adipocytes. It appears, therefore, that polyamines initiate their insulin-like effects by interacting with the cell membrane at sites which are common to biologically active polyamines and which are distinct from the insulin receptor.     

Rapid hyperpolarization of rat skeletal muscle induced by insulin

It has been proposed that the increase produced by insulin in electrical potential differences across membranes of target cells may be a mechanism by which the cell surface insulin-receptor complex causes at least some of the metabolic effects of insulin. If insulin-induced hyperpolarization is a transducer of common effector responses it must precede those responses. The problem has not been addressed previously, so that rapid responses to insulin have not been sought. Two methods were used. In one method, the bathing solution was changed rapidly so as to include insulin in supramaximal concentrations, and a series of measurements of membrane potentials, E_r, were made. Insulin hyperpolarized by 9.4 mV within 1 min. In the other method, nanoliter amounts of highly concentrated insulin solution were ejected from a micropipette onto the surface of an impaled muscle fiber. In 21 out of 32 insulin injections, hyperpolarization occurred within 1 s; in 11 control injections there was no change. This is the most rapid response to insulin yet reported, and is consistent with the hypothesis that insulin-induced hyperpolarization may transduce effector responses.     

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n=10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.     

Insulin adsorption into porous charged membranes: Effect of the electrostatic interaction

Insulin adsorption into a series of porous charged membranes was investigated by batch adsorption experiments, and the experimental results were analyzed by the homogeneous diffusion model. The membranes used in this study were prepared by pore‐surface modification of porous poly(acrylonitrile) (PAN) membranes by grafting with weak acidic and basic functional groups. The amount of insulin adsorbed into the membrane was determined from the material balance of insulin. The insulin partition coefficient K between the membrane and solution was estimated from the equilibrium adsorption amount, and the effective diffusion coefficient D was estimated by matching the model with the experimental data as a fitting parameter. The dependence of K and D on the charge properties of the insulin and membrane is observed and discussed. The partition coefficient K increased when the insulin and the membrane carried opposite charges, on the other hand, the effective diffusion coefficient D was reduced. These results indicate that the electrostatic interaction between the insulin and the membranes played an important role in the insulin adsorption. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180 000 sites/cell but this fell to 125 000 (p <0.001) in cells enriched in monounsaturated fatty acids and rose to 386 000 (p <0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants ( and ) of 0.03 and 0.01 nM⁻¹. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM⁻¹ whereas values of 0.01 and 0.005 nM⁻¹ were obtained in the cells enriched in polyunsaturated fatty acids (all comparison p <0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.     

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas

Summary We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10⁻⁶ or 10⁻⁵ M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10⁻⁶ M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10⁻⁵ M) of retinoic acid and more than three times that of the control grafts.     

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes

The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center     

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

SYSTEMATIC SIGNIFICANCE OF THE ABSENCE OF PIT-PLUG CAP MEMBRANES IN THE BATRACHOSPERMALES (RHODOPHYTA)1

Four species of the Batrachospermales were examined by transmission electron microscopy to determine whether or not cap membranes, a typical structural component of pit plugs in several orders of red algae, were present. Routine specimen preservation methods used in past studies led to contradictory reports, so Batrachospermum keratophytum Bory, B. sirodotii Skuja ex Reis, Sirodotia suecica Kylin, and S. tenuissima (Collins) Skuja ex Flint were prepared by secondary fixation in potassium permanganate or a combination of potassium ferrocyanide-osmium tetroxide to enhance membrane contrast. These fixation procedures produced clear, well-contrasted images in which cap membranes were absent. The absence of cap membranes in S. suecica and S. tenuissima and the presence of cap membranes in two members of the Nemaliales was confirmed by freeze-substitution methods. Absence of cap membranes in representatives of the Batrachospermales further distinguishes the Batrachospermales from the Nemaliales, the order in which they long resided, and demonstrates the value of this character in elucidating ordinal alliances among the Rhodophyta.     

Characterization of small GTP-Binding proteins in plant cell

To identify and characterize small GTP-binding proteins in plant cells, GTP-binding studies were performed with electroblotted plant proteins following SDS-polyacrylamide gel electrophoresis using [α-³²P]GTP. Three species of small GTP-binding protein (21, 23, and 27 kD) which have a specific GTP-binding property were identified in the membrane and cytosolic fractions of both monocotyledons (Zea mays) and dicotyledons (Glycine max). Moreover, these three species of small GTP-binding protein were gradually decreased when membranes were treated with hydroxylamine. This result indicates that these small GTP-binding proteins in plant cells are fatty acylated to the membrane lipids. The 27 kDa component was partially purified from hypocotyl membranes of Glycinemax, following S-300 gel filtration, phenylsepharose CL-4B, hydroxyapatite, and Q-sepharose column chromatography. This 27 kD protein was found to have both GTP-binding and GTPase activities.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 μg/ ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 μg/ml (P < 0.01) but the staining intensity at 0 μg/ml was not different from that at 3 μg/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo⁻ mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

The effect of placement of tryptophan residues in selected A-chain positions on the biological profile of insulin

In continuation of our efforts to study the solution structure and conformational dynamics of insulin by time-resolved fluorescence spectroscopy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in the A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using rat liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in receptor binding assays in both liver membranes and in adipocytes, but only 0.06% in lipogenesis assays as compared to porcine insulin. [A10 Trp] insulin displays a potency ofca. 40% andca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin displays a potency ofca. 39% in rat liver receptor binding assays, but onlyca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp] insulin displays a potency ofca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a potency of 18% and 9% in rat liver receptor binding and lipogenesis assays, respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] insulin, displays a potency ofca. 0.7% in both rat liver receptor binding assays and lipogenesis assays. These data suggest two major conclusions: (1) the A3 and A15 residues lie in sensitive regions in the insulin molecule, and structural modifications at these positions have deleterious effects on biological activity of the hormone; and (2) [A13 Trp]insulin appears to be a unique case in which an insulin analogue exhibits a higher potency when assayed in liver tissue than when assayed in fat cells.     

Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium,on polyethyleneterephthalate microporous membranes,as an in vitro model

Summary Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1μg/ml insulin, 1 ng/ml epidermal growth factor, 10μg/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli. Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments of microplate inserts and cell lysates were immunoprecipitated after labeling with [³H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. [³H]transferrin is found mainly in the lower compartment and in cells; [³H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [³H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood. This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (grant 3.4551.88) and the Walloon Region.     

ALTERATIONS IN THE BINDING OF [3H]LEUCINE-ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS

—Saturation binding studies with [³H]leu-enk ([tyrosyl-3, 5-³H(N)]⁵leu-enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [³H]leu-enk to the high affinity component (K_D= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (K_D= 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose-dependent inhibition of the binding of [³H]leu-enk to the morphine-sensitive and insensitive binding components. Investigations into the nature of the morphine-insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [³H]leu-enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine-insensitive manner, and under conditions where the binding of [³H]leu-enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine-insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [³H]leu-enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.     

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [³H]saxitoxin ([³H]STX) binding in a time-dependent manner (t_1/2 = 26 h). Insulin (100 nM for 4 days) increased the B_max of [³H]STX binding by 49% without changing the K_D value and also augmented the maximal influx of ²²Na⁺ due to 560 µM veratridine by 39% without altering the EC₅₀ value of veratridine. The stimulatory effect of insulin on ²²Na⁺ influx was concentration-dependent with an EC₅₀ of 3 nM, whereas insulin-like growth factor (IGF)-I had little effect at 1 nM. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine (100 µM)-induced ²²Na⁺ influx by approximately twofold in both insulin-treated cells and untreated cells. Veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine-induced ²²Na⁺ influx via the nicotinic receptor-ion channel complex and high-K⁺ (direct activation of voltage-dependent Ca²⁺ channels)-induced ⁴⁵Ca²⁺ influx. Stimulatory effects of insulin on [³H]STX binding and veratridine-induced ²²Na⁺ influx were nullified by simultaneous treatment with either 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na⁺ channel α-subunit. These results suggest that the binding of insulin to insulin (but not IGF-I) receptors mediates the up-regulation of functional Na⁺ channel expression at plasma membranes; this up-regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).     

The growth and partition of cell membranes during synchronized division cycle of Caulobacter crescentus

Summary The growth and division of cell membranes in Caulobacter crescentus has been studied. This microorganism divides into flagellated and stalked cells which are easily separated by centrifugation. The biosynthesis and partition of membranes was studied by labeling the proteins with [³H]-leucine and the lipids with [³²P]. The membranes were prepared from cell spheroplasts. They further purified on a sucrose gradient.The data obtained show changes of the rate of synthesis of membranes in C. crescentus during the first synchronized division cycle (110 min): the rate is faster in the first 70 min and it drops by 26% during the following 40 min. During the period of faster synthesis the flagellated cells are changing into stalked cells while doubling in size.There is also an intracellular pool of membrane precursors the quantity of which almost doubles as the rate of membrane synthesis decreases, i.e., before cell division.The macromolecules constituting the membranes are not degraded.After division, in each membrane of the two morphologically different cell types the specific radioactivity is 50% of that of the parent cell membranes.