Attachment of cells to basement membrane collagen type IV

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.     

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder

Summary In the urinary bladder of the toadBufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na⁺ transport. We have investigated whether T₃ might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T₃ (60nm) for up to 20 hr of incubation; ii) the kinetics of uptake of³H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T₃. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0°C) did not vary significantly after 18 hr of exposure to T₃, while aldosterone-dependent Na⁺ transport was significantly inhibited. In addition, T₃ did not modify the kinetics of uptake (90 min) of³H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubatedin vitro at 25°C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80nm). T₃ slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T₃ does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.     

Interaction of [3H]dipyridamole with the nucleoside transporters of human erythrocytes and cultured animal cells

Summary Equilibrium binding of [³H]dipyridamole identified high-affinity (K _i 10nm) binding sites on human erythrocytes (5×10⁵ sites/cell) and on HeLa cells (5×10⁶ sites/cell). The equilibration of dipyridamole with these sites on human erythrocytes was compatible with a second-order process which proceeded at 22°C with a rate constant of about 6×10⁶ m ^–1 sec^–1. Binding of dipyridamole to these sites correlated kinetically with the inhibition of the equilibrium exchange of 500 m uridine in these cells and was inhibited in a concentration-dependent manner by nucleosides and other inhibitors of nucleoside transport, such as nitrobenzylthioinosine, dilazep and lidoflazine, but not by hypoxanthine, which is not a substrate for the nucleoside transporter of human erythrocytes. The results indicate that the substrate binding site of the transporter is part of the high-affinity dipyridamole binding site. Bound [³H]dipyridamole became displaced from these sites on human erythrocytes by incubation with an excess of unlabeled dipyridamole or high concentrations of nucleosides and inhibitors of nucleoside transport, but neither by hypoxanthine nor sugars. Dissociation of [³H]dipyridamole behaved as a simple first-order process, but the rate constant was about one order of magnitude lower (about 3×10^–3 sec^–1) than anticipated for typical ligand-protein binding on the basis of the measured association rate and equilibrium constants. The reason for this discrepancy has not been resolved. No high-affinity dipyridamole binding sites were detected on Novikoff rat hepatoma cells, P388, L1210 and S49 mouse leukemia cells or Chinese hamster ovary cells, and their absence correlated with a greater resistance of nucleoside transport in these cells to inhibition by dipyridamole. All cells expressed considerable low affinity (K _d>0.5 m) and nonspecific binding of dipyridamole.     

Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

Summary Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na⁺, K⁺-pump, and other electrophysiological studies indicate a decrease in both K⁺ and Cl^– conductances of the oocyte plasma membrane. Measurement of [³H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K _d-4.2×10^–8 m), K⁺-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [³H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na⁺, K⁺-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-l-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer ofl-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 g/cm² of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 g/cm² also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.     

Evidence for Cell Surface Extracellular Matrix Binding Proteins in Hydra vulgaris

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [³H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreerting studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-β₁ integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified two major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of >200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian     

Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.     

Neurochemical,pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids

Specific binding ofl-[³H]glutamate ([³H]Glu) andl-[³H]asparate ([³H]Asp) to cerebellar membranes represented a time-, temperature- pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K _off/K _on=K _d) for [³H]Glu binding to this fraction indicated high-affinity (443 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [³H]Glu and [³H]Asp as receptors with differential pharmacological, specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37°C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents andl-Glu (50–1000 M) provided protection, against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8–12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8–12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Effects of thyromimetric drugs on aldosterone-dependent sodium transport in the toad bladder

Summary Aldosterone increases transepithelial Na⁺ transport in the urinary bladder ofBufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na⁺ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na⁺ transport still increases significantly but with very little change in resistance. Triiodothyronine (T₃, 6nm) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T₃ per se (up to 6nm) has no effect on base-line Na⁺ transport. The antagonist activity of T₃ is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T₃ (rT₃) is inactive and isopropyldiiodothyronine (isoT₂) is more active than T₃. The relative activity of these analogs corresponds to their relative affinity for T₃ nuclear binding sites which we have previously described. Our data suggest that T₃ might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10–20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or α1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the α1(I) chain and to all cyanogen-bromide-treated peptides tested (α1-CB2, α1-CB3, α1-CB4, α1-CB5, α1-CB6A, α1-CB7, α1-CB8, α2-CB2, α2-CB3 and α2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited intial cell attachment, an effect that persisted longer on substrates made of α1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland

Summary Scatchard analysis of³H ouabain bound to isolated rectal gland cells as a function of increasing ouabain concentrations produced a concave curvilinear plot that was resolved into two specific sites with either a high (I) or low (II) affinity for ouabain. Cyclic cAMP/theophylline (±furosemide, 10^–4 m) increased the amount of³H ouabain bound to the high-affinity site I. Vanadate, a phosphate congener which promotes formation of the ouabain-binding state of the enzyme, mimicked the effects of cAMP/theophylline at low concentrations of ouabain, suggesting that cAMP/theophylline increases binding to site I by enhancing the rate of turnover of resident enzyme. Enhanced⁸⁶Rb uptake seen following cAMP/theophylline administration was primarily associated with increased flux through the high-affinity ouabain site, and this stimulation was not obliterated by the co-administration of furosemide. A model was presented which suggested the presence of two noninteracting pools of enzyme or isozymes which exhibit either a high or low affinity for ouabain. Cyclic AMP both stimulated turnover via site I, and modified the kinetics of binding of³H ouabain to site II. The (ave)K _d of³H ouabain for site II was increased from 3.6 m (controls) to 0.5 m (cAMP/theophylline) and the Hill coefficient was modified from 0.45 (controls) to 1.12 (caMP/theophylline), suggesting a transition from a negative- to a noncooperative binding state. While furosemide reversed the effects of cAMP/theophylline on site II kinetics, it did not obliterate cAMP/theophylline effects on site I. This suggests that cAMP may alter the intrinsic turnover rate of this particular pool of Na,K-ATPase in shark rectal gland.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Integrins beta1, alpha6, and alpha3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin beta1, alpha3, or alpha6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin beta1 and alpha6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of alpha3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role.     

[3H]Desipramine Binding to Rat Brain Tissue: Binding to Both Noradrenaline Uptake Sites and Sites Not Related to Noradrenaline Neurons

Abstract The pharmacological and biochemical characteristics of [³H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [³H]desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The [³H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 μM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of ～1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromo-benzylamine (DSP4). In contrast, when 10 μM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with K_D values of 0.5 and >100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (B_max) for binding defined by 0.1 μM nisoxetine varied between brain regions, with very low density in the striatum (B_max not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of [³H]noradrenaline uptake but not with 5-[³H]hydroxytryptamine uptake. The low-affinity sites did not correlate with the regional distribution of [³H]noradrenaline uptake. Drug inhibition studies showed that noradrenaline inhibits the binding defined by 0.1 μM nisoxetine in a competitive manner. Together, these findings suggest that only a small fraction of the [³H]desipramine binding can be regarded as “specific” binding, and this binding fraction may represent the substrate recognition site for noradrenaline uptake. Assuming that one molecule of desipramine binds to each carrier molecule, the turnover number for the noradrenaline carrier was calculated to be 20/min, i.e., the duration of one transport cycle was 3 s.