ABSTRACT
BACKGROUND: Improving lipids beyond low-density lipoprotein cholesterol (LDL-C) lowering with statin monotherapy may further reduce cardiovascular risk. Niacin has complementary lipid-modifying efficacy to statins and cardiovascular benefit, but is underutilised because of flushing, mediated primarily by prostaglandin D(2) (PGD(2)). Laropiprant (LRPT), a PGD(2) receptor (DP1) antagonist that reduces niacin-induced flushing has been combined with extended-release niacin (ERN) into a fixed-dose tablet. METHODS AND RESULTS: Dyslipidaemic patients were randomised to ERN/LRPT 1 g (n = 800), ERN 1 g (n = 543) or placebo (n = 270) for 4 weeks. Doses were doubled (2 tablets/day; i.e. 2 g for active treatments) for 20 weeks. ERN/LRPT 2 g produced significant changes vs. placebo in LDL-C (-18.4%), high-density lipoprotein cholesterol (HDL-C; 20.0%), LDL-C:HDL-C (-31.2%), non-HDL-C (-19.8%), triglycerides (TG; -25.8%), apolipoprotein (Apo) B (-18.8%), Apo A-I (6.9%), total cholesterol (TC; -8.5%), TC:HDL-C (-23.1%) and lipoprotein(a) (-20.8%) across weeks 12-24. ERN/LRPT produced significantly less flushing than ERN during initiation (week 1) and maintenance (weeks 2-24) for all prespecified flushing end-points (incidence, intensity and discontinuation because of flushing). Except for flushing, ERN/LRPT had a safety/tolerability profile comparable with ERN. CONCLUSION: Extended-release niacin/LRPT 2 g produced significant, durable improvements in multiple lipid/lipoprotein parameters. The improved tolerability of ERN/LRPT supports a simplified 1 g-->2 g dosing regimen of niacin, a therapy proven to reduce cardiovascular risk.
Subject(s)
Dyslipidemias/drug therapy , Hypercholesterolemia/drug therapy , Hypolipidemic Agents/administration & dosage , Indoles/administration & dosage , Niacin/administration & dosage , Adolescent , Adult , Aged , Aged, 80 and over , Delayed-Action Preparations , Double-Blind Method , Drug Combinations , Female , Humans , Hypolipidemic Agents/adverse effects , Indoles/adverse effects , Male , Middle Aged , Niacin/adverse effects , Treatment Outcome , Young AdultABSTRACT
We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the transmembrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFP-expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time in vivo brefeldin A-induced retrograde transport of Golgi membrane protein to the ER.
Subject(s)
Actins/physiology , Endoplasmic Reticulum/physiology , Golgi Apparatus/physiology , Nicotiana/genetics , Nicotiana/ultrastructure , Plant Physiological Phenomena , Plants, Toxic , Amino Acid Sequence , Animals , Arabidopsis/genetics , Biological Transport, Active , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Immunoelectron , Molecular Sequence Data , Movement , Plants, Genetically Modified , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sialyltransferases/geneticsSubject(s)
GTP-Binding Proteins/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Luteinizing Hormone/metabolism , Pituitary Gland, Anterior/physiology , Animals , Cell Line , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/pharmacology , Kinetics , Mice , Pituitary Gland, Anterior/drug effects , Thionucleotides/pharmacologyABSTRACT
The investigation of the role of inhibin in the regulation of fertility is hindered by the lack of a routine, specific assay. The pituitary cell bioassay is time-consuming and the existing RIAs, based on either purified bovine 32-kDa inhibin or synthetic alpha-subunit peptides, are not specific for the biologically active inhibin molecules. We have used monoclonal antibodies, one specific for the N-terminal region of the human inhibin alpha chain, and the other raised to a peptide sequence close to the C-terminal of the human beta A-inhibin chain, to create a two-site sandwich ELISA specific for alpha beta-inhibin molecules. This was used to estimate levels of inhibin in crude bovine and human follicular fluids and fractions concentrated from them. Comparison of the values obtained with the ELISA and those obtained with the pituitary cell bioassay, suggests that the ELISA measures biologically active inhibin. Compared with the peptide-based RIA, the ELISA gave much lower (as little as 100-fold lower) values for the inhibin content of these samples, e.g., bovine follicular fluid 0.375 micrograms/ml (ELISA) compared with 41.0 micrograms/ml (RIA). Such large differences, possibly due to the presence of relatively large amounts of biologically inactive forms of inhibin such as the pro-alpha c or free alpha forms, suggest that those RIAs, which essentially measure the level of alpha-inhibin, considerably overestimate the levels of the active forms of inhibin in the samples and that results obtained using these assays may need reinterpretation.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Inhibins/analysis , Animals , Antibodies, Monoclonal , Antibody Specificity , Biological Assay , Cattle , Epitopes/immunology , Humans , Inhibins/immunology , Pituitary Gland , RadioimmunoassayABSTRACT
A monoclonal antibody was made after immunization of mice with the 1-32 amino terminal peptide of the alpha subunit of 32K human ovarian inhibin. The IgG2a mouse antibody reacted 6 times better with bovine 1-32 peptide than it did with 32K bovine inhibin. By contrast sheep polyclonal antibodies made by a similar method had a 29 fold bias in reactivity towards the immunizing peptide. Relative to homologous 1-32 peptide standards, the monoclonal antibody measured apparently higher amounts of immunoreactive material(s) in human (13.5 fold) and bovine (27 fold) follicular fluids than did the polyclonal anti 1-32 peptide antibodies. Immunochemical studies revealed that the epitope recognized by the monoclonal antibody was different from the major epitope recognized by the polyclonal antibodies. The monoclonal antibody reacted much better with human inhibin 1-32 sequences than with bovine (73 fold) or porcine (23 fold). Although the 32K form of human inhibin has not yet been purified, it can be inferred that the monoclonal antibody would be able to detect as little as 2 ng/ml of 32K human inhibin in competitive radioimmunoassays. The antibody must also react with some of the multiple molecular forms of inhibin found in human follicular fluids, and it was shown to function well in the quantitative immunoaffinity extraction of inhibin-like immunoreactivity from follicular fluid. It seems likely that this monoclonal antibody will prove a useful tool for research on human inhibin.
Subject(s)
Antibodies, Monoclonal , Inhibins/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Epitopes , Female , Humans , Molecular Sequence Data , Molecular Weight , Ovarian Follicle/immunology , Peptide Fragments/immunologyABSTRACT
The alpha-2-L-fucosyltransferase in human plasma has been freed from alpha-3-L-fucosyltransferase activity and purified approximately 200,000-fold by a series of steps involving ammonium sulphate precipitation, hydrophobic chromatography on Phenyl Sepharose 4B and affinity chromatography first on GDP-adipate-Sepharose and then on GDP-hexanolamine-Sepharose. The purified alpha-2-L-fucosyltransferase had a M(r) on gel filtration HPLC of 158,000 and showed optimal activity in the pH range 6.5-7.0. The enzyme transferred fucose equally well to Type 1 (Gal beta 1-3GlcNAc) and Type 2 (Gal beta 1-4GlcNAc) substrates but Type 3 (Gal beta 1-3GalNAc) structures were less efficient acceptors. Competition experiments indicated that a single enzyme species in the purified preparation was responsible for reactivity with the Type 1 and Type 2 structures. Thus the differences in conformation between the Type 1 and Type 2 disaccharides do not appear to influence the capacities of their terminal non-reducing beta-D-galactosyl residues to function as acceptor substrates for the alpha-2-L-fucosyltransferase expressed by the blood group H gene in haemopoietic tissue.
Subject(s)
Fucosyltransferases/blood , Binding, Competitive , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Fucosyltransferases/chemistry , Fucosyltransferases/genetics , Fucosyltransferases/isolation & purification , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Substrate Specificity , Galactoside 2-alpha-L-fucosyltransferaseABSTRACT
Homogenates of rabbit stomach mucosa were examined for enzymes catalysing the transfer of D-galactose from UDP-D-galactose to various low-molecular-weight acceptors of known structure. Treatment of the products with alpha and beta-D-galactosidases revealed that D-galactose was transferred in both alpha and beta-anomeric linkages. The beta-D-galactosyltransferase used N-acetylglucosamine and compounds containing terminal nonreducing beta-N-acetylglucosaminyl residues as acceptor substrates. The compounds accepting D-galactose in alpha-anomeric linkage had unsubstituted terminal non-reducing beta-D-galactosyl units or a fucose substituent on the carbon-2 position of a subterminal beta-D-galactosyl unit. Methylation analysis of the products formed with N-acetyllactosamine [beta-D-Galp(1 leads to 4)D-GlcNAcp] and 2'fucosyllactose [alpha-L-Fucp(1 leads to 2)-beta-D-Galp(1 leads to 4)D-Glcp] revealed that D-galactose was transferred to the carbon-3 position of the beta-D-galactosyl residue in both of these acceptor substrates. Competition experiments with the two substrates indicated that the transfer of D-galactose was catalysed in each case by a different alpha-3-D-galactosyltransferase. Differences were also observed in the solubility properties of the enzymes: the alpha-3-D-galactosyltransferase using acceptor substrates with unsubstituted beta-D-galactosyl residues was more readily soluble both in the presence and absence of detergents than the transferase using beta-D-galactosyl residues substituted at carbon-2 with L-fucose. These findings demonstrate that rabbit stomach mucosa has two distinct alpha-3-D-galactosyltransferases: one, which is more tightly membrane-bound, resembles the human B-gene-specified transferase in its acceptor specificity, and the second, which is a more soluble enzyme, transfers D-galactose to the same positional linkage in unsubstituted beta-D-galactosyl residues.
Subject(s)
Galactosyltransferases/isolation & purification , Gastric Mucosa/enzymology , Animals , Centrifugation, Density Gradient , Galactosyltransferases/classification , Galactosyltransferases/metabolism , Rabbits , Solubility , Substrate SpecificityABSTRACT
In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.
Subject(s)
1-Methyl-3-isobutylxanthine/pharmacology , Calcium/physiology , Cyclic AMP/analogs & derivatives , Cyclic GMP/physiology , Pituitary Gland, Anterior/metabolism , Prostaglandins E/biosynthesis , Theophylline/analogs & derivatives , 8-Bromo Cyclic Adenosine Monophosphate , Animals , Arachidonic Acids/metabolism , Calcimycin/pharmacology , Cyclic AMP/pharmacology , Cyclic AMP/physiology , Growth Hormone/metabolism , In Vitro Techniques , Male , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Potassium/pharmacology , RatsABSTRACT
The effect of insulin was tested on the rate of synthesis and release of growth hormone in cultured rat anterior pituitary cells. Concentrations of insulin between 10(-9) and 10(-7) mol/l (6--600 ng/ml or 0.15--15 mu./ml) inhibited synthesis of growth hormone; 10(-8) mol insulin/l was most effective. The effect was observed after a time-lag of at least 1 h. Insulin at concentrations between 3 x 10(-9) mol/l and 3 x 10(-7) mol/l also inhibited growth hormone secretion in 30 min incubations. The most effective insulin concentration in this case was 3 x 10(-8) mol/l. Insulin (10(-9)-10(-7) mol/l) also decreased the intracellular content of prostaglandins E and F. The effect was rapid, reaching a maximum after 30 min. Indomethacin, an inhibitor of prostaglandin synthetase, dramatically lowered the concentration of prostaglandins in the cells within 30 min; growth hormone synthesis was also decreased, but not until after 2 h of incubation. The results suggest that an initial response to insulin treatment is a lowering of intracellular levels of prostaglandins, which may then mediate a decrease in growth hormone synthesis, after a 1--2 h delay.
Subject(s)
Growth Hormone/biosynthesis , Insulin/pharmacology , Pituitary Gland, Anterior/metabolism , Prostaglandins E/physiology , Prostaglandins F/physiology , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Growth Hormone/metabolism , Indomethacin/pharmacology , Leucine/metabolism , Male , Pituitary Gland, Anterior/drug effects , Prostaglandins E/metabolism , Prostaglandins F/metabolism , Rats , Secretory Rate/drug effects , Time FactorsABSTRACT
The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.
Subject(s)
Growth Hormone/biosynthesis , Phospholipases/pharmacology , Pituitary Gland, Anterior/metabolism , Prostaglandins E/metabolism , Animals , Arachidonic Acids/pharmacology , Fatty Acids/pharmacology , Growth Hormone/metabolism , In Vitro Techniques , Male , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Rats , Secretory Rate/drug effectsABSTRACT
The addition of luteinizing hormone releasing hormone releasing hormone (LH-RH) to cultures of monolayers of rat anterior pituitary cells was shown to increase both the concentrations of prostaglandins E1 and E2 (PGE) in the cells and the release of LH over similar ranges of concentrations of LH-RH (10(-6) to 10(-10) mol/l). The peak concentration of PGE was observed after 2.5 h. The stimulation of the level of PGE in the cells by LH-RH was completely inhibited by two inhibitors of prostaglandin synthetase, which only partially inhibited the stimulation of LH release. Therefore the increased concentration of PGE was not obligatory for the effect of LH-RH on LH release. It was also shown that monobutyryl cyclic AMP stimulated the intracellular concentration of PGE and it is suggested that the stimulation of PGE levels may be mediated by increased levels of cyclic AMP in the cells after the addition of LH-RH.
Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , Luteinizing Hormone/metabolism , Pituitary Gland, Anterior/metabolism , Prostaglandins E/metabolism , Animals , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Female , Flufenamic Acid/pharmacology , Indomethacin/pharmacology , RatsSubject(s)
8,11,14-Eicosatrienoic Acid/pharmacology , Arachidonic Acids/pharmacology , Aspirin/pharmacology , Cyclic AMP/metabolism , Fatty Acids, Unsaturated/pharmacology , Indomethacin/pharmacology , Pituitary Gland, Anterior/drug effects , Pituitary Gland/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Growth Hormone/metabolism , Luteinizing Hormone/metabolism , Pituitary Gland, Anterior/metabolism , Thyrotropin/metabolismSubject(s)
Glucose/pharmacology , Growth Hormone/biosynthesis , Pituitary Gland, Anterior/metabolism , Pituitary Gland/metabolism , Animals , Carbon Radioisotopes , Deoxyglucose/pharmacology , Electrophoresis, Disc , Fructose/metabolism , Fructose/pharmacology , Galactose/metabolism , Galactose/pharmacology , Glucose/metabolism , In Vitro Techniques , Leucine/metabolism , Male , Mannose/metabolism , Mannose/pharmacology , Phlorhizin/pharmacology , Pituitary Gland, Anterior/drug effects , Protein Biosynthesis , Rats , Regression Analysis , TritiumABSTRACT
The effect of insulin on the incorporation of radioactive leucine into growth hormone was investigated by using rat anterior pituitary glands incubated in vitro. A 50% stimulation over control values was observed at insulin concentrations above 2mum (280munits/ml). The effect was specific for growth hormone biosynthesis, over the range 1-5mum-insulin (140-700munits/ml). Lower more physiological concentrations had no significant effect in this system. Above 10mum (1.4 units/ml) total protein synthesis was also increased. The stimulation of growth hormone synthesis could be partially blocked by the addition of actinomycin D, suggesting that RNA synthesis was involved. Insulin was found to stimulate the rate of glucose utilization in a similar way to growth hormone synthesis. 2-Deoxyglucose and phloridzin, which both prevented insulin from stimulating glucose utilization, also prevented the effect of insulin on growth hormone synthesis. If glucose was replaced by fructose in the medium, the effect of insulin on growth hormone synthesis was decreased. We conclude that the rate of utilization of glucose may be an important step in mediating the effect of insulin on growth hormone synthesis.