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Biomedical subjects

M Wibo

Publications and source records attributed to M Wibo.

At least 37 records · Page 2Linked to original sources
PubMed1988

Pharmacologic relevance of dihydropyridine binding sites in membranes from rat aorta: kinetic and equilibrium studies.

The kinetic features of the interaction of dihydropyridines with rat aortic smooth muscle were investigated in parallel mechanical and binding studies. The inhibitory action of (+)-PN200-110 and nisoldipine on contractions evoked by potassium chloride depolarization was characterized by a pronounced time dependency, in which the inhibition increased slowly after depolarization to attain a steady-state value, while with (-)-PN200-110 and (+)-Bay K8644 the inhibition was almost instantaneous. To explain these observations, specific binding sites for dihydropyridines were studied in membranes isolated from rat aorta, using [3H](+)-PN200-110 as a radioligand. We found that the time course of the development of inhibition of potassium chloride-evoked contractions by various concentrations of (+)-PN200-110 paralleled the time course of [3H](+)-PN200-110 binding to isolated membranes and that the level of inhibition was predictable from the level of occupation of these binding sites. These results indicate that depolarization increases the affinity of calcium channels for dihydropyridines in vascular smooth muscle and that the time course of the inhibitory effect on contraction is determined by the time course of association with the high-affinity state of the channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

Subcellular localization of [3H]-nitrendipine binding sites in guinea-pig ileal smooth muscle.

The binding of [3H]-nitrendipine was studied in microsomal fractions isolated from guinea-pig ileal smooth muscle. Only one class of specific binding sites was detected, with a KD of 0.4 nM. For various dihydropyridine derivatives, including the stereoisomers of nimodipine and the 'Ca agonist' Bay K 8644, the potency for inhibition of [3H]-nitrendipine binding correlated well with the reported pharmacological potency in smooth muscle preparations. To establish the subcellular localization of [3H]-nitrendipine binding sites, untreated and digitonin-treated microsomal fractions were subfractionated by isopycnic density gradient centrifugation. The density distribution of [3H]-nitrendipine binding was markedly shifted by digitonin towards higher densities, as were the distributions of 5'-nucleotidase and [3H]-ouabain binding, whereas the distributions of NADPH:cytochrome c reductase and NADH:cytochrome c reductase were hardly modified by digitonin. It is concluded that most, if not all, [3H]-nitrendipine binding sites in guinea-pig ileal smooth muscle are present in the plasma membrane, in agreement with the postulated mode of action of dihydropyridines as inhibitors of plasmalemmal Ca channels.

5'-Nucleotidase↗
PubMed1983

Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

Acid Anhydride Hydrolases↗
PubMed1981

Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle.

ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5'-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivity of K+, vanadate and calmodulin. In this respect, the oxalate-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.

Animals↗
PubMed1981

A calmodulin-stimulated Ca2+ pump in rat aorta plasma membranes.

An ATP-driven Ca2+-transport system has been characterized in a microsomal fraction from rat aorta. Calmodulin enhanced 2.5-fold 45Ca accumulation by EGTA-treated microsomes incubated with 10 microM Ca2+ (in the absence of oxalate) by increasing markedly the apparent affinity of the transport system for Ca2+. The ionophore A23187 induced a rapid release of the sequestered 45Ca. The vesicles that took up 45Ca were distributed like plasmalemmal marker enzymes when the microsomal fraction was subfractionated by density gradient centrifugation. In particular, these vesicles were markedly shifted towards higher equilibrium densities after addition to the microsomes of 0.2 mg digitonin/mg protein before isopycnic centrifugation. We conclude that the calmodulin-stimulated Ca2+ pump associated with the microsomal fraction is located in plasmalemmal elements.

Animals↗
PubMed1981

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes.

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.

Animals↗
PubMed1980

Subcellular location of semicarbazide-sensitive amine oxidase in rat aorta.

With tyramine as substrate, a considerable part of the amine oxidase activity of rat aorta was inhibited by 0.1 mM semicarbazide. The residual activity was little affected by 1 mM semicarbazide. Oxidation of 5-hydroxytryptamine was not inhibited by 0.1 mM semicarbazide. The subcellular location of the semicarbazide-sensitive and semicarbazide-resistant amine oxidases was investigated by analytical density gradient centrifugation. The semicarbazide-resistant enzyme was identified with the mitochondrial monoamine oxidase, located in the outer envelope of mitochondria. The semicarbazide-sensitive amine oxidase was ascribed to the plasma membrane because it was distributed like 5'-nucleotidase and (oligomycin-insensitive) Mg2+-ATPase in various fractionation experiments, and markedly shifted by digitonin towards higher equilibrium densities in sucrose gradient.

Animals↗
PubMed1979

The dolichol pathway of protein glycosylation in rat liver. Stimulation by GTP of the incorporation of N-acetylglucosamine in endogenous lipids and proteins of rough microsomes treated with pyrophosphate.

Incorporation of N-acetylglucosamine into endogenous lipid and protein acceptors was investigated on heavy microsomes from rat liver, incubated with UDP-N-acetyl[14C]glucosamine and GDP-mannose in the absence of detergent. This subcellular preparation derived for 95% or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N-acetylglucosamine units to glycoproteins. The label was found almost exclusively in dolichyl diphosphate N-acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N-acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N'-diacetylchitobiose, with dolichyl diphosphate oligosaccharides and with proteins. The time-course of N-acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP-mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N-acetylglucosamine in protein. It is concluded that rough microsomes are capable of assembling dolichol-linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N'-diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N'-diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N'-diacetylchitobiose from dolichyl diphosphate N-acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaccharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts.

Acetylglucosamine↗
PubMed1977

Incorporation of N-acetylglucosamine into endogenous acceptors of rough microsomes from rat liver: stimulation by GTP after treatment with pyrophosphate.

Upon incubation of UDP-N-acetyl[14C]glucosamine and GDP-mannose with rough microsomes from rat liver, GlcNAc incorporation occurred mostly into acid-labile products extractable by chloroform/methanol that were presumed to be dolichol pyrophosphate GlcNAc and, for a small part, dolichol pyrophosphate N,N'-diacetylchitobiose. The production of this latter glycolipid was greatly enhanced when rough microsomes, previously treated with pyrophosphate, were incubated in the presence of GTP. Furthermore, under these particular conditions, high levels of radioactivity were obtained in products precipitated by trichloroacetic acid and not extracted by chloroform/methanol. The label appeared in acid-labile and in acid-resistant products, presumed to be dolichol pyrophosphate mannosylated oligosaccharides, and glycoproteins, respectively. From these results, dolichol pyrophosphate N,N'-diacetylchitobiose appears to be an obligate metabolic intermediate in protein glycosylation by this system. Whether or not dolichol pyrophosphate N,N'-diacetylchitobiose must be mannosylated prior to transfer of the N,N'-diacetylchitobiose moiety to protein is an open matter, because GlcNAc was also incorporated into acid-resistant products when the reaction was carried out in absence of GDP-mannose.

Acetylglucosamine↗