Bonaventura Ruiz-Montasell

CHARACTERIZATION OF TWO DISTINCT INTRACELLULAR GLUT4 MEMBRANE POPULATIONS IN MUSCLE FIBER. DIFFERENTIAL PROTEIN COMPOSITION AND SENSITIVITY TO INSULIN

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide ...

Early induction of Na'-dependent uridine uptake in the regenerating rat liver

Na+‐dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham‐operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased V max of uridine uptake (3.7 vs. 1.4 pmol/mg prot/3 s) without any change in Km (6μM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na+ gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.

Uridine transport in basolateral plasma membrane vesicles from rat liver.

SummaryThe characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were KM 8.9 μm and Vmax 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na+ counterions determined uridine transport rates (SCN− > NO3−> Cl− > SO42−). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.

Up-regulation of system A activity in the regenerating rat liver

System A activity for neutral amino acid transport, measured as the MeAIB‐sensitive Na+‐dependent l‐alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na+‐dependent transporters, like system ASC (MeAIB‐insensitive Na+‐dependent l‐alanine transport) and the nucleoside carrier show similar inductions. Up‐regulation of system A is not explained by changes in the dissipation rate of the Na+ transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N‐ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up‐regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na+ transmembrane gradient.

Na+,K(+)-ATPase expression during the early phase of liver growth after partial hepatectomy.

Na+,K+‐ATPase expression has been studied in the early phase of liver growth after partial hepatectomy to ascertain whether its increased activity is due to stable effects, involving de novo synthesis and insertion of pumps into the plasma membrane. Na+,K+‐ATPase activity progressively increases after partial hepatectomy, reaching a three‐fold induction above basal values 12 h after surgery. mRNA amounts of both α 1 and β 1 subunits are rapidly increased up to two‐fold for α 1 and nearly three‐fold for β 1, at 9 and 12 h post‐hepatectomy, respectively. This correlates with increased abundance of both subunit proteins. The results prove that the increase of Na+,K+‐ATPase activity correlates with higher expression of both subunit proteins and mRNAs, although the characteristics of the induction suggest that some translational and post‐translational events may be equally involved in the increased activity of the pump.

Coordinate induction of Na(+)-dependent transport systems and Na+,K(+)-ATPase in the liver of obese Zucker rats.

Solute uptake into liver plasma membrane vesicles from either lean or obese Zucker rats was monitored. D-Glucose and L-leucine uptakes at physiological concentrations of the substrate were not different in lean and obese Zucker rats. In agreement with a previous report (Ruiz et al. (1991) Biochem. J. 280, 367-372) L-alanine uptake was significantly enhanced in those preparations from obese animals. Na(+)-coupled uridine transport was markedly enhanced also in obese rats. The effect was due to an increase in Vmax (5.5 +/- 0.6 vs. 2.1 +/- 0.2 pmol/mg protein per 3 s, P < 0.01) without any significant change in Km (11.0 +/- 2.8 vs. 9.0 +/- 2.7 microM for obese and lean rats, respectively). Na+,K(+)-ATPase activity was also higher in liver plasma membrane vesicles from rat liver and it correlated with a higher amount of alpha 1-subunit protein in both, plasma membrane vesicles and homogenates from obese rat livers. In summary, in the hypertrophic liver of obese Zucker rats a coordinate induction of several Na(+)-dependent transport systems occurs and, in order to sustain the metabolic pressure associated with this adaptation, a significant induction of the Na+,K(+)-ATPase expression is also found. These data also provide new evidence for regulation of the recently characterized Na(+)-dependent nucleoside transporter.

Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms.

Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co‐localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP‐25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.