cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes

1997 ◽  
Vol 273 (4) ◽  
pp. G842-G848 ◽  
Author(s):  
Sunil Mukhopadhayay ◽  
M. Ananthanarayanan ◽  
Bruno Stieger ◽  
Peter J. Meier ◽  
Frederick J. Suchy ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Plasma Membrane Vesicles ◽  
Short Term ◽  
Kinase A

Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles

2021 ◽  
Author(s):  
Nikolas K. Teiwes ◽  
Ingo Mey ◽  
Phila C. Baumann ◽  
Lena Strieker ◽  
Ulla Unkelbach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
Author(s):  
Christie Cefaratti ◽  
Andrea Romani ◽  
Antonio Scarpa
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Channel Blockers ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Two Phases Of Ferricyanide Reductase Activity In Ehrlich Cell Plasma Membranes

1992 ◽  
Vol 47 (11-12) ◽  
pp. 929-931 ◽  
Author(s):  
Antonio del Castillo-Olivares ◽  
Javier Márquez ◽  
Ignacio Núñez de Castro ◽  
Miguel Angel Medina
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Cell Plasma Membrane ◽  
Ferricyanide Reductase ◽  
Ehrlich Cell ◽  
Cell Plasma ◽  
Two Phases

Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases

scholarly journals Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

2015 ◽  
Vol 36 (3) ◽  
pp. 866-883 ◽  
Author(s):  
Annika Sommerfeld ◽  
Roland Reinehr ◽  
Dieter Häussinger
Keyword(s):  
Bile Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Rat Hepatocytes ◽  
Β1 Integrin ◽  
Dependent Manner ◽  
Mitogen Activated Protein ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
Kinase A

Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

scholarly journals Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

2004 ◽  
Vol 3 (1) ◽  
pp. 190-199 ◽  
Author(s):  
Alejandro Cassola ◽  
Marc Parrot ◽  
Susana Silberstein ◽  
Beatrice B. Magee ◽  
Susana Passeron ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Double Mutant ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Gfp Fusion Protein ◽  
Kinase A

ABSTRACT The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

scholarly journals Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Peter J. A. van den BROEK ◽  
Angeline E. van GOMPEL ◽  
Marijke A. H. LUTTIK ◽  
Jack T. PRONK ◽  
Carla C. M. van LEEUWEN
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Transport Systems ◽  
Sugar Accumulation ◽  
Plasma Membrane Vesicles ◽  
Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

scholarly journals The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

1991 ◽  
Vol 273 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Darnanville ◽  
R Bredoux ◽  
K J Clemetson ◽  
N Kieffer ◽  
N Bourdeau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Glycoprotein Ib ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

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