The Association of the Adaptor Complexes AP1 and AP3 with GLUT4 Vesicles: Implications for GLUT4 Compartmentalisation

1999 ◽  
Vol 27 (3) ◽  
pp. A100-A100
Author(s):  
Alison K. Gillingham ◽  
Françoise Koumanov ◽  
Paul R. Pryor ◽  
Barbara J. Reaves ◽  
Geoffrey D. Holman
Keyword(s):  
Plasma Membrane ◽  
Western Blotting ◽  
Subcellular Localisation ◽  
Density Gradients ◽  
Transferrin Receptors ◽  
Rat Adipocytes ◽  
In Vitro Effects ◽  
Adaptor Recruitment

By using glycerol density gradients of post-plasma membrane supernatants from rat adipocytes, we have been able to separate the compartment containing GLUT4 from that containing transferrin receptors. The major pool of GLUT4 (≈ 80%) is localised to dense fractions, which also contain VAMP2, while the remainder is in lighter fractions, some of which also contain transferrin receptors. AP1 and AP3 complexes are detected in two fractions, the heavier of which co-sediments with the major pool of GLUT4. Nycodenz gradients have been used to examine the in vitro effects of GTP-γ-S on adaptor recruitment. On addition of GTPγS, GLUT4 vesicles fractionate as a heavier population of vesicles, which we suggest is due to a coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with API, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of AP1 and AP3 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased 3-fold by the addition of GTP-γ-S and an ATP regenerating system to the post-plasma membrane lysate. and 4-fold by carrying out the incubation at 37°C compared with 0°C. These data demonstrate that GLUT4 subcellular localisation can be studied in isolated fractions and that GLUT4 compartments are associated with at least two distinct adaptor complexes.

Association of AP1 adaptor complexes with GLUT4 vesicles

1999 ◽  
Vol 112 (24) ◽  
pp. 4793-4800 ◽  
Author(s):  
A.K. Gillingham ◽  
F. Koumanov ◽  
P.R. Pryor ◽  
B.J. Reaves ◽  
G.D. Holman
Keyword(s):  
Temperature Dependence ◽  
Western Blotting ◽  
Brefeldin A ◽  
Intracellular Sorting ◽  
In Vitro Effects ◽  
Gamma S ◽  
Isolated Cell

Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes.

In Vitro Effects of Glucocorticoid on Glucose Transport in Rat Adipocytes: Evidence of a Post-Receptor Coupling Defect in Insulin Action1

1984 ◽  
Vol 96 (6) ◽  
pp. 1893-1902 ◽  
Author(s):  
Nobuaki WATANABE ◽  
Masashi KOBAYASHI ◽  
Hiroshi MAEGAWA ◽  
Osamu ISHIBASHI ◽  
Yasumitsu TAKATA ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Rat Adipocytes ◽  
Receptor Coupling ◽  
In Vitro Effects

scholarly journals OR09-05 Expression of SLC35F1 in the Plasma Membrane of Cells of Aldosterone Producing Cell Clusters (APCCs) and Its Possible Role in Aldosterone Synthesis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Emily Goodchild ◽  
Jan Stoetaert ◽  
William Drake ◽  
Kenneth Linton ◽  
Morris Jonathan Brown
Keyword(s):  
Plasma Membrane ◽  
Adrenal Cortex ◽  
Ex Vivo ◽  
Subcellular Localisation ◽  
Serial Sections ◽  
Nucleotide Sugar ◽  
Pathological Subtype ◽  
Aldosterone Production ◽  
H295r Cells

Abstract Background Genetic mutations and histological appearances suggest that APCCs are precursors to some aldosterone producing adenomas (APA). They are hypothesised to contribute to post-operative non-cure and recurrence of primary aldosteronism (PA) but are currently undetectable pre-operatively. SLC35F1 is a possible nucleotide sugar transporter. On microarray it is highly expressed in APCCs, but not in the rest of the adrenal cortex (1). Our aim was to investigate the role of SLC35F1 in APCCs, determine its subcellular localisation and establish whether expression is consistent with pathological APCC subtypes (as suggested by recent evidence from in situ metabolomic studies (2)). Methods Comparative bioinformatic analysis of the SLC35F1 amino acid sequence was carried out. Ex vivo 4uM adrenal sections, from 6 PA patients in the MATCH study, were stained on serial sections with anti-CYP11B2 (Gomez-Sanchez) or anti-SLC35F1 (Novus NBP1-86755). In vitro, H295R cells were transfected with SLC35F1 cDNA. Subcellular localisation of SLC35F1-GFP was studied by comparison to organelle markers (golgin 97, RAB11, wheat germ agglutinin, calnexin and Tom20) using confocal microscopy. Overexpression and siRNA knock-down in H295R cells was correlated to aldosterone production. Results SLC35F1 is a decamembrane-spanning transporter molecule predicted to have a negatively charged pocket in the substrate binding site, implying a transport substrate with a positive charge. Strong staining of CYP11B2 in clusters of cells in adrenal cortex, consistent with APCCs, were present in all six adrenal glands (25 APCCs). Serial sections showed specific SLC35F1 staining of APCCs, in cytoplasm and plasma membrane. SLC35F1 staining was absent from normal cortex. 12 APCCs (48%) were SLC35F1-negative. Visualisation of transiently expressed SLC35F1 demonstrated localisation to the endoplasmic reticulum in H295R cells (Pearson’s coefficient r=0.758) with no plasma membrane localisation (r=-0.07). Preliminary transfection data suggest direct involvement in aldosterone production. Conclusions Expression of SLC35F1 in the plasma membrane and cytosol of APCC cells supports a role in pathological aldosterone production by APCCs. The inferred transport substrate of SLC35F1 is the NAD(P)(H) precursor, nicotinamide riboside, a positively-charged nucleotide sugar. If confirmed, the essential requirement for NAD(P)H in steroidogenesis, and heterogeneity of SLC35F1-staining in APCCs, is consistent with a metabolically active, possibly pathological, subtype of APCCs. Detection of SLC35F1 in vivo may therefore facilitate sub-classification of PA patients. 1). Nishimoto et. al. PNAS. 2015 Aug 18;112(33):E4591-9. 2). Murakami et. al. 2019 Hypertension. In press.

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

1992 ◽  
Vol 84 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Marta Roldan ◽  
Andres Belver ◽  
Pilar Rodriguez-Rosales ◽  
Nuria Ferrol ◽  
Juan Pedro Donaire
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
In Vitro Effects ◽  
Plasma Membrane Proton Pump

Effects of Atrazine and Fenoxaprop-Ethyl on Capacitation and the Acrosomal Reaction in Boar Sperm

2009 ◽  
Vol 28 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Ramiro Maravilla-Galván ◽  
Reyna Fierro ◽  
Humberto González-Márquez ◽  
Sandra Gómez-Arroyo ◽  
Irma Jiménez ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Human Health ◽  
Endocrine Disruptor ◽  
Acrosome Reaction ◽  
Acrosomal Reaction ◽  
Boar Sperm ◽  
In Vitro Effects ◽  
Membrane Destabilization

Atrazine is a herbicide of the chloro-s-triazine family. It inhibits photosynthesis in plants and is an endocrine disruptor, but its effects on human health are controversial. Fenoxaprop-ethyl, an aryloxy phenoxyalkanoic acid herbicide, inhibits the biosynthesis of fatty acids and provokes depolarization of membranes. The aim of this study is to evaluate the in vitro effects of both herbicides on capacitation, spontaneous acrosome reaction (SAR) and progesterone-induced acrosome reaction (PIAR) in boar sperm. Sperm capacitation is done in TALP-HEPES media for 4 hours. Capacitation and SAR are evaluated immediately; PIAR, 30 minutes later. LC50 for fenoxaprop-ethyl is 60 mM and 40 mM for atrazine. Fenoxaprop-ethyl induces capacitation at 60 mM and SAR at all concentrations, also increases significantly PIAR. Atrazine decreased capacitation whereas increase significantly SAR and PIAR at all concentrations. It seems that fenoxaprop-ethyl and atrazine accelerate the capacitation and the acrosomal reaction, possibly via plasma membrane destabilization.

The Anti-Allodynic Gabapentinoids

2016 ◽  
Vol 23 (1) ◽  
pp. 40-55 ◽  
Author(s):  
Sascha R. A. Alles ◽  
Peter A. Smith
Keyword(s):  
Neuropathic Pain ◽  
Plasma Membrane ◽  
Nerve Terminals ◽  
Membrane Expression ◽  
Release Process ◽  
Voltage Gated ◽  
Plasma Membrane Expression ◽  
Accessory Subunits ◽  
In Vitro Effects

The gabapentinoids (pregabalin and gabapentin) are first line treatments for neuropathic pain. They exert their actions by binding to the α2δ accessory subunits of voltage-gated Ca2+ channels. Because these subunits interact with critical aspects of the neurotransmitter release process, gabapentinoid binding prevents transmission in nociceptive pathways. Gabapentinoids also reduce plasma membrane expression of voltage-gated Ca2+ channels but this may have little direct bearing on their therapeutic actions. In animal models of neuropathic pain, gabapentinoids exert an anti-allodynic action within 30 minutes but most of their in vitro effects are 30-fold slower, taking at least 17 hours to develop. This difference may relate to increased levels of α2δ expression in the injured nervous system. Thus, in situations where α2δ is experimentally upregulated in vitro, gabapentinoids act within minutes to interrupt trafficking of α2δ subunits to the plasma membrane within nerve terminals. When α2δ is not up-regulated, gabapentinoids act slowly to interrupt trafficking of α2δ protein from cell bodies to nerve terminals. This improved understanding of the mechanism of gabapentinoid action is related to their slowly developing actions in neuropathic pain patients, to the concept that different processes underlie the onset and maintenance of neuropathic pain and to the use of gabapentinoids in management of postsurgical pain.

scholarly journals In vitro effects of palmitylcarnitine on cardiac plasma membrane Na,K-ATPase, and sarcoplasmic reticulum Ca2+-ATPase and Ca2+ transport.

1979 ◽  
Vol 254 (24) ◽  
pp. 12404-12410
Author(s):  
R.J. Adams ◽  
D.W. Cohen ◽  
S. Gupte ◽  
J.D. Johnson ◽  
E.T. Wallick ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
In Vitro Effects
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