[38] Preparation of synaptosomes and mitochondria from mammalian brain

López‐Pérez, Manuel J.

doi:10.1016/0076-6879(94)28040-1

Cited by 16 publications

(8 citation statements)

References 9 publications

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“…Rat cortical synaptosomes were prepared essentially as described by Lopez‐Perez (1994). In short, the method employs conventional differential centrifugations in a continuous sucrose gradient followed by a purification procedure based on a two‐phase aqueous system of dextran 500 and polyethylene glycol 4000 (Brooks and Norris‐Jones 1994).…”

Section: Methodsmentioning

confidence: 99%

Alanine metabolism, transport, and cycling in the brain

Bröer¹,

Bröer²,

Hansen³

et al. 2007

Journal of Neurochemistry

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Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na(+)-dependent transporters with properties similar to system B(0) isoform B(0)AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.

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Section: Methodsmentioning

confidence: 99%

Alanine metabolism, transport, and cycling in the brain

Bröer¹,

Bröer²,

Hansen³

et al. 2007

Journal of Neurochemistry

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“…Isolation and purification of rat brain mitochondria and microsomes Isolation and purification of brain mitochondria was performed as described [26]. Brains were removed, washed, and homogenized in Medium A [320 mM sucrose, 1 mM potassium EDTA, 0.1 % fatty acid-free BSA (FAF-BSA), 10 mM Tris-HCl pH 7.40].…”

Section: Fluorescent Image Acquisition and Processingmentioning

confidence: 99%

Mitochondrial association of alpha-synuclein causes oxidative stress

Parihar¹,

Parihar²,

Fujita

et al. 2008

Cell. Mol. Life Sci.

299

245

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Alpha-synuclein is a neuron-specific protein that contributes to the pathology of Parkinson's disease via mitochondria-related mechanisms. The present study investigated possible interaction of alpha-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing alpha-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that alpha-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of alpha-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by alpha-synuclein.

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“…Mouse cortical synaptosomes were prepared essentially as described by Lopez-Perez [17]. Briefly, the method employs differential centrifugation in a continuous sucrose gradient followed by a purification procedure based on a two-phase aqueous system of dextran 500 and PEG [poly(ethylene glycol)] 4000 [18].…”

Section: Synaptosomesmentioning

confidence: 99%

“…The amino-acid-induced currents (rows [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] in the mB 0 AT1 column are taken from [20]. Amino-acid-induced currents were determined 3-6 days after injection, by discharging substrate-containing solutions directly into the bath, close to the oocyte surface.…”

Section: Substrate Specificity Of Mb 0 At2 (V7-3)mentioning

confidence: 99%

The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2)

Bröer

Tietze

Kowalczuk

et al. 2005

Biochemical Journal

123

112

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Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 microM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na+-dependent, Cl--independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na+-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na+/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed.

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[38] Preparation of synaptosomes and mitochondria from mammalian brain

Cited by 16 publications

References 9 publications

Alanine metabolism, transport, and cycling in the brain

Alanine metabolism, transport, and cycling in the brain

Mitochondrial association of alpha-synuclein causes oxidative stress

The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2)

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