Mitochondria from Hamster Brown‐Adipose Tissue

Abstract

1.The oxidation of palmitoyl-L-carnitine by mitochondria from the brown adipose tissue of cold-adapted hamsters is highly dependent on the nature of the incubation medium. No respiration occurs in media containing 200 mM sucrose or 100 mM choline chloride, while 100 mM NaCl allows rapid respiration.2. Media containing 100 mM KCl allow steadily increasing rates of palmitoyl-carnitine oxidation. High initial rates may be obtained by adding valinomycin, or by decreasing the concentration of KC1.3. Respiration can proceed in media containing sucrose or choline chloride if they are made sufficiently hypotonic. Pyruvate and 2-oxoglutarate oxidation is also sensitive to tonicity, whereas ~-glycerol-3-phosphate oxidation is unaffected.4. A close correlation was found between the sucrose-impermeable space and the rate of palmitoyl-carnitine oxidation under all conditions examined, a low space determination being associated with respiratory inhibition.5. Mitochondria stored in 250 mM sucrose have a very low sucrose impermeable space. Evidence is presented that this space must be increased by hypotonic conditions or by ion permeation before respiration can occur. Mitochondria stored in 100 mM KC1, 20 mM K-TES have a much larger sucrose-impermeable space and can immediately oxidize palmitoyl-carnitine. 6.The ion permeability properties of these mitochondria are discussed, together with the nature of the respiratory inhibition associated with matrix condensation.This work arose from observations that the oxidation of palmitoyl-carnitine by freshly prepared, uncoupled [l --51 hamster brown-adipose-tissue mitochondria was highly dependent on the nature of the major component of the incubation medium. The effects were so striking that a systematic study was undertaken, in the course of which it became apparent that all conditions which resulted in a high degree of condensation of the matrix compartment resulted in an inhibition of palmitoylcarnitine oxidation. This paper presents these results, together with evidence for a causal relationship between matrix condensation and respiratory inhibition. While such inhibition is unlikely to occur under physiological conditions, its importance as a factor complicating the interpretation of experimentation in vitro will be discussed. Abbreviation. K-TES, potassium N-tris(hydroxymethy1)-Enzyme. Adenylate kinase (EC 2.7.4.3).2-aminoethane sulphonate. MATERIALS AND METHODSSyrian hamsters were acclimatised for a t least 15 days a t 4 f 1 "C, with food and water ad libitum. The animals were decapitated, and pooled interscapular, cervical and axillary brown adipose tissue was rapidly excised into ice-cold 250 mM sucrose. Mitochondria were then prepared as described by Hittelman et al. [4] for rat brown adipose tissue and stored a t 0 "C in 250 mM sucrose. Alternatively, the mitochondria were taken before the final centrifugation, resuspended in 100 mM KCl, 20mM K-TES, pH 7.2, and resuspended h a l l y in this medium. Protein was determined by the biuret method.All experiments were performed ...

On the mechanism of uncoupler‐dependent inhibition of acyl‐carnitine oxidation by rat liver mitochondria

“…This has led us to suggest that the inhibitory effect of the uncoupler, which is expressed only in sucrose containing media, is primarily due to a collapse of the osmotic potential of the matrix. Analogous conclusions have been reached both with respect to the dinitrophenol (DNP) + valinomycin induced inhibition of glutamate/malate oxidation by rat liver mitochondria in sucrose media [6], and the slow rate of palmitoyl-carnitine oxidation by brown * 2(N-hydr oxyethylpiperazin-N-yl)ethanesulphonate adipose tissue mitochondria suspended in iso-osmolar sucrose media [7]. The presence of a proton gradient across the inner membrane is therefore at best of secondary significance.…”

“…(mMl between decreased respiratory rates and sucrose impermeable spaces [7]. Increasing concentrations of sucrose have also been associated with decreased glutamate/malate induced NADH fluorescence [12], which has been taken as evidence that substrate transport into the matrix has been inhibited.…”

The Relationship between Extramitochondrial Ca2+ Concentration, Respiratory Rate, and Membrane Potential in Mitochondria from Brown Adipose Tissue of the Rate

“…The incubation medium consisted of 125 mM purified sucrose (purified on a Dowex 50 WX8 ionexchange column), 20 mM Tris, 5 mM glycerol 3-phosphate (dicyclohexylammonium salt), 4 pM rotenone and 0.2 % fattyacid-free bovine serum albumin. The low sucrose concentration was used in order to avoid osmotic condensation of the mitochondria [8].…”

The bioenergetics of brown adipose tissue mitochondria

“…One consequence of this conductance is that the mitochondria show a decreased ability to maintain potassium or phosphate in the matrix during preparation [22,35], and as a result they display an extremely condensed matrix in 0.25 M sucrose [37]. This is manifested in a reduced ability to oxidise NADlinked substrates, which may be countered by decreasing the osmolarity of the incubation medium [37].…”