This study was designed to elucidate the mechanism of action of norepinephrine in brown fat. Previous studies have shown that norepinephrine can increase brown adipose tissue respiration rates up to 40-fold. One explanation of hormone action involves stimulation of (Na+,K+)-ATPase in the cell membrane followed by increased mitochondrial respiration coupled to the synthesis of ATP. An alternate hypothesis suggests that brown fat respiration is uncoupled from ATP synthesis by norepinephrine.

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... rt for this theory comes from the discovery of a protein uniquely present in the membranes of brown fat mitochondria which can uncouple respiration from ATP synthesis. The protein, which is a hydroxyl ion carrier, is inactive in the presence of ATP and ADP. Since the nucleotide binding constant of the protein is in the micromolar concentration range, control of coupling via physiological fluctuations of cytosolic nucleotides seemed unlikely. Data from the present study suggest that the uncoupling protein is regulated by cytosolic ATP. Assay of total cellular adenine nucleotides and the nucleotides in isolated brown fat mitochondria indicates that 68% of the adenine nucleotides of brown fat cells are compartmented within the mitochondria, whereas 40% of the aqueous intracellular volume is mitochondrial. The measured Ki of external ATP for inhibition of mitochondrial respiration is not in the micromolar concentration region but is about 1 m~ because of the presence of 1 m~ Mg+ in the media and an excess of the uncoupling protein in the mitochondrial membranes. When the mitochondria are coupled, the electrogenic character of the adenine nucleotide transporter excludes external ATP from the mitochondrial matrix. Calculations indicate that when mitochondria are coupled most tissue ATP is cytosolic and the concentration is 2.5 times the Ki of ATP. When isolated brown fat mitochondria are uncoupled and media ATP/ADP ratios are over 5, mitochondrial matrix ATP levels increase 3-fold. When mitochondria are uncoupled in the cell, cellular ATP levels may remain high, but cytosolic ATP levels may fall well below 1 m~ due to redistribution of ATP. Addition of norepinephrine causes a 20% decrease in total cellular ATP, possibly due to activation of (Na+,K+)-ATPase. This may partially activate the uncoupling protein, allowing some ATP into the matrix, further lowering cytosolic ATP and continuously amplifying the initial decrease in cytosolic ATP. Estimation of cytosolic nucleotides in the basal and hormonally stimulated states of brown fat cells indicates that regulation of the uncoupling protein by ATP is possible.