1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 pM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicated by a dramatic increase in the phosphorylation state of adenine nucle-'otides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix.2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 pM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20 -50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP.3. GDP (100 pM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier.4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro ; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.Investigations on the mechanism of non-shivering heat production in brown adipose tissue have revealed a close linkage between the thermogenic activity of the tissue and the degree of energy coupling I in isolated brown fat mitochondria [l, 21, suggesting a hormone-regulated uncoupling-recoupling-device of respiratory chain phosphorylation as the basis mechanism of the calorigenic process [3,4]. In view of the high energy dissipation in the tissue mitochondria during increased thermogenesis, cellular ATP-production should be considerably restricted. Actually a significant decrease in the ATP level is found in brown adipose tissue during active thermogenesis and in isolated adipocytes after addition of norepinephrine [5-71; on the other hand the ATP-formation in brown fat is obviously sufficient to maintain ATPdependent metabolic functions of the tissue, especially fatty acid activation.Two reasons may be advanced for the unexpectedly high ATP level in brown fa...
To determine whether the release of acrolein from oxazaphosphorinane-cytostatics contributes to their cytotoxic action, the effect of 4-hydroperoxycyclophosphamide, 4-hydroperoxy-semi-cyclophosphamide, 4-hydroperoxy-dechloro-cyclophosphamide, and acrolein on murine L 1210 leukemia cells in vitro was compared by measuring the median survival time (MST) after transplantation of the tumor cells in DBA2/Han mice. We found that only 4-hydroperoxycyclophosphamide, which is able to release both acrolein and the alkylating metabolite phosphoramide-mustard, decreased the transplantability of L 1210 cells, while the structurally analogous 4-hydroperoxy-dechloro-cyclophosphamide and 4-hydroperoxy-semi-cyclophosphamide, which under physiological conditions only release acrolein but no alkylating split products showed no cytotoxicity. Acrolein itself showed only a marginal effect, when administered in concentrations equivalent to the release of acrolein from the oxazaphosphorinane-derivatives in test. In this case, however, significant lysis of the L 1210 cells was observed by estimating dye exclusion, while acrolein released intracellularly from 4-hydroperoxy-oxazaphosphorinane-compounds did not. This points to a different mechanism of the cytotoxic action of extracellular acrolein and acrolein released intracellularly from activated oxazaphosphorinane-compounds. The results suggest that the cytotoxic effect of activated cyclophosphamide is based on the alkylating moiety of the molecule. Neither the 4-hydroperoxy-group nor the activated oxazaphosphorinane-ring itself, nor acrolein released intracellularly during toxification of activated cyclophosphamide exert a direct cytotoxic effect. Thus, the release of acrolein from activated CP apparently does not contribute to the cytotoxicity of CP in vivo.
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