Mitochondrial and Cytosolic NADPH Systems and Isocitrate Dehydrogenase Indicator Metabolites during Ureogenesis from Ammonia in Isolated Rat Hepatocytes

Abstract

1. Citrate, isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions thereof, obtained by the digitonin and silicone oil fractionation technique.2. Calculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV.3. Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, as demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reversed.4. It is concluded that during ureogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 pM, below the K, (isocitrate) of the NADlinked enzyme but in the range of that for the NADP-linked enzyme.5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondria1 inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate.6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.Isocitrate dehydrogenases occur both intramitochondrially and extramitochondrially in rat liver [l] ; NADP-dependent enzymes are present in both spaces, whereas an NAD-dependent enzyme is present only intramitochondrially (see [2] for a review). The clear concept, put forward by Goebell and Klingenberg [3], of the dichotomy of mitochondrial isocitrate oxidation into a non-equilibrium, regulated (or regulatory) pathway catalyzed by the NAD-dependent enzyme and an equilibrium pathway catalyzed by the NADP-dependent enzyme implies that the compoEnzymes. Isocitrate dehydrogenase (NAD+) (EC 1.1.1.41); isocitrate dehydrogenase (NADP+) (EC 1.1.1.42).nents of the mitochondrial isocitrate dehydrogenase system may be utilized as 'indicator metabolites' for the mitochondrial NADP system, provided that the activity is high enough (see below). Regardins the extramitochondrial (cytosolic) space, there are no major complications to utilize the isocitrate dehydrogenase system as an indicator for the redox potential of cytosolic free NADPH. Veech etal. [4] have, in fact, utilized the isocitrate dehydrogenase system for this purpose ; however, their analytical data were the total tissue levels rather than the...

Effect of ammonia on mitochondrial and cytosolic NADH and NADPH systems in isolated rat liver cells

“…The mitochondrial free-NAD redox-state (table 2) The data in table 4 shows that the presence of ammonia has no effect on the free-NADP redox-state potential difference (37-38 mV) across the mitochondrial membrane. In similar experiments, Sies et al [23] found that the NADP redox potential difference in the absence of ammonia was 10 mV more negative in the mitochondria, while ammonia addition produced a potential 15 mV more negative in the cytosol. However, Sies et al [23] used cells isolated from fed rats with fixed lactate/pyruvate and /3-hydroxybutyratel acetoacetate ratios in the absence of oleate, which may account for the conflicting data.…”

“…In similar experiments, Sies et al [23] found that the NADP redox potential difference in the absence of ammonia was 10 mV more negative in the mitochondria, while ammonia addition produced a potential 15 mV more negative in the cytosol. However, Sies et al [23] used cells isolated from fed rats with fixed lactate/pyruvate and /3-hydroxybutyratel acetoacetate ratios in the absence of oleate, which may account for the conflicting data. Although the mitochondrial data of Sies et al was calculated at pH 7.0, instead of pH 7.4, the major discrepancy results from their lower isocitrate gradients: 10 and 2 in the absence and presence of ammonia, respectively and a non-equilibrium of aconitase (see table 5).…”

“…We thank Drs Sies, Akerboom and Tager for permitting us to read their manuscript (ref. [23]) prior to its publication.…”

Proton‐translocating transhydrogenase and NAD‐ and NADP‐linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria

“…The concentrations of IC and KG in isolated heart/ liver mitochondria are in the range 0.02-0.2 and 0.2-2 mM, respectively [9,11,40]. The CO, concentration in vivo is about 1.5 mM [53].…”

Drug Oxidations Dependent on Cytochrome P‐450 in Isolated Hepatocytes

“…[8] except that the final concentration of digitonin (Sigma, Munchen) was 1.67 mM instead of 2.0 mM. The lower concentration of digitonin in the separation medium was found to give an improved separation, possibly due to a lower cholesterol content in the liver plasma membrane of the phenobarbital-pretreated animals.…”