The operation of the malate-aspartate shuttle in the reoxidation of glycolytic NADH in slices of fetal rat liver.

Lactate production by liver slices from fetal rats (17th--18th day of gestation) is enhanced about two fold by aminooxyacetate, an inhibitor of aspartate transaminase (EC 2.6.1.1). Such an effect is consistent with an increase of the cytosolic NAD-redox state owing to the parallel fall in the pyruvate level, whereas the glycolytic flux does not seem to be influenced appreciably. Indeed, although the inhibitor causes a marked increase of fructose 1,6-diphosphate, glucose-6-phosphate decreases only slightly. These results suggest that in fetal rat liver the malate-aspartate shuttle is operative in the reoxidation of cytosolic NADH produced during aerobic glycolysis.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Evidence for the existence of phosphoenolpyruvate carboxykinase ferroactivator activity in adult and fetal guinea pigs.

Newborn (24--72 h) guinea pig liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) activity is increased by incubation of the cytosol with the metal salts FeCl2, MnCl2, CoCl2 and CdCl2. FeCl2 at 30 micromol/l concentration is the most effective activator causing a 3.5-fold increase in activity. Purified rat liver cytosolic PEPCK is activated by 30 mumol/l FeCl2 in the presence of liver cytosol of fetal and newborn guinea pigs. These results confirm the existence of PEPCK ferroactivator in the guinea pig which has properties similar to the one found in rat liver. The tissue distribution of ferroactivator activity parallels that of cytosolic PEPCK, being highest in the gluconeogenic organs liver and kidney. Hepatic PEPCK ferroactivator activity can be demonstrated by day 45 of gestation, increasing linearly in specific activity to adult levels at term (65 days). The distribution and development of the ferroactivator is consistent with the hypothesis that it may play a role in the physiologic control of PEPCK.     

Ontogeny of cytosolic proteins capable of modulating sarcoplasmic reticulum calcium transport in heart muscle

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca² uptake by sarcoplasmic reticulum (SR); further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53–66, 1983). The present study investigated the ontogenetic expression of the activities of Ca² transport inhibitor and inhibitor antagonist in heart cytosol during fetal and postnatal development of the rat. The SR Ca² transport inhibitor activity was undetectable in the cytosol of fetal (15- or 20-days gestation) rat heart but was manifested in the cytosol as early as one day after birth and increased progressively thereafter to reach almost adult levels within the first two weeks of postnatal development. The activity of the SR Ca² transport inhibitor antagonist was barely detectable in the near-term (20 days gestation) fetus but increased substantially during early postnatal development, in parallel with the rise in activity of the inhibitor. The ontogenetic appearance and increase in the activities of the Ca² transport inhibitor and its antagonist correlated well with the concurrent appearance and increase in the amounts of two polypeptides of apparent molecular weights 43 kDa and 64 kDa, which we have tentatively identified as the inhibitor and inhibitor antagonist, respectively. The co-ordinated expression of both the inhibitor and inhibitor antagonist activities in the cytosol during the early postnatal period parallels the morphogenesis and functional maturation of SR in cardiac muscle suggesting likely involvement of these cytosolic proteins in the physiological regulation of SR function.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Premature appearance of hepatic phosphoenolpyruvate carboxykinase in fetal rats, not mediated by adenosine 3':5'-monophosphate.

Injection of streptozotocin in utero to fetuses elicited a premature appearance of cytosolic hepatic activity of phosphoenol pyruvate carboxykinase. This was due to a precocious initiation of the synthesis of the enzyme. The streptozotocin-induced appearance of enzyme activity was not mediated by adenosine 3':5'-monophosphate since the concentration of the cyclic nucleotide in the liver was unaffected by the antibiotic, the administration of dibutyryladenosine 3':5'-monophosphate to streptozotocin-treated fetuses elicited an additive increase in enzyme activity, and insulin administration in utero repressed the streptozotocin effect while the effect due to dibutyryladenosine 3':5'-monophosphate was not inhibited by simultaneous insulin injection. Streptozotocin treatment also caused a small but consistent retardation of fetal growth and a marked reduction of liver wet weight. Histological analysis of the liver demonstrated a premature loss of some hematopoietic elements, while hepatocytes appeared normal. Hepatic protein synthesis was unaffected by the streptozotocin treatment. Streptozotocin treatment had no effect on fetal renal phosphoenol pyruvate carboxykinase activity or kidney wet weight.     

Possible cyclic AMP-dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.     

The regulation of cytosolic epoxide hydrolase in mice

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.     

The role of the cytosolic fraction and of initiation factor eIF-2 for changes of the rate of protein synthesis during liver regeneration

The protein synthesis-stimulating activity of the cytosolic fraction from regenerating rat liver was tested in a cell-free system using washed polysomes from normal rat liver. This activity undergoes significant changes during liver regeneration after partial hepatectomy (p.h.). An initial decrease until 16 h after p.h. is followed by a significant increase until 24 h after p.h. Beyond 32 h after p.h. the activity begins to decline again. Evidence is presented that these changes of the cytosolic activity may not be due to alterations in the distribution of protein synthesis-stimulating factors between the microsomal and the cytosolic fraction. The Met-tRNAf-binding activity of the cytosolic fraction changes during liver regeneration analogously to the protein synthesis-stimulating activity measured in the polysomal assay. This indicates that initiation factor eIF-2 is involved in the observed changes of the cytosolic activity. This conclusion could be confirmed by addition of purified eIF-2 to the polysomal assay system. Addition of eIF-2 to cytosolic fractions of low endogenous protein synthesis-stimulating activity (16 h after p.h.) enhances amino-acid incorporation to a significantly higher extent than addition to highly active cytosolic fractions (24 h and 32 h after p.h.). From these results it is concluded that changes in eIF-2 plays an essential role in the described alterations of the cytosolic activities during liver regeneration.     

Androgen metabolism by hepatic and renal tissues of the fetal rhesus monkey

Liver and kidney from fetal monkeys (day 125 of gestation) were fractionated into low speed pellets, microsomal and cytosolic fractions. Liver cytosols converted as much testosterone (T) to 5 beta-androstane-3 alpha,17 beta-diol (5 beta-diol) at 0 degrees C as at 4 degrees-45 degrees C without exogenous cofactors. The principal product formed from 5 alpha-dihydrotestosterone (5 alpha-DHT) was 5 alpha-diol. A 1000-fold molar excess of radioinert 5 beta- or 5 alpha-DHT inhibited 5 beta-diol formation from [3H]T by cytosols and increased 5 beta-DHT formation. Similarly, using 5 alpha-DHT as substrate, 5 alpha-diol formation was inhibited. Microsomal and low speed pellets with added cofactors formed products which recrystallized with either etiocholanolone or androsterone from [3H]T or [3H]DHT, respectively. Little product was formed without cofactor. Whole liver homogenates produced 5 beta-reduced products from [3H]T in the presence of an NADPH generating system whereas kidney homogenates produced 5 alpha-reduced products. These data provide new information on the capacity of fetal monkey liver and kidney to metabolize androgens. The 3 alpha-reductases are cytosolic. The 5 alpha- and 5 beta-reductases are mostly in the low speed pellet but are sufficiently represented in cytosols to mediate diol formation. The 17-hydroxysteroid dehydrogenases are in the microsomal fraction. Our results suggest that 5 alpha-DHT is the active androgen in fetal liver since testosterone is metabolized to 5 beta-DHT and 5 beta-diol which are inactive androgens.     

Metabolite concentrations in the liver of the adult and developing guinea pig and the control of glycolysis in vivo.

A range of metabolite concentrations have been determined in the liver of the adult and fetal guinea pig during the latter half of gestation. Adenine nucleotides showed little change during development of the fetal liver and the only major difference from the adult was a low ADP concentration. The hexose phosphates, particularly fructose 1,6-diphosphate, were higher and the triose phosphates in the glycolytic pathway after glyceraldehyde 3-phosphate were lower in the fetal liver. Cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratios were comparable in both adult and fetal livers as were cytosolic $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ ratios for the last 15–20 days of gestation. The metabolite concentrations have been used to indicate that glycolysis in the fetal guinea pig liver is controlled largely by hexokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate kinase.     

Effects of fructose 6-phosphate and adenylates on the activities of rabbit liver fructose bisphosphatase and phosphofructokinase.

The kinetics of induction of cytosolic DT-diaphorase (NAD(P)H dehydrogenase-quinone, EC 1.6.99.2) by benzo(a)pyrene (BP) in the liver of the 8-day-old rat has been studied. After a lag phase of 8 h, DT-diaphorase reaches its maximum activity in three waves, with plateau levels of activity between 15–18, 26–36, and 40 h after administration of BP, at 4, 15, and 26 times the basal activity, respectively. A lower degree of induction of DT-diaphorase could be observed in the kidney cortex of the young rat and in the liver of the adult rat. No induction was observed in the fetal liver and in the adult kidney cortex. Lead acetate treatment of the adult rat resulted in induction of DT-diaphorase by BP in the liver and in the kidney cortex. Induction could not be observed in the regenerating liver of the adult rat. Experiments with picolinic acid (PA)—as a G₁ inhibitor—administered simultaneously or at different time intervals after BP administration resulted in an inhibition of induction, depending on the time of administration of picolinic acid. It is concluded that a mitotic cell cycle is necessary for DT-diaphorase induction by BP. Evidence is presented that BP acts in late G₁. The kinetics of induction of aryl hydrocarbon hydroxylase (AHH) by BP in liver microsomes of the 8-day-old rat has been compared with the induction of cytosolic DT-diaphorase. The effect of PA on the induction of AHH has also been studied. In view of the differences in kinetics of induction and in the effects of PA, it is concluded that the induction of AHH and that of DT-diaphorase are dissociated. AHH induction may take place in all hepatocytes, in contrast to DT-diaphorase induction.     

Influence of H-2-linked genes on glucocorticoid receptors in the fetal mouse palate

The binding of ³H-dexamethasone to cytosolic receptors in fetal jaws and in cytosols and nuclei of primary cell cultures of fetal palates was studied in various congenic strains of mice. The amount of specific binding was greater in palatal tissues from B10.A and BlO.A(2R) mice than in B10 or B10.A(5R) preparations. These differences were not observed in the liver. Since the strains with higher levels of glucocorticoid receptor are known to be more susceptible to cortisone-induced cleft palate than the strains with low receptor levels, it is suggested that quantitative variation in receptor levels may be involved in determining H-2-linked differences in cleft-palate susceptibility. Whether or not this is the case, it appears that an H-2-linked gene affects the quantity of a cytosolic glucocorticoid-binding protein which translocates to the nucleus.     

Human rhinovirus HRV14 uncoats from early endosomes in the presence of bafilomycin

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin-Sepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.     

Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro.

Dephosphorylation of 1D-myo-inositol 1,4-bisphosphate [Ins(1,4)P2] in rat liver is catalysed by a cytosolic phosphatase that removes the 1-phosphate group. The Km for Ins(1,4)P2 is approx. 17 microM. Li+ (100 mM) causes 50% inhibition of Ins(1,4)P2 phosphatase activity when activity is measured at the very low substrate concentration of 10 nM, but on raising the substrate concentration to 100 microM there is a greater than 10-fold increase in sensitivity to Li+, suggesting that Li+ acts mainly, but not entirely, as an uncompetitive inhibitor of Ins(1,4)P2 phosphatase. In addition, rat liver cytosol shows Li+-sensitive phosphatase activity against 1D-myo-inositol 1-,3- and 4-monophosphates. The Ins(1,4)P2 1-phosphatase and inositol monophosphatase activities all share an apparent Mr of 47 x 10(3), as determined by gel-filtration chromatography. However, the Ins(1,4)P2 1-phosphatase is more sensitive to inactivation by heat, and can be separated from inositol monophosphatase activity by anion-exchange chromatography. We conclude that rat liver cytosol contains an Ins(1,4)P2 1-phosphatase that is distinct from, but in many ways similar to, inositol monophosphatase.     

Mechanism of aminobisphosphonate action: characterization of alendronate inhibition of the isoprenoid pathway

Alendronate (ALN), an aminobisphosphonate compound used for the treatment of osteoporosis and other disorders of bone resorption, has been suggested to act by inhibition of the formation of GGPP. In the present study we used an S(10) homogenate fraction of rat liver to show that ALN causes a dose-dependent inhibition of [(3)H]MVA incorporation into sterols and a concomitant increase in incorporation of radiolabel into IPP and DMAPP. We further show that ALN is a potent inhibitor of cytosolic trans-prenyltransferase (FPP synthase). The inhibition is competitive with respect to allylic pyrophosphate substrates, but not IPP, suggesting that ALN acts as an allylic pyrophosphate analog and binds to the free enzyme. The K(i) is in the 0.5 microM range.     

Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.