Incorporation of cytosol δ-aminolevulinate synthase of rat liver into the mitochondria in vitro

When ¹²⁵I-labeled cytosol δ-aminolevulinate synthase was incubated in suspensions of rat liver mitochondria, the enzyme was incorporated into the mitochondira at the rate that was linear with time and with the [¹²⁵I]enzyme added. Subfractionation of the mitochondria using a digitonin technique revealed that the [¹²⁵I]enzyme was incorporated into the innermembrane-matrix fraction where endogeneous δ-aminolevulinate synthase is located.     

The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.     

A model for the regulation of δ-aminolaevulinate synthetase induction in rat liver

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.     

Tumor necrosis factor-α in macrophages of heart,liver, kidney,and in the pituitary gland

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n=54; 9±3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean±S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112±69 vs. 34±21x10³ m³) than fast and slow soleus fibers (40±20 vs. 30±14x10³ m³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 m) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 m) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116±51 vs. 55±22 and 44±23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

The features of activation of free oxidation by α,ω-tetradecanedioic acid in liver mitochondria

It was found that α,ω-tetradecanedioic acid (TDA) at the concentration of 0–500 μM doubles the rate of nonphosphorylating respiration (free oxidation) of liver mitochondria in a dose-dependent manner. This effect of TDA is observed in the presence of the excess of EGTA, which eliminates the induction of the Ca²⁺-dependent nonspecific permeability of the mitochondrial inner membrane (pore opening). An unusually high concentration of cyclosporin A (10 mM) completely eliminates this effect when added to the mitochondria before or after TDA. The stimulatory effect of TDA is not accompanied by inhibition of oxidative ATP synthesis and decrease in the ADP/O ratio, in contrast to the effects of other activators of free oxidation, such as protonophore uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and palmitic acid. It was shown that neither oligomycin, an inhibitor of H⁺-ATP synthase, nor ADP, ATP and P_i affected the activity of TDA. This is seen as an evidence that the effect of TDA is not associated with the influence on H⁺-ATP synthase and it differs from the action of membranotropic uncouplers. In the presence of the lipophilic cation tetraphenylphosphonium (TPP⁺) cyclosporin A does not affect the TDA-stimulated respiration of mitochondria, but carboxyatractylate and glutamate added after TDA do inhibit the respiration. In addition, under these conditions TDA decreases the rate of oxidative ATP synthesis and reduces the ADP/O ratio. It is assumed that the mechanism of the TDA-induced activation of free oxidation in liver mitochondria in the absence of TPP⁺ is similar to that of the so-called decouplers and is associated with the switching of the respiratory chain complexes to the idle mode (inner uncoupling).     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Activities of purine converting enzymes in heart,liver and kidney mice LDLR−/− and Apo E−/−

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis.

Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5′-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC.

The activity of ecto5′-nucleotidase differ in hearts of LDLR^?/? and ApoE^?/? mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models.

This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.     

The synthesis and processing of β-glucuronidase in normal and egasyn deficient mouse kidney

The presence of a precursor form of β-glucuronidase, with a subunit molecular weight of 75,000 was demonstrated in mouse kidney. This was later processed to the mature form, with subunit molecular weight of 71,500. Tissue fractionation revealed that the precursor was associated with the microsomes whereas the mature form was associated with the lysosomes. In mice lacking egasyn both forms of β-glucuronidase were present, but the rate of processing was elevated compared to normal.     

A comparison of the in vitro binding of α-tocopherol to microsomes of lung,liver, heart and brain of the rat

The in vitro binding of α-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of α-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of α-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for α-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain > liver ≈ heart > lung. The differences in affinity did not correlate with total fatty acid content (r = − 0.39), total unsaturated fatty acid content (r = − 0.26), or with the content of fatty acids containing two or more double bonds (r = − 0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = + 0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.     

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse,rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970)208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F^?, malonate, and arsenite), only F^? (100 mm final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.     

Modulation of myocardial stiffness by β-adrenergic stimulation--its role in normal and failing heart

The acute effects of beta-adrenergic stimulation on myocardial stiffness were evaluated. New-Zealand white rabbits were treated with saline (control group) or doxorubicin to induce heart failure (HF) (DOXO-HF group). Effects of isoprenaline (10(-10)-10(-5) M), a non-selective beta-adrenergic agonist, were tested in papillary muscles from both groups. In the control group, the effects of isoprenaline were also evaluated in the presence of a damaged endocardial endothelium, atenolol (beta(1)-adrenoceptor antagonist), ICI-118551 (beta(2)-adrenoceptor antagonist), KT-5720 (PKA inhibitor), L-NNA (NO-synthase inhibitor), or indomethacin (cyclooxygenase inhibitor). Passive length-tension relations were constructed before and after adding isoprenaline (10(-5) M). In the control group, isoprenaline increased resting muscle length up to 1.017+/-0.006 L/L(max). Correction of resting muscle length to its initial value resulted in a 28.5+/-3.1 % decrease of resting tension, indicating decreased muscle stiffness, as confirmed by the isoprenaline-induced right-downward shift of the passive length-tension relation. These effects were modulated by beta(1)- and beta(2)-adrenoceptors and PKA. In DOXO-HF group, the effect on myocardial stiffness was significantly decreased. We conclude that beta-adrenergic stimulation is a relevant mechanism of acute neurohumoral modulation of the diastolic function. Furthermore, this study clarifies the mechanisms by which myocardial stiffness is decreased.     

The protective role of Coenzyme Q10 in metallothionein-3 expression in liver and kidney upon rats’ exposure to lead acetate

Molecular Biology Reports - Metallothionein-3 (MT3) is an antioxidant protein that alters after exposure to heavy metals. In this study, we investigated the hepatic and renal expression of MT3 gene...     

Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.