Activation of cytosol aldosterone receptors in rat kidney

Cytosolic aldosterone-protein complexes are isolated from rat kidney slices after incubation with [3H]aldosterone and dexamethasone. Activated and unactivated forms of the complex are characterized by gel electrophoresis and hydroxyapatite chromatography after incubation at 4 degrees C and 25 degrees C respectively. It is found that the activated form reaches a maximum after 30 min at 25 degrees C and can be separated as an homogeneous peak by electrophoresis. Intermediate forms can also be identified. In the presence of 10 mM ATP, activation immediately occurs at 4 degrees C and is almost complete. In the presence of 10 mM molybdate, the activation is strongly enhanced and the increase in activated form may be about fifteen-fold whether molybdate is added during kidney homogenization or just before incubation at 25 degrees C. On the other hand molybdate reduces to one third the binding of the aldosterone-receptor complexes to nuclei. In the presence of the steroid RU 26988 which is a pure glucocorticoid, experiments done on aldosterone-receptors complexes and their binding to nuclei are confirmed. This proves that aldosterone is specific for mineralocorticoid sites. The general pattern of the mineralocorticoid receptor activation is discussed and its resemblance to the case of other steroid hormones is emphasized.     

The kinetics of rat liver citrate synthase in situ.

The kinetics of citrate synthase in situ in toluene-treated rat liver mitochondria were studied. The V_max, K_m, and kinetic pattern for oxaloacetate were the same as those for the pure rat liver citrate synthase. The K_m, for acetyl-CoA for the in situ enzyme was increased compared to pure enzyme (8.5 to 77 μm), and the sensitivity of the in situ enzyme to inhibition by ATP, NADPH, or tricarballylate was decreased. The change seen with ATP was not due to problems of small molecule diffusion.     

Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney

The distribution of the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) was examined in the rat kidney by immunolocalization with anti-C1-THF synthase serum using the peroxidase-antiperoxidase method. C1-THF synthase immunoreactivity was detected in both distal and proximal epithelial cells. Staining of the distal tubule epithelia was more intense and granular whereas staining of the proximal tubule epithelia was diffuse. All cells of the cortical collecting duct showed positive granular staining. In the outer medullary collecting duct, the intercalated cells showed intense granular cytoplasmic staining and the principal cells were either negative or weakly positive. The ascending thick limb of Henle's loop was also positive. Glomeruli and the inner medulla showed no staining for C1-THF synthase.     

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution ¹³C NMR. The oxidation of [1,2,3-¹³C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS^?) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The ¹³C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the ¹³C spectra of cells presented with [3-¹³C] propionate or [2-¹³C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.     

Melatonin inhibits Gram-negative pathogens by targeting citrate synthase

Bacterial infections caused by Gram-negative pathogens represent a growing burden for public health worldwide. Despite the urgent need for new antibiotics that effectively fight against pathogenic bacteria, very few compounds are currently under development or approved in the clinical setting. Repurposing compounds for other uses offers a productive strategy for the development of new antibiotics. Here we report that the multifaceted melatonin effectively improves survival rates of mice and decreases bacterial loads in the lung during infection. Mechanistically, melatonin specifically inhibits the activity of citrate synthase of Gram-negative pathogens through directly binding to the R300, D363, and H265 sites, particularly for the notorious Pasteurella multocida. These findings highlight that usage of melatonin is a feasible and alternative therapy to tackle the increasing threat of Gram-negative pathogen infections via disrupting metabolic flux of bacteria.

     

Role of sodium and potassium in aldosterone binding of kidney receptors in the rat.

An increase of the sodium concentration produced a diminished binding of labeled aldosterone to kidney receptors in both in vivo and in vitro conditions. The elevation of potassium level did not change the binding capacity of kidney nuclear receptors for aldosterone. Hypernatraemia-induced decrease of aldosterone uptake was significant in the nuclear fraction of both medullary and cortical region of the kidney but did not show remarkable changes in the uptake of cytosol fraction. The transfer of aldosterone from cytosol into the nuclear compartment seems to be changed by the alteration of extracellular sodium concentration.     

Developmental changes of cytochrome c oxidase and citrate synthase in rat heart homogenate

Activity of cytochrome c oxidase and citrate synthase in rat heart homogenates was determined in 5-, 15-, 28- and 60-day-old rats. The activity of both enzymes increased during postnatal development but their changes followed different kinetics. The membrane-bound cytochrome c oxidase reached its adult values during the early postnatal period, i.e. between days 5 and 15, whereas soluble matrix-localized citrate synthase also continued to increase between days 15 and 60. Our data indicate a relative excess of cytochrome c oxidase in neonatal cardiocytes.     

Controls of citrate synthase activity

The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an $\text{in vitro}$ catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration $\text{in vivo}$ compared to its concentration $\text{in vitro}$. In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available $\text{in vitro}$ so that little can be said concerning the $\text{in situ}$ control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes $\text{in vitro}$ can only serve as a guideline for parameters to study when techniques are available to study control of enzymes $\text{in situ}$.