Aromatic sulphonamides of aziridine-2-carboxylic acid derivatives as novel PDIA1 and PDIA3 inhibitors.

In this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compounds in low micromolar to low nanomolar concentrations showed the effective in vitro inhibitory properties of PDIA1 with weaker effects on PDIA3. Complexes of 15N- and 15N,13C- uniformly labelled recombinant human PDIA1a with two PDIA1 inhibitors were produced and investigated by a protein nuclear magnetic resonance (NMR) spectroscopy. It was found that both C53 and C56 of the PDIA1 enzyme were involved in covalent binding. Finally, in a range of pharmacological studies, we demonstrated that investigated compounds displayed anti-cancer and anti-thrombotic activity. These findings demonstrate that sulphonamides of Az-COOH derivatives are promising candidates for the development of novel anti-cancer and anti-thrombotic agents.

Inhibition of L-carnitine biosynthesis and transport by methyl-γ-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction.

BACKGROUND AND PURPOSE: The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury. KEY RESULTS: In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC50 3 µM) and organic cation transporter 2 (OCTN2, IC50 3 µM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20 mg·kg(-1)) attenuated the infarct size by 45% and improved 24 h survival of rats by 20-30%. CONCLUSIONS AND IMPLICATIONS: Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.

[Role of nitric oxide (NO) in the mechanism of anesthetic action]. / Role oksida azota (NO) v mekhanizme deistviia sredstv dlia narkoza.

Published reports on modifications of nitric oxide (NO) synthase (NOS) activity and NO production in the brain during development of anesthesia induced by the most common inhalational (halothane, isoflurane, sevoflurane, enflurane) and intravenous (ketamine, barbiturates, propofol, etomidate) anesthetics are reviewed. According to a popular universally acknowledged hypothesis, inhibition of NOS activity and blockade of NO neurotransmitter function are important steps in the mechanism of action of anesthetics. There are data which confirm the validity of this hypothesis for all above-listed drugs, but there are also data which disagree with it. Some scientists find that anesthesia has no effect on NOS activity and NO production, others found that the enzyme activity and NOS gene expression increased under the effect of anesthesia. Published reports and authors' data on a drastic increase of NO content in the cerebral cortex in halothane anesthesia are discussed. The effects of narcotics on NO-mediated changes in vascular tone are analyzed.

Modulation of Na, K-ATPase activity by immunoglobulins--1. Effects of IgM on the Na, K-ATPase activity in glomerulonephritis.

BACKGROUND: In the cases of acute glomerulonephritis unfavourable outcome is related to increased blood concentration of IgM. We assumed that pathologic changes in glomerulonephritis might result from direct binding of IgM antibodies to Na, K-ATPase in the nephron channels. METHODS: The study involved 30 patients suffering from glomerulonephritis. Na, K-ATPase activity was studied in the erythrocytes of these patients and healthy controls at different concentrations of Mg2+ ions in the incubation media. RESULTS: Na, K-ATPase activity in the erythrocytes of healthy subjects was shown to gradually diminish at the increasing concentrations of Mg2+ ions. Another pattern was displayed for the enzyme activity in glomerulonephritis patients--a sharp decrease of the enzymatic activity at 6 mM Mg2+, as compared with that at 3 mM, and restoration to essentially the starting level at 12 mM. Incubation of erythrocytes from healthy subjects with plasma from glomerulonephritis patients changed the response of Na, K-ATPase activity to Mg2+ concentration to that typical for glomerulonephritis patients. Analogous changes were caused by adding IgM antibodies to whole blood. It was demonstrated in vivo, that hypotonic artificial mineralised water "Marina" restores Na, K-ATPase activity in erythrocytes of glomerulonephritis patients and reduces proteinuria. CONCLUSIONS: We have postulated, that the changes in the Na, K-ATPase activity, typical for glomerulonephritis patients are caused by the interaction of the enzyme with immunoglobulin M antibodies. The lack of difference between the Na, K-ATPase activity in erythrocyte ghosts from healthy subjects and glomerulonephritis patients suggests that the immunoglobulin M attachment site is on the outer membrane and that the toxic factor could be removed.

Modulation of Na,K-ATPase activity by immunoglobulins--II. Na,K-ATPase activity in erythrocytes from patients with various chronic disturbances.

BACKGROUND: The condition of various organs can be characterized by the Na,K-ATPase activity therein. The present investigation was aimed at determining of Na,K-ATPase activity in erythrocytes from patients with chronic disturbances, resulting from viral infection and disturbances of non-viral origin. METHODS: The functional activity of Na,K-ATPase was investigated in erythrocytes of patients with chronic disturbances resulting from viral infections: chronic glomerulonephrits, chronic pancreatitis, chronic active hepatitis B, diabetes mellitus, and disturbances of non-viral origin: malignant tumours, post-radiation syndrome, and atherosclerosis. ATPase activity was determined in erythrocytes, sampled from patients and from healthy persons, both before and after adding monoclonal antibodies immunoglobulin M (IgM) and G (IgG) to blood samples. RESULTS: Two types of abnormal Na,K-ATPase activity pattern were registered for each of the investigated disturbances. Addition of IgM and IgG to the blood samples from healthy subjects caused analogous abnormalities in the functional activity of Na,K-ATPase, each of the two patterns attributable to a specific antibody. CONCLUSIONS: It has been postulated, that the toxic factors, modifying the functional activity of Na,K-ATPase, are immunoglobulins of various types.

Modulation of Na, K-ATPase activity by immunoglobulins--III. Influence of an ATPase activity regulating agent "Marina" on Na, K-ATPase activity in patients with various chronic disturbances.

BACKGROUND: In some earlier work we have demonstrated that hypotonic mineral water can change the functional activity of Na, K-ATPase both in vitro and in vivo. The current investigation was aimed at measuring of the functional activity of ATPase in erythrocytes from blood samples of patients before and after treatment with the artificially mineralised water "Marina". METHODS: "Marina"--an artificial mineral water containing K+ and Mg2+ ions, was investigated by double-blind method. Treatment lasted 7 days, daily intake of "Marina" was 4 x 200 ml before meals. Na, K-ATPase activity in erythrocytes and a number of clinical indices was determined before and after the treatment. RESULTS: After the intake of the preparation "Marina" the normal activity pattern of the Na, K-ATPase in chronic active hepatitis or atherosclerosis patients was restored. The restoration of the Na, K-ATPase activity brought about the normalization of vital clinical indices. CONCLUSIONS: The summarized data from the in vitro and in vivo experiments point to formation of Na, K-ATPase and immunoglobulin complexes as possible cause of cell disfunction and source of organic disturbances.

Drastic increase in nitric oxide content in rat brain under halothane anesthesia revealed by EPR method.

A drastic increase in nitric oxide (NO) content was revealed by the EPR method in rat brain cortex and cerebellum under halothane anesthesia. The NO scavenger diethyldithiocarbamate sodium salt (DETC) and ferrous citrate were injected into adult rats 30-60 min before anesthesia. Rats were anesthetized by inhalation of a halothane-oxygen mixture (1%, 1.5%, 2%, or 4%). After different times of anesthesia, rats were decapitated, and brain cortex and cerebellum were dissected, frozen in liquid nitrogen, and subjected to EPR spectroscopy. The concentration of NO was determined from the NO-Fe-DETC radical spectrum. In control animals, NO content in the cerebellum was only 68% of that in the cortex. We observed a time-dependent increase in NO content in the cortex and cerebellum of rats anesthetized with 1.5% halothane. In brain cortex, the NO level increased to six times that of waking animals after 30 min and remained at this level up to 60 min of anesthesia. In cerebellum the changes were less drastic, the NO level showing only a 2-fold increase. The same effect was produced by 1% and 2% halothane. Ketamine, chloral hydrate, and pentobarbital were used as reference drugs. None of these anesthetics produced effects similar to those of halothane. In ketamine-anesthetized rat brain, the NO content slightly decreased. Pentobarbital and chloral hydrate produced an insignificant increase in NO. Data are discussed in the context of possible interference of halothane in the regulation of nitric oxide synthase activity.

3-(2,2,2-Trimethylhydrazinium)propionate (THP)--a novel gamma-butyrobetaine hydroxylase inhibitor with cardioprotective properties.

A protein fraction containing gamma-butyrobetaine hydroxylase (sp.act. 1.54 mU/mg) was isolated from the rat liver by differential precipitation with ammonium sulphate. 3-(2,2,2-Trimethylhydrazinium)propionate (THP), a noncompetitive enzyme inhibitor, when administered orally to rats for 10 days (150 mg/kg) elicited a reduction in myocardial free carnitine and long-chain acyl carnitine content by 63.7 and 74.3%, respectively. This reduction in free carnitine concentration causes a suppression of the free fatty acid oxidation, as measured by the production of 14CO2 and ketone bodies. The inhibition of fatty acid oxidation is particularly manifest when their metabolism is stimulated by feeding a fat-rich diet to the animals or in fasting rats. The inhibition of fatty acid metabolism at the stage of activation (acyl carnitine formation) can account for the cardioprotective effect of THP, which is assessed by its ability to prevent a decrease in ATP level and myocardial energy charge as well as to prevent a rise in creatine phosphokinase and lactic dehydrogenase (myocardium-specific isozyme) activity in rat blood serum in response to isoproterenol and epinephrine. Regulation of the carnitine-dependent fatty acid metabolism in ischaemia is a pathogenetically justified approach to pharmacological treatment of ischaemic myocardium. In its biochemical mechanism, THP significally distinguishes itself from other known inhibitors of fatty acid oxidation.