Biochemical parameters of endothelial dysfunction in cardiological syndrome X.

UNLABELLED: The endothelial dysfunction in cardiological syndrome X has been studied mainly by invasive methods and by measuring vasoactive mediator (nitric oxide (NO), endothelin-1) levels. Other parameters evaluating this dysfunction (defined as an imbalance between vascular relaxing and contracting factors, between procoagulant and anticoagulant or growth-inhibiting and growth-promoting substances) have not been used. METHODS: Twenty-five non-diabetic patients (16 men, 9 women) with cardiological syndrome X and 10 healthy volunteers (5 men, 5 women) were examined. Biochemical parameters: ET-1, the end products of nitric oxide metabolism (NOx), VEGF, vWF, betaTG, tPA, PAI-1 were measured before and during an ECG exercise tolerance test. The blood concentrations of testosterone and estradiol in men and LH, FSH and estradiol in women were tested. RESULTS: A significantly lower basal concentration of NOx (p=0.01), lower basal NOx/ET-1 ratio (p<0.05) and higher levels of VEGF (p<0.05) were observed in patients with cardiological syndrome X. The male patients also had higher concentrations of estradiol (p<0.05). A significant decrease in tPA concentration and increase in betaTG was noticed during exercise, but with no differences between the study groups. CONCLUSIONS: Endothelial dysfunction in cardiological syndrome X manifests mainly in the regulation of vessel wall tonus. which was revealed by the decrease of NOx level and NOx/ET-1 ratio. VEGF elevation in syndrome X may result from chronic tissue ischaemia due to endothelial dysfunction. Exercise augments the prothrombotic activity of the blood, since a significant elevation in betaTG and decrease in tPA were observed after exercise.

[Vascular endothelium--function, disorders and clinical modification probes]. / Sródblonek naczyniowy--funkcje, ich zaburzenie oraz kliniczne próby modyfikacji.

According to contemporary views, the endothelium is not only a barrier separating blood from surrounding tissues, but a dynamic, heterogeneous organ, which possesses many secretory, metabolic and immunologic functions. Endothelial cells produce mediators, which regulate blood flow, influence platelet adhesion and aggregation, coagulation and fibrinolysis and also immunological response. Endothelial dysfunction is defined as an imbalance between vascular relaxing and contracting factors, between procoagulant and anticoagulant mediators or growth-inhibiting and growth-promoting substances. The definition is often confined to dysfunction of the vessel wall tonus control. The endothelial dysfunction frequently proceeds structural changes in vessels, as e.g. atherosclerotic plaque formation, neointima formation and vessel wall remodelling. This dysfunction has been confirmed in systemic hypertension, atherosclerosis, cardiac syndrome X, heart failure, using various invasive and non-invasive techniques. There are pharmacologic and non-pharmacologic methods to modify endothelial functions. It is obligatory to reduce risk factors of atherosclerosis, which lead to endothelial cell damage, i.e. hypertension, hyperlipidemia, cigarette smoking, estrogen deficiency and elevated levels of homocysteine. The role of physical exercise, low-cholesterol diet, discontinuation of smoking is emphasised. Among drugs statins, angiotensin-converting enzyme inhibitors and hormone replacement therapy are considered particularly beneficial. The importance of angiotensin receptor antagonists, endothelin receptor antagonists, L-arginine, growth factors and calcium-channel blockers for the improvement of endothelial function is studied.

Molecular mechanism of hydrogen peroxide conversion and activation by Cu(II)-amikacin complexes.

The interactions between Cu(II)-amikacin complexes [Cu(II)-Ami] and hydrogen peroxide were studied by spectroscopy (EPR, UV-vis, CD, XAS) and cyclic voltammetry. A monomer-dimer equilibrium was detected at complex concentrations above 5 mM (log K(dim) = 1.84 +/- 0.03). The dimeric complex undergoes easy, although irreversible oxidation (ca. 0.5-0.6 V) to a Cu(III) species on platinum electrode. However, the monomeric complexes are able to catalyze hydrogen peroxide disproportionation reaction at pH 7.4 in a multistep process, mediated by hydroxyl radicals and involving both Cu(I)/Cu(II) and Cu(II)/Cu(III) redox pairs.

Coordination mode and reactivity of copper(II) complexes with kasugamycin.

Protonation and Cu(II) coordination of kasugamycin were studied by potentiometry, UV-vis, CD, EPR, 13C NMR, and 1H NMR. Mononuclear complexes with stoichiometries ranging from CuHL to CuH(-1)L were found. The aminoamidine moiety provides the coordination site in the CuHL species. The additional axial coordination of the amino nitrogen of the aminosugar ring is present in CuL. Finally, the CuH(-1)L complex is formed as a result of a deprotonation and coordination of the hydroxyl group of the inositol ring. The non-planar arrangement of the chelate rings results in the relative stabilization of a Cu(I) species. As a consequence, Cu(I) and superoxide radicals are involved in the redox mechanism of H(2)O(2) activation by the Cu(II) complex of kasugamycin.

Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant.

D,L-Dithiothreitol (DTT), known also as Cleland reagent, is a thiol group protectant, used commonly in peptide and protein chemistry. Therefore, it is often added at high concentrations in preparations of proteins relevant to heavy metal biochemistry. The coordination of five of these metal ions, Zn(II), Cd(II), Pb(II), Ni(II) and Cu(I) to DTT was studied by means of potentiometric titrations, and UV-Vis and NMR spectroscopies. It was found that DTT forms specific and very stable polymeric and monomeric complexes with all of these metal ions, using both of its sulfur donors. The quantitative description of these complexes in solution and the solid state provides the basis for predictions of interference from DTT in studies of metal ion binding of thiol-containing biomolecules.

Copper(II)-lincomycin: complexation pattern and oxidative activity.

Coordination of Cu(II) to lincomycin was studied by potentiometry, UV-Vis, circular dichroism (CD), EPR, NMR, cyclic voltammetry (CV) and ESI-MS. Only mononuclear complexes of stoichiometries ranging from CuL to CuH(-3)L were found. In the main species present at neutral pH, CuH(-2)L, lincomycin bonds Cu(II) through both of its nitrogen donors, and a deprotonated oxygen donor at C4 of the sugar moiety. High pressure liquid chromatography (HPLC) of products of 2'-deoxyguanosine (dG) oxidation and agarose gel electrophoresis of plasmid DNA confirmed that lincomycin complexes effectively facilitate dG oxidation by H2O2, but are not able to cleave double-stranded plasmid DNA.

Upstream stimulatory factor is involved in the regulation of the human calcyclin (S100A6) gene.

Calcyclin (S100A6) is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity. The calcyclin gene promoter fragment -361/-167 activates transcription several fold when compared to the basal -167/+134 promoter fragment indicating the presence of enhancer element within -361/-167 bp region. By means of the electrophoretic mobility shift assay (EMSA) we found that this region contains a protein-binding site and mapped it to an E-box sequence at position -283/-278. Using antibodies against USF1 we identified the upstream stimulatory factor as the transcription factor bound to the E-box sequence in EMSA. This factor was also enriched in protein fractions obtained from Ehrlich ascites tumor cells nuclear extract by affinity chromatography using the E-box sequence as a ligand. Cotransfection of the USF1 expression vector with a plasmid carrying the luciferase gene under control of the -361/+134 calcyclin gene promoter fragment resulted in several fold activation of luciferase activity. On the other hand, mutations within the E-box led to a marked decrease in the efficiency of calcyclin gene promoter fragment. The results indicate that USF1 binds to an E-box sequence of the calcyclin gene promoter and enhances its transcription activity. This mechanism might be responsible for the upregulation of calcyclin gene expression in response to various stimuli and in tumors.

Regulation of cell specific expression of calcyclin (S100A6) in nerve cells and other tissues.

Many of the small, acidic, calcium binding S100 proteins present in the brain specifically map different anatomical regions and cell types and their overexpression is implicated in pathological changes. Similarly to other members of the S100 protein family, calcyclin (S100A6) is expressed in a cell specific manner and is found in subpopulations of neurons and astrocytes in the brain and in epithelial cells and fibroblasts. In this article we review data concerning the cell specific expression of S100 protein genes and present experimental results on the regulation of the calcyclin gene. We have performed promoter deletion studies to locate regions within the calcyclin gene promoter responsible for transcriptional regulation. The results demonstrate that the 3 kb long calcyclin gene promoter lacks a cell specific cis-acting element and drives the expression of the reporter gene also in cells that do not express endogenous calcyclin. The expression is modulated by positive and negative elements acting uniformly in the four different cell lines studied. The first intron of the calcyclin gene was found to have an inhibitory influence on expression regardless of cell type. It was also shown that calcyclin expression can be induced in calcyclin-negative cells by treatment with 5-azacytidine suggesting the involvement of gene methylation in its cell specific expression. The results are discussed in light of the data available on the regulation of other S100 genes.

[Regulation of protein S100 expression during transcription]. / Regulacja ekspresji bialek S100 na poziomie transkrypcji.

The S100 protein family is a group of homologous, small, calcium-binding proteins expressed in a cell specific manner. In man, the genes coding the majority of these proteins are localized in a cluster on chromosome 1. The cell specific expression of these proteins is mainly attained at the transcriptional level. It appears that the promoters of some S100 genes (rat S100A4, mouse and human S100B and rat CALB3) contain inhibitory sequences which reduce transcription level. Their effect may be counteracted by an interaction with cell specific transcription factor(s), which allows transcription in certain cell types. In other cases (human S100A2, mouse S100A4 and human S100A6), the main mechanism controlling cell specific expression seems to be methylation of the promoter or intronic sequences leading to gene silencing in some cells. The level of protein expression might be further modulated by different regulatory sequences that respond, via interactions with specific transcription factors, to various extracellular stimuli.

Ca2+-dependent interaction of calcyclin with membrane.

The presence of calcyclin in the microsomal fraction of Ehrlich ascites tumor cells was detected using polyclonal antibodies. Association of calcyclin with the microsomes depended on the presence of calcium ions in the buffer used for cell fractionation. The interaction of calcylcin with Ehrlich ascites tumor cells microsomes was confirmed in the in vitro conditions by cosedimentation assay using exogenous calcyclin. It was shown that phospholipids extracted from natural membranes and purified phosphatydylserine or phosphatydylcholine were not involved in the binding. Instead, several low molecular weight polypeptides in the Triton X-100 resistant membrane fraction were found to interact with calcyclin.

Interaction of calcyclin and its cyanogen bromide fragments with annexin II and glyceraldehyde 3-phosphate dehydrogenase.

The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.

Effect of alpha adrenergic stimulation and carnitine palmitoyl transferase I inhibition on hypertrophying adult rat cardiomyocytes in culture.

Long-term, serum supplemented cultures of rat adult ventriculocytes were utilized to study the tropic effects of the alpha-agonist phenylephrine and of the carnitine palmitoyltransferase I inhibitor etomoxir. Cell protein and the rate of incorporation of phenylalanine were measured, corrected for cellular DNA content and utilized as an index for hypertrophy and of anabolic activity of the cells, respectively. The mRNA level of ANF was utilized as an index for the pathological phenotypic change (i.e., switch to fetal gene program), and that of the Na-channel--a constantly expressed gene in normal and hypertrophic cardiomyocytes--served as an internal control. Both mRNAs were quantified at various stages in culture by competitive reverse transcriptase PCR. The size of control myocytes steadily increased for over 3 weeks. The cells were completely redifferentiated and reached a maximum of anabolic activity 2 weeks after plating. Secretion and mRNA levels of ANF were increased severalfold after 7-8 days. Addition of 10 microM phenylephrine considerably speeded up cell growth. Maximum anabolic activity and complete redifferentiation were reached already after 1 week. Levels of mRNA and of ANF release increased 30-40 fold. Interestingly, induction of ANF gene transcription lagged behind the redifferentiation of the cells. Ten microM etomoxir inhibited the oxidation of palmitic acid and stimulated that of exogenous glucose by adult cardiomyocytes. In spite of its clear effect on fuel utilization, etomoxir had no direct hypertrophic effect on the myocytes in culture and did not inhibit the stimulatory action of alpha-agonists. Reactivation of the fetal gene program, as visualized by ANF production, was not reversed by etomoxir.

Reversible inactivation of the sarcoplasmic reticulum Ca(2+)-ATPase coupled to rearrangement of cytoplasmic protein domains as revealed by changes in trypsinization pattern.

Repetitive homogenization of skeletal muscle sarcoplasmic reticulum (SR) membranes in the presence of chelating agents at low ionic strength leads to the loss of the Ca-ATPase activity. This inactive state of the enzyme is coupled to an extensive rearrangement of the cytosolic domains as visualized by a completely different trypsinization pattern of the enzyme. In addition to the primary cleavage site (Arg 505), a novel trypsinization site (Arg 334), just N-terminal of the phosphorylation domain and localized on the primary tryptic fragment A, becomes exposed. Cleavage at the latter site yields a soluble fragment of M(r) 20,117 and the membrane-bound N-terminal one-third of the ATPase of M(r) 35,279. Two additional trypsinization sites C-terminal of the nucleotide binding domain become exposed in the inactive Ca(2+)-ATPase conformation. Rapid cleavage at these sites yields two soluble fragments of about 15 and 10 kDa. All together, the three soluble fragments comprise most of the large cytosolic loop of the Ca(2+)-ATPase. The inactivation and the change in trypsinization pattern can be reversed by rehomogenization of the extracted membranes in the presence of divalent cations. The results suggest the presence of an occluded site for divalent cations which can be depleted or refilled during application of sheer forces. Occupation of this site is essential to confer to the enzyme an active conformation.

Effect of carbodiimides on the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal membranes.

1. The hydrophobic N,N'-dicyclohexylcarbodiimide (DCCD) inhibits the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal fraction. 2. The inhibition is dependent on time and concentration, with half-maximal inhibition occurring at 0.4 mM concentration of carbodiimide after a 0.5 hr incubation at room temperature. 3. ATP does not protect against the inhibition. 4. Two water-soluble carbodiimides, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide (CMCD) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCD), are not inhibitory. 5. Inhibition of Mg(2+)-ATPase activity by DCCD is accompanied by covalent incorporation of the radioactive agent into the partially purified enzyme preparation.