Cigarette smoke-induced alterations in endothelial nitric oxide synthase phosphorylation: role of protein kinase C.

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.

Cigarette smoke and its formaldehyde component inhibit bradykinin-induced calcium increase in pig aortic endothelial cells.

Bradykinin-induced increase in the intracellular concentration of free calcium evokes an activation of the endothelial nitric oxide synthase (eNOS) enzyme, producing nitric oxide (NO). Cigarette smoke inhibits the eNOS-NO-cGMP signaling pathway. The pathomechanism of this deleterious effect of smoke on NO production is unknown. The aim of this study was to investigate the effect of gas phase smoke trapped in a buffer (smoke buffer, SB) on the bradykinin-induced calcium increase in cultured endothelial cells. FURA-2-AM was used to detect bradykinin-induced calcium increase. A sensitive, fluorescent method using O-phthaldialdehyde was used for the determination of intracellular reduced glutathione (GSH) and protein-thiol levels. SB caused a time- and concentration-dependent inhibition of bradykinin-induced calcium increase. Formaldehyde, a component of SB, inhibited bradykinin-induced calcium increase in concentrations characteristic for SB. SB decreased both the intracellular GSH (0.22 +/- 0.06 vs. 2.23 +/- 0.32 mumol/g protein, SB vs. control, p < .001) and protein-thiol levels (4.98 +/- 0.54 vs. 7.31 +/- 0.97 microEqu GSH/g protein, SB vs. control, p < .05) in the endothelial cells. Intracellular GSH and protein-thiol levels were not changed by 80 microM formaldehyde. GSH (4 mM) prevented the effect of SB (p < .001) and formaldehyde (p < .05) on the bradykinin-induced calcium increase. Our data support the premise that SB inhibits bradykinin-induced calcium increase. This inhibition is partially due to protein-thiol oxidation but may also be caused by the formaldehyde content of SB, which inhibits calcium increase in a protein-thiol-independent manner.

Role of iron in the interaction of red blood cells with methylglyoxal. Modification of L-arginine by methylglyoxal is catalyzed by iron redox cycling.

Diabetes mellitus is characterized by increased methylglyoxal (MG) production. The aim of the present study was to investigate the role of iron in the cellular and molecular effects of MG. A red blood cell (RBC) model and L-arginine were used to study the effects of MG in the absence and presence of iron. Intracellular free radical formation and calcium concentration were measured using dichlorofluorescein and Fura-2-AM, respectively. Effects of MG were compared to the effect of ferrous iron. Reaction of L-arginine with MG was investigated by electron spin resonance (ESR) spectroscopy and by a spectrophotometric method. MG caused an iron dependent oxidative stress in RBCs and an elevation of the intracellular calcium concentration due to formation of reactive oxygen species. Results of co-incubation of MG with ferrous iron in the RBC model suggested an interaction of MG and iron; one interaction was a reduction of ferric iron by MG. A role of iron in the MG-L-arginine reaction was also verified by ESR spectroscopy and by spectrophotometry. Ferric iron increased free radical formation as detected by ESR in the MG-L-arginine reaction; however, ferrous iron decreased it. The reaction of MG with L-arginine yielded a brown product as detected spectrophotometrically and this reaction was catalyzed at a lower rate with ferric iron but at a higher rate with ferrous iron. These data suggest that MG causes oxidative stress in cells, which is due at least in part to ferric iron reduction by MG and to the modification of amino acids e.g. L-arginine by MG, which is catalyzed by iron redox cycling.

N(epsilon)-(carboxymethyl)lysine levels in patients with type 2 diabetes: role of renal function.

Advanced glycation end products (AGEs) such as N(epsilon)-(carboxymethyl)lysine (CML) have been implicated in the development and progression of diabetic nephropathy. The aim of the present study is to investigate AGE levels in patients with type 2 diabetes with special regard to the role of renal impairment. Serum and urine CML levels (using a newly developed enzyme-linked immunosorbent assay), as well as serum AGE-fluorescence, were measured in 109 patients with type 2 diabetes. Patients were divided into groups with normal and impaired renal function. We found elevated serum fluorescent AGE and CML levels, as well as decreased urinary CML excretion rates, in patients with diabetes with renal impairment, but not those with normal renal function. In the presence of impaired renal function, serum CML and fluorescent AGE levels showed a significant inverse relation with creatinine clearance and a significant direct correlation with each other. No relationship could be found between serum AGE levels and parameters of blood glucose control or the presence of the following clinical complications: ischemic heart disease, diabetic retinopathy, and neuropathy. We conclude that the decline in renal function leads to increased serum AGE levels in patients with type 2 diabetes.

[Detection of glycation end products in the urine of diabetic patients]. / Glikációs végtermékek kimutatása diabetes mellitusban szenvedö betegek vizeletében.

Advanced glycation end products play an important role in the development of tissue damage in diabetes mellitus. The aim of the present study was the investigation of the excretion of different glycation end products in the urine. Methylglyoxal, an intermediate product of the glycation, formed with L-arginine in an in vitro model two fluorescent peaks. These peaks can be characteristic for imidazolone-like product(s) which are produced also in the reaction of methylglyoxal with proteins described in the literature, suggesting modification of proteins with methylglyoxal at the guanidino group of the L-arginine amino acid. Using the fluorescent characteristics of these (excitation/emission: 320/400 nm and 340/425 nm) and the generally accepted wavelength of the so called non-specific advanced glycation end product (370/440 nm) could be identified these glycation end products in the urine of 98 patients with diabetes mellitus (21 type I., 77 type II., 51 female and 47 male, mean age: 56.6 years). These three particular glycation end products showed significant intercorrelations in the urine (p < 0.001). Concentrations of these glycation end products in the urine correlated negatively with the serum creatinine in the range between 120-240 mumol/l (p < 0.001). Data presented here verify that non-specific glycation end product and imidazolone-like glycation end products can be detected in the urine of diabetic patients. Elimination of these products by the urine is markedly decreased in the stage of early renal insufficiency. These decrease in the secretion can cause an elevation of the advanced glycation end products in the circulation leading progression of diabetic complication.

[Detection of carbonyl stress markers in the urine of diabetic patients]. / Karbonil stressz-metabolitok kimutatása diabetes mellitusban szenvedö betegek vizeletében.

Carbonyl stress-induced tissue damage is caused by reactive aldehydes produced by non-enzymatic glycation, oxidative stress and metabolic processes. The aim of this study was the detection of the major markers of carbonyl stress in the urine of diabetic patients (21 type 1, 77 type 2, 51 female, 47 male, 56.6 +/- 13.7 year of age; mean +/- SD). Oxidative stress was detected by using the reaction of malondialdehyde, the end product of free radical damage of the tissues, with L-arginine. This reaction produced a fluorescent compound, pyrimidinyl-L-ornithine. Thus, pyrimidinyl-L-ornithine, as well as pentosidine, an advanced glycation end product, and the non-specific advanced glycation end product, which is thought to be partially as a result of lipid peroxidation, could be detected simultaneously by using the fluorescent method. Correlation coefficients among the concentrations of these products in the urine of 98 diabetic patients were as follows: pyrimidinyl-L-ornithine vs. non-specific advanced glycation end product: r = 0.72, p < 0.001; pentosidine vs. non-specific advanced glycation end product: r = 0.68, p < 0.001; pentosidine vs. pyrimidinyl-L-ornithine: r = 0.60, p < 0.001. Strong negative correlations were found between the serum creatinine levels of these patients, between 120-240 mumol/l, and the urinary concentration of these products: r = -0.88 for non-specific advanced glycation end products, r = -0.86 for pentosidine and r = -0.89 for pyrimidinyl-L-ornithine (p < 0.001 for all three). These data support a closer relation of the so-called non-specific glycation end product to oxidative stress than to non-enzymatic glycation. Results presented here suggest an early retention of the products of carbonyl stress in the patients with moderate renal insufficiency, which can play a role in the development of diabetic complications.

Possible role of free radicals generated by pseudohypoxia in the regulation of hepatic glucose output. An in vitro model using rat liver microsomal glucose 6-phosphatase.

Hepatic glucose output is decreased by hyperglycaemia through an unknown mechanism. We hypothesize that free radicals generated by hyperglycaemic pseudohypoxia might cause glucose output to decrease by inhibiting glucose 6-phosphatase - a key enzyme of gluconeogenesis. To prove this a model experiment was performed on a microsome fraction of rat liver. One of the characteristic features of pseudohypoxia due to hyperglycaemia is an increase in the ratio of NADH/NAD+, so in the present study the changes in NADH - induced glucose 6-phosphatase activity were investigated as related to the release of inorganic phosphate (Pi) derived from glucose 6-phosphate. After incubation for 50 min, Pi release was significantly reduced by NADH (4.026 +/- 0.189 vs 2.696 +/- 0.429 micromol x l(-1) x mg protein(-1), control vs NADH samples, p < 0.01). The decrease in the activity of glucose 6-phosphatase generated by NADH was prevented by using desferrioxamine, an irreversible ferric chelator, butylated hydroxytoluene and Trolox, two agents which inhibit lipid peroxidation, and reduced glutathione, a non-specific radical scavenger. Superoxide dismutase, catalase and the hydroxyl radical scavenger dimethyl sulphoxide proved to be ineffective. When the above investigations were carried out in the presence of a ferric-EDTA complex the inhibition of glucose 6-phosphatase was found to be inducible by hydrogen peroxide and/or hydroxyl free radicals. These investigations seem to indicate that pseudohypoxia due to hyperglycaemia can inhibit the activity of glucose 6-phosphatase both by lipid peroxidation and by inducing hydrogen peroxide and/or hydroxyl free radicals and thus it may play a part in the glucose-induced decrease of hepatic glucose output.