The intracellular concentration of homocysteine and related thiols is negatively correlated to sperm quality after highly effective method of sperm lysis.

The aims of this study were to determine concentrations of total homocysteine, cysteine, cysteinylglycine and glutathione in spermatozoa, seminal fluid and blood plasma and to analyse their relationships with sperm parameters. For this reason, a new highly effective method of spermatozoa lysis was developed, using methanol, freezing and subsequent thawing in ultrasonic bath. An HPLC-FD assay was conducted on thiols concentrations in lysed spermatozoa, seminal fluid and blood plasma. Concentrations of thiols in spermatozoa were significantly lower in men with normozoospermia than in samples with pathological semen parameters. Statistical analysis found significant correlations between thiol concentrations in spermatozoa and semen parameters, while the same analysis with thiol concentrations in seminal fluid was substantially less powerful. Only cysteinylglycine concentrations in seminal fluid significantly correlated with pathological semen parameters. No significant differences or correlations were found with blood plasma concentrations.

Lack of the effect of superoxide dismutase and catalase on Na+,K+-ATPase activity in stunned rabbit hearts.

Reactive oxygen species (ROS) have been implicated in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. In this study, we examined protective effects of antioxidant enzymes, superoxide dismutase (SOD) and catalase, against ischemia/reperfusion-induced cardiac dysfunction and inhibition of Na+,K+-ATPase activity. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min of global normothermic ischemia followed by 10 min reperfusion. The hearts treated with SOD plus catalase did not show significant recovery of left ventricular (LV) end-diastolic pressure compared with untreated ischemic reperfused hearts. Treatment with antioxidants had no protective effects on developed LV pressure or its maximal positive and negative first derivatives (+/-LVdP/dt). Myocardial stunning was accompanied by significant loss in sarcolemmal Na+,K+-ATPase activity and thiol group content. Inhibition of enzyme activity and oxidation of SH groups were not prevented by antioxidant enzymes. These results suggest that administration of SOD and catalase in perfusate do not protect significantly against cardiac dysfunction in stunned rabbit myocardium.

Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts.

Reactive oxygen species and other oxidants are involved in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. The present study investigated the oxidative modification of cardiac proteins in isolated Langendorff-perfused rabbit hearts subjected to 15 min normothermic ischemia followed by 10 min reperfusion. Reperfusion under these conditions resulted in only 61.8+/-2.7 % recovery of developed pressure relative to preischemic values and this mechanical dysfunction was accompanied by oxidative damage to cardiac proteins. The total sulfhydryl group content was significantly reduced in both ventricle homogenates and mitochondria isolated from stunned hearts. Fluorescence measurements revealed enhanced formation of bityrosines and conjugates of lipid peroxidation-end products with proteins in cardiac homogenates, whereas these parameters were unchanged in the mitochondrial fraction. Reperfusion did not alter protein surface hydrophobicity, as detected by a fluorescent probe 1-anilino-8-naphthalenesulfonate. Our results indicate that oxidation of proteins in mitochondria and possibly in other intracellular structures occurs during cardiac reperfusion and might contribute to ischemia-reperfusion injury.

Ion transport systems as targets of free radicals during ischemia reperfusion injury.

Oxidative stress is a recognized pathogenic factor in ischemia/reperfusion injury (IRI). Iron induced generation of reactive oxygen species (ROS) in vitro reduces both the Na+K+-ATPase activity and Na+-Ca2+ exchanger of synaptosomal membranes, concomitantly with alteration of physical state of membranes. Oxidative insult also leads to the loss of ability of endoplasmic reticular membranes (ER) to sequester Ca2+ as well as to the increase of Ca2+ permeability. Furthermore, ROS induces both lipid peroxidation and lipid-independent modifications of membrane proteins. Acute in vivo ischemia alters kinetic parameters of Na+K+-ATPase affecting mainly the dephosphorylation step of ATPase cycle with parallel changes of Na+-Ca2+ exchanger and alterations of physical membrane environment. Subsequent reperfusion after ischemia is associated with decrease of immuno signal for PMCA 1 isoform in hippocampus. In addition, incubation of non-ischemic membranes with cytosol from ischemic hippocampus decreases level of PMCA 1 in non-ischemic tissues. Loss of PMCA 1 protein is partially protected both by calpain- and by non-specific protease inhibitors which suggest possible activation of proteases in the reperfusion period. On the other hand, ischemia does not affect the level of Ca2+ pump (SERCA 2b) and calreticulin of intracellular Ca2+ stores. However, IRI resulted in a decrease of IP3 receptor I and altered active Ca2+ accumulation into the ER. A non-specific alteration of physical properties of total membranes such as the oxidative modifications of proteins as well as the content of lipoperoxidation products can also be detected after IRI. ROS can alter physical and functional properties of neuronal membranes. We discuss our results suggesting that ischemia-induced disturbation of ion transport systems may participate in or follow delayed death of neurons after ischemia.

Myofibrillar Ca2+-stimulated Mg2+-ATPase from chronically ischemic canine heart.

Functional properties of myofibrils from chronically ischemic canine myocardium were evaluated. Ischemia was produced by tight stenosis of left anterior descending artery (LAD), followed by 40 min acute ischemia with prior preconditioning. Animals of the first group were sacrificed after 8 weeks. In the second group, angioplasty of LAD was performed after 8 weeks of ischemia and animals were kept alive for other 4 weeks. Control animals were sham operated. Activity and kinetic parameters of myofibrillar Ca2+-stimulated Mg2+-ATPase were measured in myofibrils isolated from anterior and posterior parts of all hearts. We did not find any differences in maximal velocity (Vmax), half-maximal activation constant for calcium (K(Ca2+)50) and cooperativity coefficient (n(hill)) of myofibrils from different experimental groups as compared to controls, either at pH 7, pH 6.5 (acidosis) or pH 7.5 (alkalosis). K(Ca2+)50 increased in medium simulated acidosis (12.6-33.5 times) and n(hill) decreased significantly in all groups as compared with values obtained at pH 7. These results indicate that activity and Ca2+-sensitivity of myofibrillar Mg2+-ATPase remain unchanged despite deteriorated heart function 8 weeks after LAD obstruction. Experiments have confirmed that Ca2+-stimulated-ATPase from canine heart myofibrils responded to pH decrease by a decreased sensitivity to Ca2+ and a decreased cooperativity. However, sensitivity of the enzyme to the pH changes is unaltered by 8 weeks of chronic ischemia.

Membrane ion transport systems during oxidative stress in rodent brain: protective effect of stobadine and other antioxidants.

The effect of oxidative stress in vitro induced by radical generating systems (RGS) (Fe2+-EDTA and Fe2+-EDTA plus H2O2) on synaptosomal and microsomal ion transport systems as well as on the membrane fluidity was investigated. Oxidative insult reduced Na+, K+-ATPase activity by 50.7% and Na+-dependent Ca2+ uptake measured in choline media by 46.7%. Membrane fluidity was also significantly reduced as observed with the fluorescent probe. Stobadine (ST) prevented the decrease in membrane fluidity and Na+-dependent Ca2+ uptake, however Na+, K+-ATPase activity was only partially protected, indicating a more complex mechanism of inhibition. Incubation of microsomes with RGS led to the loss of ability of membranes to sequester Ca2+, as well as to the decrease of Ca2+-ATPase activity and to the increase of Ca2+ permeability to 125.1%. The relative potency of the two RGS to decrease membrane fluidity correlated well with the system's potencies to induce lipid peroxidation. The extent of protection against depression of Ca2+ uptake values and Ca2+-ATPase activity by membrane soluble antioxidants (U-74500A, U-83836E, t-butylated hydroxytoluene-BHT and ST) was dependent on the experimental conditions and on the dose and nature of antioxidant used. ST seems to be at least as affective as BHT and 21-aminosteroids, and more potent than tocopherol acetate. Water soluble glutathione had no significant effect on the RGS induced inhibition of Ca2+-ATPase activity. Combination of ST with glutathione enhanced ST antioxidant efficacy, so drug combination might be beneficial therapeutically.

Carnosine: an endogenous neuroprotector in the ischemic brain.

1. The biological effects of carnosine, a natural hydrophilic neuropeptide, on the reactive oxygen species (ROS) pathological generation are reviewed. 2. We describe direct antioxidant action observed in the in vitro experiments. 3. Carnosine was found to effect metabolism indirectly. These effects are reflected in ROS turnover regulation and lipid peroxidation (LPO) processes. 4. During brain ischemia carnosine acts as a neuroprotector, contributing to better cerebral blood flow restoration, electroencephalography (EEG) normalization, decreased lactate accumulation, and enzymatic protection against ROS. 5. The data presented demonstrate that carnosine is a specific regulator of essential metabolic pathways in neurons supporting brain homeostasis under unfavorable conditions.

Na/K-ATPase under oxidative stress: molecular mechanisms of injury.

1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters. 2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components. 3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP K(m) value for the endogenous enzyme was about 85 microM; the Kh was equal to 800 microM. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate Vm, while the K(m) was increased slightly and interprotomer interaction was abolished. 4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly. but the sigmoidal component, characterizing the enzyme's ability to form oligomers was abolished completely. The K(m) value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another. 5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.

Protein kinase C expression and subcellular distribution in chronic myocardial ischemia. Comparison of two different canine models.

We studied protein kinase C (PKC) isozyme expression and activity distribution in two models of chronically ischemic canine myocardium: (1) single vessel obstruction (SVO), produced by tight stenosis of LAD followed by preconditioning and acute ischemia (40 min); (2) three vessel obstruction (3VO), produced by LAD-stenosis and gradual occlusion of right coronary artery and left circumflex. In both models after 8 weeks of chronic ischemia the dogs were either sacrificed or had PTCA of the LAD with a follow up of another 4 weeks. Control dogs were sham operated. PKC activity was measured in subcellular fractions of tissue samples from anterior and posterior regions in the presence of histone and gamma-[32P]-ATP. PKC isozymes were detected by Western blotting. All regions perfused by the obstructed coronaries were dysfunctional at 8 weeks when compared to baseline, with improvement of anterior wall function after PTCA of LAD. PKC activity was elevated in the membrane fraction of SVO, but unchanged in the 3VO model. PKCs alpha, epsilon, and zeta prevailed in cytosol fraction of the controls (cytosol/membrane ratios were +/- 3.34, 1.38 and 4.56 for alpha, epsilon and zeta, respectively), consistent with PKC activity distribution, while delta was not detected. There was no significant difference between the groups concerning the relative membrane amount of the isozymes. PKCs alpha and epsilon were decreased in the cytosol fraction of both models at 8 weeks (for anterior region, by 56 and 57% in SVO and by 49 and 46% in 3VO, respectively) without there being any differences between anterior and posterior regions, and were low also in the PTCA group. PKC zeta distribution however varied between the two models. The amount of PKC zeta isozyme was downregulated by 45% after 8 weeks of chronic ischemia and returned towards the control values after PTCA in the anterior region of SVO, while it did not change in anterior wall after 8 weeks in 3VO but was significantly decreased (by 47%) in posterior region after PTCA. In conclusion, our results suggest modified PKC signalling in chronically ischemic canine myocardium.

[The effect of ischemia and ischemia-reperfusion on ion transport systems]. / Vplyv ischémie a ischémie-reperfúzie na iónové transportné systémy.

Interruption of cerebral blood flow leads to dissipation of ionic gradients as the consequence of ionic channel overstimulation and ionic pump failure. The aim of this work was to study the possible effects of ischaemia and ischaemia followed by reperfusion on biochemical properties of endoplasmic calcium pump and synaptosomal sodium pump and sodium/calcium exchanger. The results presented in this study showed that 15 minute ischaemia led to the inhibition of all three ionic transport systems, however in different degrees. 60 minute reperfusion following 15 minute ischaemia led to partial recovery of calcium pump and sodium/calcium exchanger. The activity of sodium pump was still significantly depressed. Ischaemia and ischemia followed by reperfusion did not affect kinetic parameters of calcium pump. On the other side, both ischaemia and ischaemia-reperfusion led to an increase of sodium pump affinity to ATP and a decrease of the enzyme affinity to potassium. The possible causes of the changes, as the alteration of membrane structure or altered enzymes phosphorylation are discussed in the study. In addition to the inhibitory effect of ischaemia-reperfusion injury, intracellular water accumulation, as the possible consequence of altered ion homeostasis, is documented by nuclear magnetic resonance (imaging).

Iron-induced inhibition of Na+, K(+)-ATPase and Na+/Ca2+ exchanger in synaptosomes: protection by the pyridoindole stobadine.

The effect of oxidative stress, induced by Fe(2+)-EDTA system, on Na+,K(+)-ATPase, Na+/CA2+ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 microM FeSO4-EDTA per mg of protein at 37 degrees C for 30 min. The oxidative insult reduced Na+,K(+)-ATPase activity by 50.7 +/- 5.0% and Na+/Ca2+ exchanger activity measured in potassium and choline media by 47.1 +/- 7.2% and 46.7 +/- 8.6%, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na+/Ca2+ exchanger activity. The Na+,K(+)-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na+/Ca2+ exchanger and the Na+,K(+)-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.

Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium.

We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.

Synaptosomal Na, K-ATPase during forebrain ischemia in Mongolian gerbils.

We studied the activity and kinetic parameters of synaptosomal Na, K-ATPase during 15 min of forebrain ischemia and following 60 min of reperfusion produced by reversible common carotid occlusion in Mongolian gerbils. A synaptosomal fraction was obtained by both differential centrifugation of brain tissue homogenate and centrifugation of crude mitochondrial fraction at a discontinual sucrose density gradient. We found two components of ATP concentration dependence of ATP hydrolysis that represent two types of ATP-binding sites: high affinity and low affinity. Neither ischemia nor reperfusion affected kinetic parameters of a high-affinity site. However, low-affinity site parameters were affected by both ischemia and ischemia followed by reperfusion. Maximal velocity (Vmax) decreased by 43 and 42% after ischemia and after ischemia/reperfusion, respectively. The apparent Km for ATP decreased by 52% after ischemia and by 47% after ischemia/reperfusion. The apparent affinities for K+ and Na+ were determined from the ATP hydrolysis rate as a function of Na+ and K+ concentrations. We found the half-maximal activation constant for K+ (KaK+) increased by 60% after ischemia and by 146% after ischemia/reperfusion. On the other hand, we found that KaNa+ decreased significantly after ischemia/reperfusion (16%). We concluded that it is the dephosphorylation step of the ATPase reaction cycle that is primarily affected by both ischemia and ischemia/reperfusion. This might be caused by alteration of the protein molecule and/or its surroundings subsequent to ischemia.