Neuroendocrine and oxidoreductive mechanisms of stress-induced cardiovascular diseases.

The review concerns a number of basic molecular pathways that play a crucial role in perception, transmission, and modulation of the stress signals, and mediate the adaptation of the vital processes in the cardiovascular system (CVS). These highly complex systems for intracellular transfer of information include stress hormones and their receptors, stress-activated phosphoprotein kinases, stress-activated heat shock proteins, and antioxidant enzymes maintaining oxidoreductive homeostasis of the CVS. Failure to compensate for the deleterious effects of stress may result in the development of different pathophysiological states of the CVS, such as ischemia, hypertension, atherosclerosis and infarction. Stress-induced dysbalance in each of the CVS molecular signaling systems and their contribution to the CVS malfunctioning is reviewed. The general picture of the molecular mechanisms of the stress-induced pathophysiology in the CVS pointed out the importance of stress duration and intensity as etiological factors, and suggested that future studies should be complemented by the careful insights into the individual factors of susceptibility to stress, prophylactic effects of 'healthy' life styles and beneficial action of antioxidant-rich nutrition.

Regional differences in antioxidative response of rat brain after cranial irradiation.

In order to examine if differences in activity and inducibility of antioxidative enzymes in rat cerebral cortex and hippocampus are underlying their different sensitivity to radiation, we exposed four-day-old female Wistar rats to cranial radiation of 3 Gy of gamma-rays. After isolation of hippocampus and cortex 1 h or 24 h following exposure, activities of copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) were measured and compared to unirradiated controls. MnSOD protein levels were determined by SDS-PAGE electrophoresis and Western blot analysis. Our results showed that CuZnSOD activity in hippocampus and cortex was significantly decreased 1 h and 24 h after irradiation with 3 Gy of gamma-rays. MnSOD activity in both brain regions was also decreased 1 h after irradiation. 24 h following exposure, manganese SOD activity in hippocampus almost achieved control values, while in cortex it significantly exceeded the activity of the relevant controls. CAT activity in hippocampus and cortex remained stable 1 h, as well as 24 h after irradiation with 3 Gy of gamma-rays. MnSOD protein level in hippocampus and cortex decreased 1 h after irradiation with 3 Gy of gamma-rays. 24 h after exposure, MnSOD protein level in cortex was similar to control values, while in hippocampus it was still significantly decreased. We have concluded that regional differences in MnSOD radioinducibility are regulated at the level of protein synthesis, and that they represent one of the main reasons for region-specific radiosensitivity of the brain.

Alterations in hippocampal antioxidant enzyme activities and sympatho-adrenomedullary system of rats in response to different stress models.

The study deals with activity of three antioxidant enzymes, copper, zinc-superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT) in hippocampus of rats, following the exposure to single chronic (individual housing or forced swimming) and acute (immobilization or cold) stress, as well as to combined chronic/acute stress. In addition, plasma noradrenaline (NA) and adrenaline (A) concentrations were measured in the same stress conditions, because their autooxidation can add to the oxidative stress. We observed that i) long-term social isolation and repeated forced swimming had minor effects on plasma catecholamines, but in the long-term pretreated groups, acute stressors caused profound elevation NA and A levels, ii) chronic stressors activate antioxidant enzymes, iii) acute stressors decrease catalase activity, their effects on CuZnSOD appear to be stressor-dependent, whereas MnSOD is not affected by acute stressors, and iv) pre-exposure to chronic stress affects the antioxidant-related effects of acute stressors, but this effect depends to a large extent on the type of the chronic stressor. Based on both metabolic and neuroendocrine data, long-term isolation appears to be a robust psychological stressor and to induce a "priming" effect specifically on the CuZnSOD and CAT activity.

Ontogenetic profile of ecto-ATPase activity in rat hippocampal and caudate nucleus synaptic plasma membrane fractions.

An ontogenetic study of ecto-ATPase activity and the content of enzyme proteins was assessed in the caudate nucleus and hippocampal synaptic plasma membranes isolated from rats at various ages (15, 30, 90, 180 and 365 days). The ontogenetic profile revealed that the enzyme activities in both brain areas were the highest on day 30 and 365, while the ecto-ATPase protein abundance was the highest on day 15 after birth. Possible explanation for obtained ontogenetic profile and the discrepancy between activity and abundance may reside in the fact that ecto-ATPase during development could exert additional roles other than those related to metabolism of ATP. It is likely that ecto-ATPase, regulating the concentration of ATP and adenosine in synaptic cleft, has important role in the processes of brain development and aging.

The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines.

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [alpha2-macroglobulin (alpha2-M) and gamma-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and alpha2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.

Binding of estradiol to synaptosomal mitochondria: physiological significance.

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.

Effect of steroid hormone deprivation on the expression of ecto-ATPase in distinct brain regions of female rats.

Abundant evidence indicates that ATP and adenosine act as neurotransmitters or co-transmitters, influencing nerve cell physiology in various ways. Therefore, regulation of ATP-metabolizing enzymes is essential for the normal development and function of neuronal tissue. In the present study we have examined the effect of gonadal (OVX) or adrenal (ADX) steroid hormone deprivation on the activity and expression of synaptic membrane ecto-ATPase in three extrahypothalamic brain areas of female rats, primarily not associated with reproductive function. It was shown that OVX significantly increased ecto-ATPase activity and the relative abundance of this enzyme in the hippocampal (Hip) and caudate nucleus (CN), but not in brain stem (BS) membrane preparations. ADX was followed by an upregulation of the enzyme activity and its relative abundance in all the brain areas investigated. The highest enzyme activity and the most profound effects of OVX and ADX were detected in the CN. The results obtained indicate that ADX and OVX upregulate the expression of ecto-ATPase, potentiating the production of adenosine in synaptic cleft thus modulating the activity of numerous neurotransmitter systems in distinct areas of the CNS.

Chromosome aberrations, micronuclei, and activity of superoxide dismutases in human lymphocytes after irradiation in vitro.

The goal of this study was to provide data on the dose-dependent production of dicentrics and micronuclei in human lymphocytes irradiated with 22.6 MeV protons and to estimate the possible contribution of intracellular superoxide dismutases (SOD) to the relative biological effectiveness (RBE) of protons. For the dose-response study, heparinized whole blood of a healthy volunteer was irradiated with protons and X-rays employing radiation doses of 0.5-4 Gy. Three biological endpoints were analyzed: chromosomal aberrations, micronuclei, and specific activity of cytosolic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases in harvested human blood cells. Dicentric dose-response curves fit a linear-quadratic form (alpha = 0.094 +/- 0.006, beta = 0.032 +/- 0.001) induced with X-rays and (alpha = 0.119 +/- 0.057, beta = 0.029 +/- 0.014) for 22.6 MeV protons. Protons were more effective than X-rays in producing exchanges, particularly at 0.5 and 1 Gy. In contrast to X-ray irradiated samples where a significant increase in the specific activity of MnSOD was recorded (up to a radiation dose of 1 Gy), irradiation with protons markedly reduced its activity. As a consequence of the reduced activity of MnSOD, the chromosomal dose-response curve became quadratic. The RBE for dicentrics varies with dose (from 2.2 to 1.01) and reduced activity of MnSOD is an important contributor to the RBE of protons. SODs, particularly MnSOD, play an important role in defending DNA from reactive oxygen species. A reduced activity of SOD, particularly MnSOD, is an important contributor to the RBE of protons.

Role of antioxidant enzymes in radiosensitivity of human blood cells.

The marked variability in radiation response among individuals of the same age group prompted us to investigate the role of antioxidative enzyme activity. Micronuclei (MN) and enzyme assays were performed on blood samples of healthy male volunteers. The procedure consisted of micronucleus analysis and measurement of the superoxide dismutase (SOD) activity in harvested blood samples irradiated in vitro with 2 Gy gamma-rays and in unirradiated control samples for each individual. We found that the yield of radiation-induced micronuclei was in the range of 112 to 378 micronuclei per 1000 binucleated cells. The activity of cytosol superoxide dismutase (CuZnSOD) was reduced, whereas the activity of manganese superoxide dismutase (MnSOD) was markedly elevated in the blood samples harvested in lymphocyte cultures after irradiation. The analysis of our results showed that MnSOD plays the most important role in radiation-induced cellular damage. The results of this investigation showed that measurement of micronuclei and the activities of SOD in harvested human blood cells can serve as a rapid predictive assay of radiosensitivity in a clinical setting.

Identification and characterization of glucocorticoid receptors in B16 mouse melanoma cells.

OBJECTIVE: To gain better insight into the role of glucocorticoids as modulators of cell growth, as well as to investigate the presence and characteristics of glucocorticoid receptors (GR) in mouse melanoma cells. METHODS: In two different B16 mouse melanoma cell clones (B16/F10 and B16/C3) the role of synthetic glucocorticoids (triamcinolone acetonide, TA) as cell growth modulators was investigated. RESULTS: The inhibitory effect of TA on B16/F10 cell growth after 8 days in culture was observed. The same hormonal treatment applied on B16/C3 melanoma cells also provoked changes in the cell growth. Dot blot analysis, using monoclonal antirodent glucocorticoid receptor antibodies showed the presence of receptor protein in both cell clones. The analysis of glucocorticoid receptors in B16/F10 and B16/C3 cell cytosol by Scatchard assay and ion-exchange chromatography on DEAE-Sephadex A-50 minicolumn indicated that the changes in melanoma cell growth may be mediated by glucocorticoid receptors and may relieve changes in the GR itself. CONCLUSIONS: It was found that B16/C3 melanoma cells exhibited different growth pattern under TA treatment when compared to the results obtained with B16/F10 cells. Such differences may be mediated by glucocorticoid receptors.

Glucocorticoid receptors in ageing rats.

The role of the glucocorticoid receptor (GR) in senescence was studied in rats of increasing age. Statistically significant changes in the number of GRs from rat liver were detected, whereas the affinity for the ligand triamcinolone acetonide (TA) did not change with increasing age, and was in the range of 1-2 nM. In all cases the number of receptors was lower in rats treated with hormone in vivo relative to untreated animals. In addition, we have found changes in GR activation, as measured by the binding to DNA cellulose in the mentioned age groups. Furthermore, expression of the glucocorticoid hormone (GH)-inducible gene, tyrosine amino transferase (TAT) also showed age-related alterations. We conclude that receptor function shows oscillatory changes during ageing. In addition, response to GH generally declines towards the older age. This specific periodicity in functional characteristics of the GR may reconcile conflicting results about the receptor number and properties during the ageing process, and marks particular age at which individual organism shows the highest or the lowest sensitivity to the actions of GH.

Using yeast to study glucocorticoid receptor phosphorylation.

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.

Adrenalectomy and dexamethasone treatment alter the patterns of basal and acute phase response-induced expression of acute phase protein genes in rat liver.

Hormonal requirements for full hepatic expression of alpha2-macroglobulin (alpha2M), alpha1-acid glycoprotein (AGP), haptoglobin (Hp) and gamma-fibrinogen (Fb) were assessed at the level of mRNA. Prior to exposure to turpentine-induced inflammation, rats were either depleted of glucocorticoids by adrenalectomy or supplemented with an excess of dexamethasone. Adrenalectomy alone did not affect the basal level of acute phase protein (APP) expression except for alpha2M mRNA, the level of which was enhanced. In contrast, dexamethasone treatment alone promoted full induction of alpha2M, significant, but not maximal increase of AGP and Hp mRNAs and suppression of Fb. In adrenalectomized rats, acute phase (AP)-cytokines, released in response to inflammation, promoted full expression of Fb and Hp and increased the level of AGP mRNA whereas alpha2M mRNA remained at the basal level. Inflammation in dexamethasone pretreated rats elicited changes which, in comparison to mRNA values for dexamethasone unpretreated inflamed rats, were seen as overexpression of alpha2M, full expression of AGP and incomplete expression of Hp, whereas Fb mRNA remained at the basal level. These data suggest that glucocorticoids are the principal inducers of alpha2M and AP-cytokines of Fb. For full induction of AGP, additive actions of glucocorticoids and AP-cytokines are required whereas expression of Hp is predominantly controlled by AP-cytokines.

Influence of pyridine and urea on the rat brain ATPase activity.

The neurotoxicity of pyridine and urea was investigated in respect to their ability to alter the activity of synaptosomal membrane Na+/K(+)-ATPase and Mg(2+)-ATPase. In vitro treatment with pyridine and urea stimulated Na+/K(+)-ATPase activity in a dose-dependent manner up to 40% and 60%, respectively. Mg(2+)-ATPase activity increased up to 40% after pyridine treatment, while urea had no effect at all. The neuroactive potencies of pyridine and urea were evaluated by estimating parameters Km and delta Vmax for enzyme stimulation, as well as Hill coefficient to estimate the levels of cooperativity for pyridine and urea binding. The results suggest that pyridine stimulates both enzymes, probably by interacting with some neuronal membrane components, and altering the lipid micro-environment of the ATPases. In contrast, urea stimulates the Na+/K(+)-ATPase only, assumingly by acting on it directly or via some other regulatory mechanism. Stimulation of Na+/K(+)-ATPase and Mg(2+)-ATPase by the substances tested and subsequent alteration of neuronal cell functioning could contribute to the CNS dysfunction upon chronic exposure to pyridine and urea.

Effects of estradiol benzoate and progesterone on superoxide dismutase activity in the rat liver.

Activities of superoxide dismutases MnSOD and CuZnSOD were measured in appropriate subcellular fractions prepared from livers of intact and long-term gonadectomized (GX) rats of both sexes, and of GX female and male rats injected sc with a single dose of 5 micrograms estradiol benzoate (EB) or 2 mg progesterone (P). In female livers, MnSOD activity did not vary significantly during the estrous cycle, declined after gonadectomy in comparison to proestrus, and was steady in GX females treated with EB or P. The activity of CuZnSOD was lowered at proestrus and elevated after removal of the ovaries in comparison to proestrus value. EB suppressed, and P elevated CuZnSOD activity in GX females. In the liver of male rats, MnSOD was not affected by gonadectomy nor by EB and P treatments. CuZnSOD activity was reduced following orchiectomy and enhanced in GX males following treatment with P, while EB had no effect. These results suggest that P and EB modulate the activity of CuZnSOD and do not affect MnSOD in the rat liver. The modulatory effects are elicited by P in the males and by P and EB in the females.

Estradiol in vitro modulates Na+-dependent Ca2+ uptake by synaptic plasma membrane vesicles of rat brain regions.

Membrane vesicles loaded with [Na+], prepared from synaptosomal plasma membranes (SPM) of whole brains (WB), hippocampi (Hip) and caudate nuclei (NC) of female rats, were used to study Na+ -dependent Ca2+ transport across SPM vesicles under the influence of 17beta-estradiol (E2) in vitro. In concentrations near to physiologic, E2 significantly increased 45Ca2+ uptake by SPM vesicles from all the brain tissues investigated. The maximum increase was observed for WB (21%) and Hip (33%) at 10(-9) mol/l, and for NC (31%) at 5 x 10(-9) mol/l of E2. These results (a) confirm our earlier finding that E2 in vitro modulates Na+-dependent Ca2+ transport across synaptosomal membrane in rat brain regions, and (b) suggest Na+/Ca2+ exchange as principal mechanism of the E2-stimulated Na-dependent Ca2+ uptake by membrane vesicles. The involvement of any ATPases as possible mediators is discussed.

Steroid receptors in nonmalignant and malignant kidney tissue of patients with endemic (Balkan) nephropathy.

The presence, affinity, binding capacity, structure and function of receptors for estrogen (ER), progesterone (PR) and glucocorticoid (GR) were investigated in 24 autologous pairs of control and neoplastic kidney tissues of patients with endemic (Balkan) nephropathy. In control tissue, all the three steroid receptors were absent in 20.8% and present in 25.0% of samples, whereas in malignant tissues the percentage of negative samples increased to 37.5% and that of positive ones decreased to 20.8%. Ten patients had identical receptors in both, control and cancer tissues. Due to malignant transformation nine patients lost one or more receptors, while five patients acquired them. Wide ranges of values were obtained when evaluating receptor affinity (Kd) and binding capacity (N). The structure and function of steroid receptors were investigated by determining the sedimentation coefficients (S) of steroid-receptor complexes before and after the activation. The unactivated GR-complex (8 S) was detected in two of control samples only, whereas in the remaining control tissues, as well as in malignant tissues only the activated form (4 S) was found regardless of the activation. PR and ER complexes were detected at 4 S region only. These results show that in endemic nephropathy the structure of steroid receptors may be altered often in both, non malignant and malignant kidney tissue, suggesting that the analysis of receptor structure may be worthwhile for the prediction of the success of eventual hormone therapy.