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.

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.

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.

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.

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.

Sport activity and personality as elements in preventing cancer and coronary heart disease.

Results are reported for 3 groups of healthy male probands, 318 in each group, matched for age and personality type on the Personality-Stress Questionnaire. One group was actively engaged in sports, one had discontinued former sporting activities, and one group had never taken part in regular sports. Follow-up after 13 yr. showed lowest mortality in those actively engaged in sport, highest mortality in those who had given up sport, with those who had never been engaged in sport intermediate. Prophylactic behaviour therapy was shown to reduce mortality of those who had given up sport to a significant extent but not to affect degree of retinal sclerosis.

Protein kinase activity can be separated from the purified activated rat liver glucocorticoid receptor.

Previous studies have shown that the purified rat liver glucocorticoid receptor (GR) has a protein kinase activity. In this report we show that the GR-associated kinase can be partially separated from the 94-kDa steroid-binding protein by DEAE-Sepharose chromatography. The kinase elutes from the column at a higher salt concentration than the 94-kDa GR protein. This GR copurifying protein kinase phosphorylates basic substrates such as various histone fractions and protamine. The phosphorylation occurs in the presence of Mg2+ ions, and is not supported by Ca2+ ions. The amino acid residues phosphorylated by the kinase are threonine and serine. This kinase also phosphorylates the 94-kDa GR protein and thus might be of physiological relevance for the GR function.

Psychosocial factors as strong predictors of mortality from cancer, ischaemic heart disease and stroke: the Yugoslav prospective study.

We investigated the relation of psychosocial risk factors to mortality in a prospective study of 1353 inhabitants of Crvenka, 619 of whom died between 1966 and 1976. All 38 lung cancer deaths occurred in those with high scores for rationality and antiemotionality (R/A), a factor related to suppression of aggression. Compared with lower R/A, high R/A was also associated with a relative risk of mortality of 29 for other cancer, 4.3 for ischaemic heart disease and 6.5 for stroke. Standardising for R/A reduced the smoking/lung cancer association, virtually eliminated the smoking/other cancer and smoking/heart disease relationships and reduced the association of heart disease with blood cholesterol, blood sugar and hypertension. Long lasting hopelessness was also independently associated with cancer as was anger with heart disease, though not so strongly as for R/A. Psychosocial variables are important predictors of mortality and decisively modify the effect of physical risk factors such as smoking.

The role of multiply phosphorylated S6 in ribosome degradation.

Rat liver ribosomes, prepared 1-24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.

[Changes in steroid receptors during histopathologic transformation of tissue in the human uterus]. / Promene steroidnih receptora u toku histopatoloske transformacije tkiva humanog uterusa.

Estrogen (ER) and progesterone receptors (PR) were analyzed in the cytosol and nuclei prepared from specimens of human uterine tissue of patients with certain disorders identified as hyperplasia endometrii adenomatosa, myoma uteri per magnum, adenocarcinoma endometrii and adenocarcinoma corporis uteri. These investigations have revealed a different level of ER and PR in analyzed tissue specimens, as well as the existence of a relationship between changes in receptor levels and respective Kd. These changes suggest a correlation between steroid receptor levels and the type of tissue transformation. The functionality of the receptors was analyzed by the ultracentrifugation of non-activated and activated steroid-receptor complexes in sucrose density gradients, as well as by the investigation of their interaction with isolated nuclei. These results indicate that some changes in steroid receptor molecules can be detected when the tissue turns from normal to malignant transformation. On the basis of this investigation it could be proposed that the analysis of activated and non-activated steroid-receptor complexes by means of the methods used in this study can be applied as a useful clinical tool in the determination of the endocrine dependence of transformed tissues, as well as for the optimum dosing of individual treatment of patients with uterine and other tissue carcinoma.

Psychosomatic factors involved in the process of cancerogenesis. Preliminary results of the Yugoslav prospective study.

In a Yugoslav village, psychosocial, anamnestic medical, and pathophysiological data were recorded for 1,353 persons in a longitudinal study. The role of psychosocial stress in carcinogenesis, as far as we have investigated it, may be described as follows: (1) Psychosocial stress in terms of high hopelessness, high antiemotionality, etc. has a strong relevance for cancer incidence which does not act via one of our physiological variables associated with cancer. This follows from the results of our multivariate analysis. (2) Psychosocial stress is substantially associated with a low lymphocyte percentage, which in turn is a relatively strong risk factor for cancer. (3) Psychosocial stress is relatively weakly associated with the cholesterol minimum; but apart from the fact that the cholesterol characteristics of cancer subjects are more marked descriptively under stress conditions, psychosocial stress significantly enhances the efficacy of the most important physiological risk variables for cancer.

Smoking as a risk factor for lung cancer and cardiac infarct as mediated by psychosocial variables. A prospective investigation.

In 1965-66, a prospective psychosomatic investigation was started with 1,353 relatively old inhabitants of the village of Crvenka, Yugoslavia. The present article reports on the relevance of smoking for the incidence of lung cancer and cardiac infarct. The main results are: (1) The relevance of smoking is reduced, but not eliminated, by introducing psychosocial control variables, suggesting that the latter have direct influences both on smoking and on the diseases. (2) The relevance of smoking interacts very strongly with psychosocial background conditions: it is nearly reduced to zero when the latter are favorable, and is correspondingly high when they are unfavorable. The results are also interpreted in biochemical terms.