Activation of the ER stress and calcium signaling in angiomyolipoma.

Renal angiomyolipomas (AMLs) are uncommon benign tumors that occur sporadically or as a part of tuberous sclerosis complex (TSC). Risk of life threatening hemorrhage is the main clinical concern. Although several evidences suggest that hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is crucial for these tumors, modulation of other metabolic pathways might affect tumor growth and progression. Therefore, we aimed to further characterize angiomyolipoma by TSC1/TSC2 expression, hypoxic status, expression of endoplasmic reticulum (ER) stress markers and calcium transport from the ER through the inositol 1,4,5-trisphosphate (IP3) receptors. Despite our expectations, angiomyolipoma were not hypoxic, as determined by absent expression of the carbonic anhydrase IX, which is a reliable marker of hypoxia. This was in accord with very low expression of TSC1 (that is associated with HIF activation) and a high expression of TSC2. Angiomyolipoma specimens also showed a significant upregulation of an anti-apoptotic marker Bcl2 when compared to healthy kidney tissue supporting the induction of pro-survival signaling. Moreover, angiomyolipoma specimens showed the overexpression of the ER stress markers XBP1, CHOP and ATF4 as well as of the mediators of calcium metabolism, namely the type 1 and 2, but not the type 3 IP3 receptors. These data suggest that the ER stress response, survival and calcium metabolism-related pathways but not hypoxia is an important component of the angiomyolipoma pathogenesis.

Slow sulfide donor GYY4137 differentiates NG108-15 neuronal cells through different intracellular transporters than dbcAMP.

Cellular differentiation is the process, by which a cell changes from one cell type to another, preferentially to the more specialized one. Calcium fluxes play an important role in this action. Differentiated NG108-15 or PC12 cells serve as models for studying neuronal pathways. NG108-15 cell line is a reliable model of cholinergic neuronal cells. These cells differentiate to a neuronal phenotype due to the dibutyryl cAMP (dbcAMP) treatment. We have shown that a slow sulfide donor - GYY4137 - can also act as a differentiating factor in NG108-15 cell line. Calcium is an unavoidable ion required in NG108-15 cell differentiation by both, dbcAMP and GYY4137, since cultivation in EGTA completely prevented differentiation of these cells. In this work we focused primarily on the role of reticular calcium in the process of NG108-15 cell differentiation. We have found that dbcAMP and also GYY4137 decreased reticular calcium concentration by different mechanisms. GYY4137 caused a rapid decrease in type 2 sarco/endoplasmic calcium ATPase (SERCA2) mRNA and protein, which results in lower calcium levels in the endoplasmic reticulum compared to the control, untreated group. The dbcAMP revealed rapid increase in expression of the type 3 IP3 receptor, which participates in a calcium clearance from the endoplasmic reticulum. These results point to the important role of reticular calcium in a NG108-15 cell differentiation.

Sulphide signalling potentiates apoptosis through the up-regulation of IP3 receptor types 1 and 2.

AIM: To investigate an interaction between the calcium and sulphide signalling pathways, particularly effects of the slow H2 S release donor morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate (GYY4137) on the expression of inositol 1,4,5-trisphosphate receptors (IP3 R) with the possible impact on the apoptosis induction in HeLa cells. METHODS: Gene expression, Western blot analysis, apoptosis determination by Annexin-V-FLUOS and drop in mitochondrial membrane potential by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC1) and immunofluorescence were used to determine differences in control and GYY4137-treated HeLa cells. RESULTS: In HeLa cell line, GYY4137 (10 µm) up-regulated expression of the IP3 R1 and IP3 R2, but not IP3 R3 on both mRNA and protein levels. Concurrently, cytosolic calcium increased and reticular calcium was depleted in concentration-dependent manner, partially by the involvement of IP3 R. Depletion of calcium from reticulum was accompanied by increase in endoplasmic reticulum (ER) stress markers, such as X-box, CHOP and ATF4, thus pointing to the development of ER stress due to GYY4137 treatment. Also, GYY4137 treatment of HeLa cells increased the number of apoptotic cells. CONCLUSION: These results suggest an involvement of H2 S in both IP3 -induced calcium signalling and induction of apoptosis, possibly through the activation of ER stress.

Modulation of the sodium-calcium exchanger in the rat kidney by different sequential stressors.

Stress, if exaggerated, modulates a variety of metabolic pathways and results in development of serious health consequences. The cell membrane sodium-calcium exchanger (NCX) is a major calcium extrusion system and is also modulated by stress. It has been shown previously that mRNA, protein levels and activity of the type 1 NCX (NCX1) in the left ventricle of the rat heart are increased by stressors, such as immobilization or hypoxia. In this study we investigated whether exposure to a subsequent different stressor can affect gene expression, protein level and activity of the NCX1 in rat kidney compared to exposure to only one type of stressor. In these experiments, we used immobilization and cold as the model stressors.We found that cold exposure at 4 degrees C for 24 h, when applied after immobilization repeated seven times, completely abolished the immobilization-induced increase in NCX mRNA level and after 7 days cold exposure the increases in NCX1 protein and activity in rat kidney were also abolished. Permanently increased NCX1 expression can result in imbalance of cellular calcium homeostasis and thus contribute to kidney dysfunction. Based on our results, we conclude that exposure to a cold stressor can have a protective effect on the kidney in rats exposed previously to repeated immobilization stress. This might be explained by differential stimulation of sympathetic neural and adrenal medullary responses by these different stressors.

Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity.

L1210/VCR cells, which express an abundant amount of P-glycoprotein (P-gp), were found to be resistant to thapsigargin--an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In the current paper, we have studied the possible differences among L1210 and L1210/VCR cells in expression of endoplasmic reticulum proteins involved in the regulation of calcium homeostasis and calcium-dependent processes. Amounts of mRNA encoding both calcium release channels (ryanodine receptor channels--RyR and IP3-receptor channels--IP3R) were found to be at similar levels in sensitive and resistant cells. However, mRNAs encoding IP3R1 or 2 were decreased in resistant cells cultivated in the presence of VCR (1.08 micromol/l), while mRNA encoding RyR remained unchanged. The amount of mRNA for SERCA2 was decreased in resistant cells when compared with sensitive cells. This decrease was more pronounced when resistant cells were cultivated in the presence of vincristine (VCR). Calnexin was found to be less expressed at the protein level in resistant as in sensitive cells. The level of mRNA encoding calnexin was decreased only when resistant cells were cultivated in the presence of VCR. Calnexin was found to be associated with immature P-gp in resistant cells. Thus, differences exist between sensitive and resistant cells in the expression of endoplasmic reticulum proteins involved in the control of intracellular calcium homeostasis or calcium-dependent processes. These changes may be at least partially responsible for the lack of sensitivity of resistant cells to thapsigargin.

Effect of aging on the expression of intracellular Ca(2+) transport proteins in a rat heart.

Aging process is accompanied by various biological dysfunctions including altered calcium homeostasis. Modified calcium handling might be responsible for changed cardiac function and potential development of the pathological state. In the present study we compared the mRNA and protein levels of the intracellular Ca(2+)-handling proteins--inositol 1,4,5-trisphosphate receptor (IP(3)R), ryanodine receptor (RyR), sarcoplasmic reticulum Ca(2+) pump (SERCA2), and also transient receptor potential C (TRPC) channels in cardiac tissues of 5-, 15-, and 26-month-old rats. Aging was accompanied by significant increase in the mRNA levels of IP(3)R and TRPC channels in both ventricles and atria, but mRNA level of the type 2 RyR was unchanged. Protein content of the IP(3)R1 correlated with mRNA levels, in the left ventricle of 15- and 26-month-old rats the value was approximately 1.8 and 2.8-times higher compared to 5-month-old rats. No significant differences were observed in mRNA and protein levels of the SERCA2 among 5-month-old and aged rats. However, Ca(2+)-ATPase activity significantly decreased with age, activities in 5-, 15-, and 26-month-old rats were 421.2 +/- 13.7, 335.5 +/- 18.1 and 304.6 +/- 14.8 nmol P(i) min(-1) mg(-1). These results suggest that altered transporting activity and/or gene expression of Ca(2+)-handling proteins of intracellular Ca(2+) stores might affect cardiac function during aging.

Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress.

AIM: The Na(+)/Ca(2+) exchanger (NCX) is a major Ca(2+) extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. METHODS: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemia-reperfusion to compare response of control and stressed hearts. RESULTS: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. CONCLUSIONS: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.

Levels of myosin heavy chain mRNA transcripts and protein isoforms in the fast extensor digitorum longus muscle of 7-month-old rats with chronic thyroid status alterations.

We have studied the effect of chronic thyroid status alterations on the myosin heavy chain (MyHC) isoform composition (by SDS-PAGE) and on MyHC mRNA levels (by RT-PCR) in the fast extensor digitorum longus (EDL) muscle of 7-month-old inbred Lewis strain female rats and compared this with corresponding results of the previously studied slow soleus muscle. Our findings show that in the EDL muscle, all four types 1, 2a, 2x/d and 2b of MyHC mRNA transcripts and protein isoforms are present in euthyroid, hypothyroid and hyperthyroid rats, i.e. after chronic treatment with methimazole and T(3), respectively. This is in contrast with the soleus, where only MyHC1 and 2a protein isoforms are expressed under similar conditions. Except for 2x/d MyHC mRNA transcripts in the EDL muscles, there was always significant difference between hypothyroid and hyperthyroid rats both at mRNA and protein levels. From our results we can conclude that extended alteration of the thyroid status leads to typical changes in the expression of MyHC mRNA transcripts and MyHC protein isoforms in the fast EDL and the slow soleus muscles. These changes correspond to those described after shorter periods of altered thyroid status. The characteristic phenotype differences between soleus and EDL muscles remain, however, preserved even after 7 months of thyroid hormone status alteration.

Is the ApoE polymorphism associated with dilated cardiomyopathy?

Apolipoprotein E (ApoE) is 34 kDa protein involved in the modulation of cholesterol transport and homeostasis. Polymorphism of the ApoE gene has been implicated in many chronic cardiovascular and neuronal diseases. ApoE epsilon4 allele has been reported to be associated with increased risk of cardiovascular diseases such as myocardial infarction, hypertension, coronary heart disease, etc. Fifty patients with the end-stage dilated cardiomyopathy (DCM) and advanced congestive heart failure were examined in our study. For evaluation of ApoE polymorphism, novel approach of fast screening of ApoE gene polymorphism by combination of PCR and blotting (CVD StripAssay) was used. Individual genotypes were correlated with basic cardiologic clinical parameters. The reported frequency of this allele in Caucasian population is 14.7 %. Our results showed that in patients with DCM frequency of the ApoE epsilon4 allele is 40 %. Frequency of the genotype epsilon2/4 was 58 % and epsilon3/4 was 22 %. Comparison with control Caucasian groups monitored by others clearly revealed that frequency of epsilon4 alelle is increased in patients with advanced stages of DCM. This observation suggests association of ApoE polymorphism with severe form of DCM. Physiological consequences of this observation remain to be clarified.

Levels of myosin heavy chain mRNA transcripts and content of protein isoforms in the slow soleus muscle of 7 month-old rats with altered thyroid status.

Skeletal muscles of small rodents contain four main fiber types, namely type 1, 2A, 2X/D and 2B fibers containing myosin heavy chain (MyHC) 1, 2a, 2x/d and 2b isoforms. Each of these MyHC isoforms is the product of a distinct gene and their expression is believed to be primarily transcriptionally controlled. In most rat muscles, messenger RNA (mRNA) transcripts for MyHC1, 2a, 2x/d and 2b and their corresponding protein products were found with the exception of the soleus muscle, where typically only MyHC1 and 2a transcripts and protein isoforms were demonstrated under normal conditions. Here we show the expression of all four MyHC1, 2a, 2x/d and 2b mRNA transcripts in the soleus muscle under normal conditions in euthyroid, as well as in experimental hypothyroid and hyperthyroid (with the exception of 2b MyHC transcript) 7-month-old female inbred Lewis rats. This is not matched, however, by the appearance of corresponding four isoforms, as we have found that 2x/d and 2b protein isoforms are not present at levels detectable by SDS-PAGE. We also show that the chronic hypothyroid and hyperthyroid status affects the expression of MyHC isoforms both at the mRNA and protein levels.

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats.

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.

Retinoic acid increased expression of the Na+/Ca2+ exchanger in the heart and brain.

The Na+/Ca2+ exchanger (NCX) is an important calcium transport system, which regulates intracellular calcium homeostasis. In particular, NCX is highly expressed in the plasma membrane of excitable neuronal and cardiac cells. We report here that binding of retinoic acid enhances expression of the NCX1 in the rat cardiac atria, brain stem and hypothalamus, but not in cerebellum. Differences in the regulation of the NCX1 by retinoic acid might suggest that GATA4-retinoic acid inducible transcription factor is activated in the hypothalamus and brain stem, but not in the cerebellum. Our results support the idea that inducible transcription factors play an important role in the fine-tuning of local tissue calcium homeostasis.

Effect of 6-hydroxydopamine on the gene expression of Na+/Ca2+ exchanger in the rat heart.

The Na+/Ca2+ exchanger (NCX) is an important component of the process of excitation-contraction coupling in the heart muscle. The level of gene expression as well as transport activities of this membrane structure is changed under pathological conditions like ischemic injury, myocardial infarction or diabetes. In this work we focused on the question whether the adrenergic modulation affects gene expression of the NCX in rat hearts. NCX mRNA levels were studied in the left cardiac atrium (divided into ganglionic and nonganglionic part) and also in the left ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. We have shown that administration of 6-OHDA decreases mRNA levels of NCX in both ganglionic and nonganglionic part of the left atrium and also in the left ventricle. This effect was not altered under combined administration of 6-OHDA and single immobilization stress. These data suggest that an activation of the adrenergic system can potentiate gene expression of the cardiac NCX.

SB203580, a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance.

P-glycoprotein (P-gp) is the plasma membrane transport pump responsible for efflux of chemotherapeutic agents from cells and is one of the systems that secures multidrug resistance (MDR) of neoplastic cells. In the present study, drug sensitive L1210 and multidrug resistant L1210/VCR (characterized by overexpression of P-gp) mouse leukemic cell lines were used as an experimental model. We have found that SB203580, a specific inhibitor of p38-MAPK pathway, significantly reduced the degree of the vincristine resistance in L1210/VCR cells. This phenomenon was accompanied by a decrease in the LC(50) value of vincristine from 3.203+/-0.521 to 0.557+/-0.082 microM. The LC(50) value of sensitive cells for vincristine was about 0.011 microM. The effect of SB203580 on L1210/VCR cells was associated with significantly increased intracellular accumulation of [3H]-vincristine in the concentration dependent manner. Prolonged exposure of resistant cells to 30 microM SB203580 did neither significantly influence the gene expression of P-gp, nor change the protein levels of p38-MAPK. Western blot analysis revealed that the MDR phenotype in L1210/VCR cells was associated with increased level and activity of cytosolic p38-MAPK. In resistant cells, the enhanced phosphorylation of both, p38-MAPK and ATF-2 (endogenous substrate for p38-MAPK) was found as well. In conclusion we could remark that SB203580, an inhibitor of p38 kinase pathway, reversed the MDR resistance of L1210/VCR cells. MDR phenotype of these cells is connected with increased levels and activities of p38-MAPK. These findings point to the possible involvement of the p38-MAPK pathway in the modulation of P-gp mediated multidrug resistance in the L1210/VCR mouse leukemic cell line. However, the mechanisms of SB203580 action should be further investigated.

Nuclear all-trans retinoic acid receptors: in vitro effects of selenium.

The present study was undertaken to investigate the effects of selenite (SeIV) and selenate (SeVI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of SeIV on the RARalpha and the type I iodothyronine 5'-deiodinase gene expression in the GH4C1 rat pituitary tumor cells. SeIV at 1.0 micromol/L was found to reduce (p < 0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by SeIV. SeVI at 0.1 micromol/L reduced (p < 0.05) the RA specific binding to RAR in liver, as well. Seleno-L-methionine (Se-II) when compared to L-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. SeIV (up to 2.5 micromol/L) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. SeIV at 1.0 micromol/L significantly decreases the rate of mRNA synthesis and/or degradation of the alpha form of the RAR and causes the enhancement of the type I iodothyronine 5'-deiodinase gene expression in GH4C1 cells. The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARalpha, with the cell vitality and the cell growth remaining unchanged.

Nuclear all-trans retinoic acid receptors in liver of rats with diet-induced insulin resistance.

Retinoic acid receptor alpha (RAR alpha) expression and RAR binding characteristics were investigated in the liver of rats with high-sucrose (HS) diet-induced insulin resistance. Animals were fed a basal (B) or HS (63 cal%) diet with or without fish oil (FO) (30% w/w of total fatty acids) for two weeks. A significant augmentation (p < 0.01) in the RAR alpha mRNA accumulation in rats fed HS diet when compared to rats fed B diet was demonstrated. In comparison with rats fed B + FO diet, a significant increase (p < 0.005) in the RAR alpha expression was found in rats fed HS + FO diet. In [3H]-retinoic acid (RA) binding studies, Scatchard plots confirmed a significant increase (p < 0.05) in the RAR maximal binding capacity (Bmax) only in rats fed HS + FO diet when compared to rats fed B diet. No significant changes in the association constant (Ka) were found among the groups when compared to rats fed B diet. In contrast to RAR alpha, a significant decrease (p < 0.005) in nuclear thyroid hormone receptor alpha 1 (TR alpha 1) expression was found in rats fed HS diet when compared to rats fed B diet. A significant decrease (p < 0.05) in the TR alpha 1 expression was also detected in rats fed HS + FO diet in comparison with rats fed B + FO diet. In addition, an analogous pattern in the expression of the TR isoform (TR alpha 2) was evaluated as well. In conclusion, the high-sucrose diet-induced insulin resistance might be associated with an increased RAR alpha expression and RAR population, and also with a decreased TR alpha 1 and TR alpha 2 mRNA accumulation.

Angiotensin I modulates Ca-transport systems in the rat heart through angiotensin II.

The objective of this study was to determine the effect of angiotensin I (Ang I) treatment in vivo on two major Ca-transport systems-the L-type voltage dependent calcium channel (L-VDCC) and the Na/Ca exchanger in rat heart. For our experiments we used four groups of rats, treated differently with saline, Ang I, the ACE inhibitor enalapril and/or combination of both for 6 days, every 24 h. We observed an increase in the activity, and also in mRNA expression of the Na/Ca exchanger, after repeated administration of Ang I in vivo. The maximal binding capacity of Ca-antagonist PN 200-110, which binds to the alpha 1 subunit of the L-VDCC was elevated from 0.8-1.85 pg/mg protein. mRNA expression of the voltage-dependent calcium channels of L-type system was also upregulated by Ang I administration, but not when enalapril was applied simultaneously with Ang I. These results demonstrate that in vivo application of the Ang I significantly modulates not only the activity, but also expression of the Na/Ca exchanger and the L-VDCC in rat hearts through angiotensin II (Ang II). Since in the in vitro experiments on the isolated cardiomyocytes, Ang II (100 nM) increased the calcium uptake after depolarization, and the AT1 receptor agonist losartan prevented this increase, we assume that this regulation might involve the AT1 receptors.

Molecular variants of the renin-angiotensin system components in the Slovak population.

Molecular variants of individual components of the renin-angiotensin system (RAS) are reported to constitute the inherited predisposition to some cardiovascular diseases in man, e.g. essential hypertension or myocardial infarction. The frequency of these variants depends highly on the race and population. Therefore, we examined the M235T molecular variant of the angiotensinogen gene and the I/D polymorphism of the ACE gene in Slovak healthy population, in patients with diagnosed essential hypertension and in patients who had undergone myocardial infarction. DNA from 241 subjects was tested for the presence of M235T and I/D molecular variants. The frequency of both these polymorphisms in the Slovak population is similar to other Caucasian populations. In the group of hypertensive patients, the frequency of the M235T molecular variant was increased compared to controls, predominantly in males (0.45 vs. 0.28), while in the I/D polymorphism the incidence of the D allele was the same for both controls and hypertensives (0.49 vs. 0.50). A significant increase in the D allele frequency compared to the controls occurred in the group of infarcted patients (0.63). The increased frequency of the M235T allele in hypertensive patients compared to the healthy population confirms that the M235T variants associated with increased blood pressure in the Slovak population. In the Slovak population, I/D polymorphism of the ACE gene is associated with myocardial infarction rather than with hypertension.

[DNA analysis in the determination of a predisposition for hypertension]. / Vyuzitie analýzy DNA pri urcení predispozície pre hypertenziu.

Angiotensinogen gene belongs to the genes designated as hypertension candidate genes. These genes might participate in development of hypertension. The aim of this work was to establish frequency of the mutant allele M235T on angiotensinogen gene in Slovak population and compare this frequency with that obtained from the group of hypertensive patients. We tested DNA from 120 healthy individuals and 20 hypertensive patients. By polymerase chain reaction followed by restriction analysis we determined frequency of mutant allele M235T in healthy population as well as in the group of hypertensive patients. We have found that frequency of the mutant allele in Slovak population was 0.33, while among hypertensive patients 0.45. Percentage of heterozygosity for M235T allele was 44.5%. Frequency of this mutant allele was significantly higher among women compared to men (0.38 vs. 0.27). Increased frequency of M235T allele among hypertensive patients compared to healthy population confirm that M235T mutation is bound to increased blood pressure. This quick and noninvasive method should help in the future to determine the possible risk of hypertension development. (Tab. 1, Fig. 2, Ref. 9.)