J Pácha

Publications of J Pácha

• The early effect of dextran sodium sulfate administration on carbachol-induced short-circuit current in distal and proximal colon during colitis development.

  Authors: M Hock, M Soták, M Kment, J Pácha

  Physiological research / Academia Scientiarum Bohemoslovaca. 12/2011; 60(6):921-31.

  Increased colonic Cl(-) secretion was supposed to be a causative factor of diarrhea in inflammatory bowel diseases. Surprisingly, hyporesponsiveness to Cl(-) secretagogues was later described inIncreased colonic Cl(-) secretion was supposed to be a causative factor of diarrhea in inflammatory bowel diseases. Surprisingly, hyporesponsiveness to Cl(-) secretagogues was later described in inflamed colon. Our aim was to evaluate changes in secretory responses to cholinergic agonist carbachol in distal and proximal colon during colitis development, regarding secretory activity of enteric nervous system (ENS) and prostaglandins. Increased responsiveness to carbachol was observed in both distal and proximal colon after 3 days of 2 % dextran sodium sulfate (DSS) administration. It was measured in the presence of mucosal Ba(2+) to emphasize Cl(-) secretion. The described increase was abolished by combined inhibitory effect of tetrodotoxin (TTX) and indomethacin. Indomethacin also significantly reduced TTX-sensitive current. On the 7th day of colitis development responsiveness to carbachol decreased in distal colon (compared to untreated mice), but did not change in proximal colon. TTX-sensitive current did not change during colitis development, but indomethacin-sensitive current was significantly increased the 7th day. Decreased and deformed current responses to serosal Ba(2+) were observed during colitis induction, but only in proximal colon. We conclude that besides inhibitory effect of DSS on distal colon responsiveness, there is an early stimulatory effect that manifests in both distal and proximal colon.

• Circadian regulation of electrolyte absorption in the rat colon.

  Authors: M Soták, L Polidarová, J Musílková, M Hock, A Sumová, J Pácha

  American journal of physiology. Gastrointestinal and liver physiology. 09/2011; 301(6):G1066-74.

  The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport,The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (α-, β-, and γ-subunits of Enac and Cftr), transporters (Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na(+)/H(+) exchanger (NHE) regulatory factor (Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, γEnac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the subjective night. The peak γEnac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and γEnac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, αEnac, βEnac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of βEnac and γEnac during the subjective night but did not affect expression of αEnac. Similarly, colonic electrogenic Na(+) transport was much higher during the subjective night than the subjective day. These findings indicate that the transporters and channels operating in NaCl absorption undergo diurnal regulation and suggest a role of an intestinal clock in the coordination of colonic NaCl absorption.

• Reciprocal changes in maternal and fetal metabolism of corticosterone in rat during gestation.

  Authors: K Vagnerová, Z Vacková, P Klusonová, F Staud, M Kopecky, P Ergang, I Miksík, J Pácha

  Reproductive sciences (Thousand Oaks, Calif.). 10/2008; 15(9):921-31.

  Objective. The objective of this study was to investigate the role of 11beta-hydroxysteroid dehydrogenases (11HSD1 and 11HSD2) in determining the fetal concentration of glucocorticoids. Methods. TheObjective. The objective of this study was to investigate the role of 11beta-hydroxysteroid dehydrogenases (11HSD1 and 11HSD2) in determining the fetal concentration of glucocorticoids. Methods. The expression patterns for mRNA abundance, protein level, and enzyme activities of placental and fetal 11HSD1 and 11HSD2 were assessed from embryonic day 13 (E13) to day 21 (E21; term E22). The transplacental passage of maternal corticosterone and its contribution to fetal glucocorticoids was also studied. Results. Placental 11HSD1 mRNA decreased between days E13 and E14 and then remained at much lower values up to E21. Similarly, NADP+-dependent 11beta-oxidation and 11-reduction were lower in late gestation. In contrast, placental 11HSD2 mRNA and protein decreased between E13 and E21. Dithiothreitol increased the activity of 11HSD2 and the output of 11-dehydrocorticosterone into fetal circulation.The fetal activity of 11HSD1 increased and 11HSD2 decreased between E16 and E21. Conclusions. The final third of gestation is accompanied by reciprocal changes in placental and fetal metabolism of corticosterone due to changes in 11HSD1 and 11HSD2 not only at the level of transcription but also at a posttranslational level.

• Glucocorticoid availability in colonic inflammation of rat.

  Authors: P Ergang, P Leden, J Bryndová, S Zbánková, I Miksík, M Kment, J Pácha

  Digestive diseases and sciences. 09/2008; 53(8):2160-7.

  Recent in vitro studies have shown the involvement of pro-inflammatory cytokines in the regulation of the local metabolism of glucocorticoids via 11beta-hydroxysteroid dehydrogenase type 1 and type 2Recent in vitro studies have shown the involvement of pro-inflammatory cytokines in the regulation of the local metabolism of glucocorticoids via 11beta-hydroxysteroid dehydrogenase type 1 and type 2 (11HSD1 and 11HSD2). However, direct in vivo evidence for a relationship among the local metabolism of glucocorticoids, inflammation and steroid enzymes is still lacking. We have therefore examined the changes in the local metabolism of glucocorticoids during colonic inflammation induced by TNBS and the consequences of corticosterone metabolism inhibition by carbenoxolone on 11HSD1, 11HSD2, cyclooxygenase 2 (COX-2), mucin 2 (MUC-2), tumor necrosis factor alpha (TNF-alpha), and interleukin 1beta (IL-1beta). The metabolism of glucocorticoids was measured in tissue slices in vitro and their 11HSD1, 11HSD2, COX-2, MUC-2, TNF-alpha, and IL-1beta mRNA abundances by quantitative reverse transcription-polymerase chain reaction. Colitis produced an up-regulation of colonic 11HSD1 and down-regulation of 11HSD2 in a dose-dependent manner, and these changes resulted in a decreased capacity of the inflamed tissue to inactivate tissue corticosterone. Similarly, 11HSD1 transcript was increased in colonic intraepithelial lymphocytes of TNBS-treated rats. Topical intracolonic application of carbenoxolone stimulated 11HSD1 mRNA and partially inhibited 11HSD2 mRNA and tissue corticosterone inactivation and these changes were blocked by RU-486. The administration of budesonide mimicked the effect of carbenoxolone. In contrast to the local metabolism of glucocorticoids, carbenoxolone neither potentiates nor diminishes gene expression for COX-2, TNF-alpha, and IL-1beta, despite the fact that budesonide down-regulated all of them. These data indicate that inflammation is associated with the down-regulation of tissue glucocorticoid catabolism. However, these changes in the local metabolism of glucocorticoids do not modulate the expression of COX-2, TNF-alpha, and IL-1beta in inflamed tissue.

• Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.

  Authors: J Bryndová, P Klusonová, M Kucka, K Mazancová-Vagnerová, I Miksík, J Pácha

  Journal of molecular endocrinology. 01/2007; 37(3):453-62.

  The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymesThe ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

• Intestinal inflammation modulates expression of 11beta-hydroxysteroid dehydrogenase in murine gut.

  Authors: K Vagnerová, M Kverka, P Klusonová, P Ergang, I Miksík, H Tlaskalová-Hogenová, J Pácha

  The Journal of endocrinology. 12/2006; 191(2):497-503.

  The effect of glucocorticoids is controlled at the pre-receptor level by the activity of 11beta-hydroxysteroid dehydrogenase (11HSD). The isoform 11HSD1 is an NADP+ -dependent oxidoreductase, usuallyThe effect of glucocorticoids is controlled at the pre-receptor level by the activity of 11beta-hydroxysteroid dehydrogenase (11HSD). The isoform 11HSD1 is an NADP+ -dependent oxidoreductase, usually reductase, that amplifies the action of glucocorticoids due to reduction of the biologically inactive 11-oxo derivatives cortisone and 11-dehydrocorticosterone to cortisol and corticosterone. The NAD+ -dependent isoform (11HSD2) is an oxidase that restrains the effect of hormones due to 11beta-oxidation of cortisol and corticosterone to their 11-oxo derivatives. Although the immunosuppressive and anti-inflammatory effects of glucocorticoids are well known, the relationship between inflammation and local metabolism of glucocorticoids is not well understood. In this study, we demonstrated that colitis induced by dextran sulfate sodium modulates colonic 11HSD1. Experimentally induced intestinal inflammation stimulated colonic NADP+ -dependent but not NAD+ -dependent 11HSD activity. Colonic 11HSD1 mRNA was increased, whereas 11HSD2 mRNA was not changed. Additional parallel studies revealed a similar pattern of 11HSD1 mRNA induction in mesenteric lymph nodes and intestinal intraepithelial lymphocytes, but not in spleen and peritoneal macrophages. These data suggest that inflammation modulates local metabolism of glucocorticoid and support the notion that pre-receptor regulation of endogenous corticosteroids might play a role in inflammatory processes.

• Corticosterone metabolism in chicken tissues: evidence for tissue-specific distribution of steroid dehydrogenases.

  Authors: M Kucka, K Vagnerová, P Klusonová, I Miksík, J Pácha

  General and comparative endocrinology. 08/2006; 147(3):377-83.

  Glucocorticoids influence the function of numerous tissues. Although there are a very large number of studies that have investigated the local metabolism of glucocorticoids in mammals, the knowledgeGlucocorticoids influence the function of numerous tissues. Although there are a very large number of studies that have investigated the local metabolism of glucocorticoids in mammals, the knowledge of this metabolism in birds is limited. The local concentration of corticosterone is critical for both glucocorticoid- and mineralocorticoid-dependent activity, and we have therefore carried out studies of corticosterone metabolism in various chicken organs. It was found that corticosterone was metabolized to 20-dihydrocorticosterone, and in some tissues also to 11-dehydrocorticosterone and 11-dehydro-20-dihydrocorticosterone. The activity of 20-hydroxysteroid dehydrogenase (20HSD), responsible for the transformation of corticosterone to 20-hydroxy derivatives, was abundant in the kidney and intestine, with lower levels in the liver and testis. Low levels of 20HSD were detected in the brain and ovaries. In contrast, 11-hydroxysteroid dehydrogenase (11HSD) activity was only found in the kidney and intestine. No activity was observed in the brain, testis, or ovaries. The treatment of chickens with estrogens stimulated 20HSD activity in the kidney, intestine, and oviduct and 11HSD activity in the liver and oviduct. Kinetic studies for corticosterone yielded an apparent Km for 11HSD in the nanomolar (Km = 21 +/- 5 nmol.l(-1)) and for 20HSD in the micromolar range (Km = 3.7 +/- 0.3 micromol.l(-1)). When progesterone or 5alpha-dihydrotestosterone were used instead of corticosterone, the tissues reduced the former to 20beta-dihydroprogesterone and the latter to both 5alpha,3alpha- and 5alpha,3beta-dihydrotestosterone. The data presents the first evidence for corticosterone metabolism via 11beta-, 3alpha/3beta-, and 20beta-hydroxysteroid dehydrogenases in various chicken organs and provide support for the theory of prereceptor modulation of glucocorticoid signals in avian tissues.

• Colitis up-regulates local glucocorticoid activation and down-regulates inactivation in colonic tissue.

  Authors: J Bryndová, S Zbánková, M Kment, J Pácha

  Scandinavian journal of gastroenterology. 07/2004; 39(6):549-53.

  BACKGROUND: Pro-inflammatory processes are counteracted by anti-inflammatory factors such as glucocorticoids. The response of target cells to glucocorticoids depends on several factors includingBACKGROUND: Pro-inflammatory processes are counteracted by anti-inflammatory factors such as glucocorticoids. The response of target cells to glucocorticoids depends on several factors including prereceptor modulation of glucocorticoid signals via local glucocorticoid metabolism. This is determined by two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD); 11betaHSD1 operates in vivo as a reductase converting inactive 11-oxo glucocorticoids to active glucocorticoids cortisol or corticosterone, whereas 11betaHSD2 catalyses oxidation of active glucocorticoids to their inactive 11-oxo derivatives. The aim of this study was to investigate the changes in local metabolism of glucocorticoids and in the expression of 11betaHSD1 and 11betaHSD2 mRNA during colonic inflammation. METHODS: Acute colitis was induced by intracolonic administration of 2,4,6-trinitrobenzenesulphonic acid (TNBS) or by drinking a dextran sodium sulphate (DSS) solution. Metabolism of glucocorticoids was measured in tissue fragments in vitro and 11betaHSD1 and 11betaHSD2 mRNA abundance was quantified using real-time RT-PCR one week after administration of TNBS and 10 days after drinking the DSS solution. RESULTS: In both models of inflammatory bowel disease we observed down-regulation of corticosterone oxidation to 11-dehydrocorticosterone by 64% (TNBS) and 53% (DSS) and reciprocal stimulation of reduction of 11-dehydrocorticosterone to corticosterone by 83% and 54%, respectively. A similar pattern was observed at the level of mRNA; 11betaHSD1 mRNA was significantly higher (TNBS: increase by 660%; DSS: increase by 760%) and 11betaHSD2 mRNA lower (TNBS: decrease by 85%; DSS: decrease by 60%) during inflammation. CONCLUSIONS: Colitis induces local glucocorticoid activation from 11-oxo steroids and decreases glucocorticoid inactivation; i.e. inflammation increases local tissue ratio of active and inactive glucocorticoids. The results indicate that the changes in local metabolism of glucocorticoids could contribute to the control of an overshoot of inflammation processes in the colon.

• Corticosteroid regulation of colonic ion transport during postnatal development: methods for corticosteroid analysis.

  Authors: J Pácha, I Miksík, L Mrnka, Z Zemanová, J Bryndová, K Mazancová, M Kucka

  Physiological research / Academia Scientiarum Bohemoslovaca. 02/2004; 53 Suppl 1:S63-80.

  Many mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinalMany mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinal tract that undergoes abrupt transitions coinciding with birth and weaning. This review deals with the maturation of ion transport functions in colon, the intestinal segment that plays an important role in sodium and potassium absorption and secretion. The purpose of the present review is to summarize the mechanism of sodium and potassium transport pathways and show how these transport processes change postnatally and how hormones, particularly corticosteroids, modify the pattern of development. Finally we describe some of the ways, how to analyze corticosteroid metabolism in target tissue.

• Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation.

  Authors: S Jindrichová, O Nováková, J Bryndová, E Tvrzická, V Lisá, F Novák, J Pácha

  The Journal of steroid biochemistry and molecular biology. 12/2003; 87(2-3):157-65.

  Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect ofPrevious studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

• Intracellular pH regulation in colonocytes of rat proximal colon.

  Authors: I Vanecková, M Vylitová-Pletichová, S Beskid, J Zicha, J Pácha

  Biochimica et biophysica acta. 06/2001; 1536(2-3):103-15.

  The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytesThe regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.

• Development of intestinal transport function in mammals.

  Authors: J Pácha

  Physiological reviews. 11/2000; 80(4):1633-67.

  Considerable progress has been made over the last decade in the understanding of mechanisms responsible for the ontogenetic changes of mammalian intestine. This review presents the current knowledgeConsiderable progress has been made over the last decade in the understanding of mechanisms responsible for the ontogenetic changes of mammalian intestine. This review presents the current knowledge about the development of intestinal transport function in the context of intestinal mucosa ontogeny. The review predominantly focuses on signals that trigger and/or modulate the developmental changes of intestinal transport. After an overview of the proliferation and differentiation of intestinal mucosa, data about the bidirectional traffic (absorption and secretion) across the developing intestinal epithelium are presented. The largest part of the review is devoted to the description of developmental patterns concerning the absorption of nutrients, ions, water, vitamins, trace elements, and milk-borne biologically active substances. Furthermore, the review examines the development of intestinal secretion that has a variety of functions including maintenance of the fluidity of the intestinal content, lubrication of mucosal surface, and mucosal protection. The age-dependent shifts of absorption and secretion are the subject of integrated regulatory mechanisms, and hence, the input of hormonal, nervous, immune, and dietary signals is reviewed. Finally, the utilization of energy for transport processes in the developing intestine is highlighted, and the interactions between various sources of energy are discussed. The review ends with suggestions concerning possible directions of future research.

• 11Beta-hydroxysteroid dehydrogenase activity in spontaneously hypertensive and Dahl rats.

  Authors: I Pohlová, I Miksík, J Kunes, J Pácha

  American journal of hypertension : journal of the American Society of Hypertension. 09/2000; 13(8):927-33.

  The role of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) in hypertension remains unknown even if it appears that the inappropriately decreased 11betaHSD activity might be involved in aThe role of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) in hypertension remains unknown even if it appears that the inappropriately decreased 11betaHSD activity might be involved in a process that leads to high blood pressure. The possible changes of 11betaHSD were therefore investigated in rats with spontaneous or salt-induced hypertension. The adult male rats of the following genotypes were used: spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY), Dahl salt-sensitive rats fed either a high-salt diet containing 8% NaCl (DS-HS) or low-salt diet containing 0.2% NaCl (DS-LS), and Dahl salt-resistant rats fed the same diets (DR-HS, DR-LS). 11betaHSD was investigated in colon, aorta, renal cortex, and renal medulla and was assessed as percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in the presence of NAD or NADP. The results demonstrated that genotype exerts a significant effect on 11betaHSD. 11betaHSD activity was significantly increased in colon and renal medulla of SHR compared with WKY rats. No significant differences were observed in renal cortex and aorta. In Dahl rats kept on a low-salt diet, 11betaHSD activity was significantly higher in colon, renal medulla, and cortex of DS-LS than in DR-LS rats but no difference was observed in aorta. The differences disappeared in age-matched DS and DR rats fed the high-salt diet. Increased dietary sodium intake stimulated the activity of 11betaHSD in renal cortex and medulla of DR rats and decreased the activity in colon of DS rats. We conclude that the development of spontaneous and salt-induced hypertension is not associated with decreased activity of 11betaHSD. However, the results showed that salt intake is able to modulate the activity of 11betaHSD and that 11betaHSD in DS and DR rats responds to high dietary salt intake in a different manner.

• Aldosterone alters the phospholipid composition of rat colonocytes.

  Authors: L Mrnka, O Nováková, F Novák, E Tvrzická, J Pácha

  The Journal of steroid biochemistry and molecular biology. 06/2000; 73(1-2):11-7.

  Previous studies have shown that aldosterone treatment of amphibian epithelial cells results not only in stimulation of Na(+) absorption but also in changes in phospholipid composition which arePrevious studies have shown that aldosterone treatment of amphibian epithelial cells results not only in stimulation of Na(+) absorption but also in changes in phospholipid composition which are necessary for the mineralocorticoid action of aldosterone. The present study was designed to investigate the effect of aldosterone on phospholipids of mammalian epithelia. Phospholipid and fatty acid composition was examined in colonic epithelium (mineralocorticoid target tissue) and thymus (non-mineralocorticoid but glucocorticoid target tissue) of rats which had received aldosterone or vehicle by a miniosmotic pump for 7 days. Aldosterone increased the mass of colonic phospholipids relative to cellular proteins with concomitant changes in the percentage distribution of fatty acids, whereas the relative distribution of membrane phospholipds was not changed. Phosphatidylcholine increased the content of polyunsaturated and decreased that of monounsaturated fatty acids, which predominantly reflected the accretion of arachidonic and a decrease in oleic and palmitoleic acids. Within the phosphatidylethanolamine subclass, pretreatment of rats with aldosterone decreased the content of monounsaturated fatty acids (predominantly oleic and palmitoleic acid) and of n-3 fatty acids, and increased the content of saturated fatty acids (palmitic acid). The saturated-to-nonsaturated fatty acid ratio also significantly increased after aldosterone treatment. No changes in thymic phospholipids were seen. The results are consistent with the contention that aldosterone specifically modulates phospholipid concentration and metabolism in mineralocorticoid target tissue. The changes in phospholipid content and its fatty acid composition during the fully developed effect of aldosterone may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

• Permissive effect of thyroid hormones on induction of rat colonic Na+ transport by aldosterone is not localised at the level of Na+ channel transcription.

  Authors: L Mrnka, J Pácha

  Molecular and cellular endocrinology. 02/2000; 159(1-2):179-85.

  The interrelationship between thyroid hormones and aldosterone has been examined in the regulation of rat colonic amiloride-sensitive Na+ transport which translocates Na+ through apicalThe interrelationship between thyroid hormones and aldosterone has been examined in the regulation of rat colonic amiloride-sensitive Na+ transport which translocates Na+ through apical amiloride-sensitive Na+ channels and basolateral Na+, K+-ATPase. Electrogenic Na+ transport was measured in an Ussing chamber by the short-circuit current and identified by Na+ channel blocker amiloride. Na+-pumping activity of the basolateral Na+,K+-ATPase was investigated in nystatin-treated epithelium by measuring the equivalent short-circuit current after addition of mucosal Na+. The abundance of mRNA coding for alpha, beta and gamma subunits of the Na+ channel (rENaC) was estimated using Northern blot analysis. Hyperaldosteronism was induced by a low-salt diet and hypothyroidism by methimazole. The low-Na+ diet induced electrogenic Na+ transport in euthyroid rats but its effect was almost completely inhibited in hypothyroid animals even if the plasma concentration of aldosterone was high enough to stimulate this transport pathway both in euthyroid and hypothyroid rats. A kinetic study of the basolateral Na+,K+-ATPase revealed a decrease of Na+ transport capacity in hypothyroid rats kept on the low-Na+ diet in comparison with euthyroid animals fed the same diet. No significant differences in steady-state levels of alpha, beta and gamma rENaC mRNA were detected between euthyroid and hypothyroid rats. These data suggest that hypothyroidism decreases the efficacy of the basolateral Na+ pump but fails to inhibit it completely even though it inhibits the transepithelial electrogenic Na+ transport in response to aldosterone. We conclude that the permissive effect of thyroid hormones on the induction of electrogenic Na+ transport by aldosterone is localised beyond the transcriptional step of Na+ channel regulation.

• Low-salt diet alters the phospholipid composition of rat colonocytes.

  Authors: L Mrnka, O Nováková, F Novák, E Tvrzická, J Pácha

  Physiological research / Academia Scientiarum Bohemoslovaca. 02/2000; 49(2):197-205.

  The effect of low-salt diet on phospholipid composition and remodeling was examined in rat colon which represents a mineralocorticoid target tissue. To elucidate this question, male Wistar rats wereThe effect of low-salt diet on phospholipid composition and remodeling was examined in rat colon which represents a mineralocorticoid target tissue. To elucidate this question, male Wistar rats were fed a low-salt diet and drank distilled water (LS, low-salt group) or saline instead of water (HS, high-salt group) for 12 days before the phospholipid concentration and fatty acid composition of isolated colonocytes were examined. The dietary regimens significantly influenced the plasma concentration of aldosterone which was high in LS group and almost zero in HS group. Plasma concentration of corticosterone was unchanged. When expressed in terms of cellular protein content, a significantly higher concentration of phospholipids was found in LS group, with the exception of sphingomyelin (SM) and phosphatidylserine (PS). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for more than 70% of total phospholipids in both groups. A comparison of phospholipid distribution in LS and HS groups demonstrated a higher percentage of PE and a small, but significant, decrease of PC and SM in LS group. The percentage of phosphatidylinositol (PI), PS and cardiolipin (CL) were not affected by mineralocorticoid treatment. With respect to the major phospholipids (PE, PC), a higher level of n-6 polyunsaturated fatty acids (PUFA) and lower levels of monounsaturated fatty acids were detected in PC of LS group. The increase of PUFA predominantly reflected an increase in arachidonic acid by 53%. In comparison to the HS group, oleic acid content was decreased in PC and PE isolated from colonocytes of the LS group. Our data indicate that alterations in phospholipid concentration and metabolism can be detected in rats with secondary hyperaldosteronism. The changes in phospholipid concentration and their fatty acid composition during fully developed effect of low dietary Na+ intake may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

• Separation and identification of corticosterone metabolites by liquid chromatography--electrospray ionization mass spectrometry.

  Authors: I Miksík, M Vylitová, J Pácha, Z Deyl

  Journal of chromatography. B, Biomedical sciences and applications. 05/1999; 726(1-2):59-69.

  High-performance liquid chromatography coupled to atmospheric pressure ionization-electrospray ionization mass spectrometry (API-ESI-MS) was investigated for the analysis of corticosteroneHigh-performance liquid chromatography coupled to atmospheric pressure ionization-electrospray ionization mass spectrometry (API-ESI-MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C18 column (eluted with a methanol-water-acetic acid gradient) with identification using positive ion mode API-ESI-MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in avian intestines.

• Localization of Na,K-ATPase activity in developing rat distal colon: role of corticosteroids.

  Authors: Z Zemanová, J Pácha

  Mechanisms of ageing and development. 04/1998; 101(1-2):129-43.

  The role of corticosteroids in the development of Na,K-ATPase and its distribution along the crypt base-colonic surface was investigated in suckling, weanling and adult rats using a biochemical and aThe role of corticosteroids in the development of Na,K-ATPase and its distribution along the crypt base-colonic surface was investigated in suckling, weanling and adult rats using a biochemical and a histochemical approach. The cerium-based histochemical method for detection of ouabain-sensitive K+-dependent p-nitrophenylphosphatase (K-NPPase) component of the Na,K-ATPase complex was used to localize Na,K-ATPase in the epithelium. The activity of Na,K-ATPase was very low 2 days after birth and increased to a maximum in adulthood. Mature surface colonocytes and immature cells at the crypt base were similarly reactive and the reaction product was decreased by the addition of ouabain and inhibited by omission of K+. Adrenalectomy decreased colonic Na,K-ATPase activity in surface and deep crypt cells of suckling, weanling and adult animals. Mineralocorticoids (deoxycorticosterone acetate, DOCA) restored the Na,K-ATPase activity both in surface and crypt cells of adrenalectomized weanling rats and the effect of DOCA was inhibited by the mineralocorticoid receptor antagonist, spironolactone. Physiological doses of glucocorticoids (dexamethasone) stimulated Na,K-ATPase activity in surface colonocytes of adrenalectomized weanling rats; supraphysiological doses restored Na,K-ATPase probably via cross-over into mineralocorticoid receptors both in surface and crypt cells. High dietary Na+ intake during the weaning period reduced the reaction product to the level detected in adrenalectomized rats. The distribution of Na, K-ATPase activity in the epithelium of adrenalectomized rats with substitutional replacement hormone therapy was the same as in control animals or, in some animals, the surface absorptive epithelium exhibited a stronger reaction than the crypt cells. Similarly, the surface colonocytes of adult rats kept on a low-salt diet showed a stronger reaction than the crypt cells. These data indicate that postnatal development of Na,K-ATPase is regulated predominantly by aldosterone and that both surface and crypt cells are responsive to mineralocorticoids. Surface cells are also responsive to glucocorticoids.

• Metabolism of corticosterone in mammalian and avian intestine.

  Authors: M Vylitová, I Miksík, J Pácha

  General and comparative endocrinology. 04/1998; 109(3):315-24.

  11beta-hydroxysteroid dehydrogenase (11betaHSD) catalyzes the conversion of the glucocorticoids, corticosterone and cortisol, to the respective derivatives 11-dehydrocorticosterone and cortisone. The11beta-hydroxysteroid dehydrogenase (11betaHSD) catalyzes the conversion of the glucocorticoids, corticosterone and cortisol, to the respective derivatives 11-dehydrocorticosterone and cortisone. The recent findings underline the importance of this enzyme in excluding glucocorticoids from mineralocorticoid receptors. In the present study, 11betaHSD activity was compared in the intestine of herbivorous (guinea pig), omnivorous (rat), and granivorous (hen) animals, i.e., in animals in which the Na+ transport either is or is not regulated by aldosterone under normal conditions and in which the plasma levels of individual glucocorticoids are different. Slices of various intestinal segments were incubated in the presence of corticosterone or 11-dehydrocorticosterone, and the steroids were extracted and analyzed by HPLC. In the mammalian intestine, the activity of 11betaHSD was very low (approaching zero) in aldosterone-insensitive segments (duodenum, jejunum) but significant activity was revealed in aldosterone-sensitive segments (ileum, cecum, and proximal and distal colon). In comparison with the rat, the guinea pig large intestine exhibited significantly higher activity of 11betaHSD. There was no detectable reductase activity (conversion of 11-dehydrocorticosterone to corticosterone) in any intestinal segments of either species. Unexpectedly, no 11betaHSD activity was observed in the avian intestine. It was found that, in contrast to the mammalian intestine, corticosterone was metabolized to 20-dihydrocorticosterone while 11-dehydrocorticosterone was converted to 11-dehydro-20-dihydrocorticosterone. The distribution of 20-hydroxysteroid dehydrogenase (20HSD) activity in the avian intestine was homogenous along the intestine and did not correlate with the mineralocorticoid sensitivity of intestinal segments. To trace different cosubstrate dependence of 11betaHSD and 20HSD, homogenates of ileum and distal colon were incubated with NAD+/NADH or NADP+/NADPH, respectively. In accordance with slice experiments mammalian intestine displayed only oxidation of corticosterone to 11-dehydrocorticosterone and NAD+ preference. In avian intestine, the metabolite formed from corticosterone was 11-dehydrocorticosterone in the presence of NAD+ or NADP+ whereas in the presence of NADPH 11-dehydro-20-dihydrocorticosterone and 20-dihydrocorticosterone were formed. Given the wide similarity between mineralocorticoid regulation of epithelial transport in mammals and birds, the unexpected finding of differences in the metabolism of corticosterone suggests that role of 20HSD is to allow aldosterone occupancy of mineralocorticoid receptors.

• The role of 11 beta-hydroxysteroid dehydrogenase in maturation of the intestine.

  Authors: I Pohlová, I Miksík, J Pácha

  Mechanisms of ageing and development. 12/1997; 98(2):139-50.

  Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD)Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) which converts active glucocorticoids to inactive metabolites. As 11 beta HSD is also expressed in the intestine, this enzyme may be an important regulator of intestinal maturation. To investigate this, we have performed the systematic study of the development of intestinal 11 beta HSD activity and its cofactor preference as well as of the effect of 11 beta HSD inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and Na,K-ATPase in the intestine. The activity of 11 beta HSD was low in ileum of suckling rats and significantly increased during the weaning period. In colon, the activity was already high in suckling rats and gradually rose during the postnatal development. 11 beta HSD activity was undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic 11 beta HSD utilized predominantly NAD as a cofactor, but displayed significant sensitivity also to NADP. Ileal 11 beta HSD had similar sensitivity to both cofactors. With NAD as a cofactor, ileal 11 beta HSD had a Km (59 +/- 10 nM) compatible with the colonic enzyme (81 +/- 14 nM). Carbenoxolone administration to suckling and weanling rats in vivo did not result in any changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone significantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-affinity type of 11 beta HSD is expressed not only in colon but also in ileum and that 11 beta HSD is an important factor in the regulation of tissue levels of active glucocorticoids in developing colon but not in the small intestine.