Jirí Pácha

Publications of Jirí Pácha

• Local metabolism of glucocorticoids and its role in rat adjuvant arthritis.

  Authors: Peter Ergang, Pavel Leden, Karla Vagnerová, Petra Klusonová, Ivan Miksík, Jana Jurcovicová, Milan Kment, Jirí Pácha

  Molecular and cellular endocrinology. 03/2010; 323(2):155-60.

  11beta-Hydroxysteroid dehydrogenase 1 (11HSD1) regulates local glucocorticoid activity and plays an important role in various diseases. Here, we studied whether arthritis modulates 11HSD1, what is11beta-Hydroxysteroid dehydrogenase 1 (11HSD1) regulates local glucocorticoid activity and plays an important role in various diseases. Here, we studied whether arthritis modulates 11HSD1, what is the role of pro-inflammatory cytokines in this process and whether altered local metabolism of glucocorticoids may contribute to the feedback regulation of inflammation. Adjuvant arthritis increased synovial 11HSD1 mRNA and 11-reductase activity but treatments with tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) antagonists etanercept and anakinra reduced 11HSD1 upregulation. Treatment with carbenoxolone, an 11HSD inhibitor, increased expression of TNF-alpha, cyclooxygenase 2, and osteopontin mRNA without any changes in the plasma levels of corticosterone. Similar changes were observed when arthritic rats were treated with RU486, an antagonist of GR. This study suggests that arthritis upregulates synovial 11HSD1, this upregulation is controlled by TNF-alpha and IL-1beta and that the increased supply of local corticosterone might contribute to feedback regulation of inflammation.

• Expression profiles of proliferative and antiapoptotic genes in sporadic and colitis-related mouse colon cancer models.

  Authors: Jirí Svec, Peter Ergang, Václav Mandys, Milan Kment, Jirí Pácha

  International journal of experimental pathology. 02/2010; 91(1):44-53.

  Elevated levels of survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and the regulatory factors c-MYB andElevated levels of survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and the regulatory factors c-MYB and Tcf-4 are often found in human cancers including colorectal cancer (CRC) and have been implicated in the development and progression of tumorigenesis. The aim of this study was to determine the expression of these genes in mouse models of sporadic and colitis-associated CRC. To address these issues, we used qRT-PCR approach to determine changes in gene expression patterns of neoplastic cells (high-grade dysplasia/intramucosal carcinoma) and surrounding normal epithelial cells in A/J and ICR mouse strains using laser microdissection. Both strains were injected with azoxymethane and ICR mice were also given drinking water that contained 2% dextran sodium sulphate. In both sporadic (A/J mice) and colitis-associated (ICR mice) models of CRC, the levels of TERT mRNA, COX-2 mRNA and Tcf-4 mRNA were higher in neoplastic cells than in surrounding normal epithelial cells. In contrast, survivin mRNA was upregulated only in neoplastic cells from A/J mice and ILK mRNA was upregulated only in neoplastic cells from ICR mice. However, the expression of iNOS mRNA was similar in normal and neoplastic cells in both models and c-MYB mRNA was actually downregulated in neoplastic cells compared with normal cells in both models. These findings suggest that the genetic background and/or the molecular mechanisms of tumorigenesis associated with genotoxic insults and colonic inflammation influence the gene expression of mTERT, COX-2, Tcf-4, c-MYB, ILK and survivin in colon epithelial neoplasia.

• Chicken 11beta-hydroxysteroid dehydrogenase type 2: partial cloning and tissue distribution.

  Authors: Petra Klusonová, Marek Kucka, Ivan Miksík, Jana Bryndová, Jirí Pácha

  Steroids. 04/2008; 73(3):348-55.

  NAD(+)-dependent 11beta-hydroxysteroid dehydrogenase (11HSD2) converts glucocorticoids to 11-oxo derivatives and thus decreases their local concentration and prevents them from activatingNAD(+)-dependent 11beta-hydroxysteroid dehydrogenase (11HSD2) converts glucocorticoids to 11-oxo derivatives and thus decreases their local concentration and prevents them from activating corticosteroid receptors. In this paper we report the partial cloning, characterization and tissue distribution of chicken 11HSD2. A cDNA of 991bp was cloned from kidney mRNA by reverse transcription and polymerase chain reaction. At the amino acid level, the sequence of PCR product had 56-59% homology with mammalian and 46-48% with fish 11HSD2. The consensus sequences of the short-chain dehydrogenase/reductase superfamily such as the catalytic activity motif Tyr-X-X-X-Lys and cosubstrate-binding motif Gly-X-X-X-Gly-X-Gly, were found in the cloned cDNA. Analysis of the tissue expression of chicken 11HSD2 mRNA and NAD(+)-dependent 11beta-oxidase activity showed a similar tissue distribution pattern in the majority of tissues. High levels of expression and activity were found in kidney, small intestine, colon and oviduct; low in ovary and almost zero in brain, liver and testis.

• Melatonin inhibits prostaglandin E2- and sodium nitroprusside-induced ion secretion in rat distal colon.

  Authors: Libor Mrnka, Miroslav Hock, Markéta Rybová, Jirí Pácha

  European journal of pharmacology. 03/2008; 581(1-2):164-70.

  Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this workAlthough the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

• Insight into the circadian clock within rat colonic epithelial cells.

  Authors: Martin Sládek, Markéta Rybová, Zuzana Jindráková, Zdena Zemanová, Lenka Polidarová, Libor Mrnka, John O'Neill, Jirí Pácha, Alena Sumová

  Gastroenterology. 10/2007; 133(4):1240-9.

  BACKGROUND & AIMS: The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation,BACKGROUND & AIMS: The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation, suggesting the presence of endogenous circadian rhythmicity. The aim of the study was to provide insight into the circadian core clock mechanism within the rat colon. Moreover, the potency of a restricted feeding regime to shift the circadian clock in the colon was tested. The question of whether the colonic clock drives circadian expression in NHE3, an electroneutral Na(+)/H(+) exchanger, was also addressed. METHODS: Daily profiles in expression of clock genes Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbalpha, and the NHE3 transporter were examined by reverse transcriptase-polymerase chain reaction and their mRNA levels, as well as PER1 and BMAL1 protein levels, were localized in the colonic epithelium by in situ hybridization and immunocytochemistry, respectively. RESULTS: Expression of Per1, Per2, Cry1, Bmal1, Clock, Rev-erbalpha, and NHE3, as well as PER1 and BMAL1 protein levels, exhibited circadian rhythmicity in the colon. The rhythms were in phase with those in the liver but phase-delayed relative to the master clock in the suprachiasmatic nucleus. Restricted feeding entrained the clock in the colon, because rhythms in clock genes as well as in NHE3 expression were phase-advanced similarly to the clock in the liver. CONCLUSIONS: The rat colon harbors a circadian clock. The colonic clock is likely to drive rhythmic NHE3 expression. Restricted feeding resets the colonic clock similarly to the clock in the liver.

• 11beta-hydroxysteroid dehydrogenase 1 and 2 expression in colon from patients with ulcerative colitis.

  Authors: Sárka Zbánková, Jana Bryndová, Pavel Leden, Milan Kment, Alexander Svec, Jirí Pácha

  Journal of gastroenterology and hepatology. 08/2007; 22(7):1019-23.

  BACKGROUND AND AIM: 11beta-hydroxysteroid dehydrogenase (11betaHSD) is an enzyme responsible for the interconversion of active 11beta-hydroxysteroids (cortisol) into biologically inactiveBACKGROUND AND AIM: 11beta-hydroxysteroid dehydrogenase (11betaHSD) is an enzyme responsible for the interconversion of active 11beta-hydroxysteroids (cortisol) into biologically inactive 11-oxosteroids (cortisone). The isoform 11betaHSD1 operates predominantly as a reductase converting cortisone to cortisol, whereas 11betaHSD2 catalyzes oxidation of cortisol to cortisone. This mechanism of peripheral metabolism of glucocorticoids has been suggested to be involved in increasing the availability of anti- inflammatory glucocorticoids as a response to inflammatory stimuli. The aim of this study therefore was to investigate the impact of inflammatory bowel disease on the expression of colonic 11betaHSD1 and 11betaHSD2. METHODS: Quantitative real-time RT-PCR was used to assess messenger RNA for 11betaHSD1 and 11betaHSD2 in bioptic samples taken from patients with ulcerative colitis and in healthy controls, and in colon of rats with colitis induced by dextran sulfate sodium (DSS). Rat colonic fragments were used for assessment of local metabolism of glucocorticoids. RESULTS: In both human and rat specimens colitis up-regulated the expression of colonic 11betaHSD1 mRNA and down-regulated 11betaHSD2 mRNA. A similar pattern was observed at the level of local metabolism of corticosterone. Oxidation of corticosterone to 11-dehydrocorticosterone was decreased and reduction of 11-dehydrocorticosterone to corticosterone was increased in colonic tissue of rats with DSS-colitis. CONCLUSIONS: Colonic inflammation induces local glucocorticoid activation via 11betaHSD1 and impairs glucocorticoid inactivation via 11betaHSD2. The observed changes indicate a role for local metabolism of glucocorticoids in the control of colonic inflammation.

• Heterogeneous expression of melatonin receptor MT1 mRNA in the rat intestine under control and fasting conditions.

  Authors: Matús Soták, Libor Mrnka, Jirí Pácha

  Journal of pineal research. 10/2006; 41(2):183-8.

  Melatonin is found in mammalian central nervous system and various peripheral tissues including gastrointestinal tract (GIT) where it participates in the regulation of intestinal motility, bloodMelatonin is found in mammalian central nervous system and various peripheral tissues including gastrointestinal tract (GIT) where it participates in the regulation of intestinal motility, blood flow, immunomodulation, ion transport, cell proliferation and scavenging of free radicals. Some of these effects are achieved via melatonin binding to specific receptors, MT1 and MT2. As no thorough study on the expression of these receptors in the GIT has yet been done, the aim of this study was to determine the MT1 mRNA expression in the rat intestine under both control and fasting conditions. Our results suggest that MT1 mRNA is present in epithelial as well as subepithelial layer, with higher expression in the latter in all intestinal segments studied. The highest signal of the MT1 transcript along the rostro-caudal intestinal axis was found both in epithelial and subepithelial layers of the duodenum. Nevertheless, duodenal MT1 mRNA expression did not reach the level found in pituitary gland. In a 12:12-hr light:dark cycle a MT1 receptor expression in the subepithelial layer of rat distal colon did not manifest a significant diurnal rhythm. Short-term fasting increased the expression of MT1 transcript in the subepithelial layer of both the small and large intestine. During long-term fasting the increase persisted only in distal colon while a return to control levels was observed in small intestinal segments. In conclusion we demonstrated heterogeneous expression of MT1 receptor in the rat intestine and showed that its expression is up-regulated by nutritional deprivation.

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• Age-dependent effect of secretagogues during colonic maturation.

  Authors: Libor Mrnka, Sergej Beskid, Oksana Sevastyanova, Jana Bryndová, Jirí Pácha

  European journal of pharmacology. 07/2005; 516(3):268-75.

  Intestinal secretory response is altered during colonic development. The aim of this report was to study the developmental changes of the Ca(2+)- and cAMP-induced regulatory pathways with specialIntestinal secretory response is altered during colonic development. The aim of this report was to study the developmental changes of the Ca(2+)- and cAMP-induced regulatory pathways with special attention to the direct and indirect effect of secretagogues on the colonic epithelium. We investigated the effect of bethanechol, 5-hydroxytryptamine (5-HT), and histamine on Cl(-) secretion and stimulation of intracellular Ca(2+) ([Ca(2+)](i)) and cAMP in the distal colon of suckling, weanling and adult rats. In the presence of tetrodotoxin, immature colon of suckling and weanling rats displayed higher potency (EC(50)) of 5-HT to stimulate Cl(-) secretion, whereas the potency of histamine was not changed during development. The potency of bethanechol was reduced during weaning and partially restored in adulthood. 5-HT increased cAMP level similarly in both neonatal and adult colonic crypts, but the adults had higher basal level of cAMP than suckling rats. Also the effect of bethanechol on [Ca(2+)](i) was independent of colonic maturation. The results suggest that colonic Cl(-) secretion displays developmental changes of regulation depending on the non-neural secretagogue-signalling pathway and that these developmental changes seem to be localized somewhere outside colonocytes.

• 11beta-Hydroxysteroid dehydrogenase in the heart of normotensive and hypertensive rats.

  Authors: Karla Mazancová, Martin Kopecký, Ivan Miksík, Jirí Pácha

  The Journal of steroid biochemistry and molecular biology. 03/2005; 94(1-3):273-7.

  Corticosteroids have been shown to play a role in cardiac remodeling, with the possibility of a direct effect of overexpression of 11beta-hydroxysteroid dehydrogenase (11HSD) isoform 2 at the levelCorticosteroids have been shown to play a role in cardiac remodeling, with the possibility of a direct effect of overexpression of 11beta-hydroxysteroid dehydrogenase (11HSD) isoform 2 at the level of the cardiomyocytes. The aim of this study was to examine cardiac steroid metabolism in hypertensive rats with hearts that are hypertrophied and fibrotic and have structural alterations in the coronary circulation. To assess possible alterations of cardiac steroid metabolism the expression and activity of both isoforms of 11beta-hydroxysteroid dehydrogenase (11HSD) were studied in spontaneously hypertensive rats (SHR), their normotensive controls Wistar-Kyoto (WKY), and in Dahl salt-sensitive (DS) and salt-resistant rats (DR) kept on a low- or high-salt diet. Using real-time quantitative RT-PCR and enzyme activity assay we found strain-dependent differences in cardiac metabolism of glucocorticoids. In Dahl rats expression of 11HSD1 and 11HSD2 mRNA was lower in DS than in DR rats and was not influenced by dietary salt intake; 11HSD1 mRNA was expressed at higher level than 11HSD2 mRNA. NADP(+)-dependent cardiac 11HSD activity showed similar distribution as 11HSD1 mRNA-lower activity in DS than in DR rats and no effect of salt intake. In SHR and WKY strains 11HSD2 mRNA expression was significantly higher in WKY than in SHR but no differences were observed in 11HSD1 mRNA abundance and NADP(+)-dependent 11HSD activity. These results show that the heart is able to metabolize glucocorticoids and that this metabolism is strain-dependent but do not support the notion of association between cardiac hypertrophy and changes of 11HSD1 and 11HSD2 expression.

• Glucocorticoid metabolism and Na+ transport in chicken intestine.

  Authors: Karla Mazancová, Marek Kucka, Ivan Miksík, Jirí Pácha

  Journal of experimental zoology. Part A, Comparative experimental biology. 03/2005; 303(2):113-22.

  The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone andThe role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.

• Expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in colorectal cancer.

  Authors: Sárka Zbánková, Jana Bryndová, Milan Kment, Jirí Pácha

  Cancer letters. 08/2004; 210(1):95-100.

  Glucocorticoid hormones have been reported to operate as regulators of cell proliferation and differentiation and to inhibit growth of several colon tumors and adenocarcinoma cell lines. TheGlucocorticoid hormones have been reported to operate as regulators of cell proliferation and differentiation and to inhibit growth of several colon tumors and adenocarcinoma cell lines. The glucocorticoid action is regulated, in part, at the pre-receptor level through the expression of isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD1, 11betaHSD2) which are responsible for the interconversion of hormonally active cortisol to cortisone. Since both of these isoforms are expressed in the mammalian colon, we examined whether 11betaHSD1 and 11betaHSD2 are expressed in human colorectal cancer and whether their expression differs between neoplastic and autologous non-neoplastic tissue. We provide evidence that both isoforms of 11betaHSD are expressed in the colon adenocarcinoma, but their expression is not identical in neoplastic and non-neoplastic tissue. There is a significant decrease of 11betaHSD2 mRNA abundance and enzyme activity in neoplastic tissue. In contrast, 11betaHSD1 activity and mRNA abundance are increased in some but not all tumor samples. The results demonstrate that (1) neoplastic transformation is associated with decreasing steady-state levels of 11betaHSD2 mRNA and enzyme activity and in some cases also with increasing expression of 11betaHSD1, and (2) colorectal tumor cells have a decreased capability of autocrine inactivation of glucocorticoids.

• Application of liquid chromatography-electrospray ionization mass spectrometry for study of steroid-converting enzymes.

  Authors: Ivan Miksík, Katerina Mikulíková, Jirí Pácha, Marek Kucka, Zdenek Deyl

  Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 03/2004; 800(1-2):145-53.

  A high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study ofA high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol-water-acetic acid gradient) and identification of the steroids involved was done by API-ESI-MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).

• Carbenoxolone accelerates maturation of rat intestine.

  Authors: Jirí Pácha, Radka Vagnerová, Jana Bryndová

  Pediatric research. 06/2003; 53(5):808-13.

  The rat undergoes profound maturational changes in the intestinal structure and function during the third week of its life. To investigate the role of peripheral glucocorticoid metabolism in thisThe rat undergoes profound maturational changes in the intestinal structure and function during the third week of its life. To investigate the role of peripheral glucocorticoid metabolism in this process, we studied the postnatal maturation of intestinal structure and function. The peripheral metabolism of glucocorticoids depends on enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD), which is responsible for the interconversion of corticosterone to 11-dehydrocorticosterone and thus for the modulation of glucocorticoid access to corticosteroid receptors. The pups were treated with carbenoxolone (CBX), an inhibitor of 11betaHSD, for 10 d during the suckling (days 8-18) or weaning period (days 14-24 or days 20-30), and we determined the parameters of intestinal growth and activities of sucrase, alkaline phosphatase, and Na,K-ATPase. The CBX treatment increased plasma concentrations of corticosterone as a result of a significant reduction of peripheral degradation of corticosterone catalyzed by 11betaHSD. This also stimulated intestinal growth without changing somatic growth. The mucosal cell mass was significantly higher in CBX-treated suckling rats, whereas the effect of this treatment was less obvious in weanling animals. CBX increased the crypt depth and villus height in 18- and 24-d-old pups but not in 30-d-old animals. The small intestinal activities of sucrase, alkaline phosphatase, and Na,K-ATPase were not influenced by CBX. In contrast, colonic Na,K-ATPase was stimulated by CBX. We conclude that the administration of CBX results in acceleration of intestinal growth and structural maturation without any influence on the developmental pattern of brush-border hydrolases. The results indicate an important role of peripheral glucocorticoid metabolism in the regulation of intestinal growth during early postnatal life.

• Placental 11beta-hydroxysteroid dehydrogenase in Dahl and spontaneously hypertensive rats.

  Authors: Karla Mazancova, Ivan Miksik, Jaroslav Kunes, Jirí Pácha

  American journal of hypertension : journal of the American Society of Hypertension. 06/2003; 16(5 Pt 1):401-6.

  Studies in normotensive rats showed that excessive fetal exposure to maternal glucocorticoids retards growth and programs hypertension in later life. This excessive exposure is proposed to occur dueStudies in normotensive rats showed that excessive fetal exposure to maternal glucocorticoids retards growth and programs hypertension in later life. This excessive exposure is proposed to occur due to a reduction of the placental barrier to maternal glucocorticoids that is provided by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To assess the possible alterations of glucocorticoid placental barrier in two genetic models of hypertension - spontaneously hypertensive (SHR) and Dahl salt-sensitive rats (DS) and their normotensive counterparts Wistar-Kyoto (WKY) and Dahl salt-resistant rats (DR)-we performed real-time reverse transcriptase-polymerase chain reaction analysis and bioactivity measurements of placental 11betaHSD in the last third of gestation. Whereas 11betaHSD2 mRNA expression was not different among the investigated strains, 11betaHSD1 mRNA abundance was 2.4 times higher in WKY than in SHR and 9.6 times higher in DS than in DR placentae. The 11betaHSD2 activity studies performed in placental homogenates revealed activity that did not differ among the strains. Concomitant with 11betaHSD1 mRNA expression 11-oxoreductase activity was clearly evident in all strains and was higher in WKY and DS rats than in SHR and DR, respectively. Nevertheless, the net 11betaHSD activity of tissue fragments (11beta-dehydrogenase minus 11-oxoreductase) was tended toward dehydrogenase action, ie, toward corticosterone inactivation and was significantly lower in DS than in DR rats. The 11beta-dehydrogenase/11-oxoreductase ratio was less than 2:1 in SHR and WKY rats, whereas this ratio was 9:1 in DR and 4.5:1 in DS rats. These data suggest that the placental glucocorticoid barrier is not decreased in SHR rats in comparison with normotensive WKY but is lower in DS than in DR counterparts. It cannot be excluded, therefore, that the placental glucocorticoid barrier in Dahl rats influences the pathways that might lead to the sensitivity of blood pressure to high salt intake in later life.

• Sexual dimorphism of 11beta-hydroxysteroid dehydrogenase in hypertensive and normotensive rats.

  Authors: Karla Mazancová, Ivan Miksík, Jaroslav Kunes, Josef Zicha, Jirí Pácha

  Hypertension research : official journal of the Japanese Society of Hypertension. 05/2003; 26(4):333-8.

  To evaluate the role of sexually dimorphic tissue expression of 11beta-oxidase activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD) in gender-associated blood pressure differences, we haveTo evaluate the role of sexually dimorphic tissue expression of 11beta-oxidase activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD) in gender-associated blood pressure differences, we have studied female and male hypertensive rats of two different strains and their normotensive controls: spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY) and Dahl salt-sensitive (SS/Jr) and salt-resistant rats (SR/Jr). In hypertensive SHR and SS/Jr, but not in normotensive strains WKY and SR/Jr, blood pressure reached a higher level in males than in females. The activity of 11betaHSD was higher in the renal cortex, medulla, colon and aorta of males than of females in all investigated strains with the exception of aortic 11betaHSD in SHR and WKY rats, both of which had very low 11beta-oxidase activity. In contrast to gender-dependent differences, strain differences of 11betaHSD were observed in a limited number of tissues only. Renal medullary 11betaHSD showed significantly lower activity in WKY than in SHR, whereas no difference was observed in the renal cortex. Similarly, colonic 11betaHSD activity was lower in WKY than in SHR. In Dahl rats the strain differences were observed in aortic 11betaHSD that had higher activity in SR/Jr than in SS/Jr rats; no difference was observed in the kidney or colon. These data demonstrate the following. 1) Sexual dimorphism of 11betaHSD activity exists in the kidney, colon, and aorta. 2) The sexual dimorphism of 11betaHSD does not play a role in gender-associated differences in blood pressure. 3) The reduced 11betaHSD activity in the aorta of hypertensive SS/Jr compared to SR/Jr rats suggests that this enzyme might play a role in the pathogenesis of salt-sensitive hypertension in Dahl rats.

• Membrane properties of rat colonic crypts during early postnatal development.

  Authors: Sergej Beskid, Jirí Pácha

  Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 02/2003; 13(6):385-90.

  The expression patterns of plasma membrane transporters and channels undergo developmental changes during cell differentiation and postnatal development. Until now, little is known about cellThe expression patterns of plasma membrane transporters and channels undergo developmental changes during cell differentiation and postnatal development. Until now, little is known about cell membrane conductances during maturation of intestinal crypts even though it is accepted that the crypts undergo dramatic changes during this period of life. Using the whole-cell patch clamp technique, we investigated the developmental changes of potassium conductance (GK) in rat colonic crypts and its heterogeneity along the crypt-surface axis. The resting membrane potential of immature crypts was sensitive to Ba2+ and was higher than in mature crypts. The whole cell conductance during outward currents was higher in immature crypts than in the crypts of adult animals and was reduced by Ba2+ and tetraethylammonium (TEA). With Na+-containing Ringer solution in the bath and K+-containing solution in the pipette, Ba2+- and TEA-sensitive outward currents and conductances were higher in immature than in mature crypts. The TEA-sensitive conductance in immature crypts was distributed heterogenously along the crypt-surface axis with higher values in undifferentiated cells of the crypt base than in differentiated cells of the crypt surface. These data indicate that (1) both colonocyte differentiation and maturation of colonic crypt epithelium are associated with decreasing GK and (2) the axial heterogeneity already exists early in postnatal life when the colonic epithelium undergoes rapid proliferation and growth associated with elongation and bifurcation of the crypts.

• Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

  Authors: Jirí Pácha, Vera Lisá, Ivan Miksík

  Steroids. 03/2002; 67(2):119-26.

  11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.

• Apical ouabain-sensitive and ouabain-insensitive ATPases in rat colonic epithelium.

  Authors: Radomíra Vagnerová, Zdenka Zemanová, Jirí Pácha, Jaroslava Smídová

  Acta histochemica. 02/2002; 104(4):407-11.

  Active resorption of potassium ions in the colon is mediated by ouabain-sensitive and ouabain-insensitive H,K-ATPases localized in the apical membrane of colonic enterocytes (colonocytes). TheActive resorption of potassium ions in the colon is mediated by ouabain-sensitive and ouabain-insensitive H,K-ATPases localized in the apical membrane of colonic enterocytes (colonocytes). The present study was performed to investigate distribution patterns of apical ATPases using catalytic histochemistry and immunohistochemistry. Activity of the ATPases was localized both at the apical and basolateral regions of these cells. The basolateral activity was almost completely inhibited by ouabain, but apical ATPase activity was only partially inhibited by ouabain. Ultracytochemically, activity was localized at the cytoplasmic side of the apical and basolateral membrane and thus we assume that the activity represents isoforms of apical H,K-ATPases and basolateral Na,K-ATPase. With the use of a polyclonal antibody raised against H,K-ATPase alpha-subunit, we demonstrated immunostaining only in the apical region of colonic enterocytes whereas positive staining was not observed at the basolateral membrane. On the other hand, when antibodies against alpha1-subunit Na,K-ATPase were applied, immunostaining was localized only in the basolateral membrane domain. Therefore, we conclude that ATPases as demonstrated histochemically in the present study were identified immunohistochemically as colonic alpha-subunit H,K-ATPase (in the apical cell membrane of colonocytes along the entire length of crypts) and as alpha1-subunit Na,K-ATPase (in the basolateral membrane).