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298 Cholestatic conditions act on prolactin signaling in rat cholangiocytes and hepatocytes: Two ways — One direction
Posters
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and survivin silencing and processing of caspase-3 was analyzed by Western blotting. After treatment with or without 10 ngjml recombinant human TRAIL, we used 4,6-diamidino-2-phenylindole (DAPI) staining and counted cells that had nuclear condensation and fragrnentation. Furthermore, we investigated viabilities of HCC cells transfected with siRNAs after trea~.ment with chemotherapeutic agents (camptothecin, doxorubicin, or cisplatin). To assess the viability of HCC cells, Cell Titer 96 Assay kit (Promega, Madison, WI) was utilized. Results: We found that siRNA-XIAP and siRNA-survMn significantly downregnlated protein expression o f respective genes. Suppression of the IAPs resulted in a decrease in procaspase-3 levels especially by suppression of XIAR The apoptosis cell ratio of IAPs suppression to control expanded twice or three times after treatment with 10 ng/ml recombinant human TRAIL. Cytotoxicity of chemotherapeutic agent (camptothecin and doxorubicin) was increased by suppression of XIAR Conclusions: Targeting XIAP or survMn by siRNAs sensitizes HCC cells to TRAIL- and chemotherapeutic agents-induced cell death.
Category 4b: Molecular and Cellular Biology: Biliary Tract Pathophysiology
[ 2 ~ l CHOLESTATIC CONDITIONS ACT ON PROLACTIN SIGNALING IN RAT C H O L A N G I O C Y T E S A N D HEPATOCYTES: TWO WAYS - ONE DIRECTION R. Bogorad I , T. Zenkova I , P. Rubtsov 2, O. Smimova 1. 1Lab. of Endocrinology. Moscow State University. Moscow, Russia," 2Engelhardt Institute of Molecular Biology, Moscow, Russia Prolactin participates in the regulation of functional and proliferative activity of hepatocytes as well as cholangiocytes. The cholestasis shifts the functional and proliferatingjapoptotic status of all liver cell types. The participation of prolactin in the regulation of activity of liver cells under cholestaais is obscure. Prolactin effects depend on the level of prolactin receptor (PrlR) and on the ratio of receptor isoforms. Long isoform transduces its signal via JAK/STAT cascade while short isoform negatively modulates the activity" of long one and transduces "subsidiary" signals. Aim: to analyze PrlR level and its isoforms ratio in hepatocytes and cholangiocytes from the liver o f normal rat and animals with obstructive cholestaais, induced by common bile duct ligation. Hepatocytes and cholangiocytes as fragments o f intrahepatic bile ducts were differentially isolated by enzymatic/mechanical segregation. The PrlR level and the ratio of its isoforms were measured by semiquantitative RT-PCR. It is revealed that the PrlR level in cholangiocytes from liver of intact females and males is low irrespective of the animal sex, contrary to hepatocytes, where the PrlR level is higher and dependent on sex. Obstructive cholestasis induces the 4-fold augmentation of this level in cholangiocytes of both males and females, but does not notably effect on PrlR level in hepatocytes. The single PrlR isoform in cholangiocytes of intact animals is the long one, the ratio of short, to long isofoma under obstructive cholestasis is 2.5:1 irrespective of the animal sex. In rat hepatocytes cholestasis decreases the ratio of short PrlR isofoma to long one, this effect of obstructive cholestasis is also sex-independent. Thus, the cholestatic conditions act in opposite directions on the ratio of PrlR isoforms in hepatocytes and cholan~ocytes, but cause hyperstimulation of PrlR signaling via JAK/STAT cascade leading to compensatory proliferation in both cell types. While in cholangiocytes prolactin hyper-signaling is achieved due to stimulation of PrlR gene transcription, in hepatocytes cholestasis mainly shifts the direction of alternative splicing, increasing the proportion of long isoform.
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CONTRIBUTION OF E N D O G E N O U S OPIOID PEPTIDES A N D NITRIC OXIDE IN CHOLESTATIC CARDIOMYOPATHY
A.R. Dehpour, E Ebrahimi, S. Tavakoli, A.R. Hajrasouliha, E JabehdarMaralani. Department of Pharmacology, School of Medicine, Tehran
University of Medical Sciences, Tehran, Iran Background and Aims: Attenuation of cardiovascular responsiveness to
sympathetic stimulations is proposed as an important factor for development o f some postoperative complications such as renal failure and hemorrhagic shock in cholestatic patients. We examined the role of nitric oxide (NO) overproduction and endogenous opioid peptides accumulation in cardiovascular hyporesponsiveness to f3-adrenetgic stimulation in isolated papillary muscles of 7-day bile duct ligated (BDL) rats. Methods: The animals were randomly divided into 5 groups of BDL, 5 groups of sham-operated, and an additional group of control rats. The first, second and third groups of sham-operated and BDL rats were treated with daily subcutaneous administration of normal saline (1 ml/k~g), L-N,~vIE, non-sdective NO synthase inhibitor (10 mgjkg), and naltrexone (20rag/ks) for 6 consecutive days following BDL or sham-operation (chronic treamlents). The papillary muscles o f the fourth and fifth groups were pre-incubated with L-NAME (10-4M) and naltrexone (10-tM) before stimulation by isoproterenol (acute treatments). Results: The basal contractile force of papillary muscle, rate constant for papillary contraction (+dT/dt) and relaxation @dT/dt) were significantly decreased in BDL rats compared to sham-operated ones (P < 0.05). Treamlent with L-NAME and naltrexone (acute/chronic) corrected these basal abnormalities in BDL rats. Rmax of contractile force, +dT/dtmax and -dT/dtmax of papillary musde in response to isoproterenol (10 5 M) stimulation were significantly lower in BDL rats compared to shamoperated group (P<0.05, P<0.05, P<0.01; respectively). The halfmaximal effective concentrations of isoproterenol (ECs0) of contractility dose-response curve were significantly increased in BDL rats compared to sham-operated ones (P <0.05). Chronic L-NAME treamaent resulted in a significant decrease in ECs0 of contractility dose~response curve in BDL rats (P <0.05), while this was not seen in acutely L-NAME treated BDL group. Rmax of contractile force, +dT/dtmax and -dT/d-tma x of papillary muscle and ECs0 o f contractility dose~response curve of BDL rats were completely reversed by chronic naltrexone treatment but partially restored by acute treatment. Conclusion: This investigation demonstrates that 7-day BDL rats have impaired basal contractile force, +dT/dt and dT/dt of papillary muscle together with hyporesponsiveness to f3-adrenergic stimulation and also provided evidence for the involvement of increased opioidergic tone and NO overproduction in cholestasis-induced cardiomyopathy.
I300l CHOLESTASIS INDUCED NEPHROTOXICITY: THE ROLE OF E N D O G E N O U S OPIOIDS A. Farajzadeh Deroee ~, A. Hassanzadeh Salmasi t , R. Hosseini2, E. Jahanzad 3, S. Ejtemaee-mehr~, B. Afshar Irnani 2, A.R. Dehpour I .
1Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran," 2Department of Toxicology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran," 3Cancer Research Institute, School of Medicine, Tehran University of Medical Sciences, Tehmn, Iran Background and Aims: The pathophysiology of renal impairment oc-
curring in cholestasis has been extensively studied but the underlying mechanism is not well known. It was shown that disturbance of the oxidant=antioxidant balance may be responsible for cholestasis induced nephrotoxicity. It is also shown that opioids have dual effects in protecting against oxidative stresses in other organs. Endogenous opioids are also increased in cholestasis. The aim of this study was to investigate the role of endogenous opioids in cholestasis induced nephrotoxicity.
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and survivin silencing and processing of caspase-3 was analyzed by Western blotting. After treatment with or without 10 ngjml recombinant human TRAIL, we used 4,6-diamidino-2-phenylindole (DAPI) staining and counted cells that had nuclear condensation and fragrnentation. Furthermore, we investigated viabilities of HCC cells transfected with siRNAs after trea~.ment with chemotherapeutic agents (camptothecin, doxorubicin, or cisplatin). To assess the viability of HCC cells, Cell Titer 96 Assay kit (Promega, Madison, WI) was utilized. Results: We found that siRNA-XIAP and siRNA-survMn significantly downregnlated protein expression o f respective genes. Suppression of the IAPs resulted in a decrease in procaspase-3 levels especially by suppression of XIAR The apoptosis cell ratio of IAPs suppression to control expanded twice or three times after treatment with 10 ng/ml recombinant human TRAIL. Cytotoxicity of chemotherapeutic agent (camptothecin and doxorubicin) was increased by suppression of XIAR Conclusions: Targeting XIAP or survMn by siRNAs sensitizes HCC cells to TRAIL- and chemotherapeutic agents-induced cell death.
Category 4b: Molecular and Cellular Biology: Biliary Tract Pathophysiology
[ 2 ~ l CHOLESTATIC CONDITIONS ACT ON PROLACTIN SIGNALING IN RAT C H O L A N G I O C Y T E S A N D HEPATOCYTES: TWO WAYS - ONE DIRECTION R. Bogorad I , T. Zenkova I , P. Rubtsov 2, O. Smimova 1. 1Lab. of Endocrinology. Moscow State University. Moscow, Russia," 2Engelhardt Institute of Molecular Biology, Moscow, Russia Prolactin participates in the regulation of functional and proliferative activity of hepatocytes as well as cholangiocytes. The cholestasis shifts the functional and proliferatingjapoptotic status of all liver cell types. The participation of prolactin in the regulation of activity of liver cells under cholestaais is obscure. Prolactin effects depend on the level of prolactin receptor (PrlR) and on the ratio of receptor isoforms. Long isoform transduces its signal via JAK/STAT cascade while short isoform negatively modulates the activity" of long one and transduces "subsidiary" signals. Aim: to analyze PrlR level and its isoforms ratio in hepatocytes and cholangiocytes from the liver o f normal rat and animals with obstructive cholestaais, induced by common bile duct ligation. Hepatocytes and cholangiocytes as fragments o f intrahepatic bile ducts were differentially isolated by enzymatic/mechanical segregation. The PrlR level and the ratio of its isoforms were measured by semiquantitative RT-PCR. It is revealed that the PrlR level in cholangiocytes from liver of intact females and males is low irrespective of the animal sex, contrary to hepatocytes, where the PrlR level is higher and dependent on sex. Obstructive cholestasis induces the 4-fold augmentation of this level in cholangiocytes of both males and females, but does not notably effect on PrlR level in hepatocytes. The single PrlR isoform in cholangiocytes of intact animals is the long one, the ratio of short, to long isofoma under obstructive cholestasis is 2.5:1 irrespective of the animal sex. In rat hepatocytes cholestasis decreases the ratio of short PrlR isofoma to long one, this effect of obstructive cholestasis is also sex-independent. Thus, the cholestatic conditions act in opposite directions on the ratio of PrlR isoforms in hepatocytes and cholan~ocytes, but cause hyperstimulation of PrlR signaling via JAK/STAT cascade leading to compensatory proliferation in both cell types. While in cholangiocytes prolactin hyper-signaling is achieved due to stimulation of PrlR gene transcription, in hepatocytes cholestasis mainly shifts the direction of alternative splicing, increasing the proportion of long isoform.
[•
CONTRIBUTION OF E N D O G E N O U S OPIOID PEPTIDES A N D NITRIC OXIDE IN CHOLESTATIC CARDIOMYOPATHY
A.R. Dehpour, E Ebrahimi, S. Tavakoli, A.R. Hajrasouliha, E JabehdarMaralani. Department of Pharmacology, School of Medicine, Tehran
University of Medical Sciences, Tehran, Iran Background and Aims: Attenuation of cardiovascular responsiveness to
sympathetic stimulations is proposed as an important factor for development o f some postoperative complications such as renal failure and hemorrhagic shock in cholestatic patients. We examined the role of nitric oxide (NO) overproduction and endogenous opioid peptides accumulation in cardiovascular hyporesponsiveness to f3-adrenetgic stimulation in isolated papillary muscles of 7-day bile duct ligated (BDL) rats. Methods: The animals were randomly divided into 5 groups of BDL, 5 groups of sham-operated, and an additional group of control rats. The first, second and third groups of sham-operated and BDL rats were treated with daily subcutaneous administration of normal saline (1 ml/k~g), L-N,~vIE, non-sdective NO synthase inhibitor (10 mgjkg), and naltrexone (20rag/ks) for 6 consecutive days following BDL or sham-operation (chronic treamlents). The papillary muscles o f the fourth and fifth groups were pre-incubated with L-NAME (10-4M) and naltrexone (10-tM) before stimulation by isoproterenol (acute treatments). Results: The basal contractile force of papillary muscle, rate constant for papillary contraction (+dT/dt) and relaxation @dT/dt) were significantly decreased in BDL rats compared to sham-operated ones (P < 0.05). Treamlent with L-NAME and naltrexone (acute/chronic) corrected these basal abnormalities in BDL rats. Rmax of contractile force, +dT/dtmax and -dT/dtmax of papillary musde in response to isoproterenol (10 5 M) stimulation were significantly lower in BDL rats compared to shamoperated group (P<0.05, P<0.05, P<0.01; respectively). The halfmaximal effective concentrations of isoproterenol (ECs0) of contractility dose-response curve were significantly increased in BDL rats compared to sham-operated ones (P <0.05). Chronic L-NAME treamaent resulted in a significant decrease in ECs0 of contractility dose~response curve in BDL rats (P <0.05), while this was not seen in acutely L-NAME treated BDL group. Rmax of contractile force, +dT/dtmax and -dT/d-tma x of papillary muscle and ECs0 o f contractility dose~response curve of BDL rats were completely reversed by chronic naltrexone treatment but partially restored by acute treatment. Conclusion: This investigation demonstrates that 7-day BDL rats have impaired basal contractile force, +dT/dt and dT/dt of papillary muscle together with hyporesponsiveness to f3-adrenergic stimulation and also provided evidence for the involvement of increased opioidergic tone and NO overproduction in cholestasis-induced cardiomyopathy.
I300l CHOLESTASIS INDUCED NEPHROTOXICITY: THE ROLE OF E N D O G E N O U S OPIOIDS A. Farajzadeh Deroee ~, A. Hassanzadeh Salmasi t , R. Hosseini2, E. Jahanzad 3, S. Ejtemaee-mehr~, B. Afshar Irnani 2, A.R. Dehpour I .
1Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran," 2Department of Toxicology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran," 3Cancer Research Institute, School of Medicine, Tehran University of Medical Sciences, Tehmn, Iran Background and Aims: The pathophysiology of renal impairment oc-
curring in cholestasis has been extensively studied but the underlying mechanism is not well known. It was shown that disturbance of the oxidant=antioxidant balance may be responsible for cholestasis induced nephrotoxicity. It is also shown that opioids have dual effects in protecting against oxidative stresses in other organs. Endogenous opioids are also increased in cholestasis. The aim of this study was to investigate the role of endogenous opioids in cholestasis induced nephrotoxicity.