Histone deacetylase inhibition attenuates hepatic steatosis in rats with experimental Cushing's syndrome

Cushing's syndrome (CS) is a collection of symptoms caused by prolonged exposure to excess cortisol. Chronically elevated glucocorticoid (GC) levels contribute to hepatic steatosis. We hypothesized that histone deacetylase inhibitors (HDACi) could attenuate hepatic steatosis through glucocorticoid receptor (GR) acetylation in experimental CS. To induce CS, we administered adrenocorticotropic hormone (ACTH; 40 ng/kg/day) to Sprague-Dawley rats by subcutaneous infusion with osmotic mini-pumps. We administered the HDACi, sodium valproate (VPA; 0.71% w/v), in the drinking water. Treatment with the HDACi decreased steatosis and the expression of lipogenic genes in the livers of CS rats. The enrichment of GR at the promoters of the lipogenic genes, such as acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn), and sterol regulatory element binding protein 1c (Srebp1c), was markedly decreased by VPA. Pan-HDACi and an HDAC class I-specific inhibitor, but not an HDAC class II a-specific inhibitor, attenuated dexamethasone (DEX)-induced lipogenesis in HepG2 cells. The transcriptional activity of Fasn was decreased by pretreatment with VPA. In addition, pretreatment with VPA decreased DEX-induced binding of GR to the glucocorticoid response element (GRE). Treatment with VPA increased the acetylation of GR in ACTH-infused rats and DEX-induced HepG2 cells. Taken together, these results indicate that HDAC inhibition attenuates hepatic steatosis hrough GR acetylation in experimental CS.

Effect of ECQ on Iodoacetamide-Induced Chronic Gastritis in Rats

This study investigated effect of extract containing quercetin-3-O-beta-D-glucuronopyranoside from Rumex Aquaticus Herba (ECQ) against chronic gastritis in rats. To produce chronic gastritis, the animals received a daily intra-gastric administration of 0.1 ml of 0.15% iodoacetamide (IA) solution for 7 days. Daily exposure of the gastric mucosa to IA induced both gastric lesions and significant reductions of body weight and food and water intake. These reductions recovered with treatment with ECQ for 7 days. ECQ significantly inhibited the elevation of the malondialdehyde levels and myeloperoxidase activity, which were used as indices of lipid peroxidation and neutrophil infiltration. ECQ recovered the level of glutathione, activity of superoxide dismutase (SOD), and expression of SOD-2. The increased levels of total NO concentration and iNOS expression in the IA-induced chronic gastritis were significantly reduced by treatment with ECQ. These results suggest that the ECQ has a therapeutic effect on chronic gastritis in rats by inhibitory actions on neutrophil infiltration, lipid peroxidation and various steps of reactive oxygen species (ROS) generation.

Probing Regulatory Proteins for Vascular Contraction by Deoxyribonucleic Acid Microarray

BACKGROUND AND OBJECTIVES: The heat-shock response modulates contractility of vascular smooth muscles. With complementary deoxyribonucleic acid microarray, we tried to identify the novel genes that are involved in the regulation of vascular contraction after heat shock. MATERIALS AND METHODS: Human radial artery strips were mounted in organ baths, exposed at 42degrees C for 45 minutes, and returned to equilibrate at 37degrees C. This study examined gene expression profile associated with heat-shock response in radial arteries of patients with hyperlipidemia by using a microarray that contained 5763 human cDNA. The results of microarray hybridization experiments from the radial arteries of 4 different subjects were analyzed and classified by the cluster program. RESULTS: Among these differentially-expressed genes, Hsp70, Hsp10, alphaB-crystallin, and Hsp60 were significantly increased by the heat shock response. Of non-HSP genes, 15 genes increased, while 22 genes decreased. Among these 37 genes, alphaB-crystallin (CRYAB) (up 1.92-fold), myosin, light polypeptide kinase transcript variant 8, 6 (up 1.70-fold, up 1.68-fold), catenin (cadherin-associated protein, alpha-like 1) (down-0.57 fold) and tropomyosin 3 (down 0.68-fold) were thought to be related with the contraction. Real-time quantitative polymerase chain reaction showed that Hsp70, Hsp10 and alphaB-crystallin were significantly increased. CONCLUSION: Gene expression profile by heat shock provides information about genes implicated in augmentation of vascular contraction after heat shock.

MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of NG-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca2+-free buffer but reappeared in normal Ca2+-containing buffer indicating that the contraction was Ca2+ dependent. 4-aminopyridine (4-AP), voltage-dependent K+ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a Gi inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca2+ and K+ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca2+, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.

Calcium Sensitization Induced by Sodium Fluoride in Permeabilized Rat Mesenteric Arteries

It was hypothesized that NaF induces calcium sensitization in Ca2+-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with beta-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca2+ (decreased EC50 and increased E(max)). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca2+-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPgammaS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca2+-dependent manner in beta-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 micrometer) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC20), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC20 phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca2+-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 micrometer) and atropine (1 micrometer). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of 'on' and 'off' contraction and the effects of G-proteins, phospholipase, and K+ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a Gi inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, Gs inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K+ channel opener) decreased these contractions, and tetraethylammonium (TEA, K+ Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca2+ channel may be activated by G-protein alpha subunits. Furthermore, K+ Ca-channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca2+ channel and to investigate the effects of other K+ channels on EFS-induced on and off contractions.

Vascular Response of Isolated Rat Thoracic Aorta after Chemical Hypoxia/Reoxygenation / 대한마취과학회지

BACKGROUND: Hypoxia/reoxygenation (H/R) results in formation of toxic reactive oxygen species (ROS), which can impair the vascular pathophysiology. Nitric oxide (NO) is an important free radical in many physiological or pathological processes including H/R injury. The loss of NO after H/R might be one of the major causes of an impaired vascular response. METHODS: Isolated rat aortic rings were prepared and NaCN was used to induce chemical hypoxia. The NaCN concentration and the hypoxia/reoxygenation time were determined by the responsiveness of phenylephrine (Phe), sodium nitroprusside (SNP) and acetylcholine (Ach). A cumulative doses of Phe and SNP (10(-9)-10(-5.5) M) were added to construct the vascular contraction and relaxation curves. The cumulative doses of Ach (10(-9)-10(-5) M) were added to construct the relaxation after precontraction with Phe (10(-6) M). The effects of the N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and the superoxide dismutase (SOD, 50 unit) pretreatment during chemical H/R were evaluated. RESULTS: The NaCN concentration and H/R time were 1 mM, 30 minutes/5 minutes, respectively. Chemical hypoxia reduced the Phe-induced vascular contraction significantly. However chemical H/R increased the Phe-induced contraction significantly, and impaired the relaxation by SNP and Ach. A pretreatment with L-NAME increased the Phe-induced contraction and impaired the relaxation by SNP as well as Ach. The SOD pretreatment reduced the Phe-induced increased vascular contraction after NaCN-induced chemical H/R. CONCLUSIONS: NO plays a key role in endothelial-dependent relaxation and the recovery of the augmented contractility by vasoconstrictors after chemically-induced H/R.

Partial Outlet Obstruction Induced Alterations of Rat Urinary Bladder Muscle / 대한해부학회지

Partial outlet obstruction induced by benign prostatic hypertrophy or urethral stricture are common urologic conditions showing voiding dysfunction. This study was performed to compare the effects of short-term partial outlet obstruction on the contractile responses to different stimuli and to elucidate the mechanism of altered contractile response during the development of significant voiding dysfunction. Sprague-Dawley rats, 10 week old, were subjected to partial outlet obstruction for 7 days. Contractile responses of the urinary bladder detrusor muscle strips to electrical field stimulation (1, 2, 5, 10 and 20 Hz), KCl (50 and 100 mM) and carbachol (10(-9 to 5) M) were measured. Total protein and DNA contents of the bladder were measured. Connexin 43 mRNA expression was measured using RT-PCR and connexin 43 protein was observed in the fixed bladder tissue using immunohistochemistry. The electrical field stimulation-induced contractile response and KCl-induced contraction was not changed after partial outlet obstruction. Contraction induced by carbachol was enhanced by the partial outlet obstruction. Total protein and DNA contents were increased in the partial outlet obstruction group. Connexin 43 mRNA and protein expression were detectable in the normal bladders and increased after 7 days of obstruction. These results suggest that the altered contractile responses during the early stage of the partial outlet obstruction are the result of the changes of the contraction mechanisms or structure of bladder muscle. Connexin 43 may play an important role in those alterations.

Pulse exposure to ethanol augments vascular contractility through stress response

Drinking excessive alcohol has been recognized as a risk factor for hypertension. However, the mechanism by which alcohol intake causes hypertension still remains elusive. We tested the hypothesis that ethanol itself acts as a stress factor on vasculature and indirectly modulates vascular contractility. After end of exposure to 1, 2.5 and 5% ethanol for 45 min, rat aortic strips were subjected to contractile responses, immunoblot for Hsp70 and the measurement of levels of myosin light chain phosphorylation. Exposure to 5% ethanol not only augmented contractions to KCl or phenylephrine, but also increased expression of Hsp70 and the levels of myosin light chain phosphorylation. There were no significant differences in contractions produced by 1 micromol/L phorbol 12,13-dibutyrate, a protein kinase C activator, whether the tissues were exposed to 5% ethanol or not. This is the first report to show that even short exposure to ethanol has a delayed effect to increase vascular smooth muscle contractility through a modulation of thick filament regulation. It may be a mechanism by which ingestion of alcohol induces hypertension.

The Implementation of Problem-based Learning in Kyungpook National University School of Medicine and its Evaluation / 한국의학교육

The effectiveness of Problem-based Learning(PBL) in medical education has already been acclaimed widely. Representatives of the curriculum committee at Kyungpook National University School of Medicine paid a visit to McMaster University School of Medicine in Canada in May, 1994 in order to learn mechanics and effectiveness of PBL in its medical education and they were impressed by the efficacy of PBL. Soon after that the school launched a pilot PBL tutorial for two years from 1994 through 1996(4-semester) as a non-credit course for senior, junior and sophomore in medical school during one semester each, to introduce PBL to faculty members and students as well. After the pilot, opinion survey on PBL from both faculty and students revealed affirmative for PBL from 55.1% of seniors, 61.4% of juniors and 83.9% of faculty members. The faculty body at medical school was then encouraged by the pilot experience and decided to include the PBL as the part of medical education reform. During the fall semester in 1998, the senior at pre-medical course was given PBL experience to prepare for implementation of PBL at school of medicine. The PBL was implemented as an essential 2-credit-hour course in each semester commencing in 1999 to the freshmen class throughout the year; it was extended to the sophomore in 2000 and to the junior in 2001. Although there had been initial excitements of over expectations, confusion, and disappointments from faculty members and students, majority opinion of both parties on continuation of PBL was positive. The issues to be settled are preparation of study cases, students learning resources, and method of evaluating students' performance. The PBL was started as an essential course in medical school in 1999 after 4 years of preparation and on the basis of our interim evaluations the following conclusions were made: we have reached the following consensus that students seem to follow the objectives of PBL and new PBL tutorial has well been accepted by students; and enhancing the program by correcting currently known weaknesses, the PBL tutorials could further be expanded to be a major modality of teaching in our medical school.

Effects of Inhalational Anesthetics on Contractile Responses and Nitric Oxide Synthase Activity in Endotoxemic Rats / 대한마취과학회지

BACKGROUND: Recent studies revealed that inhalational anesthetics (IA) attenuate NO production. But the hemodynamic changes produced by IA in septic syndrome patient are still sufficient to threaten patient, surgeon and anesthesiologist. So we examined which IA is proper to maintain vascular contractile force and evaluated the effects of NOS inhibitors on contractile force of septic rat aorta under IA. METHODS: Aortic ring preparation was obtained from LPS-treated (1.5 mg/kg, i.p. for 18h) rats. The development of sepsis was confirmed by iNOS activity and iNOS expression using RT-PCR. Contractile responses of aorta to phenylephrine admministation in the presence or absence of halothane, enflurane and isoflurane were evaluated. We also evaluated the effects of NOS inhibitors, one is NG-nitro-L-arginine methyl ester (L-NAME) and the other is aminoguanidine. Statistical significances (p<0.05) were analyzed according to data characteristics by unpaired t-test and paired t-test. RESULTS: The contractile responses to phenylephrine admministration were attenuated in LPS-treated rings. Isoflurane, even at the dose of 2 MAC, didn't affect the contractile response while both halothane and enflurane decreased the contractile response even at the dose of 1 MAC. The potentiation of contractile responses by NOS inhibitors were not affected during administeration of IA. CONCLUSIONS: From these results, it is suggested that isoflurane is the safest inhalational anesthetic and NOS inhibitors, especially L-NAME, may be very useful in the therapy of septic shock patients during general anesthesia.

Gene expression of intrarenal renin-angiotensin system in streptozotocin-induced diabetic rats

In humans and many animal models with chronic progressive renal diseases, angiotensin-converting enzyme (ACE) inhibitor markedly attenuates the progression of nephropathy. Several studies have reported augmented gene expression and redistribution of renal renin in partial nephrectomized rats. Although precise mechanism(s) is not known, the renin-angiotensin system (RAS) may play an important role in the progression of renal diseases. Thus, this study was undertaken to examine the gene expression of renal renin, angiotensinogen, and AT-1 subtypes (AT-1A and AT-1B) in rats with diabetic nephropathy, and the influences of lipopolysaccharide (LPS)-induced septicemia on the gene expression. Four weeks after streptozotocin (STZ) treatment (55 mg/kg, i.p.), rats were randomly divided into LPS-treated (1.6 mg/kg, i.p.) and control rats. At 6 hours after LPS treatment, the rats were killed and the kidney was removed from each rat. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to detect mRNA expression. STZ treatment markedly attenuated body weight gain and significantly increased blood glucose level. Renal renin content (RRC) was significantly decreased in the STZ-treated rats compared to that in control rats. The renal ACE activity between STZ-treated and control rats was not significantly different. Renal renin mRNA level was prominently increased, while angiotensinogen and AT-1A mRNA levels were slightly decreased in STZ-treated rats compared to those in controls. AT-1B mRNA level did not differ in both groups. Acute LPS treatment did not show any significant changes of mRNA levels of intrarenal RAS components in both groups. These results suggest that intrarenal RAS components were differentially regulated in STZ-treated diabetic rats. Further studies are required to evaluate the relationship between intrarenal RAS and other vasomodulatory systems.

Renal mRNA expression of renin, AT-1 receptor, TGF-beta-1 and fibronectin in obstructive nephropathy

The present study was designed to quantify the alterations of renal renin, angiotensin type I receptor (AT-1), TGF-beta-1, and fibronectin gene expression in rats with unilateral ureteral obstruction (UUO). We also investigated the change of AT-1 density during UUO. Reverse transcription-polymerase chain reaction (RT-PCR) technique and receptor binding assay were used to detect mRNA expression and receptor density, respectively. At one day after UUO, renin mRNA level of the obstructed kidneys was decreased transiently and then subsequently increased to the level of sham kidneys. In the contralateral kidneys of the same rats, on the contrary, renin mRNA level was gradually decreased. Then, at 9 days after UUO, it was significantly lower than that of sham kidneys. The expressions of both AT-1 subtypes, called AT-1A and AT-1B, mRNAs did not change at any time. UUO led to a significant decrease in AT-1 density in the obstructed kidneys compared with the sham kidneys at 1 and 3 days (66 +/- 11.6% (p lt 0.05) and 73 +/- 4.0% (p lt 0.01), respectively). Thereafter, AT-1 density was gradually increased and at 9 days it showed a marked elevation in the obstructed kidneys compared to the sham kidneys. In contrast, in the contralateral kidneys AT-1 density was significantly reduced from 3 to 9 days after UUO. The TGF-beta-1 m-RNA level of the obstructed kidneys was unexpectedly decreased at 6 days after UUO. Then, at 9 days it was followed by a significant increase in the obstructed kidneys, whereas it showed an obvious decrease in the contralateral kidneys. In addition, fibronectin mRNA level was also significantly increased in the obstructed kidneys after UUO compared to the sham or the contralateral kidneys of the same rats. These results suggest a differential regulation of renal renin, AT-1 receptor, TGF-beta-1 and fibronectin mRNA levels at different stages of UUO.

Effects of Volatile Anesthetics on Aortic Contractile Responses in Endotoxemic Rats / 대한마취과학회지

BACKGROUND: Endotoxins play important roles in the pathophysiologic alterations associated with sepsis so we examined the effects of volatile anesthetics on vascular smooth muscle contractile function in LPS-treated rat aorta. METHODS: Fifty male Sprague-Dawley rats(250~300 gm) were made septic by intraperitoneal injection of lipopolysaccharide(1.5 mg/kg). Cumulative doses of phenylephrine and norepinephrine(10 -8~10 -5M) were added to construct a contraction response curve. Two percent of volatile anesthetics, IBMX (3-isobutyl-1-methylxanthine, phosphodiesterase inhibitor) or L-NAME(Ng-nitro-L-arginine-methylester, Nitric oxide synthase inhibitor) was added and those contractile responses were observed respectively. We also measured nitric oxide synthase (NOS) activity of liver, lung and adrenal gland after 18 hours in the LPS-treated rats. Individual values between the control rats and LPS-treated rats were compared by unpaired t-test. A p-value less than 0.05 was considered statistically significant. RESULTS: Contractile response of 2% halothane to norepinephrine was significantly decreased both in the control rats and LPS-treated rats. The NOS inhibitor enhanced the contractile responses to phenylephrine and norephinephrine in the vessels from LPS-treated rats more significantly than those of control rats. CONCLUSIONS: These results suggest that LPS-treatment impairs vasopressor-induced contractility and doesn't alter the contractile responses during administration of volatile anesthetics.