Neuroprotective effect of oral S/B remedy (Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd) on iron-induced neurodegeneration in the nigrostriatal dopaminergic system of rat brain.

AIM OF THE STUDY: S/B remedy prepared from Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd, two herbals of Xiao-Tsai-Hu-Tang or Sho-Saiko-To (TJ-9), contains active flavonoids. In this study, the protective effect of S/B remedy on iron-induced neurodegeneration was investigated in the nigrostriatal dopaminergic system of rat brain. MATERIALS AND METHODS: The antioxidative activity of S/B remedy was studied using brain homogenates incubated with ferrous citrate (iron, 1M), S/B remedy, Trolox and melatonin. Furthermore, a Parkinsonian animal model by an intranigral infusion of iron in the anesthetized rats was employed to investigate the protective effect of S/B remedy in the nigrostriatal dopaminergic system. RESULTS: Our in vitro studies showed that S/B remedy was more potent than melatonin and equal to trolox in inhibiting iron-induced lipid peroxidation of brain homogenates. Our in vivo studies found that oral administration of S/B remedy dose-dependently attenuated iron-elevated lipid peroxidation in the infused substantia nigra (SN) and iron-depleted dopamine levels in the ipsilateral striatum. Furthermore, iron-induced reductions in glutathione (GSH) content and increases in GSSG (oxidized GSH)/GSH ratio in the infused SN were inhibited in S/B remedy-treated rats. Systemic S/B remedy attenuated the iron-induced increases in heme-oxygenase-1 levels and α-synuclein aggregation in the infused SN. Moreover, S/B remedy reduced iron-induced apoptosis via attenuating mitochondrial and endoplasmic reticulum stress. In addition, S/B remedy was anti-inflammatory as indicated by the attenuation of iron-induced elevations in inducible nitric oxide synthase and cyclo-oxygenase II levels as well as glial fibrillary acidic protein (a biological marker of astrocytes) and ED-1 (a protein indicative of activated microglia) levels in the infused SN of S/B remedy-treated rats. CONCLUSIONS: These findings suggest that oral administration of S/B remedy is protective against iron-induced neurodegeneration in the nigrostriatal dopaminergic system of rat brain. Therefore, S/B remedy may be therapeutically useful for the treatment of CNS neurodegenerative diseases.

Intestinal regeneration by a novel surgical procedure.

BACKGROUND: Treatment of short bowel syndrome is problematical. Small bowel tissue engineering has achieved modest results in animal studies. The aim of this study was to investigate intestinal regeneration in a novel surgical model. METHODS: Roux-en-Y bypass procedures were performed on 40 Wistar rats weighing 250-350 g. Animals were killed at 1, 2, 3, 4, 8, 12 and 24 weeks after implantation with a 3-cm silicone tube. The spatio temporal relationship of intestinal regeneration was analysed using three-dimensional multislice computed tomography, and examination of sequential morphological changes on gross or histological findings and measurement of missing intestinal tissue (growth defects). RESULTS: Progressive intestinal regeneration on a silicone tube was identifiable in 35 animals. Most adhesions were initially localized on the tube but spread to a distal site 4 weeks after implantation. Growth defects decreased with time, with a marked reduction in the first 4 weeks and a gradual reduction to week 24 after implantation. Luminal patency shown radiologically as well as sequential histological findings, such as mucosal lining, matrix remodelling and muscular regeneration, suggested that regeneration of intestinal tissue took place, not merely scar contraction. CONCLUSION: Non-invasive as well as histomorphological assessment followed intestinal regeneration over time in this model, which provides scope for further studies.

Modulation of HER2 expression by ferulic acid on human breast cancer MCF7 cells.

BACKGROUND: The molecular mechanisms underlying the mitogenic effect of ferulic acid (FA), an active compound derived from Angelica sinensis, have never been elucidated. It was the aim of this study to investigate the proliferative effect of FA on human breast cancer cell lines and to elucidate its modulation mechanism on HER2 expression in MCF7 line. MATERIALS AND METHODS: By using MCF7 (oestrogen receptor-positive; ER+, HER2-low), BT474 (ER+, HER2-high), MDAMB231 (ER-, HER2-low) and SKBR3 (ER-, HER2-high) human breast cancer cell lines as in vitro models, the mitogenic effects of FA were assessed by trypan blue dye exclusion assay and DNA flow cytometry. Ferulic acid-modulated cell signalling and HER2 gene expression were evaluated in MCF7 line by Western blot and real-time RT-PCR analysis. RESULTS: Ferulic acid ER-dependently stimulated cell proliferation on MCF7 cells in a concentration-dependent manner. The HER2 oncogene (one of the prognostic factors of breast cancer) and ESR1 gene (oestrogen receptor-alpha; ERalpha) transcription were markedly up-regulated by FA treatment. Besides, HER2 signalling and its downstream molecules such as AKT and ERK1/2 were involved in FA-modulated ERalpha and cyclin D1 synthesis. Addition of anti-HER2 antibody, trastuzumab, abrogated FA-enhanced proliferative effect on MCF7 cells, indicated a positive feedback control for the action of HER2 in this setting. The fact that the ER antagonist blocked most of the FA-up-regulated HER2 expression, and that trastuzumab down-regulated ERalpha gene expression, suggested a cross-talk between ERalpha and HER2 signalling on MCF7 cells. CONCLUSION: The authors' conclude that FA causes human breast cancer cell proliferation by up-regulation of HER2 and ERalpha expression.

Epimedium brevicornum Maxim extract relaxes rabbit corpus cavernosum through multitargets on nitric oxide/cyclic guanosine monophosphate signaling pathway.

Epimedium brevicornum Maxim (EbM) has been reputed to have sexual stimulation effects on males. The study is aimed to test the hypothesis that EbM extracts relaxed the corpus cavernosum (CC) smooth muscle through activation of multitargets on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. Water extract of EbM and its subfraction (EP-20) were prepared and standardized by high-performance liquid chromatography. Isolated rabbit CC strips were mounted in organ baths and isometric tension was recorded in the presence or absence of specific inhibitors related to NO/cGMP signaling such as L-N(G)-nitro-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor) or phosphodiesterase 5 (PDE 5) inhibitors. cGMP level was determined in EP-20-treated CC strips. The results showed that EP-20 enriched the content of L-arginine in the process of purification and relaxed the CC smooth muscle precontracted with phenylephrine (PE, 1 microM) in a concentration-dependent manner. Besides, EP-20 increased the amount of cGMP production in rabbit CC tissues. Coincubation with EP-20 and L-NAME or ODQ significantly decreased EP-20-induced relaxation whereas EP-20 increased sodium nitroprusside-induced relaxation in PE-precontracted CC strips. Besides, EP-20 increased the potency and the duration of the relaxation effects caused by electrical field stimulation. Finally, EP-20 could potentiate PDE 5 inhibitors in relaxation of PE-precontracted CC strips. We concluded that extract of EbM relax the CC smooth muscle through multitargets in NO/cGMP/PDE 5 pathway and might bring into perspective the treatment strategy for those patients with erectile dysfunction.

Single vs. two steroid injections for carpal tunnel syndrome: a randomised clinical trial.

We investigated the efficacy of a single vs. double steroid injections in the treatment of carpal tunnel syndrome (CTS) in a randomised double-blind controlled trial. Patients with idiopathic CTS were randomised into (i) one group receiving a baseline methylprednisolone acetate injection plus a saline injection 8 weeks later and (ii) a second group receiving methylprednisolone acetate injection at baseline and at 8 weeks. The primary outcome was the Global Symptom Score (GSS). Forty patients were recruited. By 40 weeks, the mean GSS improved from 25.6 to 14.1 in the single-injection group whereas from 26.7 to 12.6 in the reinjection group, but there was no significant difference in GSS between the two groups (p = 0.26). There were also no significant differences in terms of electrophysiological and functional outcomes. The results suggest that an additional steroid injection confers no added benefit to a single injection in terms of symptom relief.

Prognostic significance of nm23-H1 expression in oral squamous cell carcinoma.

Recent studies indicated nm23-H1 played a role in cancer progression. Therefore, we investigated clinical significance of nm23-H1 expression in oral squamous cell carcinoma (OSCC). In total, 86 OSCC specimens were immunohistochemically stained with nm23-H1-specific monoclonal antibodies. Immunohistochemical staining of nm23-H1 was confirmed by immunoblotting. The relations between nm23-H1 expression and clinicopathologic variables were evaluated by chi(2) analysis. As increased size of primary tumour could escalate metastatic potential and the data of patients at the late T stage might confound statistical analyses, we thus paid special attention to 54 patients at the early T stage of OSCC. Statistical difference of survival was compared by a log-rank test. Immunohistochemically, nm23-H1 expression was detected in 48.8% (42 out of 86) of tumorous specimens. It positively correlated with larger primary tumour size (P=0.03) and inversely with cigarette-smoking habit (P=0.042). In patients at the early T stage, decreased nm23 expression was associated with increased incidence of lymph node metastasis (P=0.004) and indicated poor survival (P=0.014). Tumour nm23-H1 expression is a prognostic factor for predicting better survival in OSCC patients at the early T stage, which may reflect antimetastatic potential of nm23. Therefore, modulation of nm23-H1 expression in cancer cells can provide a novel possibility of improving therapeutic strategy at this stage. In addition, our results further indicated cigarette smoking could aggravate the extent of nm23-H1 expression and possibly disease progression of OSCC patients.

Expression of aminopeptidase N in bile canaliculi: a predictor of clinical outcome in biliary atresia and a potential tool to implicate the mechanism of biliary atresia.

BACKGROUND: Only a few studies on extrahepatic biliary atresia (BA) have reported that the morphological changes of bile canaliculi could predict the clinical outcome after portoenterostomy and provide differential diagnosis of neonatal jaundice. Aminopeptidase N (APN) is an ectoenzyme of bile canaliculi that is involved in bile secretion. In this study, we tried to see whether APN of bile canaliculi had a significant role in BA. PATIENTS AND METHODS: We used monoclonal antibody 9B2 to compare the expression of APN in livers with BA, neonatal hepatitis, and choledochal cysts, as well as in nontumorous portions of pediatric hepatic livers with tumors. The expression of APN in fetuses, preterm babies, and term neonates was also studied. RESULTS: A high degree of 9B2 expression in BA was closely related to poor outcome. Cholestasis in choledochal cysts, rather than neonatal hepatitis, made 9B2 expression stronger. Increasing expression of 9B2 from fetuses to neonates was noted and the degree of 9B2 expression was similar between term neonates and nontumorous portions of pediatric livers with tumors. Interestingly, some cases of BA had 9B2 expression like that of preterm babies. CONCLUSIONS: APN of bile canaliculi progressively develops from fetuses to neonates and is well developed in neonates. APN can be induced to stronger expression by obstructive jaundice. The amount of expression of APN of bile canaliculi in BA is a predictor of clinical outcome and may be a tool for implicating the mechanism of BA.

Preconditioning somatothermal stimulation on right seventh intercostal nerve territory increases hepatic heat shock protein 70 and protects the liver from ischemia-reperfusion injury in rats.

Hyperthermic preconditioning attenuates the heat-induced cellular response to a subsequent severe heat challenge. However, it is impractical to perform whole-body hyperthermia in humans. This study was designed to test the hypotheses that hepatic heat shock protein 70 (Hsp70) could be induced by local somatothermal stimulation (LSTS) on right seventh intercostal nerve territory and that preconditioning the rats with LSTS protects the liver from subsequent ischemia-reperfusion injury. LSTS was brought about by application of a heating rod above right seventh intercostal nerve territory in male Sprague-Dawley rats. Hepatic gene expression of Hsp70 was assessed by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Finally, serum ALT and AST and the lipid peroxidation product malondialdehyde (MDA) were evaluated in ischemic-reperfused rats preconditioned by application of LSTS on right seventh intercostal nerve territory. The results showed that hepatic gene expression of Hsp70 was upregulated in rats treated with LSTS. When animals were preconditioned with LSTS, followed by subsequent ischemia-reperfusion injury of the liver, there were significant decreases in liver enzymes (ALT/AST) and MDA formation in rats pretreated with one dose of LSTS (LSTS-1 group) as compared with those not treated with LSTS (control group) or treated with three doses of LSTS (LSTS-3 group). We conclude that mild local heat stress (one dose) on right seventh intercostal nerve territory upregulates hepatic gene expression of Hsp70 and protects the liver from subsequent ischemia-reperfusion injury. This might provide an easily applicable method for those patients facing ischemia-reperfusion challenge of the liver, as in liver resection and liver transplantation.

Idiopathic pulmonary hemosiderosis in a child: report of one case.

Idiopathic pulmonary hemosiderosis (IPH) is uncommon in children. We report a 3-year-old girl who was presented with acute pale-looking appearance, hemoptysis, hematemesis and shortness of breath. This patient was confirmed to have pulmonary hemorrhage by the presence of hemosiderin-laden macrophages in the bronchoalveolar lavage fluid using a flexible bronchoscope. Other causes of PH including glomerular, cardiac and immunological disorder were excluded by normal laboratory studies. She was primarily treated by oral prednisolone, but due to recurrent hemoptysis, immunosuppressive agent was added for maintenance therapy. Pediatricians should consider PH in a patient who has recurrent dyspnea, hemoptysis and iron deficiency anemia.

Electroacupuncture-induced neural activation detected by use of manganese-enhanced functional magnetic resonance imaging in rabbits.

OBJECTIVE: To investigate the effects of acupuncture on neural activity detected by use of manganese-enhanced functional magnetic resonance imaging (fMRI) and elucidate the relationship between somatic acupoint stimulation and brain activation. ANIMALS: 40 New Zealand White rabbits. PROCEDURE: Manganese-enhanced fMRI was performed in anesthetized rabbits manipulated with electroacupuncture (EA) on Zusanli (ST-36) and Yanglingquan (GB-34) acupoints. Image acquisition was performed on a 1.5T superconductive clinical scanner with a circular polarized extremity coil. T1-weighted images were acquired sequentially as follows: baseline, after mannitol injection, after manganese infusion, and 5 and 20 minutes after initiation of EA. RESULTS: Changes in focal neural activity were detected by use of manganese-enhanced fMRI. Stimulation on Zusanli (ST-36) for 5 minutes resulted in activation of the hippocampus, whereas stimulation on Yanglingquan (GB-34) resulted in activation of the hypothalamus, insula, and motor cortex. Activation became less specific after 20 minutes of EA. Furthermore, stimulation on ipsilateral acupoints led to bilateral brain activation. CONCLUSIONS AND CLINICAL RELEVANCE: Each acupoint has a corresponding cerebral linkage, and stimulation on these points resulted in time-dependent neural activation. Understanding the linkage between peripheral acupoint stimulation and central neural pathways may provide a useful guide for clinical applications of acupuncture.

Analysis of integrated hepatitis B virus DNA and flanking cellular sequence by inverse polymerase chain reaction.

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral-cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral-cellular junctions.

Expression of fas ligand on bile ductule epithelium in biliary atresia--a poor prognostic factor.

PURPOSE: The aim of this study was to investigate the possible role of Fas and Fas ligand system in biliary atresia. METHODS: Immunohistochemical stains of Fas and Fas ligand (FasL) and in situ hybridization of Fas ligand messenger RNA (mRNA) were performed on paraffin-embedded liver specimens of 36 biliary atresia, 6 choledochal cysts, and 14 nontumorous parts of pediatric liver tumors. Apoptosis was detected by terminal deoxynucleotidyl transferase deoxy-UTP nick end labeling (TUNEL). The grade of liver fibrosis and results of bile drainage on the patients with biliary atresia were compared with the results of FasL expression. RESULTS: Fas protein was positive on the hepatocytes and bile ductule epithelia of all the livers examined and also positive on some monocytes around the portal area in all the biliary atresia patients. FasL protein was positive on bile ductule epithelia in 10 biliary atresia patients and also positive on some monocytes in most of the biliary atresia patients. Positive signals of FasL mRNA were noted on hepatocytes in 4 biliary atresia, bile ductule epithelia in 19 biliary atresia patients, and some monocytes in most of the biliary atresia patients. Apoptotic nuclei were present among monocytes in all the biliary atresia livers but present among bile ductule epithelia only on the BA with positive FasL mRNA signals on ductule epithelium. The fibrosis grade was similar between biliary atresia with positive FasL mRNA signals and negative signals. The bile drainage was better in the biliary atresia without positive FasL mRNA signals. CONCLUSIONS: Fas ligand expression on bile ductule epithelia in biliary atresia may be induced to counterattack the infiltrating lymphocytes. Although the factors for post-Kasai bile drainage are multiple, the authors suggest Fas ligand expression on bile ductule epithelia may be a poor prognostic factor by playing a role in the continuous damage and obliteration of intrahepatic bile ducts after Kasai operation.

Tumor necrosis factor gene polymorphism and septic shock in surgical infection.

OBJECTIVES: To evaluate the relationship of the genotype distribution of the tumor necrosis factor (TNF)-alpha polymorphism with regard to the plasma TNF-alpha concentration and the development of septic shock as well as mortality of infected patients in a surgical intensive care unit (SICU). DESIGN: A total of 112 postoperative critically ill infected patients were prospectively enrolled. SETTING: SICU of a tertiary university-affiliated medical center. PATIENTS: Patients who were consecutively admitted to the SICU because of surgical infection with sepsis. INTERVENTION: Blood sampling. MEASUREMENTS AND MAIN RESULTS: Blood sample was obtained 24 hrs after intensive care unit (ICU) admission or within 2 hrs after the onset of septic shock to determine the plasma TNF-alpha level and to analyze the genotype of the biallelic polymorphism of the TNF-alpha. RESULTS: The allele frequency of the TNF2 in our infected ICU patients was 12%. Forty-two (37.5%) patients admitted fulfilled the criteria of septic shock during their ICU stay. Patients carrying the TNF2 allele were not more likely to develop septic shock, nor did they have a higher mortality rate. In the patients with septic shock, those carrying the TNF2 allele had a significantly higher mortality rate than those with the homozygous TNF1 genotype (92% vs. 62%, p < .05). In those who developed septic shock, the TNF2 allele was significantly associated with higher TNF levels. CONCLUSION: In patients admitted to SICU with surgical infection, the frequency of TNF2 allele was higher than in the general population. SICU patients with TNF2 allele did not show a higher incidence of developing septic shock, nor was there a higher baseline TNF-alpha level after infection. However, once septic shock had developed, the mortality rate was higher in those patients carrying the TNF2 allele.

In vitro relaxation of rabbit and human internal anal sphincter by rutaecarpine, an alkaloid isolated from Evodia rutaecarpa.

Rutaecarpine, a compound extracted from the Chinese medicinal herb Evodia rutaecarpa, has been shown to possess relaxing action on vascular smooth muscle from rat thoracic aorta. The internal anal sphincter is a specialized smooth muscle regulating important anorectal physiology. To investigate the effect and underlying mechanisms of rutaecarpine on internal anal sphincter, muscle strips from rabbit internal anal sphincter were used. The results showed that rutaecarpine (1 x 10(-10) M to 1 x 10(-4) M) produced a concentration-dependent muscular relaxation effect in our preparations, which were precontracted with acetylcholine. This muscular relaxation effect was not affected by treatment with L-N(G)-nitro-arginine methyl ester (a nitric oxide synthase inhibitor), methylene blue (a guanylate cyclase inhibitor), N-ethylmaleimide (an adenylate cyclase inhibitor), or by removal of the mucosa and submucosa tissue. Pretreatment with nifedipine (a calcium channel blocker) or extracellular Ca+2 removal by ethylenediaminetetraacetic acid (EDTA) greatly attenuated the relaxation effect, suggesting that calcium ion might be involved. In experiments using strips from human internal anal sphincter, an even more prominent relaxation effect was shown. It is thus concluded that rutaecarpine caused relaxation on internal anal sphincter from rabbits and human subjects. The relaxation action was not related to NO-cGMP pathway, instead calcium ion might play an important role and shed insight into clinical implications for those anorectal disorders with hyperactive anal tone.

Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide.

PURPOSE: A somatoanal reflex had been demonstrated in our previous work. Because nitric oxide plays an important role in mediating relaxation of the internal anal sphincter, our purpose was to examine whether and how local somatothermal stimulation inhibits the function of the internal anal sphincter by stimulating nitric oxide release via nitrergic neurons and to elucidate the possible mechanism. METHODS: The activity of the internal anal sphincter in anesthetized rabbits was measured by use of continuously perfused, open-tip manometric methods. Local somatothermal stimulation was achieved by applying an electroheating rod 1 cm away from the skin area at the right popliteal region. The responses were further manipulated by pre-treating the rabbits with agonists or antagonists linked to nitric oxide synthesis. RESULTS: The motility of the internal anal sphincter before and during local somatothermal stimulation was significantly different (tonic pressure (mean +/-standard error of the mean), 5.4 +/- 0.3 vs. 4.9 +/- 0.3 mmHg, P = 0.0195; phasic pressure, 3.9 +/- 0.6 vs. 2.9 +/- 0.4 mmHg, P = 0.0002; frequency distribution of the phasic contractions (peak-to-peak interval), 28.9 +/- 3.7 vs. 65.3 +/- 10.4 seconds, P = 0.0001). The response began at approximately one minute after local somatothermal stimulation when the skin temperature was 41 +/- 0.3 degrees C. No anal response was observed when local somatothermal stimulation was applied at the control area. The local somatothermal stimulation-induced internal anal sphincter relaxation was not inhibited by pretreatment with atropine, propranolol, or phentolamine (tonic pressure, 5.8 +/- 1 vs. 5.2 +/- 0.8 mmHg, P = 0.038; phasic pressure, 4.2 +/- 0.9 vs. 3.1 +/- 0.6 mmHg, P = 0.020; peak-to-peak interval, 27.2 +/- 4.3 vs. 52.9 +/- 14.5 seconds, P = 0.043) but was completely blocked by pretreatment with a nitric oxide synthesis inhibitor. The effect of the nitric oxide synthesis inhibitor could be reversed by pretreatment with L-arginine (tonic pressure, 6 +/- 0.7 vs. 5.6 +/- 0.7 mmHg, P = 0.047; phasic pressure, 4.7 +/- 0.7 vs. 3.9 +/- 0.5 mmHg, P = 0.048; peak-to-peak interval, 23.8 +/- 3 vs. 33 +/- 3.7 seconds, P = 0.048), but not by D-arginine. CONCLUSION: Local somatothermal stimulation inhibits internal anal sphincter motility through the activation of nonadrenergic noncholinergic neural release of nitric oxide. This procedure may represent a simplified approach for the treatment of anorectal diseases with hypofunction of the L-arginine/nitric oxide pathway. [Key words: Local somatothermal stimulation; Nitric oxide; Internal anal sphincter; Motility; Moxibustion] Jiang J-K, Chiu J-H, Lin J-K. Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide.

Human peritoneal mesothelial cells produce nitric oxide: induction by cytokines.

OBJECTIVE: To investigate the induction of nitric oxide synthase type II (iNOS) in human peritoneal mesothelial cells (HPMC) using cytokines and bacterial lipopolysaccharide (LPS). DESIGN: Confluent monolayers of HPMC were exposed to cytokines [tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), interferon gamma (IFNgamma)] or LPS, individually or in various double and triple combinations, for 24-72 hours. Concentrations of nitrate and nitrite in the media were quantified using the Griess reaction and used as indirect indices of nitric oxide (NO) production. The expression of iNOS was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Neither single cytokines nor LPS was able to induce iNOS mRNA or NO production. Both double combinations of TNFalpha + IFNgamma and IL-1beta + IFNgamma were able to induce iNOS mRNA expression, but only TNFalpha + IFNgamma induced significant NO production. The triple combination of TNFalpha + IFNgamma + IL-1beta induced even more NO production than TNFalpha + IFNgamma. There was no constitutive NO synthase type III (eNOS) expression in HPMC. CONCLUSIONS: Certain combinations of cytokines could stimulate cultured HPMC to produce NO, and HPMC might be a source of intraperitoneal NO production during peritonitis.

Somatic electrical nerve stimulation regulates the motility of sphincter of Oddi in rabbits and cats: evidence for a somatovisceral reflex mediated by cholecystokinin.

Cholecystokinin (CCK) plays an important role in regulating the biliary motility in herbivorous and carnivorous animals. Little is known about how the motility of the sphincter of Oddi (SO) is regulated through a somatic stimulation. It was our aim to test the hypothesis that somatic electrical nerve stimulation (SENS) affects SO motility in animals with different types of SO through CCK-related mechanisms. The activity of SO in anesthetized rabbits and cats was measured by using a continuously perfused open-tip manometric method. SENS was brought about by applying an electric current (2/15 Hz alternatively, 20 min) to two needles positioned near spinal nerves in the 6th and 7th intercostal space in the right midclavicular line. The SO motility before and X min after the start of SENS, designated as pre-SENS and SENS-X respectively, were recorded and saved in a computer equipped with off-line analysis software. The SO activity in rabbits, in terms of phasic contraction pressure and duration of summation peak during SENS were significantly higher than that before SENS. The phasic contraction pressure of pre-SENS, SENS-10, and SENS-16 were 6.83 +/- 0.39 mm Hg, 9.23 +/- 0.83 mm Hg and 10.46 +/- 0.81 mm Hg, respectively (P < 0.03, N = 13). The duration of summation peak in pre-SENS, SENS-10, and SENS-16 were 7.26 +/- 0.41 sec, 10.22 +/- 0.46 sec, and 13.49 +/- 2.31 sec, respectively (P < 0.05, N = 13). The SENS-induced SO hyperactivity was not inhibited by pretreatment with atropine, propranolol, phentolamine, or naloxone, but was blocked by pretreatment with the CCK receptor antagonist, proglumide, and by injection of anti-CCK-8 antibody during SENS in a dose-dependent manner. In contrast, SENS induced an inhibitory SO response in cats. However, in both circumstances, an obvious elevation of plasma CCK level determined by radioimmunoassay was noted after SENS. We conclude that SENS causes secretion of CCK, which in turn affects biliary tract motility in animals with different types of SO. This provides an easily applicable method for those patients who have hyperactive SO function.

Expression of IsK protein mRNA in cultured rat strial marginal cells.

A cell culture system of marginal cells (MC) of the rat stria vascularis was established by the explant method. When grown on plastic dishes, cultured MC showed a polygonal "cobblestone-like" appearance. Dome formation, composed of several hundreds to thousands of cells, occurring after confluence suggested that vectorial transport of ion(s) with accompanying fluid developed in the cultured MC. Transmission electron microscopy demonstrated junctional complexes formed of tight junctions and desmosomes at the upper lateral membranes. The polymerase chain reaction (PCR) product, amplified with primers made from the cDNA reverse transcribed from cultured MC, yielded a distinct band compatible with the expected size of the PCR products amplified from cDNA of positive control groups containing IsK protein, indicating that cultured MC expressed the IsK protein mRNA. The results show that cultured MC can form large domes and express the most characteristic IsK protein, indicating that they maintain their vectorial electrolyte transport function and, possibly, the ability to secrete K+ in this condition.