Mine Kinoshita

Expression profiling of Peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Ecabet sodium, a locally acting antiulcer drug, inhibits urease activity of Helicobacter pylori

In order to clarify the mechanism of the anti-Helicobacter pylori action of ecabet sodium (ecabet), a locally acting antiulcer drug, we evaluated the effects of ecabet on H. pylori urease activity in vitro. H. pylori was cultured and a crude preparation of urease was made. Urea-dependent survival of H. pylori at acid pH was significantly inhibited by ecabet. The urease activity of intact cells and a crude enzyme preparation from H. pylori had two pH optima: pH 4.5-5.0 and 8.0. Ecabet (1-4 mg/ml) concentration dependently inhibited the urease activity of both preparations at pH 5.0, but there was no inhibition at pH 8.0. The enzyme activity was inhibited by ecabet gradually and was not restored by dilution, in contrast to the inhibition elicited by benzohydroxamic acid, a specific and reversible urease inhibitor. These results suggest that irreversible inhibition of H. pylori urease activity contributes to the anti-H. pylori action of ecabet.

Kv7.2–7.5 voltage-gated potassium channel (KCNQ2–5) opener, retigabine, reduces capsaicin-induced visceral pain in mice

K(v)7.2-7.5 voltage-gated potassium channels (KCNQ2-5) are associated with M-current and known to distribute in the nociceptive sensory pathway (e.g., dorsal root ganglia and spinal cord). Opening of these channels leads to cell membrane hyperpolarization that results in decreased neuronal action potentials. Since, KCNQ/M-current is located in the visceral sensory system, we examined the anti-nociceptive effect of the KCNQ opener, retigabine, on visceral pain induced by an intracolonic injection of capsaicin in mice. Intraperitoneal administration of retigabine (1, 3 and 10 mg/kg) dose-dependently suppressed visceral pain behavior (i.e., the number of licking) induced by the capsaicin treatment and prolonged the latency to first licking. These data provide the first evidence that increased KCNQ channel conductance plays an inhibitory role in the visceral pain pathway.

Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis.

Background: Recent advances in fibrosis biology have identified transforming growth factor (TGF)-β type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression. Aims: To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis. Methods: Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson’s trichrome stain, and scored. TGF-β1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry. Results: The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-β1 expression in infiltrating macrophages. With the progression in fibrosis (day 7–14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells. Conclusions: The time-course data of TGF-β1 and R-Smads indicate that progressive enhancement of TGF-β1 signalling via R-Smad is activated in the process of fibrosis progression.

Analysis of change patterns of microcomputed tomography 3-dimensional bone parameters as a high-throughput tool to evaluate antiosteoporotic effects of agents at an early stage of ovariectomy-induced osteoporosis in mice.

Objectives:The purposes of this study were to develop an osteoporosis model in a short period of 2 weeks after ovariectomy in mice and to investigate whether analysis of microcomputed tomography (&mgr;CT) 3-dimensional bone parameters could provide useful information on the mechanism of action of antiosteoporotic agents. Materials and Methods:Mice were ovariectomized (OVX) or sham-operated, and the OVX mice were treated daily with 17β-estradiol (E2), parathyroid hormone (PTH[1-34]), raloxifene, rolipram, or vehicle for 2 weeks. On day 14 post-OVX, the left femur bones were removed and then the distal metaphyseal bone was analyzed by both &mgr;CT and histomorphometry. Results:The trabecular bone volume, thickness, number, and connectivity significantly decreased and the number of osteoclasts increased in OVX mice. Treatment of OVX animals with each of the 4 antiosteoporotic agents significantly increased the bone volume and improved the bone architecture. However, the improvement of trabecular thickness in the rolipram-treated group and that of cortical thickness in the PTH(1-34)-treated group were the most marked, whereas the improvement of connectivity in the rolipram-treated group was the least among the drug-treated groups. These different improving effects of agents on the bone parameters reflect the differential effects of these agents on bone formation and bone resorption. Conclusions:This study demonstrated the feasibility of evaluating the effect of the antiosteoporotic agents within 2 weeks after ovariectomy in mice. The &mgr;CT analysis may serve as a valuable tool, specifically in a high-throughput pharmacological screening test, offering useful information regarding the effects of test compounds on both bone resorption and formation.

A new method for producing urinary bladder hyperactivity using a non-invasive transient intravesical infusion of acetic acid in conscious rats

INTRODUCTION Animal models that closely resemble the pathophysiology of human overactive bladder are important for evaluating novel therapeutics to treat the disorder. We established a non-invasive hyperactive bladder model that is sensitive to anti-muscarinic drugs and without bladder inflammation. METHODS Acetic acid solution was infused into the bladder for 5 min via the urethral orifice without any surgical procedures under isoflurane anaesthesia. After washing the bladder with saline, voiding frequency (VF) and total urine volume were determined for 9 h under conscious conditions. RESULTS Infusion of a 0.5% acetic acid solution caused a significant increase in VF, without influencing total urine volume or inducing significant histopathological inflammatory alterations in the bladder urothelium. Oral administration of oxybutynin (3 and 10 mg/kg) significantly ameliorated increases in VF induced by 0.5% acetic acid. Infusion of 0.75% acetic acid induced intensive urinary inflammation and a decrease in total urine volume as well as an increase in VF. Oral treatment with oxybutynin (10 mg/kg) did not significantly improve the increased VF due to 0.75% acetic acid. Acetic acid (0.5%) infusion evoked bladder hyper-responsiveness whether applied at night or during the day. However, VF was increased more by the nighttime application of acetic acid, while there were no significant differences in basal levels of VF between daytime and nighttime. DISCUSSION In this study, the non-invasive rat urinary hyperactive bladder model indicated minimizes the secondary effects of experimental procedures such as surgical operations and anesthesia on bladder function and is sensitive to oxybutynin. Thus, the model may be useful for investigating novel therapeutics for OAB treatment.

Role of salivary mucin in the protection of rat esophageal mucosa from acid and pepsin-induced injury

The mucosal defensive mechanisms of the esophagus against acid and pepsin remain to be elucidated. In the present study, we investigated the contribution of the salivary mucin in maintaining the integrity of the esophageal mucosa. When an everted esophageal sac, isolated from normal rat, was treated with N-acetyl-l-cysteine, a mucolytic agent, the amount of glycoprotein in the gel layer adherent to the epithelium was completely depleted and the susceptibility of the mucosa against acidified pepsin-induced digestion increased. In sialoadenectomized rats, 7 days after extirpation, the amount of glycoprotein adherent to the esophageal epithelium was definitely reduced, and the esophageal mucosa was significantly vulnerable to acidified pepsin-induced digestion compared with the sham-operated rats. Induction of regurgitation of the gastric juices into the esophagus resulted in the development of severe hemorrhagic esophageal lesions only in the sialoadenectomized rats but not in the sham-operated rats. In conclusion, the glycoprotein in the adherent gel layer in rat esophagus, which mainly derives from salivary glands, plays an important role in the preepithelial defense to maintain the integrity of the esophageal mucosa against acid and pepsin.

Possible Mechanism of Increase in Gastric Mucosal PGE2 and PGI2 Generation Induced by Ecabet Sodium, a Novel Gastroprotective Agent

The gastroprotective agent ecabet sodium(ecabet, 12-sulfodehydroabietic acid monosodium salt)increases the formation of prostaglandin (PG)E2 and I2 by gastric mucosa. Inthe present study, we examined the effect of ecabet on metabolism ofarachidonic acid (AA) in rat gastric mucosal cells.Ecabet (0.1-10 mM) concentration- and time-dependentlypotentiated the release of [14C]AA fromgastric mucosal cells prelabeled with [14C]AA andsimultaneously increased the production ofPGE2 and PGI2. The ecabet-mediatedincreases in [14C]AA release andPGE2 production were both partly depressed bymepacrine (30 and 100 μM) and Ca2+ chelation.Ecabet, however, showed no effect on gastricphospholipase A2 (PLA2) activityand [Ca2+]i in the gastric mucosalcells. Ecabet and other dehydroabietic acid derivatives, 12-carboxydehydroabietic acid monosodium saltand mono[16-(12-sulfodehydroabietyl)]succinic acidmonosodium salt, which potentiated the liberation of[14C]AA, increased the membrane fluidity ofgastric mucosal cells assessed by usingdiphenylhexatrienepropionic acid (DPH-PA) as the probe,while 12-sulfamoyldehydroabietic acid showed no effecton either the AA liberation or the membrane fluidity.Ecabet (0.1-10 mM) increased the membrane fluidityconcentration- and time-dependently in accordance withits facilitating effect on AA release. In conclusion,ecabet increases the synthesis of PGE2 andPGI2 by gastric mucosal cells through promoting the release ofAA, which is partly dependent on PLA2 andCa2+. The ecabet-induced increase in membranefluidity may be involved in part 2 in the liberation ofAA from the gastric mucosal cells.

Pharmacologically distinctive behaviors other than burying marbles during the marble burying test in mice.

In the marble burying test, we focused on the 5 distinctive behavioral parameters of mice other than burying marbles, i.e. digging, latency to the first digging, exploration around marbles, rearing and locomotor activity. Typical anxiolytics or antidepressants with different mechanisms, fluvoxamine (30 mg/kg, selective serotonin reuptake inhibitor), bupropion (60 mg/kg, noradrenaline and dopamine reuptake inhibitor), imipramine (60 mg/kg, tricyclic antidepressant) and diazepam (10 mg/kg, benzodiazepine) were used to examine whether these behavioral parameters are sensitive to pharmacological treatments. Each of the drugs demonstrated an individual action pattern on the 4 behavioral parameters (latency to the first digging, exploration around marbles, rearing and locomotor activity). On the other hand, all 4 drugs reduced burying marbles and digging, which were correlated with each other. These results suggest that the former 4 behavioral parameters are sensitive to pharmacological treatment and that pharmacological regulation mechanisms of them may be different from burying marbles and digging. They could be useful to identify the type of action of a test drug like selective serotonin reuptake inhibitor, noradrenaline and dopamine reuptake inhibitor, tricyclic antidepressant or benzodiazepine.

Histopathological study of time course changes in inter-renal aortic banding-induced left ventricular hypertrophy of mice

The left ventricular hypertrophy (LVH) in response to pressure overload is an important risk factor in cardiac morbidity and mortality. To investigate the time course of histopathological alterations in the LVH in response to pressure overload, histopathological and immunohistochemical examination was performed using the aortic banding‐induced mouse LVH model. Five‐week‐old male CD‐1 mice were subjected to the inter‐renal aortic banding. Major organs were sampled on 3, 10, 14, 21, 28 or 42 days after banding. Haematoxylin and eosin (H&E) staining, Massons trichrome staining and immunohistochemistry for proliferating cell nuclear antigen (PCNA), alpha‐smooth muscle actin (aSMA), ICAM‐1, type I collagen and CD31 was performed and microscopically examined. Three days after aortic banding, acute inflammatory changes, such as macrophages/neutrophil infiltration and vascular wall injury were observed on/around the coronary arteries/arterioles of both ventricles. Intense ICAM‐1 immunostaining was observed on the endothelium of the coronary arteries/arterioles. After day 10, vascular wall thickening and perivascular fibrosis was induced on the coronary arteries/arterioles. Immunohistochemistry for aSMA and PCNA demonstrated the proliferation of vascular smooth muscle cells in the media. After day 28, minimal cardiomyocyte hypertrophy was observed at the light microscope level. In the inter‐renal aortic banding LVH model, histopathological alterations in early phase were mainly observed on coronary arteries/arterioles. These early phase alterations were thought to be hypertension‐related changes in the coronary vasculatures. The cardiomyocyte hypertrophy observed in later phase was minimal at the light microscope level. These evidences would facilitate the understanding of pathophysiology of pressure overload LVH.