Approach for Elucidating the Molecular Mechanism of Epithelial to Mesenchymal Transition in Fibrosis of Asthmatic Airway Remodeling Focusing on Cl- Channels.

Airway remodeling caused by asthma is characterized by structural changes of subepithelial fibrosis, goblet cell metaplasia, submucosal gland hyperplasia, smooth muscle cell hyperplasia, and angiogenesis, leading to symptoms such as dyspnea, which cause marked quality of life deterioration. In particular, fibrosis exacerbated by asthma progression is reportedly mediated by epithelial-mesenchymal transition (EMT). It is well known that the molecular mechanism of EMT in fibrosis of asthmatic airway remodeling is closely associated with several signaling pathways, including the TGF-ß1/Smad, TGF-ß1/non-Smad, and Wnt/ß-catenin signaling pathways. However, the molecular mechanism of EMT in fibrosis of asthmatic airway remodeling has not yet been fully clarified. Given that Cl- transport through Cl- channels causes passive water flow and consequent changes in cell volume, these channels may be considered to play a key role in EMT, which is characterized by significant morphological changes. In the present article, we highlight how EMT, which causes fibrosis and carcinogenesis in various tissues, is strongly associated with activation or inactivation of Cl- channels and discuss whether Cl- channels can lead to elucidation of the molecular mechanism of EMT in fibrosis of asthmatic airway remodeling.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and ß-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

Cesium Treatment Depresses Glycolysis Pathway in HeLa Cell.

BACKGROUND/AIMS: Cesium (Cs) is an alkali metal element that is of no essential use for humans; it has no known beneficial function that is verified by clinical research. When used as an alternative cancer therapy, it even causes toxicity in high doses. Thus, before using Cs as treatment in clinical settings, it is important to clearly determine its biological effects on cells. However, Cs was found to suppress the proliferation of human cervical cancer cells in a dose-dependent manner, and it was assumed that Cs inhibits the glycolysis pathway. In this study, we clearly determined the step of the glycolysis pathway that is affected by Cs. METHODS: The glycolytic enzyme expressions, activities, and metabolite concentrations in HeLa cells were measured by PCR, western blotting, and enzymatic methods, after treating the cells with Cs for 3 days. RESULTS: Cs treatment decreased transcriptional and expression levels of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase (PK), and lactate dehydrogenase and the activity of PK. Analysis of glycolysis pathway metabolites revealed that Cs treatment reduces lactate level and increases the level of nicotinamide adenine dinucleotide (oxidized form, NAD+); however, it did not affect the levels of pyruvate and nicotinamide adenine dinucleotide (reduced form, NADH). Increase of the [NAD+]/[NADH] ratio and decrease of the [lactate]/[pyruvate] ratio indicate that Cs treatment inhibits the aerobic glycolysis pathway. CONCLUSION: Cs treatment inhibits PK activity and increases the [NAD+]/[NADH] ratio. Hence, Cs has been determined to inhibit glycolysis, especially the aerobic glycolysis pathway. These results suggest that suppression of HeLa cell proliferation following Cs treatment was caused by inhibition of aerobic glycolysis by Cs.

Dysfunction of Cl- channels promotes epithelial to mesenchymal transition in oral squamous cell carcinoma via activation of Wnt/ß-catenin signaling pathway.

Oral squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a high incidence of recurrence and distant metastasis. However, the mechanism of epithelial to mesenchymal transition (EMT) during tumor progression and metastasis in OSCC has not yet been fully elucidated. It is well known that the Cl- channel controls cell volume and activates several signaling pathways for cell differentiation. The aim of the present study was to investigate the role of the Cl- channel on EMT in the OSC 20 cell line, which is an OSCC line. OSC-20 cells were cultured with low serum medium containing a Cl- channel blocker NPPB. Morphological changes, gene expression, immunoreactivity, cell volume, and signaling pathway of the NPPB-treated OSC-20 cells were evaluated. The NPPB-treated OSC-20 cells showed typical morphology of mesenchymal cells. The expression levels of the epithelial marker E-cadherin in the NPPB-treated OSC-20 cells were lower than those of the untreated and TGF-ß1-treated OSC-20 cells. On the other hand, mesenchymal markers such as vimentin, ZEB1, and Snail, in the NPPB-treated OSC-20 cells were higher than those in the untreated and TGF-ß1-treated OSC-20 cells. Furthermore, a large number of vimentin-positive cells also appeared in the NPPB-treated OSC-20 cells. Additionally, the cell volume of these cells was significantly increased compared to that of the untreated and TGF-ß1-treated cells. Interestingly, NPPB did not activate the TGF-ß/smad signaling pathway, but activated the Wnt/ß-catenin signaling pathway. These results suggest that Cl- channel dysfunction promoted EMT via activation of the Wnt/ß-catenin signaling pathway in OSCC.

Cl- channels regulate lipid droplet formation via Rab8a expression during adipocyte differentiation.

Several studies have shown that Cl- channels regulate the differentiation of some cell types. Thus, we investigated the role of Cl- channels on adipocyte differentiation using adipose tissue-derived stem cells (ASCs) and Cl- channel blocker. We induced rabbit ASCs into adipocytes using Cl- channel blocker. The expression levels of adipocyte markers were no significant difference between the cells treated with a Cl- channel blocker NPPB and untreated cells. However, when the cells were treated with NPPB, lipid droplets (LDs) sizes decreased compared with the untreated control. Interestingly, the expression levels of Rab8a, which is known as a regulator of LD fusion, were also decreased in the cells treated with NPPB. Other Cl- channel blockers, DIDS and IAA-94, also inhibited large LDs formation and Rab8a expression. These results demonstrate that Cl- channels do not regulate the adipocyte differentiation, but do regulate the LDs formation via Rab8a expression.Abbreviations: ASCs: adipose tissue-derived stem cells; LDs: lipid droplets; RUNX2: runt-related transcription factor 2; CFTR: cystic fibrosis transmembrane conductance regulator; TG: triacylglycerol; FA: fatty acid; GLUT4: glucose transporter type 4; ER: endoplasmic reticulum; ADRP: adipose differentiation-related protein; TIP47: tail-interacting protein of 47 kD; HSL: hormone sensitive lipase; PBS: phosphate-buffered saline; DMEM: Dulbecco's modified Eagle Medium; FBS: fetal bovine serum; SMA: smooth muscle actin; FAS: fatty acid synthase; ZONAB: ZO-1 associated nucleic acid binding protein; PPAR-γ: peroxisome proliferator-activated receptor-γ; C/EBPα: CCAAT/enhancer binding protein α; CE: cholesteryl ester; V-ATPase: vacuolar H+ ATPase.

Functional characterization of various channel-expressing central airway epithelial cells from mouse induced pluripotent stem cells.

Functional central airway epithelial cells (CAECs) from induced pluripotent stem cells (iPSCs) are an attractive potential cell source for central airway regeneration. The central airway epithelium, such as the tracheal epithelium, is composed of ciliated cells, goblet cells, and basal cells and has physiologically important functions such as the regulation of water volume on the airway surface by Cl- and water channels and the elimination of particles inhaled from the external environment by ciliary movement. Previous work from our group and from other research groups has reported the generation of airway epithelial cells from iPSCs. However, it remains unclear whether iPSC-derived CAECs express the various channels that are required for the regulation of water volume on the airway surface and whether these channels function properly. In this study, we generated CAECs from iPSCs supplemented with activin and bFGF using air-liquid interface culture. We then evaluated the physiological functioning of the iPSC-derived CAECs by examining the gene expression and transport functions of Cl - channels using a halide ion-sensitive yellow fluorescent protein and ciliary movement. Reverse-transcription polymerase chain reaction and immunohistochemistry indicated that various channel markers such as cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin (AQP) were present in iPSC-derived CAECs. Furthermore, the transport functions of Cl - channels and CFTR were successfully confirmed. Finally, ciliary movement was measured, and a ciliary beating frequency (CBF) of approximately 10 Hz was observed. These results demonstrate that CAECs generated by our method have physiological functions similar to those of native CAECs.

Carrageenan delays cell cycle progression in human cancer cells in vitro demonstrated by FUCCI imaging.

BACKGROUND: Carrageenan is a sulfated polysaccharide that exists in red seaweeds recently shown to have anticancer properties. Previous findings show various effects of carrageenan suppressing tumor cell growth. One of the hallmarks of cancer is uncontrolled proliferation, a consequence of loss of normal cell-cycle control, that underlies tumor growth. Recently there is an increasing interest in potential anticancer agents that affect cell cycle in cancer cells. Thus, in this study we investigated the effects of carrageenan on the tumor cell cycle. METHODS: Using human cervical carcinoma cells (HeLa) cells as and human umbilical vein endothelial cells (HUVEC), the cytotoxic effects of kappa carrageenan (k-CO) and lambda carrageenan (λ-CO) at the concentrations of 250-2500 µg/mL were observed. Cell viability was determined using the MTT assay while cell death rates were determined using staining with calcein-AM/propidium iodide. Cell-cycle profile and progression were demonstrated with HeLa cells expressing FUCCI (fluorescence ubiquitination-based cell-cycle indicator) probes (HeLa-FUCCI). RESULTS: Carrageenan had no significant effect on HUVEC (normal cells). In contrast both forms of carrageenan were cytotoxic towards HeLa cells (cancer cells). Furthermore, according to cell-cycle analysis with FUCCI cells, the cell cycle of HeLa cells was delayed in specific phases due to different carrageenan treatments. CONCLUSION: Considering these results, it could be suggested that carrageenan affects the cell-cycle of HeLa cells not only by arresting the cell cycle in specific phases but also by delaying the time needed for the cell to progress through the cell cycle. Additionally, different types of carrageenans have different effects on cell cycle progression. This effect of carrageenan towards cancer cells could possibly be developed into a tumor cell-specific anticancer agent.

Povidone-iodine-induced cell death in cultured human epithelial HeLa cells and rat oral mucosal tissue.

Although povidone-iodine (PVP-I) has been used as a gargle since 1956, its effectiveness and material safety have been remained controversial. The aim of this study was to investigate the toxicity of PVP-I to epithelial cells in a concentration range significantly lower than that used clinically. Study design was in vitro laboratory investigations and in vivo histological and immunologic analysis. We examined the effects of PVP-I at concentrations of 1 × 10(-2) to 1 × 10(3) µM and 1 × 10(-4) to 1 × 10 µM on HeLa cells as a model of epithelial cells and rat oral mucosa, respectively, after 1 or 2 days of exposure. Annexin V/FLUOS was used to distinguish live, apoptotic and necrotic cells. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was also used to observe whether apoptotic epithelial cells exist in rat oral mucosa after 1 day of exposure of PVP-I. HeLa cells developed concentration-dependent cytotoxicity, and epithelium of rat oral mucosa was thinned in a concentration-dependent manner. HeLa cell apoptosis increased after 1 × 10(0) µM of PVP-I exposure for 2 days. In the TUNEL method, many apoptotic epithelial cells were observed in the rat oral mucosa after 1 day of exposure to diluted 1 × 10(-2) µM of PVP-I, but minimal apoptotic epithelial cells were observed using 1 × 10(-3) µM of PVP-I. Our findings suggest that exposure to PVP-I, of which concentrations are even lower than those used clinically, causes toxicity in epithelial cells. This knowledge would help us better understand the risk of the use of PVP-I against mucosa.

Arterial stiffness of the aorta and iliofemoral artery and their responses to nitroglycerin administration in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits.

OBJECTIVES: The role of hypercholesterolemia in arterial stiffness, which usually reflects the progression of atherosclerosis has not been fully investigated. To clarify the meaning of arterial stiffness in hypercholesterolemia, we evaluated arterial stiffness in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits by using new arterial stiffness indices of the aorta and common iliac to femoral artery. The new arterial stiffness indices of both arteries were determined by the application of the theory of cardio-ankle vascular index (CAVI) to the aorta (aBeta) and ilio-femoral artery (ifBeta). Furthermore, the responses of both indices to nitroglycerin (NTG) administration were compared between WHHHMI and normal rabbits. DESIGN AND METHODS: aBeta and ifBeta of WHHLMI and normal rabbits were measured under anesthesia. Pulse wave velocity in the whole aorta (aPWV) and ilio-femoral artery (ifPWV), blood pressure, and other parameters were measured before and after administration of NTG (50-120 µg/kg/min) every 1 for 5 min. RESULTS: Atherosclerotic lesions were observed in the aorta, but a little in the ilio-femoral artery in WHHLMI rabbits. Compared with normal rabbits, aBeta was significantly higher, but ifBeta was lower in WHHLMI rabbits. When NTG was administered, ifBeta decreased significantly in both groups; however, aBeta increased in normal rabbits, but remained unchanged in WHHIMI rabbits. CONCLUSION: These findings suggested that hereditary hypercholesterolemia in rabbits did not uniformly enhance arterial stiffness in elastic artery and muscular artery. The responses to NTG were also different between two arteries. The mechanism of these different responses needs further studies.

Reducing radiation exposure using commonly available objects.

OBJECTIVES: One and a half years have passed since the Fukushima Daiichi nuclear power plant disaster. The environmental radiation dose rate was not critical, but an existing exposure situation has been identified in a large part of Fukushima Prefecture. Although people continue to live and work in the contaminated area, they are not provided with sufficient information to reduce their exposure to radiation by themselves. In this study, we attempt to evaluate the efficiency of radiation shielding by using everyday items widely available to people. METHODS: NaI scintillation and Geiger-Müller survey meters were used to measure the radiation dose of (1) contaminated soil and (2) soil covered with commonly available items. RESULTS: In the soil at a depth of 10 cm from the surface, the radiation dose rate decreased from 3.36 to 0.65 µSv/h, and the count rate decreased from 3,120 to 352 cpm. Both the radiation dose rate and count rate reduced when the soil was covered with everyday items, such as a magazine more than 20 mm thick, a polystyrene foam board, and a wooden board of the same thickness. CONCLUSIONS: To protect residents from unnecessary radiation exposure in the existing exposure situation, covering contaminated soil with a wooden board or a magazine, either of them 20 mm thick, is useful to reduce the radiation dose.

Estimation of Central Systolic Blood Pressure from Peripheral Pressure Waves using a Novel Second Systolic Pressure-Based Method in Normal and Heritable Hypercholesterolemic Rabbits.

AIM: Central systolic blood pressure (cSBP) was closely related to hypertension-related organ damage rather than peripheral systolic blood pressure (pSBP). We aimed to estimate cSBP from pSBP without generalized transfer function in normal and Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits aged 12 months. METHODS: Two catheter-tip transducers were advanced into the ascending aorta (AA) and distal end of the right brachial artery (Br) through the right common carotid and right radial arteries, respectively, under pentobarbital anesthesia. Pressure waves in response to the intravenous administration of angiotensin II and sodium nitroprusside were simultaneously recorded in AA and Br under regular cardiac pacing. RESULTS: The first (pSBP) and second peaks (pSBP2) of the brachial blood pressure and their average (pSBPm) were significantly correlated with cSBP, despite Murgo's wave pattern of central pressure waves in both rabbit groups. In Bland-Altman plot and its modification as a function of the peripheral augmentation index (pAI) analyses, the differences between pSBP and cSBP decreased, and those between pSBP2 and cSBP increased significantly in their average- or pAI-dependent manner, with undeniable mean biases in both rabbit groups. When the same analyses for SBPm were performed instead, the mean bias was around zero, with reduced variance in the two rabbit groups. The observed pressure or pAI-dependent systematic biases for pSBP and pSBP2 disappeared, representing the precise feature of pSBPm as a cSBP estimate. CONCLUSIONS: We conclude that pSBPm could be more precise than pSBP2 as a cSBP estimate, irrespective of blood pressure levels, pAI, or the presence of atherosclerosis.

Aquaporin AqpZ is involved in cell volume regulation and sensitivity to osmotic stress in Synechocystis sp. strain PCC 6803.

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.

Plasma membrane aquaporin AqpZ protein is essential for glucose metabolism during photomixotrophic growth of Synechocystis sp. PCC 6803.

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism.

An assessment of radiation doses at an educational institution 57.8 km away from the Fukushima Daiichi nuclear power plant 1 month after the nuclear accident.

OBJECTIVES: On 11 March 2011, the Great East Japan Earthquake occurred. Due to this earthquake and subsequent tsunami, malfunctions occurred at the Fukushima Daiichi nuclear power plant. Radioactive material even reached the investigated educational institution despite being 57.8 km away from the power station. With the goal of ensuring the safety of our students, we decided to carry out a risk assessment of the premises of this educational institution by measuring radiation doses at certain locations, making it possible to calculate estimated radiation accumulation. METHODS: Systematic sampling was carried out at measurement points spaced at regular intervals for a total of 24 indoor and outdoor areas, with 137 measurements at heights of 1 cm and 100 cm above the ground surface. Radiation survey meters were used to measure environmental radiation doses. RESULTS: Radiation dose rates and count rates were higher outdoors than indoors, and higher 1 cm above the ground surface than at 100 cm. Radiation doses 1 cm above the ground surface were higher on grass and moss than on asphalt and soil. The estimated radiation exposure for a student spending an average of 11 h on site at this educational institution was 9.80 µSv. CONCLUSIONS: Environmental radiation doses at our educational institution 57.8 km away from the Fukushima Daiichi nuclear power plant 1 month after the accident were lower than the national regulation dose for schools (3.8 µSv/h) at most points. Differences in radiation doses depending on outdoor surface properties are important to note for risk reduction.

Opposing Responses of the Calcium Channel Blocker Nicardipine to Vascular Stiffness in the Elastic and Muscular Arteries in Rabbits.

AIM: The cardio-ankle vascular index (CAVI) consists of intrinsic and functional arterial stiffness mainly regulated by vasoactive compounds. A new stiffness index of the aorta (aBeta) and iliac-femoral arteries (ifBeta) was determined by applying the CAVI theory to the whole aorta and iliac-femoral arteries. We investigated the changes in aBeta and ifBeta in response to decreased blood pressure (BP) induced by the Ca2＋ channel blocker nicardipine to elucidate the involvement of Ca2＋ in aBeta and ifBeta. METHODS: Pressure waves at the origin of the aorta (oA), distal end of the abdominal aorta (dA), and left femoral artery (fA) as well as flow waves at the oA were simultaneously recorded before and after the infusion of nicardipine (50 µg/kg/min) for 2 min in 12 male rabbits under pentobarbital anesthesia. Beta was calculated using the following formula: Beta=2ρ / PP×ln (SBP / DBP)×PWV2, where ρ, SBP, DBP, and PP denote blood density and systolic, diastolic, and pulse pressures, respectively. aBeta, ifBeta, and aortic-iliac-femoral Beta (aifBeta) were calculated using aPWV, ifPWV, and aifPWV, respectively. RESULTS: SBP, mean arterial pressure (MAP), DBP, and total peripheral vascular resistance significantly decreased during the administration of nicardipine, whereas cardiac output significantly increased. aBeta and ifBeta significantly increased and decreased, respectively, whereas aifBeta did not change despite the decrease in BP. ifBeta and aBeta positively and negatively correlated with BP, respectively, whereas aifBeta did not correlate with SBP. CONCLUSIONS: There were contradictory arterial responses to nicardipine between the elastic and muscular arteries. Unknown vasoconstriction mechanisms that are not involved in Ca2＋ influx may function in the aorta in response to decreased BP.

Different Responses of Arterial Stiffness between the Aorta and the Iliofemoral Artery during the Administration of Phentolamine and Atenolol in Rabbits.

AIM: The mechanism underlying the stiffness of the aorta and iliofemoral artery that is required to maintain blood pressure (BP) is unclear. A new stiffness index of the aorta (aBeta) and iliac-femoral arteries (ifBeta) was defined by applying the cardio-ankle vascular index (CAVI). We compared changes in stiffness of the two arteries in response to reduced BP, due to the non-selective α adrenergic blocker phentolamine and the ß1 adrenergic blocker atenolol, in rabbits. METHODS: Pressure waves at the origin (oA) and distal ends of the aorta (dA) and the distal end of the left femoral artery (fA) were recorded simultaneously using three pressure sensors in 25 anesthetized rabbits. Phentolamine (50 µg/kg/min) and atenolol (10 mg/kg/min) were infused for 2 min. The pulse wave velocity (PWV) in each artery was determined; aBeta, ifBeta, and whole Beta (aifBeta) were calculated by the following formula; Beta=2ρ/PP×ln(SBP/DBP)×PWV2 (ρ: blood density; SBP, SBP, and PP: systolic, diastolic, and pulse pressures, respectively). RESULTS: SBP and DBP at oA, dA, and fA decreased by the administration of phentolamine and atenolol, with and without decreased total peripheral vascular resistance. After phentramine infusion, cardiac output (CO), aBeta, and aifBeta increased, while ifBeta decreased. After infusion of atenolol, CO decreased, while aBeta, ifBeta, and aifBeta remained unchanged. CONCLUSION: The contradictory reactions of aBeta and ifBeta to phentolamine suggest that the stiffness of the aorta and ilio-femoral artery is regulated separately during decreased BP induced by phentolamine, but not by atenolol.

The deletion of glucagon-like peptide-1 receptors expressing neurons in the dorsomedial hypothalamic nucleus disrupts the diurnal feeding pattern and induces hyperphagia and obesity.

BACKGROUND: Feeding rhythm disruption contributes to the development of obesity. The receptors of glucagon-like peptide-1 (GLP-1) are distributed in the wide regions of the brain. Among these regions, GLP-1 receptors (GLP-1R) are expressed in the dorsomedial hypothalamic nucleus (DMH) which are known to be associated with thermogenesis and circadian rhythm development. However, the physiological roles of GLP-1R expressing neurons in the DMH remain elusive. METHODS: To examine the physiological role of GLP-1R expressing neurons in the DMH, saporin-conjugated exenatide4 was injected into rat brain DMH to delete GLP-1R-positive neurons. Subsequently, locomotor activity, diurnal feeding pattern, amount of food intake and body weight were measured. RESULTS: This deletion of GLP-1R-positive neurons in the DMH induced hyperphagia, the disruption of diurnal feeding pattern, and obesity. The deletion of GLP-1R expressing neurons also reduced glutamic acid decarboxylase 67 and cholecystokinin A receptor mRNA levels in the DMH. Also, it reduced the c-fos expression after refeeding in the suprachiasmatic nucleus (SCN). Thirty percent of DMH neurons projecting to the SCN expressed GLP-1R. Functionally, refeeding after fasting induced c-fos expression in the SCN projecting neurons in the DMH. As for the projection to the DMH, neurons in the nucleus tractus solitarius (NTS) were found to be projecting to the DMH, with 33% of those neurons being GLP-1-positive. Refeeding induced c-fos expression in the DMH projecting neurons in the NTS. CONCLUSION: These findings suggest that GLP-1R expressing neurons in the DMH may mediate feeding termination. In addition, this meal signal may be transmitted to SCN neurons and change the neural activities.

The effect of topical amiloride eye drops on tear quantity in rabbits.

PURPOSE: To investigate the presence of epithelial sodium channels (ENaC) in rabbit and human conjunctival epithelium and to test the effects of topical amiloride, a potassium-sparing diuretic that blocks the ENaC, on tear quantity in rabbits. METHODS: Both healthy normal human and rabbit conjunctival tissues underwent immunohistochemistry staining for ENaC-α and γ subunits as well as for reverse transcription-polymerase chain reaction (RT-PCR) for detection of ENaC-α and ENaC-γ subunit mRNA expression. Rabbits were instilled topical amiloride eye drops and tear function tests were performed before and after instillations. RESULTS: Immunohistochemical staining for ENaC-α subunit in all rabbit eyes showed positive staining in apical and basal conjunctival epithelial cells. Human conjunctival epithelia revealed positive staining with ENaC-α antibody especially in the apical and basal layers. Immunohistochemistry staining with ENaC- γ antibody also revealed positive staining of the conjunctival epithelial cells especially in the basal layers. The ENaC-α mRNA was detected in samples from healthy white rabbit conjunctival epithelia, and ENaC-α and ENaC- γ mRNAs were detected in samples from healthy human conjunctival epithelia. The mean tear quantity showed a significant increase at 15 and 30 min compared to the pre-instillation value in eyes assigned to amiloride eye drops. The mean tear quantity at 15 and 30 min was significantly higher in the amiloride group compared to the control eyes. CONCLUSIONS: Topical amiloride application appears to increase the quantity of preocular tears owing to inhibition of conjunctival epithelial sodium channels.

Cesium suppresses fibroblast proliferation and migration.

During wound healing, fibroblasts proliferate from the margin, and migrate into the provisional matrix where they differentiate into myofibroblasts resulting in wound contraction; however, fibroblasts are hyperproliferative during chronic tissue damage. We previously reported that cesium chloride inhibited a human cancer cell proliferation; therefore, cesium is also presumed to suppress fibroblast proliferation. We here investigated the effects of cesium chloride on the proliferation and migration of murine embryotic fibroblast cells, NIH/3T3 cells. Cultured NIH/3T3 cells with 0-10 mM sodium and cesium chloride were counted using trypan blue dye-exclusion method, then cell growth and viability were evaluated. The percentage of wound closure was calculated by scratch assay. The number of the cells was decreased by application of 1-10 mM cesium in a dose-dependent manner, whereas the viability of the cells was unchanged. The treatment with 3-10 mM cesium inhibited the proliferation rate and % of wound closure compared with controls. These results suggested that cesium inhibits the proliferation and migration of fibroblast cells. This study indicates a possible therapeutic role of cesium chloride in the treatment of wound healing and fibrosis.

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR.

BACKGROUND: Lubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs). METHODS: We induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses. RESULTS: Gene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor. CONCLUSION: These results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.