Bezafibrate improves bacterial lipopolysaccharide-induced dyslipidemia and anorexia in rats.

Bacterial endotoxin/lipopolysaccharide (LPS)-induced cachexia is characterized by weight loss, anorexia, and a disturbance in lipid metabolism, namely, hypertriacylglycerolemia. The aim of this study in rats with acute endotoxicity induced by an injection of LPS was to investigate whether bezafibrate, a ligand for peroxisome proliferator-activated receptor alpha and a lipoprotein lipase (LPL) activator, improved cachectic conditions, including impaired lipid metabolism. Short-term administration of LPS in the rats resulted in impairment of triacylglycerol clearance in plasma after the intake of fresh cream. In addition, LPS increased whole-body energy expenditure, reduced fasting body weight and caused anorexia in the rats. Bezafibrate treatment resulted in significant improvements in LPS-induced dyslipidemia and anorexia, but had no effect on energy expenditure, respiratory quotient, or fasting body weight in the endotoxic rats. Administration of LPS was also associated with a decrease in the level of messenger RNA (mRNA) expression for LPL in white adipose tissue and skeletal muscle and an increase in the mRNA levels for uncoupling protein 3 in skeletal muscle. Bezafibrate treatment reversed the decline in LPL mRNA levels in white adipose tissue but not in the skeletal muscle tissue of the rats. The enhanced uncoupling protein 3 mRNA level in the endotoxic rats was not affected by bezafibrate treatment. Plasma concentration of leptin was increased by short-term LPS treatment. Bezafibrate decreased the level of plasma leptin significantly without affecting the level of leptin mRNA expression. These results suggest that bezafibrate may be an effective drug not only for impaired triacylglycerol metabolism, but also for anorexia in cachectic states induced by bacterial infections.


Transcription factors Csx/Nkx2.5 and GATA4 distinctly regulate expression of Ca2+ channels in neonatal rat heart.

The cardiac transcription factors Csx/Nkx2.5 and GATA4 play important roles in vertebrate heart development. Although mutations of Csx/Nkx2.5 or GATA4 are associated with various congenital heart diseases, their mechanism of action on cardiomyocyte function is not completely elucidated. In this study, we therefore investigated the actions of these transcription factors on the electrophysiological features and expression of ion channels in cardiomyocytes. Genes for transcription factors Csx/Nkx2.5 and GATA4 were transfected into rat neonatal cardiomyocytes by adenoviral infection. Action potentials, L-, T-type Ca(2+) channels and hyperpolarization-activated cation current (I(h)) of rat neonatal myocytes were recorded by patch clamp technique after adenoviral infection. Expression of ion channels was confirmed by real-time PCR. In Csx/Nkx2.5 overexpression myocytes, the spontaneous beating rate was markedly increased with an up-regulation of the Ca(v)3.2 T-type Ca(2+) channel, while in GATA4 overexpression myocytes, the T-type Ca(2+) channel was unchanged. On the other hand, the L-type Ca(2+) channel was down-regulated by both Csx/Nkx2.5 and GATA4 overexpression; the level of Ca(v)1.3 mRNA was dramatically decreased by Csx/Nkx2.5 overexpression. These results indicate that Csx/Nkx2.5 and GATA4 play important roles on the generation of pacemaker potentials modulating voltage-dependent Ca(2+) channels in the neonatal cardiomyocyte.

Journal of Molecular and Cellular Cardiology


Modulation of Pathogenicity with Norepinephrine Related to the Type III Secretion System of Vibrio parahaemolyticus.

Norepinephrine (NE) controls the functions of the gastrointestinal tract, but its role in the pathogenicity of enteropathogens is not clear. We examined the effect of NE on the pathogenicity of Vibrio parahaemolyticus with regard to its type III secretion systems (TTSSs). To evaluate the effect of NE on pathogenicity of V. parahaemolyticus, we examined the cytotoxic activity to Caco-2 cells and enterotoxicity by use of the rat ileal loop model. It has been reported that TTSS1 causes cytotoxicity and that TTSS2 causes enterotoxicity in the animal ileal loop model. Our results showed that, although NE alone did not affect the viability of Caco-2 cells, NE stimulated the cytotoxic activity of V. parahaemolyticus. Furthermore, NE increased the transcription of the TTSS1-related genes vscQ and vscU. These results indicate that NE regulates V. parahaemolyticus cytotoxic activity. The enterotoxicity of V. parahaemolyticus was increased by NE through interaction with alpha (1)-adrenergic receptors. These results indicate that alpha (1)-adrenergic receptors on the intestinal epithelium appear to interact with V. parahaemolyticus enterotoxicity. The present findings suggest that enteric NE may modulate V. parahaemolyticus pathogenicity.


Angiotensin II inhibits insulin-induced actin stress fiber formation and glucose uptake via ERK1/2.

There is crosstalk in intracellular signaling between Angiotensin II (Ang II) and insulin. We hypothesized that the underlying mechanism might be related to changes in cytoskeleton. In the presence of 100 nM of Ang II, insulin-induced glucose uptake was decreased and insulin-induced actin filament organization was inhibited. PKC inhibitors, including GF109203x and p38MAPK inhibitor (SB203580) neither improved insulin-induced actin reorganization nor glucose uptake. In contrast, the Ang II-induced inhibition of glucose uptake and actin filament disorganization was reversed by 10 micromol ERK 1/2 MAPK inhibitor (PD98059). Pretreatment of Ang II increased ERK1/2 phosphorylation and inhibited insulin-induced Akt phosphorylation. The effect of Ang II on ERK1/2 phosphorylation was blocked by Ang II type 1 receptor antagonists, RNH6270 and PD98059 but not by SB203580 or Guanosine-5'-O-(2-ThioDiphosphate), a G-protein inhibitor. We next tested the effect of broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) on Ang II-inhibition of insulin signaling pathway. GM6001 did not improve Ang II-induced actin filament disorganization and did not inhibit ERK1/2 phosphorylation. From these data in L6 myotube, we conclude that Ang II negatively regulates the insulin signal not through MMP signaling pathway but specifically through MMP-independent ERK1/2 activation pathway, providing an alternative molecular mechanism for angiotensin-induced insulin resistance.

The Journal of Medical Investigation : JMI


Molecular cloning of a murine glycerol-3-phosphate acyltransferase-like protein 1 (xGPAT1).

A novel murine glycerol-3-phosphate acyltransferase-like protein 1 (named xGPAT1) has been cloned. The mouse xGPAT1 gene is located on mouse Chromosome 2, spans >19 kb, and consists of at least 23 exons. The protein is 32% identical and 72% similar to mouse mitochondrial GPAT (mtGPAT) on the amino acid level. Sequencing analysis confirmed that xGPAT1 has a 2403-bp open reading frame (ORF) that encodes an 801-amino acid protein with an estimated molecular mass of 89.1 kDa. A hydropathy plot of the deduced xGPAT1 protein showed a high degree of similarity with that of the mtGPAT protein. Using 5'-rapid amplification of cDNA ends, two alternate, untranslated exon 1 (1a and b) isoforms were obtained, generating variants xGPAT1-v1 and xGPAT1-v2. xGPAT1-v1 is expressed in mouse heart, liver, spleen, kidney and murine inner medullary collecting duct 3 (mIMCD3) cells, while xGPAT1-v2 is expressed in mouse liver, spleen, kidney, white and brown adipose tissues and 3T3-L1 pre- and post-adipocytes. xGPAT1 was distributed in the membrane fraction and showed GPAT activity when epitope-tagged xGPAT1 was expressed in Chinese hamster ovary (CHO)-K1 cells.


NO-1886, a lipoprotein lipase activator, attenuates vascular smooth muscle contraction in rat aorta

The chemical compound [4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester (NO-1886) is a lipoprotein lipase activator having beneficial effects on both diabetes control and the cardiovascular system. Preventing accumulation of lipids in the cell wall, in addition to improving insulin actions on vasculature, may indirectly contribute to the reducing effect of NO-1886 on vascular resistance. However, the direct effect of NO-1886 on vascular resistance, i.e., whether NO-1886 directly modulates the function of vascular endothelium and/or smooth muscle cells has not been investigated. In this study we therefore investigated the direct effect of NO-1886 on vascular contractility using rat aortic rings and cultured smooth muscle cell-line A10. The results show that administration of NO-1886 attenuated aortic contraction induced by phenylephrine and/or a high K(+) environment, in both the presence and absence of aortic endothelium. 1-(5-Chloronaphthalene-1-sulfonyl)homopiperazine hydrochloride (ML-9), a myosin light chain kinase (MLCK) inhibitor, blocked this inhibitory effect of NO-1886, whereas inhibitors of other signaling molecules such as calmodulin, protein kinase C and Rho-kinase had no effect. The vasorelaxant effect of NO-1886 was blocked in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel inhibitor, verapamil. NO-1886 attenuated smooth muscle contraction induced by the cumulative addition of CaCl(2). In A10 cells, NO-1886 inhibited the membrane depolarization-induced initial peak of [Ca(2+)](i) in the presence of extracellular Ca(2+). This inhibition did not occur in the absence of extracellular Ca(2+). Taken together these results demonstrate that NO-1886 attenuates smooth muscle contraction and causes vasorelaxation by an extracellular Ca(2+)- and MLCK-dependent mechanism.


Haemolysin produced by Vibrio mimicus activates two Cl secretory pathways in cultured intestinal-like Caco-2 cells.

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.


Pore formation of thermostable direct hemolysin secreted from Vibrio parahaemolyticus in lipid bilayers.

Vibrio parahaemolyticus secretes thermostable direct hemolysin (TDH), a major virulence factor. Earlier studies report that TDH is a pore-forming toxin. However, the characteristics of pores formed by TDH in the lipid bilayer, which is permeable to small ions, remain to be elucidated. Ion channel-like activities were observed in lipid bilayers containing TDH. Three types of conductance were identified. All the channels displayed relatively low ion selectivity, and similar ion permeability. The Cl- channel inhibitors, DIDS, glybenclamide, and NPPB, did not affect the channel activity of pores formed by TDH. R7, a mutant toxin of TDH, also forms pores with channel-like activity in lipid bilayers. The ion permeability of these channels is similar to that of TDH. R7 binds cultured cells and liposomes to a lower extent, compared to TDH. R7 does not display significant hemolytic activity and cell cytotoxicity, possibly owing to the difficulty of insertion into lipid membranes. Once R7 is assembled within lipid membranes, it may assume the same structure as TDH. The authors propose that the single glycine at position 62, substituted with serine in the R7 mutant toxin, plays an important role in TDH insertion into the lipid bilayer.


Aeromonas sobria hemolysin causes diarrhea by increasing secretion of HCO3-

Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a causative agent of diarrhea in humans. We investigated the effects of ASH on anion transport in human colonic epithelial cells. ASH increased short circuit currents across the intestinal epithelia, which were suppressed by anion channel antagonists, such as carbonic anhydrase inhibitors, and by the removal of external HCO3-. Iliac fluid accumulation was also inhibited by carbonic anhydrase inhibitors. The results suggest that ASH activates HCO3- secretion, whose level correlates with the severity of diarrhea.


Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway.

Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [3H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca2+ mobilization in HBF. The HAT-induced increase in Ca2+ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts.

American Journal of Physiology. Lung Cellular and Molecular Physiology


Monoamine Oxidase A Activity and Norepinephrine Level in Hippocampus Determine Hyperwheel Running in SPORTS Rats.

An understanding of neurological mechanisms for wheel running by rodents, especially with high exercise activity, would be applicable to a strategy for promotion of exercise motivation in humans. One of several brain regions that are candidates for the regulation of physical exercise is the hippocampus. Here we examined the running activity of Spontaneously-Running-Tokushima-Shikoku (SPORTS) rat, a new animal model for high levels of wheel-running activity, and its relation with the hippocampal norepinephrine (NE) system including the levels of NE, adrenergic receptors, and degradation enzymes for monoamines. In the hippocampus of SPORTS rats, the level of NE in extracellular fluid was augmented, whereas the level in the homogenate of the whole tissue was decreased even for sedentary conditions. Elevated extracellular NE caused downregulation of alpha(2)-adrenergic receptors in the hippocampus of SPORTS rats. Local administration of alpha(2)-adrenergic receptor antagonist yohimbine, but not of alpha(2)-agonist clonidine, into the hippocampus suppressed high running activity in SPORTS rats. The protein expression and the activity levels of monoamine oxidase A (MAOA), a critical enzyme for the degradation of NE, were decreased in the hippocampus of SPORTS rats to increase extracellular NE level. Thus, inhibition of oxidase activity in normal Wistar rats markedly increased wheel-running activity. These results indicate that decreased MAOA activity, elevation of extracellular NE, and alpha(2)-adrenergic receptors in the hippocampus determine the neural basis of the psychological regulation of exercise behavior in SPORTS rats.


高運動モデルラットSPORTSの海馬におけるノルエピネフリン動態と自発運動量

高運動モデルラットSPORTSの海馬におけるノルエピネフリン動態と自発運動量


Approach to novel functional foods for stress control 2. Microarray assessment of exercise in healthy volunteers.

DNA microarray was used to measure stress response by exercise in peripheral blood leukocytes. Aerobic exercise did not alter mRNA pattern or urinary secretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Strenuous exercise increased urinary secretion of 8-OHdG and altered mRNA pattern in microarray. These results suggest that moderate exercise, i. e. aerobic exercise, did not show any change in 8-OHdG, an oxidative stress marker, or mRNA expression in the leukocytes, which might reflect whole body neurohormanal changes. In addition, strenuous exercise produced quite different expression pattern from those of psychological stress.

The Journal of Medical Investigation : JMI


Listeriolysin O-induced membrane permeation mediates persistent IL-6 production in Caco-2 cells during Listeria monocytogenes infection in vitro.

Listeriolysin O (LLO), a major virulence factor of Listeria monocytogenes, is a member of the cholesterol-dependent cytolysin family and plays important roles not only in survival of this bacterium in phagocytes but also in induction of various cellular responses, including cytokine production. In this work, we examined the involvement of LLO in induction of the cytokine response in intestinal epithelial cells, the front line of host defense against food-borne listeriosis. Infection of Caco-2 cells with wild-type L. monocytogenes induced persistent expression of interleukin-6 (IL-6) mRNA. In contrast, IL-6 expression was observed only transiently during infection with non-LLO-producing strains. A sublytic dose of recombinant LLO (rLLO) induced the expression of IL-6 via formation of membrane pores. Under conditions of LLO-induced pore formation without extensive cell lysis, Ca2+ influx was observed, and the IL-6 expression induced by rLLO was inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), an intracellular Ca2+ chelator. LLO secreted by cytoplasmic L. monocytogenes appeared to induce pore formation in the membrane and to enable the trafficking of intracellular and extracellular molecules. Pretreatment with BAPTA-AM inhibited persistent IL-6 expression in Caco-2 cells infected with wild-type L. monocytogenes. These results suggest that LLO is involved in IL-6 production in the late phase of infection through the formation of Ca2+-permeable pores and subsequent Ca2+-dependent modulation of signaling and gene expression.


A pore-forming toxin produced by Aeromonas sobria activates Ca2+ dependent Cl- secretion.

Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca2+ levels in the extracellular solution or by pretreatment with the Ca2+ chlelator BAPTA-AM. Intracellular Ca2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca2+. Peak 2 but not peak 1 was related to Cl- secretion. These results indicate that ASH activates Ca2+-dependent Cl- secretion.


Cloning and establishment of a line of rats for high levels of voluntary running

We generated an original Wistar line of rats that displayed increased levels of wheel running, which we named SPORTS (Spontaneously-Running-Tokushima-Shikoku). Male SPORTS rats ran voluntarily in a running wheel almost six times longer than male control Wistar rats, established without selection for their running activity. The running phenotype of female SPORTS rats was the same as female control Wistar rats. However, male offspring from the cross-mating between a female SPORTS rat and a male control rat also showed a similar level of hyper-running activity as the original SPORTS line. Compared to control rats, male SPORTS rats had lower levels of mean body weight, abdominal fat and plasma insulin after 4 weeks of running. It is likely that all these beneficial changes observed in the SPORTS rats reflected the increases in glucose disposal we observed in oral glucose tolerance tests carried out on the animals. We also found hyper-running caused a significant increase in skeletal muscle oxidative capacity, measured as the ratio of malate dehydrogenase to phosphofructokinase activity, an index of aerobic metabolism. These results indicate that the SPORTS rat may be a good animal model for determining the mechanisms responsible for up-regulation of running motivation, in addition to investigating changes in nutrient metabolism induced by high intensity exercise.


A pore-forming toxin produced by Aeromonas sobria activates cAMP-dependent Cl- secretory pathways to cause diarrhea

Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a human pathogen that causes diarrhea. We investigated the effects of ASH on Cl(-) transport in human colonic epithelial cells. ASH increased short-circuit currents (Isc) and (125)I efflux from Caco-2 cells, indicating ASH activate Cl(-) secretion. Additions of inhibitors of cyclic AMP dependent Cl(-) channels, glybenclamide and NPPB suppressed the Isc and (125)I efflux increases induced by ASH. And ASH increased the intracellular cyclic AMP concentration. Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation. Thus, cAMP-dependent Cl(-) secretory pathway could be related with diarrhea induced by A. sobria.


Placental and intestinal alkaline phosphatases are receptors for Aeromonas sobria hemolysin

The Aeromonas sobria hemolysin causes diarrhea following infection by this enteropathogenic bacterium. We previously identified the putative receptor for A. sobria hemolysin as a p66 protein on Intestine 407 cells (Microb. Pathog. 27 (1999) 215-221). Here, we have partially purified and obtained a peptide mass fingerprint of p66 which revealed its identity with placental alkaline phosphatase (PLAP). Recombinant PLAP expressed in 293T cells was also found to bind to hemolysin and the binding was found not to be dependent on the N-linked glycosylation of PLAP. By immunohistochemical analysis, PLAP expression was detected in human intestinal mucosa, the target tissue in disease. In addition to PLAP, hemolysin also binds to intestinal alkaline phosphatase (IAP), an enzyme that is also abundantly expressed in intestine. Thus, both PLAP and IAP are very likely involved in the pathogenesis of diarrhea caused by this bacterial toxin.

International Journal of Medical Microbiology


Molecular mechanisms underlying ketamine-mediated inhibition of sarcolemmal adenosine triphosphate-sensitive potassium channels.

Ketamine inhibits adenosine triphosphate-sensitive potassium (KATP) channels, which results in the blocking of ischemic preconditioning in the heart and inhibition of vasorelaxation induced by KATP channel openers. In the current study, the authors investigated the molecular mechanisms of ketamine's actions on sarcolemmal KATP channels that are reassociated by expressed subunits, inwardly rectifying potassium channels (Kir6.1 or Kir6.2) and sulfonylurea receptors (SUR1, SUR2A, or SUR2B). The authors used inside-out patch clamp configurations to investigate the effects of ketamine on the activities of reassociated Kir6.0/SUR channels containing wild-type, mutant, or chimeric SURs expressed in COS-7 cells. Ketamine racemate inhibited the activities of the reassociated KATP channels in a SUR subtype-dependent manner: SUR2A/Kir6.2 (IC50 = 83 microM), SUR2B/Kir6.1 (IC50 = 77 microM), SUR2B/Kir6.2 (IC50 = 89 microM), and SUR1/Kir6.2 (IC50 = 1487 microM). S-(+)-ketamine was significantly less potent than ketamine racemate in blocking all types of reassociated KATP channels. The ketamine racemate and S-(+)-ketamine both inhibited channel currents of the truncated isoform of Kir6.2 (Kir6.2DeltaC36) with very low affinity. Application of 100 mum magnesium adenosine diphosphate significantly enhanced the inhibitory potency of ketamine racemate. The last transmembrane domain of SUR2 was essential for the full inhibitory effect of ketamine racemate. These results suggest that ketamine-induced inhibition of sarcolemmal KATP channels is mediated by the SUR subunit. These inhibitory effects of ketamine exhibit specificity for cardiovascular KATP channels, at least some degree of stereoselectivity, and interaction with intracellular magnesium adenosine diphosphate.


Endothelin-1(1 31) levels are increased in atherosclerotic lesions of the thoracic aorta of hypercholesterolemic hamsters

The novel vaso-constricting 31-amino acid-length endothelin-1 [ET-1(1-31)] is selectively produced by human mast cell chymase via its action on big ET-1. However, the pathological role of ET-1(1-31) in atherosclerosis remains unclear. The aim of this study was to clarify vasoconstrictive response and expression of ET-1(1-31) in atherosclerotic aorta. Syrian golden hamster, was used for preparing the atherosclerotic models by the administration of a high cholesterol diet (HC), treatment with the nitric oxide synthase inhibitor (Nomega-nitro-L-arginine methylester, L-NAME) alone, or both (HC and L-NAME) for 40 weeks. Early atherosclerosis was observed in the case of HC or L-NAME alone treatments respectively and severe atherosclerosis was observed in the case of combined HC and L-NAME treatment. Vasoconstriction induced by ET-1(1-31) was not altered by the atherosclerotic changes, but the expression pattern of ET-1(1-31) was different at each stage of the atherosclerotic aorta. ET-1(1-31) was observed rarely in normal aortas or in early atherosclerotic lesions, but ET-1(1-31) expression was dramatically increased in aortic neointima and adventitia in a state of atherosclerosis with severe inflammation. ET-1(1-31) might play in a role of promoting atherosclerosis, and especially be involved in inflammatory mediation during the progression of atherosclerosis.


Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels in the Cardiovascular System.

Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.


Taurine alters respiratory gas exchange and nutrient metabolism in type 2

To assess the effect of taurine supplementation on respiratory gas exchange, which might reflect the improved metabolism of glucose and/or lipid in the type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Male OLETF rats (16 weeks of age) were randomly divided into two groups: unsupplemented group and taurine-supplemented (3% in drinking water) group. After 9 weeks of treatment, indirect calorimetry and insulin tolerance tests were conducted. The amounts of visceral fat pads, tissue glycogen, the blood concentrations of glucose, triacylglycerol, taurine, and electrolytes, and the level of hematocrit were compared between groups. A nondiabetic rat strain (Long-Evans Tokushima Otsuka) was used as the age-matched normal control. The indirect calorimetry showed that the treatment of OLETF rats with taurine could reduce a part of postprandial glucose oxidation possibly responsible for the increase of triacylglycerol synthesis in the body. Taurine supplementation also improved hyperglycemia and insulin resistance and increased muscle glycogen content in the OLETF rats. Supplementation with taurine increased the blood concentration of taurine and electrolyte and fluid volume, all of which were considered to be related to the improvement of metabolic disturbance in OLETF rats. Taurine supplementation may be an effective treatment for glucose intolerance and fat/lipid accumulation observed in type 2 diabetes associated with obesity. These metabolic changes might be ascribed, in part, to the alteration of circulating blood profiles, where the improved hyperglycemia and/or the blood accumulation of taurine itself would play roles.


Molecular mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate-sensitive potassium channels.

Both propofol and thiamylal inhibit adenosine triphosphate-sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes. The authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits. Propofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 microm), SUR2A/Kir6.2 (EC50 = 72 microm), and SUR2B/Kir6.2 (EC50 = 71 microm) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2DeltaC36) channels (EC50 = 78 microm) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2DeltaC36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 microm), SUR2A/Kir6.2 (EC50 = 248 microm), SUR2B/Kir6.2 (EC50 = 183 microm), SUR2B/Kir6.1 (EC50 = 170 microm), and Kir6.2DeltaC36 channels (EC50 = 719 microm). None of the mutants significantly affects the sensitivity of thiamylal. These results suggest that the major effects of both propofol and thiamylal on KATP channel activity are mediated via the Kir6.2 subunit. Site-directed mutagenesis study suggests that propofol and thiamylal may influence Kir6.2 activity by different molecular mechanisms; in thiamylal, the SUR subunit seems to modulate anesthetic sensitivity.


Localization of the 31-amino-acid endothelin-1 in hamster tissue

Endothelin (ET)-1(1-31) is a novel vasoconstrictor peptide produced by human mast cell chymase, which selectively cleaves big ET-1 at the Try(31)-Gly(32) bond. We investigated the localization of ET-1(1-31) in various hamster tissues by immunohistochemistry and compared it to the distribution of ET-1(1-21). We found that the localization and amount of ET-1(1-31) were different from those of ET-1(1-21) in each tissue. ET-1(1-31)-like immunoreactivities (IR) in the heart, lung, and adrenal gland were observed in the same areas as ET-1(1-21) but were significantly weaker, suggesting that ET-1(1-31) might play a role only in mast cell/chymase-related pathological conditions in these tissues. In the liver, ET-1(1-31)-like IR was strongly detected in Kupffer cells where ET-1(1-21)-like IR was seen more weakly. In the kidney, ET-1(1-31)-like IR was slightly higher than ET-1(1-21). These results suggest that ET-1(1-31) might have physiological roles distinct from those of ET-1(1-21) in some hamster tissues.


Decreased blood levels of lactic acid and urinary excretion of 3-methylhistidine after exercise by chronic taurine treatment in rats

Taurine is reported to increase contractility of skeletal muscle and cardiac myocyte, which can increase exercise performance. The present study aimed to clarify taurine's effect on chronic endurance exercise, especially accumulation of lactic acid (LA), a marker of fatigue and ability of aerobic exercise, and urinary secretion of 3-methylhistidine (3-MH), a marker of muscle breakdown in rats. After exercise blood levels of LA and urinary excretion of 3-MH were significantly increased and this increase was significantly less in those with chronic treatment of taurine. Taurine treatment also significantly decreased fat accumulation and blood levels of cholesterol and triglyceride, which might improve insulin resistance and utilization of fat and glucose. These results indicate taurine treatment is useful for reducing physical fatigue and muscle damage during exercise training in rats, presumably due to antioxidant property and improvement of muscle and cardiac functions by taurine.

Journal of Nutritional Science and Vitaminology


Effect of human airway trypsin-like protease on intracellular free Ca2+ concentration in human bronchial epithelial cells

It has been shown that human airway trypsin-like protease (HAT) is localized in human bronchial epithelial cells (HBEC), and trypsin activates protease-activated receptor-2 (PAR-2). Activation of PAR-2 activates G-protein followed by an increase of intracellular free Ca2+, [Ca2+]in. This study was undertaken to clarify whether HAT can activate PAR-2 in HBEC or not. RT-PCR showed that HAT mRNA is expressed in HBEC, and PAR-2 mRNA is the most strongly expressed of the known PARs in HBEC. Both PAR-2 agonist peptide (PAR-2 AP) and HAT increased [Ca2+]in in HBEC in a biphasic fashion; a prompt, sharp increase (peak I) and a sustained low plateau (peak II). PAR-2 AP over 100-200 microM and HAT over 200-300 mU/ml (0.08-0.12 microM) induced both peak I and II, and PAR-2 AP below 100 microM and HAT below 200 mU/ml induced only peak II. Both PAR-2 AP-induced and HAT-induced peak I were induced by Ca2+ mobilization from intracellular stores, because they appeared even in Ca2+-free medium. Both PAR-2 AP-induced and HAT-induced peak II were induced by an influx of extracellular Ca2+, because they were abolished in Ca2+-free medium. The Ca2+ response to HAT was desensitized by exposure of HBEC to PAR-2 AP. These results indicate that HBEC have a functional PAR-2, and HAT regulates cellular functions of HBEC via activation of PAR-2.

The Journal of Medical Investigation : JMI


Severe catabolic state after prolonged fasting in cirrhotic patients: effect of oral branched-chain amino-acid-enriched nutrient mixture

Cirrhotic patients frequently undergo various medical procedures, such as diagnostic gastrointestinal endoscopy, without taking breakfast. The aim of the present study was to clarify the effect of longer fasting (> 12 h) on energy metabolism, and to test whether supplementation of an oral branched-chain amino-acid-enriched nutrient mixture (BCAA mixture), which contains various nutrients in addition to BCAA, could improve the catabolic state. Metabolic measurement was performed in 30 cirrhotic patients and 13 normal subjects, using indirect calorimetry. Compared with that in the normal subjects, the respiratory quotient (RQ) was significantly lower after an overnight fast in the cirrhotic patients, indicating accelerated fat oxidation and a catabolic state. In addition, RQ in cirrhotic patients (n = 7) decreased rapidly with longer fasting, whereas that in the normal subjects (n = 5) showed relatively stable values. These results indicate that special care should be taken with medical procedures that are carried out in patients who have fasted. The effect of oral glucose, a carbohydrate-rich snack (rice ball), and the BCAA mixture (each, 210 kcal) on RQ was studied in 6 normal subjects and 6 patients with liver cirrhosis after an overnight fast. Supplementation of the carbohydrate-rich snack and the BCAA mixture (210 kcal each) elevated RQ and blood glucose levels to a similar degree in the cirrhotic patients. Oral administration of glucose (210 kcal) led to significantly greater elevation of blood glucose levels than the other snacks, which may be unfavorable for cirrhotic patients, who frequently have glucose intolerance. In the 30 cirrhotic patients, supplementation with the BCAA mixture in the late evening significantly improved RQ in the early morning. Carbohydrate-rich meals are used as a late evening snack in cirrhotic patients, but our study indicates that supplementation with a BCAA mixture can also be used to reduce fat oxidation in the early morning, with results similar to those with carbohydrate-rich snacks.


Combination Therapy of Exercise and Angiotensin-Converting Enzyme Inhibitor Markedly Improves Insulin Sensitivities in Hypertensive Patients With Insulin Resistance

The contraction of muscle enhances the release of bradykinin (BK) and improves glucose uptake by the muscle. Angiotensin-converting enzyme inhibitor (ACEI) slows the breakdown of BK, thus the effect of BK is augmented in the presence of ACEI. The present study investigated whether the combination of exercise (increased production of BK) and ACEI (delay in breakdown of BK) might further improve insulin sensitivity in hypertensive patients with insulin resistance (HOMA-R>1.8). Patients were assigned either to increased walking distance (Walking group) or taking 2mg remocapril, an ACEI, daily (ACEI group) for 8 weeks. Then both interventions were given to all patients for 8 weeks (ACEI+Walking group). Blood concentrations of triglycerides were slightly lower in the ACEI+Walking group than at baseline, although there were no significant differences in total cholesterol or high density lipoprotein-cholesterol among the 2 groups. Blood glucose was not significantly different with each treatment, but blood concentrations of insulin and HOMA-R were significantly lower in the Walking and ACEI groups compared with the Control group. The combination of walking and ACEI further lowered blood concentrations of insulin and HOMA-R, which suggests that this treatment is beneficial for hypertensive patients with insulin resistance.


Clinically relevant concentrations of propofol have no effect on adenosine triphosphate-sensitive potassium channels in rat ventricular myocytes.

Activation of adenosine triphosphate-sensitive potassium (K(ATP)) channels produces cardioprotective effects during ischemia. Because propofol is often used in patients who have coronary artery disease undergoing a wide variety of surgical procedures, it is important to evaluate the direct effects of propofol on K(ATP) channel activities in ventricular myocardium during ischemia. The effects of propofol (0.4-60.1 microg/ml) on both sarcolemmal and mitochondrial K(ATP) channel activities were investigated in single, quiescent rat ventricular myocytes. Membrane currents were recorded using cell-attached and inside-out patch clamp configurations. Flavoprotein fluorescence was measured to evaluate mitochondrial oxidation mediated by mitochondrial K(ATP) channels. In the cell-attached configuration, open probability of K(ATP) channels was reduced by propofol in a concentration-dependent manner (EC(50) = 14.2 microg/ml). In the inside-out configurations, propofol inhibited K(ATP) channel activities without changing the single-channel conductance (EC(50) = 11.4 microg/ml). Propofol reduced mitochondrial oxidation in a concentration-dependent manner with an EC(50) of 14.6 microg/ml. Propofol had no effect on the sarcolemmal K(ATP) channel activities in patch clamp configurations and the mitochondrial flavoprotein fluorescence induced by diazoxide at clinically relevant concentrations (< 2 microm), whereas it significantly inhibited both K(ATP) channel activities at very high, nonclinical concentrations (> 5.6 microg/ml; 31 microm).


Growth-related renal Type II Na/Pi cotransporter.


Production of L-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein

We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.


Properties of Ca2+-dependent K+ channels of human gingival fibroblasts


Blood flow velocity in the commom carotid artery in humans during graded exercise on a treadmill


Changes in caritid blood flow and electrocardiogram in humans during and after walking on a treadmill

Blood flow velocity in the common carotid artery and the electrocardiogram were measured simultaneously by telemetry in seven male subjects during 20-min walking on a treadmill at an exercise intensity corresponding to a mean oxygen uptake of 26.0 (SD 2.9) ml.kg-1.min-1. The mean cardiac cycle was shortened from 0.814 (SD 0.103) s to 0.452 (SD 0.054) s during this exercise. Of this shortening, 73% was due to shortening of the diastolic period and 27% to shortening of the systolic period. In the relatively small shortening of the mean systolic period [from 0.377 (SD 0.043) s to 0.268 (SD 0.029) s], the isovolumetric contraction time was shortened by 56%. During exercise, the heart rate (fc) increased by 79.4% [from 74.3 (SD 9.3) beats.min-1 to 133.3 (SD 14.8) beats.min-1], and the peak blood velocity (S1) in the common carotid artery increased by 56.1% [from 0.82 (SD 0.10) m.s-1 to 1.28 (SD 0.11) m.s-1]. After exercise, the S1 decreased rapidly to the resting level. The fc decreased more slowly, still being higher than the initial resting level 5 min after exercise. The diastolic velocity wave and the end-diastolic foot decreased during exercise. The blood flow rate in the carotid artery increased transiently by 13.5% at the beginning of exercise [from 5.62 (SD 0.63) ml.s-1 to 6.38 (SD 0.85) ml.s-1] and by 26.5% at the end of the exercise period [from 5.62 (SD 0.63) ml.s-1 to 7.11 (SD 1.34) ml.s-1].(ABSTRACT TRUNCATED AT 250 WORDS)

European Journal of Applied Physiology and Occupational Physiology


Arrest of cell cycle progression of HeLa cells in the early G1 phase in K +-depleted conditions abd its recovery upon addition of insulin and LDL


Kinetic study on the effects of intracellular K+ and Na+ on Na+,K+,Cl- cotransport of HeLa cells by Rb+ influx determination


Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study.

Pancreaticobiliary maljunction (PBM), a disease with reflux of pancreatic and bile juice in the pancreaticobiliary tract, is a high-risk factor for biliary tract cancer. The aim of this study was to investigate the mechanism of carcinogenesis in PBM using a metabolomics analysis of bile sampled during surgery. Three patients with PBM without biliary tract cancer, four patients with extrahepatic bile duct cancer (EHBC), and three controls with benign disease were enrolled. Metabolomics analysis of bile samples was performed using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to discriminate the amino acid and lipidomic profiles. The principal component analysis in the capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry revealed similar metabolites in patients with PBM and those with EHBC; furthermore, there was a clear difference between patients with PBM or EHBC compared to controls. The amino acid profiles revealed the following 20 potential carcinogenic candidates for PBM: isoleucine, phenylalanine, tyrosine, leucine, tryptophan, arginine, lysine, valine, asparagine, methionine, aspartic acid, serine, threonine, histidine, glutamine, alanine, proline, glutamic acid, and pyruvic acid. The lipidomic profiles revealed the following 11 carcinogenic candidates: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl glycerol, lysophosphatidyl glycerol, triacylglycerol, diacylglycerol, ceramide, sphyngomyeline, fatty acid, hyperforin, and vitamin D. Among these characteristic metabolites, the branched-chain amino acids, methionine and lysophosphatidylcholine are known to be related to carcinogenesis. The bile metabolites were extremely similar in patients with PBM and those with EHBC. Furthermore, amino acid and lipid metabolism was markedly different in patients with PBM or EHBC compared to healthy controls.


Effect of light irradiation by light emitting diode on colon cancer cells.

Recent studies have demonstrated the efficacy of irradiation from light emitting diodes (LED) for wound healing, anti-inflammation and anticancer therapies. However, little is known about the effects of visible light in colon cancer cells. The purpose of this study was to evaluate the biological response (including gene expression changes) of human colon cancer cells to different wavelengths of LED irradiation. Human colon cancer cells (HT29 or HCT116) were seeded onto laboratory dishes that were then put on LED irradiation equipment with a 465 nm-, 525 nm-, or 635 nm-LED. Irradiation at 15 or 30 mW was performed 10 min/day, each day for 5 days. The cell counting kit8 was then used to measure cell viability. Apoptosis and expression of several mRNAs (caspase, MAPK and autophagy pathway) in HT29 cultures irradiated with 465 nm LED were evaluated via AnnexinV/PI and RT-PCR, respectively. Viability of HT29 and HCT116 cells was lower in 465 nm-LED irradiated cultures than in control cultures, but viability of HT29 cells did not differ between control cultures and 525 nm-LED or 635 nm-LED irradiated cultures. Moreover, the expression of FAS, caspase-3, capase-8, and JUK were significantly higher in 465 nm-LED irradiated cultures than in control cultures, and expression of ERK1/2 and LC3 was lower in blue-irradiated cells. LED irradiation at 465 nm inhibited the proliferation of HT29 cells and of HCT116 cells. Notably, LED irradiation at 465 nm promoted apoptosis inHT29 cultures via the extrinsic apoptosis pathway and the MAPK pathway.


Tripsin-like activities and human airway trypsin-like protease (HAT) levels in airway secretions from healthy subjects and patients with chronic airway diseases.