Dual signaling pathways of arterial constriction by extracellular uridine 5'-triphosphate in the rat.

We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 µM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,ß-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,ß-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,ß-methylene ATP (10 µM) elicited similar conductance single channel activities. UTP (≥10 µM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 µM) or nifedipine (10 µM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 µM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.

The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells.

1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

Ca(2+) channel properties in smooth muscle cells of the urinary bladder from pig and human.

Ca(2+) channel properties of pig and human bladder smooth muscle were investigated utilizing standard whole-cell patch clamp techniques. Both the amplitude obtained and the current density of Ca(2+) channel current evoked by step depolarization were larger in human than in pig myocytes. The inward currents were sensitive to an L-type Ca(2+) channel antagonist, nifedipine, the effects of which were not significantly different between species. In both species, prior application of ATP (0.1 mM) had no effect on activation of this voltage-sensitive channel current, while a muscarinic receptor agonist, carbachol (0.1 mM), significantly attenuated the amplitude of this current. Furthermore, inclusion of GDP-beta-S or Heparin in the pipette abolished or had no effect on the suppression of Ca(2+) current by carbachol, respectively. These results forward the pig as a good model for the human in detrusor Ca(2+) channel properties, especially with regard to neural modulation, although voltage-sensitive Ca(2+) channels seem to make greater contribution in human bladder physiology.

[Questionnaire about impression and knowledge of tuberculosis in employees and students in a university hospital].

The aim of the study is to search the efficient way for the prevention of nosocomial tuberculosis (TB) infection in a university teaching hospital. Through a questionnaire, informations on the degree of interest in TB, on the way how they try to learn about TB infection, and on basic knowledge of TB epidemiology were obtained. The study subjects were most employees including physicians, nursing staffs, medical technicians, pharmacists, clerks as well as medical and dental students, who were younger than 40 years. The study was done from 1999 through 2001, and a total of 2,159 questionnaires in which age, sex and occupational category were completely described were analyzed. Out of total participants, 61.8% participants showed interest in TB, however, only 3.0% had actually attended lecture meeting or collected materials on TB infection. Out of 619 nursing staffs, 431 (69.6%) felt anxiety for TB infection and the disease, and it was significantly higher than the other occupational groups. Of 2,159 participants, 1,472 (68.2%) participants desired to have health examination for TB. On the other hand, less than 50% participants including physicians answered correctly to questions about basic knowledge of TB epidemiology. Through the present study, it was suggested that employees and students in a university hospital do not voluntarily learn about TB, or do not have enough knowledge on TB in spite of their anxiety or interest, but that they are well prepared to obtain essential informations on the prevention of TB infection. Thus, it would be worthwhile to establish a system of education and health examination for the prevention of nosocomial TB infection. On the other hand, the degree of interest in and anxiety about TB in clerical employees was relatively low. Since they have some risk of TB infection through a service at window, the strengthening of health education on TB for them would be necessary.

[Postoperative bleeding after tooth extractions in patients controlled with warfarin--a clinico-statistical study on the factors influencing postoperative bleeding].

There are many reports about tooth extractions of patients taking warfarin, however PT-INR level is used to examine the postoperative bleeding in patients. To investigate other factors, postoperative bleeding, age, gender, PT-INR level, combined use of anti-platelet drugs, conditions of extracted tooth, a number of tooth extractions at a treatment, methods of management for warfarin therapy, degree of the alveolar bone loss and size of radiolucency of apical region were examined in this study. To apply Mann-Whitney U-test and chi2-test, ninety-three patients (38 male and 55 female) who took warfarin and visited our clinic for tooth extractions from April 1994 to November 2002 were classified into 2 groups: One group showed hemostasis by the next day (77 patients), the other showed the continuous bleeding after the next day (16 patients). These analyses indicated that PT-INR level, a number of tooth extractions at a treatment, methods of management for warfarin therapy, and size of radiolucency of apical region influenced postoperative bleeding. In addition, stepwise logistic regression analysis was applied to all of the factors, obtained from 77 patients out of 93 patients. This data showed that PT-INR level, a number of tooth extractions at a treatment and methods of management for warfarin therapy influenced postoperative bleeding. These results suggest that before the tooth extractions not only PT-INR level but methods of management for warfarin therapy and size of wound could be important to control the postoperative bleeding in warfarin taking patients.

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides.

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.

Dual modulation of airway smooth muscle contraction by Th2 cytokines via matrix metalloproteinase-1 production.

Altered contractility of airway smooth muscle (SM) is one of the main causes of allergic asthma, in which the predominance of Th2 over Th1 cytokines plays a central role. In the present study, we examine the effects of Th2 cytokines on airway SM contraction. Treatment with a low concentration of IL-4 (0.2 ng/ml) for 6 h augmented, whereas higher concentrations (2-20 ng/ml) inhibited, agonist-induced contractions of collagen gels containing bovine tracheal SM cells. Another Th2 cytokine (IL-13) showed an augmentation of gel contraction in the concentration range of 20-200 ng/ml. IL-4 and IL-13 increased mRNA expression and protein secretion of matrix metalloproteinase (MMP)-1, but these cytokines did not affect Ca(2+)-mobilizing properties and phosphorylation levels of myosin L chain in bovine tracheal SM cells. These changes were sensitive to wortmannin, an inhibitor of PI3K, but not to leflunomide, an inhibitor of STAT6. Scanning electron microscope observation revealed that collagen fibers twining around SM cells were completely dissolved in 20 ng/ml IL-4-treated gels and reorganized into basket-like structure in 20 ng/ml IL-13-treated gels. Exogenous application of high and low concentrations of MMP-1 also induced the inhibition and augmentation of gel contraction, respectively. Furthermore, nonselective MMP inhibitor galardin suppressed the effects of IL-4 and IL-13 on gel contraction, and MMP-1-targeted small-interfering RNA reversed the inhibitory effects of IL-4 on gel contraction to the augmentation. This indicates that Th2 cytokines modulate airway contraction without affecting cellular contractility but by secreting MMP-1 from the SM cells via PI3K activation and changing cell-to-matrix interactions.

Membrane stretch-induced activation of a TRPM4-like nonselective cation channel in cerebral artery myocytes.

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (> or =20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na(+) and Cs(+) and inhibited by Gd(3+) (30 microM) and DIDS (100 microM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca(2+) (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca(2+)-stores, first activated and then abolished SACs. Tetracaine (100 microM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca(2+) and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation.

Possible involvement of M5 muscarinic receptor in the enhancing actions of the novel gastroprokinetic agent Z-338 on nifedipine-sensitive voltage-dependent Ca2+ currents in guinea pig stomach.

We investigated the effects of the novel gastroprokinetic agent Z-338 (N-(N-N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxybenzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate) on L-type voltage-dependent Ca2+ currents (ICa) in guinea pig gastric myocytes by using the whole-cell patch clamp technique. Bath-applied acetylcholine (ACh) produced biphasic effects on ICa, i.e., enhancement (1-100 nM) and inhibition (1-100 microM), both of which were abolished by pretreatment with atropine (10 microM) or intracellular perfusion of GDPbetaS (500 microM). Z-338 (> or = 1 nM, ED50: 120 nM) mimicked the enhancing effects of ACh, but did not inhibit ICa. The effects of Z-338 and ACh were non-additive and blocked by atropine and GDPbetaS, but not by pertussis toxin (PTX) pretreatment (500 ng/ml). ACh (> or = 1 microM) induced slow inward currents via activation of the muscarinic receptor/PTX-sensitive G-protein pathway, but Z-338 was devoid of these effects. Neither pirenzepine (1 microM), AF-DX116 (1 microM), nor oxybutynin (100 nM) could prevent Z-338 (1 microM) and ACh (10 nM) from enhancing ICa, whilst 4-DAMP (100 nM) blocked the effects of Z-338 and ACh. Bath-application of protein kinase C (PKC) activator PDBu (phorbol-12,13-dibutyrate) (250 nM) enhanced ICa, and conversely, pipette inclusion of PKC inhibitor peptide (150 microM) abolished the effects of ACh and Z-338 on ICa. These results collectively suggest that although contribution of the M3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of ICa through activation of M5-like receptor.

Inward current oscillation underlying tonic contraction caused via ETA receptors in pig detrusor smooth muscle.

Endothelin-1 (ET-1) is a powerful vasoconstricting peptide. Recent studies showed synthesis of ET-1 and the presence of ET receptors in urinary bladder smooth muscle cells. In the present study, we investigated the possible role of ET-1 in detrusor contraction and its underlying mechanisms in terms of electrical activity. ET-1 caused dose-dependent tonic contraction of bladder smooth muscle strips. Whole cell patch-clamp experiments revealed that ET-1 induced a single transient inward current in the majority of detrusor cells and that additional inward current oscillations were induced in one-third of the cells. The inward current oscillation and tonic contraction shared several characteristic features: 1) both activities lasted for a considerable time after ET-1 washout and 2) only prior application of ETA receptor antagonists, not ETB receptor antagonists, significantly suppressed ET-1-induced contractions and the oscillating inward currents. It was concluded that the inward current oscillation underlies ET-1-induced tonic contraction. Experiments with ion substitution and channel blockers suggested that periodic activation of Ca2+-activated Cl- channels caused the oscillating inward currents.

Purification, gene cloning, gene expression, and mutants of Dps from the obligate anaerobe Porphyromonas gingivalis.

The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR. The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.