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Fluorescent Indicator (fluorescent + indicator)
Selected AbstractsA Novel Fluorescent Indicator for Ba2+ in Aqueous Micellar Solutions.CHEMINFORM, Issue 17 2004Yoshio Nakahara No abstract is available for this article. [source] Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2010Sriram Govindan Background and purpose:, P2Y receptors evoke Ca2+ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved. Experimental approach:, Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca2+] in cultured ASMC evoked by each P2Y receptor subtype. Key results:, The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca2+ signals that require activation of phospholipase C and comprise both release of Ca2+ from stores and Ca2+ entry across the plasma membrane. Conclusions and implications:, Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca2+ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors. [source] SKELETAL MUSCLE FUNCTION: ROLE OF IONIC CHANGES IN FATIGUE, DAMAGE AND DISEASECLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2004DG Allen SUMMARY 1.,Repeated activity of skeletal muscle causes a variety of changes in its properties: muscles become weaker with intense use (fatigue), may feel sore and weak after repeated contractions involving stretch and can degenerate in some disease conditions. The present review considers the role of early ionic changes in the development of each of these conditions. 2.,Single fibre preparations of mouse muscle were used to measure ionic changes following activity induced changes in function. Single fibres were dissected with intact tendons and stimulated to produce force. Fluorescent indicators were microinjected into the fibres to allow simultaneous ionic measurements with determination of mechanical performance. 3.,One theory to explain muscle fatigue is that fatigue is caused by the accumulation of lactic acid, producing an intracellular acidosis that inhibits the myofibrillar proteins. In contrast, we found that during repeated tetani there was little or no pH change, but that failure of calcium release was a major contributor to fatigue. Currently, it is proposed that precipitation of calcium and phosphate in the sarcoplasmic reticulum contributes to the failure of calcium release. 4.,Muscles can be used to shorten and produce force or they can be used to de-accelerate loads (stretched or eccentric contractions). One day after intense exercise involving stretched contractions, muscles are weak, sore and tender, and this damage can take a week to recover. In this condition, sarcomeres are disorganized and there are increases in resting intracellular Ca2+ and Na+. Recently, we demonstrated that the elevation of Na+ occurs through a stretch-activated channel that can be blocked by either gadolinium or streptomycin. Preventing the increase in [Na+]i with gadolinium also prevented part of the muscle weakness after stretched contractions. 5.,Duchenne muscular dystrophy is a lethal degenerative disease of muscles in which the protein dystrophin is absent. Dystrophic muscles are more susceptible to stretch-induced muscle damage and the stretch-activated channel seems to be one pathway for the increases in intracellular Ca2+ and Na+ that are a feature of this disease. We have shown recently that blockers of the stretch-activated channel can minimize some of the short-term damage in muscles from the mdx mouse, which also lacks dystrophin. Currently, we are testing whether blockers of the stretch-activated channels given systemically to mdx mice can protect against some features of the disease. [source] Blockade by ferrous iron of Ca2+ influx through N -methyl- d -aspartate receptor channels in immature cultured rat cortical neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Noritaka Nakamichi Abstract Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo-3 for assessment of intracellular free calcium ion (Ca2+) concentrations with the aid of a confocal laser-scanning microscope. In the absence of added MgCl2, the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration-dependent manner at a concentration range of 1,100 µm with the increase by KCl being transient. The addition of FeCl2, but not FeCl3, markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10,200 µm, without affecting that by KCl. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCl2 was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCl2 invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain. [source] The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to EndotoxinALCOHOLISM, Issue 2004Nobuyuki Enomoto Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-, (TNF-,) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-, should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca2+]i) is required for LPS-induced TNF-, production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca2+]i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin. Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 ,mol/liter). After addition of LPS (10 ,g/ml) to culture media, [Ca2+]i was measured using a fluorescent indicator, fura-2. Results: LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 ,g/ml)-induced TNF-, production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05). Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin. [source] Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene deliveryTHE JOURNAL OF GENE MEDICINE, Issue 5 2004Naoki Kobayashi Abstract Background The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. Methods Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. Results PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. Conclusions These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability. Copyright © 2004 John Wiley & Sons, Ltd. [source] Direct evidence of nitric oxide release from neuronal nitric oxide synthase activation in the left ventricle as a result of cervical vagus nerve stimulationTHE JOURNAL OF PHYSIOLOGY, Issue 12 2009Kieran E. Brack Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect of vagal stimulation on ventricular myocardial function is controversial. We have recently provided indirect evidence that the anti-fibrillatory effect of vagus nerve stimulation on the ventricle is mediated by nitric oxide (NO). The aim of this study was to provide direct evidence for the release of nitric oxide in the cardiac ventricle during stimulation of the efferent parasympathetic fibres of the cervical vagus nerve. The isolated innervated rabbit heart was employed with the use of the NO fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) during stimulation of the cervical vagus nerves and acetylcholine perfusion in the absence and presence of the non-specific NO synthase inhibitor NG -nito- l- arginine (l- NNA) and the neuronal NO synthase selective inhibitor 1-(2-trifluormethylphenyl)imidazole (TRIM). Using the novel fluorescence method in the beating heart, we have shown that NO-dependent fluorescence is increased by 0.92 ± 0.26, 1.20 ± 0.30 and 1.91 ± 0.27% (during low, medium and high frequency, respectively) in the ventricle in a stimulation frequency-dependent manner during vagus nerve stimulation, with comparable increases seen during separate stimulation of the left and right cervical vagus nerves. Background fluorescence is reduced during perfusion with l- NNA and the increase in fluorescence during high frequency vagal stimulation is inhibited during perfusion with both l- NNA (1.97 ± 0.35% increase before l- NNA, 0.00 ± 0.02% during l- NNA) and TRIM (1.78 ± 0.18% increase before TRIM, ,0.11 ± 0.08% during TRIM). Perfusion with 0.1 ,m acetylcholine increased NO fluorescence by 0.76 ± 0.09% which was blocked by l- NNA (change of 0.00 ± 0.03%) but not TRIM (increase of 0.82 ± 0.21%). Activation of cardiac parasympathetic efferent nerve fibres by stimulation of the cervical vagus is associated with NO production and release in the ventricle of the rabbit, via the neuronal isoform of nitric oxide synthase. [source] Increased nitric oxide production in nasal epithelial cells from allergic patients , RT-PCR analysis and direct imaging by a fluorescence indicator: DAF-2 DA*CLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2001S. Takeno Background Nitric oxide (NO) is believed to participate in the regulation of airway clearance and non-specific cellular immunity. Recent studies have suggested that airway epithelial cells of allergic and non-allergic individuals may differ in their ability to produce this molecule. Objective The aim of this study was to detect the difference in NO production in human nasal epithelial cells between normal subjects and patients with perennial allergic rhinitis (AR), and to assess the relationship between the expression of nitric oxide synthase (NOS) isoforms and the severity of the disease. Methods Nasal epithelial cells were obtained from the inferior turbinate. The expression of mRNAs encoding constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Direct NO production in living cells was visualized and quantified by a fluorescent indicator, DAF-2 DA. Results RT-PCR analysis demonstrated that AR patients with a RAST score of 5 or 6 showed significant increases in the levels of iNOS mRNA and slight reductions in those of eNOS mRNA. Patients with a RAST score of 2,4 also revealed the same tendency however, the difference was not significant. DAF-2 DA imaging demonstrated that epithelial cells, especially the ciliated cells, produced a larger amount of NO than non-epithelial inflammatory cells. Preincubation with L-NAME resulted in an approximate 40% decrease in both groups. Conclusion These results directly indicate that nasal epithelial cells of AR patients overall produce higher levels of NO through the concomitant expression of different NOS isoforms. Continuous NO production by the epithelial cells in normal subjects further support the hypothesis that NO derived from epithelium may play dual roles in the regulation of nasal airway clearance and in the host defense. In addition, the use of DAF-2 DA provides a reliable method to visualize and quantify the direct NO production of living cells. [source] Mechanisms Associated with the Negative Inotropic Effect of Deuterium Oxide in Single Rat Ventricular MyocytesEXPERIMENTAL PHYSIOLOGY, Issue 2 2000K. Hongo Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle. [source] | |||||||||||||