Potential-sensitive Fluorescent Dye Research Articles

Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity

To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K(+) efflux and Na(+) influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K(+) efflux and Na(+) influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections.

Transmembrane Potential of Red Blood Cells Under Low Ionic Strength Conditions

Background/Aims: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. Methods: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC₄(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. Results: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. Conclusions: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.

Rapid Cytotoxicity Screening Platform for Amyloid Inhibitors Using a Membrane-Potential Sensitive Fluorescent Probe

The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-β(1-42) (Aβ(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aβ(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue(1,2). GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate. GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders(3). However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations(3). Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry(4) or radiotracer analysis(5), the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

Analyzing Cellular Apoptosis Through Monitoring Mitochondrial Membrane Potential Changes with JC-10

Apoptosis is one main type of programmed cell death which involves a series of biochemical events leading to specific cell morphology characteristics and ultimately death of cells. Mitochondrial membrane potential change is a key indicator of cell health and mitochondrial permeability transition is an important step in the induction of cellular apoptosis. JC-10, a lipophilic cationic dye, can be readily used to detect mitochondrial de-polarization. It has been successfully used in several different cell types (e.g., primary rat hepatocytes, CHO-K1, HeLa, Jurkat and HepG2 cell lines). JC-10 has improved water solubility. The consistent results were obtained with flow cytometry, fluorescence plate reader and florescence microscopy. In most of the cell lines JC-10 demonstrates excellent signal to background ratios. In addition, two other membrane potential-sensitive fluorescent dyes, the near infrared red Mito NIR, and orange color Mito Orange have also showed similar results as JC-10 for measuring mitochondria membrane potential changes in Jurkat and Hela cells. In conclusion JC-10, Mito NIR, and Mito Orange are robust tools for evaluating apoptosis targets and screening their agonists and antagonists with fluorescence microplate readers, fluorescence microscopes or flow cytometers.

4-Aminopyridine-sensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery

The present study aimed at examining the role of potassium channels and endothelium in relaxations induced by sodium hydrogen sulphide (NaHS), which is the donor of gaseous hydrogen sulphide (H2S) and the effect of NaHS on endothelium-dependent relaxations in rat coronary arteries. Rat coronary arteries were suspended in a myograph for force measurement and changes of the membrane potential in arteries were determined by membrane potential-sensitive fluorescence dye. NaHS relaxed coronary arteries pre-contracted by U46619 and the relaxation was significantly less in high KCl-contracted rings. NaHS-induced relaxations were reduced by 4-aminopyridine (4-AP) but unaffected by glibenclamide, iberiotoxin, NG-nitro-L-arginine methyl ester, ODQ, indomethacin or by endothelium removal. The inhibitory effect of 4-AP was absent in NaHS-induced relaxations in high KCl-contracted rings. Addition of NaHS caused membrane hyperpolarization and this effect was inhibited by 4-AP but not by glibenclamide. NaHS causes endothelium-independent relaxations in rat coronary arteries partially through activation of 4-AP-sensitive potassium channel and ensuring hyperpolarization. Other potassium channels, Na+–K+ pump or endothelium-derived relaxing factors play little role.

Impaired Na +-dependent glutamate uptake in platelets during depolarization of their plasma membrane

Blood platelets contain neuronal Na +-dependent glutamate transporters and are able to accomplish glutamate uptake. Applying high-KCl, we have demonstrated dose-dependent depolarization of the plasma membrane of rabbit platelets that was registered as an increase in the fluorescence of the potential-sensitive fluorescent dye rhodamine 6G. The initial velocity of L-[ 14C]glutamate uptake (10 μM) in platelets was decreased by 20% during 35 mM KCl-evoked depolarization and consisted of 1.2 ± 0.09 pmol × min −1 × mg −1 protein in control and 0.96 ± 0.08 pmol × min −1 × mg −1 protein during depolarization. Confocal laser scanning microscopy and flow cytometry revealed that these changes in glutamate uptake were not a result of platelet aggregation/activation. Also, addition of high-KCl did not change acidification of platelet secretory granules that was found with pH-sensitive fluorescent dye acridine orange, thereby showing that changes in their proton gradient could not cause glutamate uptake alteration. This malfunction of glutamate transporters has to take place under: (i) the conditions of pseudohyperkalemia or hyperkalemia, i.e. activation and clotting of platelets, haemolysis, leucocytosis, acute renal failure, hypofunction of adrenal cortex, lack of aldosterone, stroke, trauma and (ii) depolarization of the plasma membrane of platelets during their activation by ADP, thrombin, platelet-activating factor. Weak glutamate uptake might have considerable consequences for platelets per se (and thus for haemostatic system) and glutamate homeostasis in the CNS.

Abstract 5515: Mitochondrial Membrane Hyperpolarization and Reduced Mitochondrial Mass Characterize the Arteriolar Endothelium and Mononuclear Cells of Humans With Type 2 Diabetes Mellitus- in vivo and in vitro Assessment of Endothelial Function

Background: Endothelial dysfunction and inflammation are central to the vascular pathology of Type 2 diabetes (DM). In vitro data suggest excessive mitochondrial reactive oxygen species production contributes to endothelial dysfunction in part due to pathological hyperpolarization of mitochondrial membrane potential (ΔΨ m ). ΔΨ m hyperpolarization in leukocytes is also associated with activation of the inflammatory response. Mitochondrial mass and morphology are also central to mitochondrial function. The relevance of these mechanisms to the human vasculature in DM is unknown. Methods: Blood mononuclear cells were isolated from 30 DM patients and 32 non-diabetics. Subcutaneous arterioles were harvested by gluteal adipose tissue biopsy in a subset (15 DM, 17 controls). ΔΨ m was measured in cells and arterioles using the membrane potential-sensitive fluorescent dye JC-1. Mitochondrial mass was measured using the cardiolipin-specific fluorophore nonyl acridine orange. Endothelium dependent vasodilation was assessed using video microscopy (arterioles) and ultrasonography (brachial artery). Arteriolar vasoreactivity was also determined in the setting of low-dose (100 nM) CCCP to reduce ΔΨ m magnitude. Results: ΔΨ m magnitude of the arteriolar endothelium and mononuclear cells were significantly greater in DM patients [P=0.01 (endothelium), P < 0.0001 (mononuclear cells)]. Mitochondrial mass was significantly lower in DM mononuclear cells (P=0.004), and in DM endothelium following age adjustment (P=0.003). Endothelium dependent vasodilation was impaired in DM in the brachial artery and subcutaneous arterioles (both P<0.001). CCCP treatment of the DM arteriolar endothelium reversed microvascular endothelial dysfunction (P<0.001 vs. untreated). Conclusions: ΔΨ m is hyperpolarized and mitochondrial mass is lower in the arteriolar endothelium and mononuclear cells of DM patients with endothelial dysfunction. Pharmacological normalization of ΔΨ m reverses microvascular endothelial dysfunction in DM. These findings are consistent with experimental studies suggesting a link between mitochondria and vascular disease. Interventions that restore mitochondrial membrane potential and mass in DM may have therapeutic potential.

Insulin Activates ATP-Sensitive Potassium Channels via Phosphatidylinositol 3-Kinase in Cultured Vascular Smooth Muscle Cells

The effects of insulin on the vasculature are significant because insulin resistance is associated with hypertension. To increase the understanding of the effects of insulin on the vasculature, we analyzed changes in potassium ion transport in cultured vascular smooth muscle cells (VSMCs). Using the potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC₄(3)], we found that insulin induced membrane hyperpolarization after 2 min in A10 cells. Insulin-induced hyperpolarization was suppressed by glibenclamide, an ATP-sensitive potassium (K_ATP) channel blocker. Using a cell-attached patch clamp experiment, the K_ATP channel was activated by insulin in both A10 cells and isolated VSMCs from rat aortas, indicating that insulin causes membrane hyperpolarization via K_ATP channel activation. These effects were not dependent on intracellular ATP concentration, but wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, significantly suppressed insulin-induced K_ATP channel activation. In addition, insulin enhanced phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and protein kinase B (Akt) after 2 min. These data suggest that K_ATP channel activation by insulin is mediated by PI3-K. Furthermore, using a nitric oxide synthase (NOS) inhibitor, we found that NOS might play an important role downstream of PI3-K in insulin-induced K_ATP channel activation. This study may contribute to our understanding of mechanisms of insulin resistance-associated hypertension.

Translesion stimulus-excitation delay indicates quality of linear lesions produced by radiofrequency ablation in rabbit hearts

Failure of cardiac antiarrhythmic ablation to block action potential conduction produces poor outcomes which lead to repeat procedures. To overcome this, an intraoperative index of the quality of an ablation lesion is needed. We hypothesized that a rise in the translesion stimulus-excitation delay (TED) can indicate a continuous, transmural, linear lesion, and that the TED is related to the path length in the viable tissue around the lesion. Rabbit hearts were isolated, perfused with a warm physiological solution and stained with transmembrane potential-sensitive fluorescent dye. Radiofrequency (RF) ablation was performed on ventricular epicardium with a vacuum-assisted coagulation device to produce either a complete or incomplete lesion. Complete lesions were both transmural and continuous. Incomplete lesions were noncontinuous or nontransmural. The TED was determined with bipolar stimulation at one side of the lesion and either a bipolar electrogram at the other side or optical mapping on both sides. Hearts were then stained with tetrazolium chloride and examined histologically to estimate minimum path lengths of viable tissue from the stimulation site to the recording site. Complete lesions increased the TED by factors of 2.6–3.1 (p < 0.05), whereas incomplete lesions did not significantly increase the TED. Larger minimum path lengths were found for cases that had an increased TED. The TED was quantitatively predictable based on a conduction velocity of 0.38–0.49 m s−1, which is typical of rabbit hearts. The TED significantly increases when a linear lesion is complete, suggesting that an intraoperative measurement of the TED may help to improve ablation lesions and outcomes. Predictability of the TED based on the viable tissue path suggests that quantitative TEDs for clinical lesions may be anticipated provided that the conduction velocity is considered.

FlexStation examination of 5-HT 3 receptor function using Ca 2+- and membrane potential-sensitive dyes: Advantages and potential problems

The FlexStation is a 96 or 384 fluorescent plate reader with the capability of adding solutions during readings; it therefore has the potential to provide high throughput analyses of the functional characteristics of neurotransmitter-gated ion channels that can be examined using changes in fluorescence. The 5-HT 3 receptor is one such protein, as its activation results in a change in membrane potential due to the opening of a Ca 2+-permeant, cation-selective channel; it can therefore be studied using both Ca 2+- and membrane potential-sensitive fluorescent dyes. Here we have used the FlexStation to examine the function of recombinant 5-HT 3 receptors expressed in HEK293 cells using both these classes of dye. The results show that the pharmacological characteristics of the receptor obtained using the FlexStation is similar to those reported using other functional methods, although caution must be applied when using the membrane potential dye as large changes in membrane potential can yield inaccurate EC 50s. Modifying the constituents of the buffer, however, so that the change in membrane potential was reduced, yielded EC 50 values that were similar to previously reported data. We conclude that the FlexStation is a useful tool for high throughput studies when examining the function of neurotransmitter receptors that result in either a change in Ca 2+ concentration or membrane potential.

Evaluation of a High-Throughput Fluorescence Assay Method for hERG Potassium Channel Inhibition

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z' factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependent activity of the antagonists.

Spectroscopic properties of Langmuir–Blodgett films containing a potential-sensitive dye

This paper reports the incorporation of a potential-sensitive fluorescent dye, Di-8-ANEPPS, in Langmuir–Blodgett (LB) films mixed with amphiphiles of different nature. We used dihexadecylphosphate (DHP) and octadecyltrimethylammonium bromide (OTMA) as lipid matrices. Surface pressure versus molecular area isotherms reveal the presence of small interactions between the components in the mixture, resulting in the formation of microcrystallites and aggregates in the LB films. The optical characteristics of the mono- and multilayers were evaluated by UV–VIS absorbance and fluorescence emission spectroscopy and they were controlled for different mixing ratio and number of LB layers.

Cytochrome P-450 metabolites but not NO, PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels.

The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI2), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. Studies have reported the hyperpolarizing effects of these factors are increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P-450 metabolites and H2O2 are candidates for EDHF. With the use of the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethione oxonol [DiBAC4(3)], we examined whether NO, PGI2, cytochrome P-450 metabolites, and H2O2 contribute to ACh-induced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (60-356 mum internal diameter) were cannulated and pressurized at 60 cmH2O in a vessel chamber perfused with physiological salt solution containing DiBAC4(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. The inhibitors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid, a cytochrome P-450 monooxygenase inhibitor, to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase, an enzyme that dismutates H2O2 to form water and oxygen, did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI2 do not contribute to the ACh-induced hyperpolarization and that the cytochrome P-450 metabolites but not H2O2 are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels.

Ion sensing and inhibition studies using the transmembrane ion channel peptide gramicidin A entrapped in sol-gel-derived silica.

The development of new, targeted drugs relies heavily on innovative technologies that allow for high-throughput screening of drug libraries against biologically relevant targets, particularly membrane-associated receptors. Therefore, immobilization of natural receptors is of the utmost importance to allow for screening of small molecule libraries. Herein, we describe the immobilization of liposomes containing the transmembrane peptide ion-channel gramicidin A into sol-gel-derived silicate materials. Steady-state fluorescence measurements of the intrinsic tryptophan residues of reconstituted gramicidin A in phospholipid vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained in solution and following entrapment in diglyceryl silane (DGS)-derived silicate to examine the effects of entrapment on the conformation of the ion channel. Only minor deviations were observed in the fluorescence properties of gramicidin following entrapment in DGS-derived silicate. DOPC vesicles containing a 50 microM internal solution of the potential sensitive fluorescent dye safranine O were used to study ion flux through the membrane ion channel. The dependence of ion flux on both ion concentration and amount of gramicidin embedded in the membrane were examined before and after entrapment in sol-gel-derived silicate. It was found that ion channel activity upon entrapment in DGS-derived silicate mirrored very closely that observed in solution. Moreover, the ability to inhibit ion flux through gramicidin A due to blockage by calcium ions was retained after the immobilization procedure. The implications for development of drug-screening and -sensing platforms are discussed.

A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Nitrate-selective optical sensor applying a lipophilic fluorescent potential-sensitive dye

An optical sensor has been developed for continuous determination of nitrate that is based on a polymer-stabilized emulsion system consisting of a hydrogel with entrapped plasticizer droplets. The droplets contain a cationic potential-sensitive fluorescent dye (PSD) located near its surface. The cationic PSD also acts as an anion-exchange catalyst that extracts nitrate out of the aqueous solution to form a complex (PSD+/NO3−) that moves into the plasticizer droplet due to its lipophilicity. This process causes a decrease of micro-polarity near the dye, resulting in an increase in the fluorescence intensity of the PSD. In a second detection scheme, the fluorescence intensity information of the PSD is converted into a fluorescence phase shift by mixing into the membrane particles that contain an inert long-lifetime reference luminophore. The second sensing scheme is referred to as “dual lifetime referencing”. Both schemes can be used for reversible sensing of nitrate in the 0.1–50mM range, with response times in the order of 3min.

NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Cl−cotransporter activity

First published August 9, 2001; 10.1152/ajprenal.0075.2001.—We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption ( JCl−). NaCl transport in the THAL depends on apical Na+-K+-2Cl−cotransporters, apical K+channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl−by inhibiting the Na+-K+-2Cl−cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl−]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]ifrom 13.5 ± 1.2 to 9.6 ± 1.6 mM ( P &lt; 0.05) and also decreased [Cl−]i( P &lt; 0.01). We next tested whether NO decreases Na+-K+-2Cl−cotransporter activity by measuring the initial rate of Na+transport. In the presence of SPM in the bath, initial rates of Na+entry were 49.6 ± 6.0% slower compared with control rates ( P &lt; 0.05). To determine whether NO inhibits apical K+channel activity, we measured the change in membrane potential caused by an increase in luminal K+from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+permeability could affect NO-induced inhibition of THAL JCl−. In the presence of luminal valinomycin, which increases K+permeability, addition of SPM decreased THAL JCl−by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (Qo2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased Qo2to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2· mg protein−1· min−1in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased Qo2by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl−by inhibiting Na+-K+-2Cl−cotransport rather than inhibiting apical K+channels or the sodium pump.

NO decreases thick ascending limb chloride absorption by reducing Na(+)-K(+)-2Cl(-) cotransporter activity.

We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption (J(Cl(-))). NaCl transport in the THAL depends on apical Na(+)-K(+)-2Cl(-) cotransporters, apical K(+) channels, and basolateral Na(+)-K(+)-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL J(Cl(-)) by inhibiting the Na(+)-K(+)-2Cl(-) cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na(+)](i)) and chloride concentrations ([Cl(-)](i)) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na(+)](i) from 13.5 +/- 1.2 to 9.6 +/- 1.6 mM (P < 0.05) and also decreased [Cl(-)](i) (P < 0.01). We next tested whether NO decreases Na(+)-K(+)-2Cl(-) cotransporter activity by measuring the initial rate of Na(+) transport. In the presence of SPM in the bath, initial rates of Na(+) entry were 49.6 +/- 6.0% slower compared with control rates (P < 0.05). To determine whether NO inhibits apical K(+) channel activity, we measured the change in membrane potential caused by an increase in luminal K(+) from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K(+) concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K(+) permeability could affect NO-induced inhibition of THAL J(Cl(-)). In the presence of luminal valinomycin, which increases K(+) permeability, addition of SPM decreased THAL J(Cl(-)) by 41.2 +/- 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na(+)-K(+)-ATPase by measuring oxygen consumption rate (QO(2)) in THAL suspensions in the presence of nystatin in varying concentrations of Na(+). In the presence of 10.5 mM Na(+), nystatin increased QO(2) to 119.1 +/- 19.2 and 125.6 +/- 23.4 nmol O(2). mg protein(-1). min(-1) in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na(+), nystatin increased QO(2) by 104 +/- 7 and 94 +/- 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL J(Cl(-)) by inhibiting Na(+)-K(+)-2Cl(-) cotransport rather than inhibiting apical K(+) channels or the sodium pump.