Membrane Potential-sensitive Dye Research Articles

A pharmacodynamic comparison of piperidine and pyridine alkaloid actions at fetal muscle‐type nicotinic acetylcholine receptors (nAChR)

Piperidine and pyridine alkaloids are found in many species of plants including Lobelia spp., Conium spp., Nicotiana spp., and Lupinus spp. Some of these alkaloids cause multiple congenital contracture deformities (MCC) and cleft palates in cattle, pigs, sheep, and goats. The mechanism behind MCC defect formation in livestock is thought to be the inhibition of fetal movement due to stimulation followed by desensitization of fetal muscle‐type nAChR. A pharmacodynamic comparison of piperidine and pyridine alkaloids was made in TE‐671 cells that express fetal muscle‐type nAChR. TE‐671 cells were cultured in DMEM supplemented with fetal bovine serum and penicillin/streptomycin. The membrane depolarization responses were measured by changes in fluorescence of a membrane potential‐sensitive dye. Alkaloids with a carbon side‐chain of at least three carbons or larger attached to the carbon alpha to the piperidine nitrogen were more potent and effective. The presence of a double bond adjacent to the nitrogen of the piperidine or pyridine ring increased potency. The rank order of potency was: anabasine > nicotine > coniine > lobeline > myosmine > ammodendrine. This research supported by ARS‐USDA.

Varenicline is a potent agonist of the human 5-hydroxytryptamine3 receptor.

Varenicline, a widely used and successful smoking cessation agent, acts as a partial agonist at nicotinic acetylcholine receptors. Here, we explore the effects of varenicline at human and mouse 5-Hydroxytryptamine(3) (5-HT(3)) receptors. Application of varenicline to human 5-HT(3) receptors expressed in Xenopus laevis oocytes reveal it is almost a full agonist (R(max) = 80%) with an EC(50) (5.9 μM) 3-fold higher than 5-HT. At mouse 5-HT(3) receptors varenicline is a partial agonist (R(max) = 35%) with an EC(50) (18 μM) 20-fold higher than 5-HT. Displacement of the competitive 5-HT(3) receptor antagonist [(3)H]granisetron reveals similar IC(50) values for varenicline at mouse and human receptors expressed in human embryonic kidney 293 cells, although studies in these cells using a membrane potential-sensitive dye show that again varenicline is a 4- or 35-fold less potent agonist than 5-HT in human and mouse receptors, respectively. Thus the data suggest that the efficacy, but not the affinity, of varenicline is greater at human 5-HT(3) receptors compared with mouse. Docking studies provide a possible explanation for this difference, because they suggest distinct orientations of the ligand in the mouse versus human 5-HT(3) agonist binding sites. Additional binding selectivity studies in a broad panel of recombinant receptors and enzymes confirmed an interaction with 5-HT(3) receptors but revealed no additional interactions of varenicline. Therefore, activation of human 5-HT(3) receptors may be responsible for some of the side effects that preclude use of higher doses during varenicline treatment.

HTDP-2, a New Synthetic Compound, Inhibits Glutamate Release through Reduction of Voltage-Dependent Ca2+ Influx in Rat Cerebral Cortex Nerve Terminals

Aim: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism. Methods: The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²⁺ indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²⁺ concentrations ([Ca²⁺]_c). Results: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²⁺]_c. Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP37157. Conclusion: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²⁺ channel activity and, in so doing, inhibits the evoked glutamate release.

Potentiation of the actions of acetylcholine, epibatidine, and nicotine by methyllycaconitine at fetal muscle-type nicotinic acetylcholine receptors

Methyllycaconitine (MLA) is a norditerpenoid alkaloid found in high abundance in toxic Delphinium (larkspur) species. It is a potent and selective antagonist of α7-nicotinic acetylcholine receptors, but has not been well investigated for activity aside from receptor antagonism. The aim of this study was to investigate the effects of MLA alone and in combination with acetylcholine, epibatidine, nicotine, and neostigmine for actions other than receptor antagonism in TE-671 cells expressing (α1)2β1γδ nicotinic acetylcholine receptors. Ligand activity was assessed through measurements of membrane potential changes in TE-671 cells using a fluorescent membrane potential-sensitive dye and normalized to the maximum response to epibatidine (10μM). MLA was ineffective in changing cell membrane potential in the absence of other receptor agonists. However at nanomolar concentrations, it acted as a co-agonist to potentiate TE-671 cell responses to acetylcholine, epibatidine, nicotine, and neostigmine. These results suggest that the poisoning of cattle by norditerpenoid alkaloids found in larkspur may be more complex than previously determined.

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.

Targeting GIRK Channels for the Development of New Therapeutic Agents

G protein-coupled inward rectifier K+ (GIRK) channels represent novel targets for the development of new therapeutic agents. GIRK channels are activated by a large number of G protein-coupled receptors (GPCRs) and regulate the electrical activity of neurons, cardiac myocytes, and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the patho-physiology of neuropathic pain, drug addiction, cardiac arrhythmias, and other disorders. However, the pharmacology of these channels remains largely unexplored. In this paper we describe the development of a screening assay for identifying new modulators of neuronal and cardiac GIRK channels. Pituitary (AtT20) and cardiac (HL-1) cell lines expressing GIRK channels were cultured in 96-well plates, loaded with oxonol membrane potential-sensitive dyes and measured using a fluorescent imaging plate reader. Activation of the endogenous GPCRs in the cells caused a rapid, time-dependent decrease in the fluorescent signal; indicative of K+ efflux through the GIRK channels (GPCR stimulation versus control, Z′-factor = 0.5–0.7). As expected this signal was inhibited by addition of Ba2+ and the GIRK channel toxin tertiapin-Q. To test the utility of the assay for screening GIRK channel blockers, cells were incubated for 5 min with a compound library of Na+ and K+ channel modulators. Ion transporter inhibitors such as 5-(N,N-hexamethylene)-amiloride and SCH-28080 were identified as blockers of the GIRK channel at sub-micromolar concentrations. Thus, the screening assay will be useful for expanding the limited pharmacology of the GIRK channel and in developing new agents for the treatment of GIRK channelopathies.

Isolation and functional characterization of pericytes derived from hamster skeletal muscle

At the interface of tissue and capillaries, pericytes (PC) may generate electrical signals to be conducted along the skeletal muscle vascular network, but they are functionally not well characterized. We aimed to isolate and cultivate muscle PC allowing to analyse functional properties considered important for signal generation and conduction. Pericytes were enzymatically isolated from hamster thigh muscles and further selected during a 16-30 days' cultivation period. PC markers were studied by fluorescence activated cell scanning (FACS) and immunocytochemistry. Electrical properties of the cultured PC were investigated by patch clamp technique as well as the membrane potential sensitive dye DiBAC(4) (3). The cultured cells showed typical PC morphology and were positive for NG2, alpha smooth muscle actin, PDGFR-β and the gap junction protein Cx43. Expressions of at least one single or combinations of several markers were found in 80-90% of subpopulations. A subset of the patched cells expressed channel activities consistent with a Kv1.5 channel. In vivo presence of the channels was confirmed in sections of hamster thigh muscles. Interleukin-8, a myokine known to be released from exercising muscle, increased the expression but not the activity of this channel. Pharmacologic stimulation of the channel activity by flufenamic acid induced hyperpolarization of PC alone but not of endothelial cells [human umbilical vein endothelial cells (HUVEC)] alone. However, hyperpolarization was observed in HUVEC adjacent to PC when kept in co-culture. We established a culture method for PC from skeletal muscle. A first functional characterization revealed properties which potentially enable these cells to generate hyperpolarizing signals and to communicate them to endothelial cells.

A Real-Time Screening Assay for GIRK1/4 Channel Blockers

The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.

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.

The actions of piperidine alkaloids at fetal muscle‐type and autonomic‐type nicotinic acetylcholine receptors

Piperidine alkaloids are found in many species of plants including Conium maculatum, Nicotiana spp., and Lupinus spp. A pharmacodynamic comparison was made of the alkaloids ammodendrine, anabasine, anabaseine, and coniine in; SH‐SY5Y cells which express autonomic‐type nicotinic acetylcholine receptors (nAChR), and in TE‐671 cells which express fetal muscle‐type nAChR. Both cell lines were cultured in DMEM supplemented with fetal bovine serum and penicillin/streptomycin. Membrane depolarization responses were measured by changes in fluorescence of a membrane potential‐sensitive dye. The alkaloids and their enantiomers were more effective in depolarizing TE‐671 cells relative to SH‐SY5Y cells. The rank order of potency in TE‐671 cells was: anabaseine > (+)‐anabasine > (−)‐anabasine > (±)‐anabasine > (−)‐coniine > (±)‐coniine > (+)‐coniine > (±)‐ammodendrine > (+)‐ammodendrine. The rank order potency in SH‐SY5Y cells was: anabaseine > (+)‐anabasine > (−)‐coniine > (+)‐coniine > (+)‐ammodendrine > (−)‐anabasine > (±)‐coniine > (±)‐anabasine > (−)‐ammodendrine. The actions of these alkaloids at nAChRs in both cell lines could be distinguished by their maximum effects in depolarizing cell membrane potential. It is possible that the mechanism behind the toxic potential of these compounds is the activation of muscle‐type nAChR followed by desensitization. Research supported by ARS‐USDA.

The effect of GHRH antagonists on human glioblastomas and their mechanism of action

The effects of new growth hormone-releasing hormone (GHRH) antagonists JMR-132 and MIA-602 and their mechanism of action were investigated on 2 human glioblastoma cell lines, DBTRG-05 and U-87MG, in vitro and in vivo. GHRH receptors and their main splice variant, SV1 were found on both cell lines. After treatment with JMR-132 or MIA-602, the cell viability decreased significantly. A major decrease in the levels of phospho-Akt, phospho-GSK3β and phosho-ERK 1/2 was detected at 5 and 10 min following treatment with the GHRH antagonists, whereas elevated levels of phospho-p38 were observed at 24 hr. The expression of caspase-3 and poly(ADP-ribose) (PARP), as the downstream executioners of apoptosis were found to be significantly elevated after treatment. Following treatment of the glioblastoma cells with GHRH antagonists, nuclear translocation of apoptosis inducing factor (AIF) and Endonuclease G (Endo G) and the mitochondrial release of cytochrome c (cyt c) were detected, indicating that the cells were undergoing apoptosis. In cells treated with GHRH antagonists, the collapse of the mitochondrial membrane potential was shown with fluorescence microscopy and JC-1 membrane potential sensitive dye. There were no significant differences between results obtained in DBTRG-05 or U-87MG cell lines. After treatment with MIA-602 and JMR-132, the reduction rate in the growth of DBTRG-05 glioblastoma, xenografted into nude mice, was significant and tumor doubling time was also significantly extended when compared with controls. Our study demonstrates that GHRH antagonists induce apoptosis through key proapoptotic pathways and shows the efficacy of MIA-602 for experimental treatment of glioblastoma.

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.

A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential–Sensitive Dye and FLIPR

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

Potential-modulated fluorescence spectroscopy of the membrane potential-sensitive dye di-4-ANEPPS at the 1,2-dichloroethane/water interface

The spectrofluorometric behavior of a membrane potential-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta-{2-(di-N-butylamino)-6-naphthyl}vinyl]pyridinium betaine (di-4-ANEPPS), at the polarized 1,2-dichloroethane/water interface was studied by means of potential-modulated fluorescence (PMF) spectroscopy. The results, combined with those from cyclic and alternating current voltammetry, clearly suggested that the dye adsorbed at the interface underwent a reorientation with increasing the interfacial potential, giving a well-developed PMF response as well as a voltammetric response. In addition to the PMF response, another PMF response was observed by addition of dilauroyl phosphatidylcholine (DLPC). This additional response was well explained in terms of a reorientation of di-4-ANEPPS at the interface, which would be induced by the potential-dependent desorption of DLPC from the interface. Thus, the present study supported the reorientation/solvatochromic mechanism for the membrane potential-sensitive dye rather than the electrochromic mechanism.

Distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

Mitochondria play a vital role during oocyte maturation, fertilization, and embryo development. In this study, confocal microscopy with the mitochondrial membrane potential–sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) was used to investigate mitochondria distribution and activity of stage III zebrafish ovarian follicles. To support the mitochondrial origin of the fluorescence obtained by JC-1, a second mitochondrial probe, MitoTracker Green FM, was used. Cryo-scanning and transmission electron microscopy were also used to validate the distribution and localization of mitochondria obtained by mitochondrial staining. The mitochondrial probes were unable to penetrate the oocyte, and as a result it was not possible to observe stained mitochondria in the oocyte cytoplasm. However, mitochondrial staining of the granulosa cell layer surrounding the stage III zebrafish oocyte exhibited a contiguous aggregation pattern of mitochondria. Cryo-scanning electron microscopy studies also showed the oocyte surface to be covered by polygonal patterns of ridges of the same dimensions as the distributional arrangement of mitochondria in the granulosa cells. Though the results suggested the need for defolliculation to assess mitochondrial distribution and activity in the stage III zebrafish oocyte cytoplasm, the findings of this study will contribute to our understanding of oogenesis and folliculogenesis processes in fish.

Fasting enhances the response of arcuate neuropeptide Y-glucose-inhibited neurons to decreased extracellular glucose

Fasting increases neuropeptide Y (NPY) expression, peptide levels, and the excitability of NPY-expressing neurons in the hypothalamic arcuate (ARC) nucleus. A subpopulation of ARC-NPY neurons ( approximately 40%) are glucose-inhibited (GI)-type glucose-sensing neurons. Hence, they depolarize in response to decreased glucose. Because fasting enhances NPY neurotransmission, we propose that during fasting, GI neurons depolarize in response to smaller decreases in glucose. This increased excitation in response to glucose decreases would increase NPY-GI neuronal excitability and enhance NPY neurotransmission. Using an in vitro hypothalamic explant system, we show that fasting enhances NPY release in response to decreased glucose concentration. By measuring relative changes in membrane potential using a membrane potential-sensitive dye, we demonstrate that during fasting, a smaller decrease in glucose depolarizes NPY-GI neurons. Furthermore, incubation in low (0.7 mM) glucose enhanced while leptin (10 nM) blocked depolarization of GI neurons in response to decreased glucose. Fasting, leptin, and glucose-induced changes in NPY-GI neuron glucose sensing were mediated by 5'-AMP-activated protein kinase (AMPK). We conclude that during energy sufficiency, leptin reduces the ability of NPY-GI neurons to sense decreased glucose. However, after a fast, decreased leptin and glucose activate AMPK in NPY-GI neurons. As a result, NPY-GI neurons become depolarized in response to smaller glucose fluctuations. Increased excitation of NPY-GI neurons enhances NPY release. NPY, in turn, shifts energy homeostasis toward increased food intake and decreased energy expenditure to restore energy balance.

Analysis of dye binding by and membrane potential in spores of Bacillus species.

To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential. Staining by four dyes and autofluorescence of B. subtilis spores that lack some (cotE, gerE) or most (cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC6(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC6(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC6(3) bound well to all germinated spores. The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate. The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.

TRPC Channels Function Independently Of STIM1 And Orai1

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor, and calcium channel, respectively, for CRAC channels, channels underlying store-operated Ca2+ entry (SOCE). However, the roles of these proteins in signaling and constructing other channels with biophysical properties distinct from CRAC channels are not known. We examined the hypothesis that STIM1 or Orai1 can interact with and regulate a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signaling of TRPC channels. Specifically, Ca2+ entry seen after carbachol treatment in cells expressing TRPC1, 3, 5, or 6 were not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to published reports. Disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of ICRAC. This suggests that TRPC signaling and STIM1/Orai1 signaling occur in distinct plasma membrane domains. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents, and single channel events recorded in cell-attached configuration from these cells detected a current with a slope conductance of 33.65 pS, similar to that published for TRPC6. Further, RT-PCR analysis of TRPC transcripts in A10 cells revealed the predominant expression of TRPC1 and TRPC6 mRNA. Using a membrane potential-sensitive dye as an assay, we determined that knockdown of either STIM1 or Orai1 had no effect on the function of this AVP-activated current, while store-operated entry was substantially reduced. Thus, both STIM1 and Orai1 appear to be specific molecular components of the ICRAC pathway and in our studies did not influence the function of exogenously or endogenously expressed TRPC channels.

Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3

Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell derived α-defensin HD5 and the major colonic β-defensins HBD-1–3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD-1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteremia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species- but also strain-specific and may be clinically relevant in the host–bacteria interaction influencing mucosal translocation and systemic infection.

HDT-1, a new synthetic compound, inhibits glutamate release in rat cerebral cortex nerve terminals (synaptosomes)

Excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. In this study, we investigated the effect of HDT-1 (3, 4, 4a, 5, 8, 8a-hexahydro-6,7-dimethyl-4a-(phenylsulfonyl)- 2-tosylisoquinolin-1(2H)-one), a novel synthetic compound, on glutamate release in rat cerebrocortical nerve terminals and explored the possible mechanism. The release of glutamate was evoked by the K+ channel blocker 4-aminopyridine (4-AP) or the high external [K+] and measured by one-line enzyme-coupled fluorometric assay. We also determined the loci at which HDT-1 impinges on cerebrocortical nerve terminals by using membrane potentialsensitive dye to assay nerve terminal excitability and depolarization, and Ca2+ indicator Fura-2 to monitor Ca2+ influx. HDT-1 inhibited the release of glutamate evoked by 4-AP and KCl in a concentration-dependent manner. HDT-1 did not alter the resting synaptosomal membrane potential or 4-APevoked depolarization. Examination of the effect of HDT-1 on cytosolic [Ca2+] revealed that the diminution of glutamate release could be attributed to reduction in voltage-dependent Ca2+ influx. Consistent with this, the HDT-1-mediated inhibition of glutamate release was significantly prevented in synaptosomes pretreated with the N- and P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC. In rat cerebrocortical nerve terminals, HDT-1 inhibits glutamate release through a reduction of voltage-dependent Ca2+ channel activity and subsequent decrease of Ca2+ influx into nerve terminals, rather than any upstream effect on nerve terminal excitability.