Hypo-osmotic Stress Research Articles

Immobilizing live Escherichia coli for AFM studies of surface dynamics

Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-l-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-l-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-l-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-l-lysine surfaces in a lower ionic strength buffer supplemented with Mg(2+) and Ca(2+) was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-l-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations for membrane stabilization while minimizing binding interference. The data also indicates that monitoring cell viability as a function of sample preparation is important and should be an integral part of the work flow for determining immobilization parameters. A method for immobilizing a less adherent E. coli mutant for AFM imaging in nutrient broth is presented here in addition to a proposed work flow for developing and optimizing immobilization strategies.

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ~30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.

Escherichia coli multiple [Ni–Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs

Escherichia coli evolves H2 via multiple [Ni–Fe]-hydrogenases (Hyd). This activity under hyper- and hypo-osmotic stress was investigated with mutants lacking different Hyd enzymes during glycerol fermentation. Inhibitory effects of hypo-stress on H2 production was stronger at pH 6.5 in wild type and mutants except fhlA, which encodes a transcriptional activator for Hyd-3, compared with the effects of N,N′-dicyclohexylcarbodiimide. These results indicate that Hyd-3 and Hyd-4 are osmosensitive at pH 7.5. Hyd-4 and FhlA are implicated in osmotic stress response at pH 6.5. Hyd-1 and FhlA might be osmosensitive at pH 5.5. Thus, osmosensitivity of Hyd enzymes is a novel property that depends on pH. This is significant for mechanisms of cell osmoregulation and H2 production biotechnology when glycerol is used as a fermentation substrate.

Involvement of the phosphatidylinositol kinase pathway in augmentation of ATP-sensitive K+ channel currents by hypo-osmotic stress in rat ventricular myocytes

The objective of this study was to investigate the mechanisms of increase in the efficacy of ATP-sensitive K(+) channel (KATP) openings by hypo-osmotic stress. The whole-cell KATP currents (IK,ATP) stimulated by 100 μmol/L pinacidil, a K(+) channel opening drug, were significantly augmented during hypo-osmotic stress (189 mOsmol/L) compared with normal conditions (303 mOsmol/L). The EC50 and Emax value for pinacidil-activated IK,ATP (measured at 0 mV) was 154 μmol/L and 844 pA, respectively, in normal solution and 16.6 μmol/L and 1266 pA, respectively, in hypo-osmotic solution. Augmentation of IK,ATP during hypo-osmotic stress was attenuated by wortmannin (50 μmol/L), an inhibitor of phosphatidylinositol 3- and 4-kinases, but not by (i) phalloidin (30 μmol/L), an actin filament stabilizer, (ii) the absence of Ca(2+) from the internal and external solutions, and (iii) the presence of creatine phosphate (3 mmol/L), which affects creatine kinase regulation of the KATP channels. In the single-channel recordings, an inside-out patch was made after approximately 5 min exposure of the myocyte to hypo-osmotic solution. However, the IC50 value for ATP under such conditions was not different from that obtained in normal osmotic solution. In conclusion, hypo-osmotic stress could augment cardiac IK,ATP through intracellular mechanisms involving the phosphatidylinositol kinase pathway.

Role of Epithelial Sodium Channel in the Realization of Homeostatic Effects of Aldosterone on the Volume of Principal Cells of Cortical Collecting Ducts in Rats after Hypoosmotic Stress

The mechanism of aldosterone effects (10 nM) on the volume of the principal cells of cortical collecting ducts after hypoosmotic stress (280/140 mOsm/kg) was studied using fluorescence microscopy. The experiments with intracellular fluorescent dye calcein showed that aldosterone significantly decreases the amplitude and rate of increase in principal cell volume during hypoosmotic stress. Epithelial sodium channel blocker amiloride (10(-5)M) significantly attenuated the effects of aldosterone on the amplitude and rate of changes in cell volume. The obtained data attest to the contribution of epithelial sodium channel to the realization of rapid non-genomic effects of aldosterone on the amplitude and rate of changes in volume of the principal cells of cortical collecting ducts in rat kidney after hypoosmotic stress.

Identification of Proteins for Salt Tolerance Using a Comparative Proteomics Analysis of Tomato Accessions with Contrasting Salt Tolerance

Tomato (Solanum lycopersicum) has a wide variety of genotypes differing in their responses to salinity. This study was performed to identify salt-induced changes in proteomes that are distinguishable among tomatoes with contrasting salt tolerance. Tomato accessions [LA4133 (a salt-tolerant cherry tomato accession) and ‘Walter’ LA3465 (a salt-susceptible accession)] were subjected to salt treatment (200 mm NaCl) in hydroponic culture. Salt-induced changes in the root proteomes of each tomato accession were identified using the isobaric tags for relative and absolute quantitation (iTRAQ) method. In LA4133, 178 proteins showed significant differences between salt-treated and non-treated control root tissues (P ≤ 0.05); 169 proteins were induced (1.3- to 5.1-fold) and nine repressed (–1.7- to –1.3-fold). In LA3465, 115 proteins were induced (1.3- to 6.4-fold) and 23 repressed (–2.5- to –1.3-fold). Salt-responsive proteins from the two tomato accessions were involved in the following biological processes: root system development and structural integrity; carbohydrate metabolism; adenosine-5′-triphosphate regeneration and consumption; amino acid metabolism; fatty acid metabolism; signal transduction; cellular detoxification; protein turnover and intracellular trafficking; and molecular activities for regulating gene transcription, protein translation, and post-translational modification. Proteins affecting diverse cellular activities were identified, which include chaperonins and cochaperonins, heat-shock proteins, antioxidant enzymes, and stress proteins. Proteins exhibiting different salt-induced changes between the tolerant and susceptible tomato accessions were identified, and these proteins were divided into two groups: 1) proteins with quantitative differences because they were induced or repressed by salt stress in both accessions but at different fold levels; and 2) proteins showing qualitative differences, where proteins were induced in one vs. repressed or not changed in the other accession. Candidate proteins for tolerance to salt and secondary cellular stresses (such as hypo-osmotic stress and dehydration) were proposed based on findings from the current and previous studies on tomato and by the use of the Arabidopsis thaliana protein database. Information provided in this report will be very useful for evaluating and breeding for plant tolerance to salt and/or water deficit stresses.

MscS-like Mechanosensitive Channels in Plants and Microbes

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity.

Serotonin receptors take the TRiPV4 highway in chronic hypoxic pulmonary hypertension. Focus on “TRPV4 channel contributes to serotonin-induced pulmonary vasoconstriction and the enhanced vascular reactivity in chronic hypoxic pulmonary hypertension”

pulmonary arterial hypertension (PAH) is a rare human disease displaying a very poor prognosis of survival. The chronic increase in pulmonary vascular resistance associated with PAH eventually leads to right ventricular hypertrophy and failure, and ultimately death. It is well accepted that the

Monocytes and macrophages as biomarkers for the diagnosis of megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells.

Silicon chips detect intracellular pressure changes in living cells

The ability to measure pressure changes inside different components of a living cell is important, because it offers an alternative way to study fundamental processes that involve cell deformation. Most current techniques such as pipette aspiration, optical interferometry or external pressure probes use either indirect measurement methods or approaches that can damage the cell membrane. Here we show that a silicon chip small enough to be internalized into a living cell can be used to detect pressure changes inside the cell. The chip, which consists of two membranes separated by a vacuum gap to form a Fabry-Pérot resonator, detects pressure changes that can be quantified from the intensity of the reflected light. Using this chip, we show that extracellular hydrostatic pressure is transmitted into HeLa cells and that these cells can endure hypo-osmotic stress without significantly increasing their intracellular hydrostatic pressure.

Osmotic stress does trigger brevetoxin production in the dinoflagellate Karenia brevis

Although a number of factors may influence its production, the physiological role of brevetoxin in the dinoflagellate Karenia brevis is still open to debate. Not to be left out of the discussion, Sunda et al. (1) challenge our suggestion that salinity stress may be a possible trigger for brevetoxin production. Their “repeat” of our experiment is not an actual replication (extraction protocols, analytical method, K. brevis isolates, and culturing conditions differed among their three laboratories). Moreover, a number of experimental details, including use of internal standards and preparation of toxin standards, are omitted. Internal standards are important because extraction efficiency varies among samples and could affect results. Nevertheless, both reports show that there is no long-term increase in brevetoxin production after salinity stress (1, 2) and that low-toxin cultures [SP1 (2) and “nontoxic” Wilson (1)] do not increase brevetoxin production in response to hypoosmotic stress.

Müller Cell Reactivity in Response to Photoreceptor Degeneration in Rats with Defective Polycystin-2

BackgroundRetinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2.MethodsAge-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution.FindingsMüller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators.InterpretationCellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.

Differential regulation of TRPC4 expression in the magnocellular PVN and SON by hyperosmotic and hypoosmotic stress

Canonical Transient Receptor Potential Channel 4 (TRPC4) is expressed in the mammalian brain and has been implicated in dendritic neurotransmitter release. Our earlier reports have shown that TRPC4 is also expressed in the vasopressin (AVP) cells of the magnocellular (MNC) supraoptic nucleus (SON) and paraventricular nucleus (PVN). To investigate whether TRPC4 expression is regulated by changes in plasma osmolality, we utilized 2 models of osmotic stress ‐ bile duct ligation (BDL) induced hypoosmolality and water deprivation (WD) induced hyperosmolality. Immunoflourescent (IF) labeled TRPC4 positive cells in the SON was significantly increased after BDL (BDL 87 ± 8; SHAM 63 ± 5) and double IF for TRPC4 and AVP showed increased colocalization in the SON (BDL SON 56 ± 6; SHAM SON 36 ± 5). However, BDL did not influence TRPC4 IF in PVN. In contrast, WD did not change the number of TRPC4 IF cells in either SON or PVN. Laser capture microdissection (LCM) of 7–10 quick immunolabeled AVP neurons for RT‐PCR showed elevated TRPC4 mRNA in the SON of BDL rats, but not in the PVN (BDL 5.3 ± 1; SHAM 1.3 ± 0.6). WD reduced TRPC4 mRNA in SON and increased in PVN. Western blot analysis showed no changes in TRPC4 in either PVN or SON of WD rats, but showed increased TRPC4 in the SON but not PVN of BDL rats (BDL 3 ± 0.4; SHAM 1.8 ± 0.1). These results show that TRPC4 is differentially regulated by hypo and hyper osmotic stress in SON but not PVN. R01 HL062579.

In Vitro Modeling of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy Using Human Induced Pluripotent Stem Cells (hiPSC)

Purpose DMD is the most common an debilitating X-linked muscular dystrophy that leads to devastating skeletal and cardiac muscle damage. DMD cardiomyopathy has now emerged as the leading cause of death in DMD, however there is limited knowledge of pathophysiology of DMD cardiomyopathy. The purpose of this is to utilize patient-specific hiPSC derived cardiomyocytes (CM) to model DMD cardiomyopathy. Methods and Materials Dermal fibroblasts were obtained from a DMD patient with severe cardiomyopathy and a normal healthy control. The fibroblasts were reprogrammed to hiPSC with retrovirus containing the human transcription factors, Oct4, Sox2, Klf4, and c-Myc. The DMD and control hiPSC lines were differentiated to CM using a monolayer, extra cellular matrix, growth factor directed differentiation. Molecular and physiologic assays were performed at day 30 of differentiation. Results The DMD and control hiPSC lines were fully characterized and demonstrated pluripotent markers with qRT-PCR and immunostaining, formed teratomas with all three germ layers, and were karyotypically normal. DMD hiPSC were shown to have absence of dystrophin exons 4-43, consistent with the patient’s mutation. DMD hiPSC differentiated to beating CM sheets and initiation of beating was observed between days 10-14 in both DMD and control hiPSC CM. Increased membrane fragility was seen in d30 DMD hiPSC CM compared to control CM as assayed by hypo-osmotic stress and LDH release. Initial gene expression, demonstrated significantly lower dystrophin expression in DMD hiPSC CM as compared to control hiPSC CM, while there was significantly higher induction of beta-1-adrenergic receptor gene expression in DMD hiPSC CM. Conclusions Our results suggest that DMD hiPSC CM can be differentiated to cardiomyocytes and have a unique phenotype for disease investigation. Further evaluation of the molecular and physiologic phenotype of DMD hiPSC CM will serve as a platform for developing novel therapeutic regimens for patients with muscular dystrophy cardiomyopathies.

FN-f induced catabolic activities are dependent on oxygen tension in chondrocyte/agarose constructs subjected to biomechanical signals

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S211 Figure 1. The percentage of chondrocytes responding with single or multiple [Ca2+]i fluxes in vitro and ex vivo as a function of final osmolarity. Starting osmolarity: 380 mOsm (in vitro), 300 mOsm (ex vivo). n>26 for each osmolarity. Conclusions: More chondrocytes respond with [Ca2+]i flux in vitro compared to ex vivo and those responses are primarily multiple [Ca2+]i fluxes in vitro in contrast to single [Ca2+]i fluxes ex vivo. These findings may indicate a role for the extracellular matrix in i) shielding chondrocytes from the fluid flow occurring during media infusion/withdrawal and/or ii) disabling the chondrocyte ability to harness [Ca2+]i stores that are likely responsible for multiple/oscillating [Ca2+]i flux, perhaps via integrin attachment to the matrix. Interestingly, the effect of hypo-osmotic stress on [Ca2+]i flux was present both in vitro and ex vivo suggesting that this response, likely mediated by transient receptor potential vanilloid 4, is independent of matrix attachment. In contrast, hyper-osmotic stress failed to elicit a [Ca2+]i flux in vitro or ex vivo. Past studies have reported [Ca2+]i flux responses from chondrocytes in vitro exposed to >140 mOsm hyper-osmotic challenge, perhaps suggesting a minimum threshold needed for hyper-osmotic response to occur. In conclusion, we have shown that chondrocyte [Ca2+]i flux response to hypo-osmotic stress is independent of matrix attachment, however the matrix likely shields the chondrocytes from fluid flow, disabling the release of Ca2+ from intracellular stores. 400 FN-F INDUCED CATABOLIC ACTIVITIES ARE DEPENDENT ON OXYGEN TENSION IN CHONDROCYTE/AGAROSE CONSTRUCTS SUBJECTED TO BIOMECHANICAL SIGNALS E. Parker y, B. Pinguuan-Murphy z, D.L. Bader y, T.T. Chowdhury y. yQueen Mary, Univ. of London, London, United Kingdom; zUniv. of Malaya, Kuala

Intracellular calcium response of chondrocytes to hypo-osmotic stress is independent of matrix attachment

Intracellular calcium response of chondrocytes to hypo-osmotic stress is independent of matrix attachment

Comparative analysis of induction of osmotic-stress-dependent genes in Vibrio vulnificus exposed to hyper- and hypo-osmotic stress

Vibrio vulnificus, a halophilic pathogenic bacterium of marine environments, encounters changes in salinity in its natural habitat and in the food-processing environment. The comparative response of V. vulnificus to hyperosmotic and hypoosmotic stress in terms of gene expression was investigated. Genes belonging to the proU operon for transport of compatible solutes and compatible solute synthesis were significantly upregulated (3- to 4.7-fold) under hyperosmotic stress. Under hypoosmotic stress, upregulation of genes coding for mechanosensitive channels of small conductance (mscS) was not observed. In hyperosmotic conditions a 2.3-fold decrease in the expression of aqpZ was observed. A 2-fold induction in gyrA was observed in V. vulnificus cells on exposure to hyperosmotic stress. groEL genes, VVA1659 (1.6-fold), and VV3106 (1-fold) were induced in hypoosmotic condition. Results of this study indicate that to manage hyperosmotic stress, V. vulnificus accumulated osmoprotectants through uptake or through endogenous synthesis of compatible solutes. Expression of mscS may not be necessary for immediate protection in cells exposed to hyper- and hypo-osmotic stress. Comparative analysis of important osmotic-stress-related genes showed up- or down-regulation of 14 genes in hyperosmotic stress as compared with up- or down-regulation of only 7 genes in hypoosmotic stress, indicating that the cells respond asymmetrically to hyper- and hypo-osmotic stress.

Enhanced cell-volume regulation in cyclosporin A cardioprotection

Cyclosporin A (CsA) has been shown to protect against ischaemia/reperfusion injury presumably by its inhibition of mitochondrial permeability transition pore opening through cyclophilin D inhibition. We examine if CsA cardioprotection involves a cell-volume regulatory mechanism. To address this issue, cultured rabbit cardiomyocytes were subjected to the following protocols: (i) cardiomyocytes were treated with 200 nM CsA either given for 10 min followed by 10 min of washout prior to 30 min hypo-osmotic stress (200 mOsm) or administered throughout 75 min simulated ischaemia/60 min simulated reperfusion. Cell necrosis and cell swelling were determined by trypan blue staining and cell-volume measurements, respectively; (ii) SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium) dye loaded cardiomyocytes were treated with 200 nM CsA for 10 min followed by 10 min washout and intracellular Cl(-) concentration measured (Cl(-) efflux); (iii) 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimi-dazolylcarbocyanine iodide(JC-1) loaded cardiomyocytes were treated with 200 nM CsA to inhibit mitochondrial membrane potential (ΔΨm) dissipation (an index of mitochondria permeability transition pore opening) by either valinomycin (2 μM) or ischaemia/reperfusion injury. Cl(-) channels were blocked by indanyloxyacetic acid 94 (IAA-94, 50 μM). CsA not only significantly (P < 0.001) reduced the % of dead cells following simulated ischaemia/reperfusion but it also triggered an efflux of Cl(-), hence enhancing cardiomyocyte cell-volume regulatory response. CsA protection against cell necrosis and its effect on Cl(-) transport/volume regulation were all blocked by IAA-94. IAA-94 had no effect on ΔΨm. These data indicate that CsA protects against cell necrosis at least in part by enhancing cardiomyocyte volume regulation, and not simply by inhibiting MPTP opening.

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

Peptide Hormone Release Monitored From Single Vesicles in “Membrane Lawns” of Differentiated Male Pituitary Cells: SNAREs and Fusion Pore Widening

In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm(2), which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.