Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration.

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines.

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway.

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca2+-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca2+-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca2+-calcineurin-NFATc1 signaling pathway.

Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca2+) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca2+) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca2+ concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca2+ concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca2+ could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and double messenger system in mice hippocampus and reversal function of procyanidins extracted from lotus seedpod.

This research investigated the influence of extremely low frequency magnetic fields (ELF-MF; 50 Hz, 8 mT, 4 h per day, for 28 days) on calcium ion signaling and the double messenger system in the hippocampus of mice. Messengers that were studied included: G-protein, Ins(1,4,5)P3 (IP3 ), diacylglycerol (DAG), cAMP-dependent protein kinase (PKA), and Ca2+ -dependent protein kinase C (PKC). The results showed that ELF-MF caused an increase in the levels of Gi protein, IP3, DAG, PKA and PKC beta, calcium and calmodulin-dependent protein phosphatase calcineuring (PP2B), and intracellular Ca2+ content, and a decrease in calcium/calmodulin-dependent protein kinase II (CaMK II) and PKC alpha. In addition, ELF-MF exposure decreased the level of brain-derived neurotrophic factor (BDNF), which played a key role in hippocampal neuronal cell death. However, oral administration of procyanidins from lotus seedpod (LSPCs) (especially 90 mg kg-1 ) significantly recovered these changes, and nearly reached normal levels. All these showed that LSPCs may mediate calcium signal and double messenger system through Ca2+ /CaMK II/CREB/BDNF and DG/PKC/MAPK signaling pathways to reverse the alteration caused by ELF-MF. Bioelectromagnetics. 38:436-446, 2017. © 2017 Wiley Periodicals, Inc.

Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc.

ON and OFF pathways in Drosophila motion vision.

Motion vision is a major function of all visual systems, yet the underlying neural mechanisms and circuits are still elusive. In the lamina, the first optic neuropile of Drosophila melanogaster, photoreceptor signals split into five parallel pathways, L1-L5. Here we examine how these pathways contribute to visual motion detection by combining genetic block and reconstitution of neural activity in different lamina cell types with whole-cell recordings from downstream motion-sensitive neurons. We find reduced responses to moving gratings if L1 or L2 is blocked; however, reconstitution of photoreceptor input to only L1 or L2 results in wild-type responses. Thus, the first experiment indicates the necessity of both pathways, whereas the second indicates sufficiency of each single pathway. This contradiction can be explained by electrical coupling between L1 and L2, allowing for activation of both pathways even when only one of them receives photoreceptor input. A fundamental difference between the L1 pathway and the L2 pathway is uncovered when blocking L1 or L2 output while presenting moving edges of positive (ON) or negative (OFF) contrast polarity: blocking L1 eliminates the response to moving ON edges, whereas blocking L2 eliminates the response to moving OFF edges. Thus, similar to the segregation of photoreceptor signals in ON and OFF bipolar cell pathways in the vertebrate retina, photoreceptor signals segregate into ON-L1 and OFF-L2 channels in the lamina of Drosophila.

Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability.

In neurons, the axon initial segment (AIS) is a specialized region near the start of the axon that is the site of action potential initiation. The precise location of the AIS varies across and within different neuronal types, and has been linked to cells' information-processing capabilities; however, the factors determining AIS position in individual neurons remain unknown. Here we show that changes in electrical activity can alter the location of the AIS. In dissociated hippocampal cultures, chronic depolarization with high extracellular potassium moves multiple components of the AIS, including voltage-gated sodium channels, up to 17 mum away from the soma of excitatory neurons. This movement reverses when neurons are returned to non-depolarized conditions, and depends on the activation of T- and/or L-type voltage-gated calcium channels. The AIS also moved distally when we combined long-term LED (light-emitting diode) photostimulation with sparse neuronal expression of the light-activated cation channel channelrhodopsin-2; here, burst patterning of activity was successful where regular stimulation at the same frequency failed. Furthermore, changes in AIS position correlate with alterations in current thresholds for action potential spiking. Our results show that neurons can regulate the position of an entire subcellular structure according to their ongoing levels and patterns of electrical activity. This novel form of activity-dependent plasticity may fine-tune neuronal excitability during development.

Adaptation to steady light by intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are recently discovered photoreceptors in the mammalian eye. These photoreceptors mediate primarily nonimage visual functions, such as pupillary light reflex and circadian photoentrainment, which are generally expected to respond to the absolute light intensity. The classical rod and cone photoreceptors, on the other hand, mediate image vision by signaling contrast, accomplished by adaptation to light. Experiments by others have indicated that the ipRGCs do, in fact, light-adapt. We found the same but, in addition, have now quantified this light adaptation for the M1 ipRGC subtype. Interestingly, in incremental-flash-on-background experiments, the ipRGC's receptor current showed a flash sensitivity that adapted in background light according to the Weber-Fechner relation, well known to describe the adaptation behavior of rods and cones. Part of this light adaptation by ipRGCs appeared to be triggered by a Ca(2+) influx, in that the flash response elicited in the absence of extracellular Ca(2+) showed a normal rising phase but a slower decay phase, resulting in longer time to peak and higher sensitivity. There is, additionally, a prominent Ca(2+)-independent component of light adaptation not typically seen in rods and cones or in invertebrate rhabdomeric photoreceptors.

UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.

Human skin is constantly exposed to solar ultraviolet radiation (UVR), the most prevalent environmental carcinogen. Humans have the unique ability among mammals to respond to UVR by increasing their skin pigmentation, a protective process driven by melanin synthesis in epidermal melanocytes. The molecular mechanisms used by melanocytes to detect and respond to long-wavelength UVR (UVA) are not well understood. We recently identified a UVA phototransduction pathway in melanocytes that is mediated by G protein-coupled receptors and leads to rapid calcium mobilization. Here we report that in human epidermal melanocytes physiological doses of UVR activate a retinal-dependent current mediated by transient receptor potential A1 (TRPA1) ion channels. The TRPA1 photocurrent is UVA-specific and requires G protein and phospholipase C signaling, thus contributing to UVA-induced calcium responses to mediate downstream cellular effects and providing evidence for TRPA1 function in mammalian phototransduction. Remarkably, TRPA1 activation is required for the UVR-induced and retinal-dependent early increase in cellular melanin. Our results show that TRPA1 is essential for a unique extraocular phototransduction pathway in human melanocytes that is activated by physiological doses of UVR and results in early melanin synthesis.

Phosphorylation of rat melanopsin at Ser-381 and Ser-398 by light/dark and its importance for intrinsically photosensitive ganglion cells (ipRGCs) cellular Ca2+ signaling.

The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca(2+) response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca(2+) response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.

Low-dose photon irradiation alters cell differentiation via activation of hIK channels.

To understand the impact of ionizing irradiation from diagnostics and radiotherapy on cells, we examined K(+) channel activity before and immediately after exposing cells to X-rays. Already, low dose in the cGy range caused in adenocarcinoma A549 cells within minutes a hyperpolarization following activation of the human intermediate-conductance Ca(2+)-activated K(+) channel (hIK). The response was specific for cells, which functionally expressed hIK channels and in which hIK activity was low before irradiation. HEK293 cells, which do not respond to X-ray irradiation, accordingly develop a sensitivity to this stress after heterologous expression of hIK channels. The data suggest that hIK activation involves a Ca(2+)-mediated signaling cascade because channel activation is suppressed by a strong cytosolic Ca(2+) buffer. The finding that an elevation of H2O2 causes an increase in the concentration of cytosolic Ca(2+) suggests that radicals, which emerge early in response to irradiation, trigger this Ca(2+) signaling cascade. Inhibition of hIK channels by specific blockers clotrimazole and TRAM-34 slowed cell proliferation and migration in "wound" scratch assays; ionizing irradiation, in turn, stimulated the latter process presumably via its activation of the hIK channels. These data stress an indirect radiosensitivity of hIK channels with an impact on cell differentiation.

An essential role of NAD(P)H oxidase 2 in UVA-induced calcium oscillations in mast cells.

Solar UVA radiation (320-400 nm) is known to have immunomodulatory effects, but the detailed mechanisms involved are not fully elucidated. UVA irradiation has been shown to induce calcium oscillations in rat peritoneal mast cells due to NAD(P)H oxidase (NOX) activation, but the specific NOX isoforms have not been identified. In the present work effects of UVA irradiation were investigated in isolated rat peritoneal mast cells, in cultured rat mast cell line RBL-2H3, and in mouse bone marrow-derived mast cells (BMMC). It was found that UVA irradiation by alternate 340/380 nm (3.2-5.6 µW cm(-2)) or by LED (380 nm, 80 µW cm(-2)) induced calcium oscillations in isolated rat peritoneal mast cells, in RBL-2H3, and in BMMC. Such UVA-induced calcium oscillations resembled closely those induced by surface IgE receptor (FcεRI) activation. It was found that RBL-2H3 expressed high levels of gp91(phox) (NOX2), p22(phox), p67(phox), p47(phox), p40(phox), Rac1, Rac2, moderate levels of DUOX2, but did not express NOX1, NOX3, NOX4, or DUOX1. The specific cellular localizations of gp91(phox) (NOX2), p22(phox), p47(phox), p67(phox), p40(phox) and Rac1/2 were confirmed by immunocytochemistry. UVA-induced reactive oxygen species (ROS) production in RBL-2H3 was completely suppressed by the NOX inhibitor diphenyleneiodonium chloride (DPI) or by the antioxidant N-acetyl-l-cysteine (NAC). siRNA suppression of gp91(phox) (NOX2), p22(phox) and p47(phox) expression inhibited markedly UVA-induced calcium oscillations, ROS and IL-6/LTC4 production in RBL-2H3. Taken together these data indicate that NOX2 plays an essential role in UVA irradiation-induced calcium oscillations, ROS and mediator production in mast cells.

Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields.

We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30 min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50 Hz sine wave) at three magnetic flux densities (5 µT, 300 µT, and 500 µT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50 Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes.

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca(2+)]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca(2+)]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.

Pulsed infrared radiation excites cultured neonatal spiral and vestibular ganglion neurons by modulating mitochondrial calcium cycling.

Cochlear implants are currently the most effective solution for profound sensorineural hearing loss, and vestibular prostheses are under development to treat bilateral vestibulopathies. Electrical current spread in these neuroprostheses limits channel independence and, in some cases, may impair their performance. In comparison, optical stimuli that are spatially confined may result in a significant functional improvement. Pulsed infrared radiation (IR) has previously been shown to elicit responses in neurons. This study analyzes the response of neonatal rat spiral and vestibular ganglion neurons in vitro to IR (wavelength = 1,863 nm) using Ca(2+) imaging. Both types of neurons responded consistently with robust intracellular Ca(2+) ([Ca(2+)]i) transients that matched the low-frequency IR pulses applied (4 ms, 0.25-1 pps). Radiant exposures of ∼637 mJ/cm(2) resulted in continual neuronal activation. Temperature or [Ca(2+)] variations in the media did not alter the IR-evoked transients, ruling out extracellular Ca(2+) involvement or primary mediation by thermal effects on the plasma membrane. While blockage of Na(+), K(+), and Ca(2+) plasma membrane channels did not alter the IR-evoked response, blocking of mitochondrial Ca(2+) cycling with CGP-37157 or ruthenium red reversibly inhibited the IR-evoked [Ca(2+)]i transients. Additionally, the magnitude of the IR-evoked transients was dependent on ryanodine and cyclopiazonic acid-dependent Ca(2+) release. These results suggest that IR modulation of intracellular calcium cycling contributes to stimulation of spiral and vestibular ganglion neurons. As a whole, the results suggest selective excitation of neurons in the IR beam path and the potential of IR stimulation in future auditory and vestibular prostheses.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

ROS and SOCE: recent advances and controversies in the regulation of STIM and Orai.

Store-operated Ca(2+) entry (SOCE) is a widespread mechanism in cells to raise cytosolic Ca(2+) and to refill Ca(2+) stores. T cells critically rely on SOCE mediated by stromal interaction molecules (STIM) and Orai molecules for their activation and regulation of gene transcription; cells such as muscle cells, neurons or melanocytes probably utilize SOCE for the transmission of inducible receptor-mediated function as well as for generalized Ca(2+) homeostasis mechanisms. Exposure to environmental or cell-intrinisic reactive oxygen species (ROS) can affect several components involved in Ca(2+) homeostasis and thus alter multiple pathways. While all cells have a capacity to produce intracellular ROS, exposure of immune and skin cells to extracellular oxidative stress is particularly high during inflammation and/or with UV exposure. This review briefly summarizes cell-intrinsic sources of ROS and focuses on current findings and controversies regarding the regulation of STIM and Orai by oxidative modifications. We also introduce melanocytes as a new model system to study the function of STIM and Orai isoforms under physiological conditions that include exposure to UV light as an activating stimulus.

Suppression of Ca2+ signaling in a mouse model of Best disease.

Mutations in BEST1, encoding bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited macular degeneration characterized by a diminished electrooculogram light peak (LP), lipofuscin in retinal pigment epithelial cells (RPE), and fluid- and debris-filled retinal detachments. To understand the pathogenesis of BVMD we generated knock-in mice carrying the BVMD-causing mutation W93C in Best1. Both Best1(+/W93C)and Best1(W93C/W93C) mice had normal ERG a- and b-waves, but exhibited an altered LP luminance response reminiscent of that observed in BVMD patients. Morphological analysis identified fluid- and debris-filled retinal detachments in mice as young as 6 months of age. By 18-24 months of age Best1(+/W93C)and Best1(W93C/W93C) mice exhibited enhanced accumulation of lipofuscin in the RPE, and a significant deposition of debris composed of unphagocytosed photoreceptor outer segments and lipofuscin granules in the subretinal space. Although Best1 is thought to function as a Ca(2+)-activated Cl(-) channel, RPE cells from Best1(W93C) mice exhibited normal Cl(-) conductances. We have previously shown that Best1(-/-) mice exhibit increased [Ca(2+)](i) in response to ATP stimulation. However, ATP-stimulated changes in [Ca(2+)](i) in RPE cells from Best1(+/W93C) and Best1(W93C/W93C) mice were suppressed relative to Best1(+/+) littermates. Based on these data we conclude that mice carrying the Best1(W93C) mutation are a valid model for BVMD. Furthermore, these data suggest that BVMD is not because of Best1 deficiency, as the phenotypes of Best1(+/W93C) and Best1(W93C/W93C) mice are distinct from that of Best1(-/-) mice with regard to lipofuscin accumulation, and changes in the LP and ATP Ca(2+) responses.

Effects of depletion of glutathione on abscisic acid- and methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

Glutathione (GSH) is involved in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we examined the effects of GSH-decreasing chemicals, p-nitrobenzyl chloride (PNBC), iodomethane (IDM), and ethacrynic acid (EA), on ABA- and MeJA-induced stomatal closure in Arabidopsis. Treatments with PNBC, IDM, and EA decreased GSH contents in guard cells. Depletion of GSH by PNBC and IDM enhanced ABA- and MeJA-induced stomatal closure and inhibition of light-induced stomatal opening by ABA, whereas EA did not enhance either ABA- and MeJA-induced stomatal closure or inhibition of light-induced stomatal opening by ABA. Depletion of GSH did not significantly increase the production of the reactive oxygen species (ROS), cytosolic alkalization, or cytosolic Ca(2+) oscillation induced by ABA and MeJA. These results indicate that depletion of GSH enhances ABA- and MeJA-induced stomatal closure without affecting ROS production, cytosolic alkalization, or cytosolic Ca(2+) oscillation in guard cells of Arabidopsis.