Ушкодження кальційзахоплюючої функції мітохондрій при експериментальній хворобі Альцгеймера

Методом двохвильового вимiрювання внутрiшньоклiтинної концентрацiї кальцiю з використанням флуоресцентного барвника fura-2/AM виявлено ушкоджуючий вплив бiлка β-амiлоїду (основного компонента сенiльних бляшок при хворобi Альцгеймера) на кальцiйзахоплюючу функцiю мiтохондрiй нейронiв культури гiпокампа щурiв.Damaging action of β-amyloid peptide (main component of senile plaques in Alzheimer’s disease) on the calcium uptake function of mitochondria of rat hippocampal cell culture has been revealed by means of the calcium microfluorometry method using fluorescent dye fura-2/AM

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells

Peripheral nerve injury increases glutamate-evoked calcium mobilization in adult spinal cord neurons

BACKGROUND: Central sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain. RESULTS: Bath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals. CONCLUSIONS: Bulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn

Data on the Lignosus rhinocerotis water soluble sclerotial extract affecting intracellular calcium level in rat dorsal root ganglion cells

The data in this article contain supporting evidence for the research manuscript entitled “Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry” by Lee et al. (2018) [1]. The data were obtained by calcium imaging technique with fluorescent calcium indicator dyes, Fura 2-AM, to visualize calcium ion movement in the rat dorsal ganglion (DRG) cells. The effects of L. rhinocerotis cold water extract (CWE1) on intracellular calcium levels in the DRG cells were presented. Keywords: Lignosus rhinocerotis, Medicinal mushroom, Bronchodilators, Calcium dynamic

Phagosome-lysosome fusion is a calcium-independent event in macrophages.

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium

TNFα induces Ca2+ influx to accelerate extrinsic apoptosis in hepatocellular carcinoma cells

BACKGROUND: Tumor necrosis factor-α has been proven an effective anticancer agent in preclinical studies. However, the translation of TNFα from research to clinic has been blocked by significant systemic toxicity and limited efficacy at maximal tolerated dose, which need urgently to be solved. METHODS: The level of cytosolic Ca RESULTS: Here, we demonstrated that TNFα induced extracellular Ca CONCLUSIONS: Our study provides the evidence supporting a novel mechanism by which TNFα induces extracellular C

TRPV4 Contributes to Resting Membrane Potential in Retinal Müller Cells: Implications in Cell Volume Regulation

Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swelling is followed by a regulatory volume decrease (RVD), partially mediated by an efflux of KCl and water. The transient receptor potential channel 4 (TRPV4), a nonselective calcium channel, has been proposed as a candidate for mediating intracellular Ca2+ elevation induced by swelling. We previously demonstrated in a human Müller cell line (MIO-M1) that RVD strongly depends on ion channel activation and, consequently, on membrane potential (Vm ). The aim of this study was to investigate if Ca2+ influx via TRPV4 contributes to RVD by modifying intracellular Ca2+ concentration and/or modulating Vm in MIO-M1 cells. Cell volume, intracellular Ca2+ levels, and Vm changes were evaluated using fluorescent probes. Results showed that MIO-M1 cells express functional TRPV4 which determines the resting Vmassociated with K+ channels. Swelling-induced increases in Ca2+ levels was due to both Ca2+ release from intracellular stores and Ca2+ influx by a pathway alternative to TRPV4. TRPV4 blockage affected swelling-induced biphasic response (depolarization-repolarization), suggesting its participation in modulating Vm changes during RVD. Agonist stimulation of Ca2+ influx via TRPV4 activated K+ channels hyperpolarizing Vm and accelerating RVD. We propose that TRPV4 forms a signaling complex with Ca2+ and/or voltage-dependent K+ channels to define resting Vm and Vm changes during RVD. TRPV4 involvement in RVD depends on the type of stimuli and/or degree of channel activation, leading to a maximum RVD response when Ca2+ influx overcomes a threshold and activates further signaling pathways in cell volume regulation.Fil: Netti, Vanina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Fernández, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Kalstein, Maia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Pizzoni, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Di Giusto, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Rivarola, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Biomembranas; Argentin

Constitutive IP₃ signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP₃ receptor disruptor BIRD-2

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2′s BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy