Fura-2 Dotaz Zobrazit nápovědu
Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the NMDA receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. In this study, we analyzed the effects of PS on calcium signaling in cultured hippocampal neurons and HEK293 cells expressing NMDAR. The cells were loaded with the Ca(2+) sensor Fura-2. In agreement with previous electrophysiological experiments, PS potentiated the increases in intracellular Ca(2+) induced by an exogenous application of glutamate; however, PS also increased intracellular Ca(2+) in the absence of exogenous NMDA agonist. The agonist-independent effect of PS was induced in all neurons studied and in HEK293 cells expressing GluN1/GluN2A-B receptors in a neurosteroid-specific manner. We conclude that PS is an endogenous NMDA agonist that activates the GluN1/GluN2A-B receptors.
- MeSH
- gating iontového kanálu fyziologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- pregnenolon metabolismus MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- vápník metabolismus MeSH
- vápníková signalizace fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Natural proteins can be used in measuring intracellular Ca(2+) concentration. As one of the Ca(2+)- regulated photoproteins, aequorin has several advantages in comparison to widely used Ca(2+) fluorescence indicators (e.g., fura-2, indo-1 and fluo-3), including high dynamic range and resistance to motion artefacts. However, incorporation of aequorin into cells remains a challenge. Hypoosmotic shock treatment was optimized and used as a method for loading aequorin into the cytoplasm of follicular lymphoma cells. Measurement of aequorin luminescence in the cells was performed using a luminometer with a sensitive photomultiplier tube and the luminescence intensity was recalculated into intracellular [Ca(2+)]. The value of (0.85 ± 0.52)·10-6 M was found. We show that the optimized method of incorporation was effective for loading aequorin into follicular lymphoma cells in vitro. The cell viability remains high immediately after the procedure. This method can also be used for measuring intracellular Ca(2+) concentration in other types of non-adherent cells.
- MeSH
- aequorin metabolismus MeSH
- aniliny metabolismus MeSH
- folikulární lymfom metabolismus MeSH
- indoly metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- vápník metabolismus MeSH
- viabilita buněk fyziologie MeSH
- xantheny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In ischemic/reperfusion (I/R) injured hearts, severe oxidative stress occurs and is associated with intracellular calcium (Ca(2+)) overload. Glucagon-Like Peptide-1 (GLP-1) analogues have been shown to exert cardioprotection in I/R heart. However, there is little information regarding the effects of GLP-1 analogue on the intracellular Ca(2+) regulation in the presence of oxidative stress. Therefore, we investigated the effects of GLP-1 analogue, (liraglutide, 10 microM) applied before or after hydrogen peroxide (H(2)O(2), 50 microM) treatment on intracellular Ca(2+) regulation in isolated cardiomyocytes. We hypothesized that liraglutide can attenuate intracellular Ca(2+) overload in cardiomyocytes under H(2)O(2)-induced cardiomyocyte injury. Cardiomyocytes were isolated from the hearts of male Wistar rats. Isolated cardiomyocytes were loaded with Fura-2/AM and fluorescence intensity was recorded. Intracellular Ca(2+) transient decay rate, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) levels were recorded before and after treatment with liraglutide. In H(2)O(2) induced severe oxidative stressed cardiomyocytes (which mimic cardiac I/R) injury, liraglutide given prior to or after H(2)O(2) administration effectively increased both intracellular Ca(2+) transient amplitude and intracellular Ca(2+) transient decay rate, without altering the intracellular diastolic Ca(2+) level. Liraglutide attenuated intracellular Ca(2+) overload in H(2)O(2)-induced cardiomyocyte injury and may be responsible for cardioprotection during cardiac I/R injury by preserving physiological levels of calcium handling during the systolic and diastolic phases of myocyte activation.
- MeSH
- hypoglykemika farmakologie MeSH
- intracelulární tekutina účinky léků metabolismus MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- liraglutid farmakologie MeSH
- oxidační stres účinky léků fyziologie MeSH
- peroxid vodíku toxicita MeSH
- potkani Wistar MeSH
- vápníková signalizace účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Previous results have suggested that orexins causes a rise of intracellular free calcium ([Ca2+]i) in cultured rat dorsal root ganglion (DRG) neurons, implicating a role in nociception, but the underlying mechanism is unknown. Hence, the aim of the present study was to investigate whether the orexins-mediated signaling involves the PKC pathways in these sensory neurons. Cultured DRG neurons were loaded with 1 µmol Fura-2 AM and [Ca2+]i responses were quantified by the changes in 340/380 ratio using fluorescence imaging system. The orexin-1 receptor antagonist SB-334867-A (1 µM) inhibited the calcium responses to orexin-A and orexin-B (59.1±5.1 % vs. 200 nM orexin-A, n=8, and 67±3.8 % vs. 200 nM orexin-B, n=12, respectively). The PKC inhibitor chelerythrine (10 and 100 µM) significantly decreased the orexin-A (200 nM)-induced [Ca2+]i increase (59.4±4.8 % P<0.01, n=10 and 4.9±1.6 %, P<0.01, n=9) versus response to orexin-A). It was also found that chelerythrine dose-dependently inhibited the [Ca2+]i response to 200 nM orexin-B. In conclusion, our results suggest that orexins activate intracellular calcium signaling in cultured rat sensory neurons through PKC-dependent pathway, which may have important implications for nociceptive modulation and pain.
- MeSH
- benzoxazoly farmakologie MeSH
- bolest metabolismus MeSH
- financování organizované MeSH
- intracelulární signální peptidy a proteiny farmakologie metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- močovina analogy a deriváty farmakologie MeSH
- nervové receptory cytologie enzymologie účinky léků MeSH
- neuropeptidy farmakologie metabolismus MeSH
- neurotransmiterové látky farmakologie MeSH
- nociceptory metabolismus účinky léků MeSH
- potkani Wistar MeSH
- proteinkinasa C metabolismus MeSH
- spinální ganglia cytologie MeSH
- vápníková signalizace fyziologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Dipeptidyl peptidase-IV (DPP-IV, CD26) is a serine protease almost ubiquitously expressed on cell surface and present in body fluids. DPP-IV has been suggested to proteolytically modify a number of biologically active peptides including substance P (SP) and the chemokine stromal cell derived factor-1alpha (SDF-1alpha, CXCL12). SP and SDF-1alpha have been implicated in the regulation of multiple biological processes and also induce responses that may be relevant for glioma progression. Both SP and SDF-1alpha are signaling through cell surface receptors and use intracellular calcium as a second messenger. The effect of DPP-IV on intracellular calcium mobilization mediated by SP and SDF-1alpha was monitored in suspension of wild type U373 and DPP-IV transfected U373DPPIV glioma cells using indicator FURA-2. Nanomolar concentrations of SP triggered a transient dose dependent increase in intracellular calcium rendering the cells refractory to repeated stimulation, while SDF-1 had no measurable effect. SP signaling in DPP-IV overexpressing U373DPPIV cells was not substantially different from that in wild type cells. However, preincubation of SP with the DPP-IV overexpressing cells lead to the loss of its signaling potential, which could be prevented with DPP-IV inhibitors. Taken together, DPP-IV may proteolytically inactivate local mediators involved in gliomagenesis.
- MeSH
- chemokin CXCL12 metabolismus MeSH
- dipeptidylpeptidasa 4 analýza farmakologie klasifikace MeSH
- financování organizované MeSH
- gliom enzymologie MeSH
- inhibitory serinových proteinas farmakologie MeSH
- lidé MeSH
- lysin analogy a deriváty farmakologie MeSH
- mifepriston farmakologie MeSH
- nádory mozku enzymologie MeSH
- oligopeptidy farmakologie MeSH
- pyrrolidiny farmakologie MeSH
- substance P metabolismus MeSH
- transfekce MeSH
- U937 buňky MeSH
- vápníková signalizace účinky léků MeSH
- Check Tag
- lidé MeSH
The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.
- MeSH
- adenosintrifosfát farmakologie MeSH
- antagonisté excitačních aminokyselin farmakologie MeSH
- biofyzikální jevy účinky léků MeSH
- blokátory sodíkových kanálů farmakologie MeSH
- GABA farmakologie MeSH
- inhibitory agregace trombocytů farmakologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- messenger RNA metabolismus MeSH
- metoda terčíkového zámku MeSH
- nervový přenos účinky léků MeSH
- nervový útlum účinky léků MeSH
- neurony účinky léků MeSH
- novorozená zvířata MeSH
- nucleus suprachiasmaticus cytologie MeSH
- potkani Wistar MeSH
- purinergní látky farmakologie MeSH
- purinergní receptory P2X genetika metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- synaptické potenciály účinky léků MeSH
- techniky in vitro MeSH
- tetrodotoxin farmakologie MeSH
- vápník metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Given the potential clinical benefit of inhibiting Na+/Ca2+ exchanger (NCX) activity during myocardial ischemia reperfusion (I/R), pharmacological approaches have been pursued to both inhibit and clarify the importance of this exchanger. SEA0400 was reported to have a potent NCX selectivity. Thus, we examined the effect of SEA0400 on NCX currents and I/R induced intracellular Ca2+ overload in mouse ventricular myocytes using patch clamp techniques and fluorescence measurements. Ischemia significantly inhibited inward and outward NCX current (from -0.04±0.01nA to 0 nA at -100 mV; from 0.23±0.08 nA to 0.11±0.03 nA at +50 mV, n=7), Subsequent reperfusion not only restored the current rapidly but enhanced the current amplitude obviously, especially the outward currents (from 0.23±0.08 nA to 0.49±0.12 nA at +50 mV, n=7). [Ca2+]i, expressed as the ratio of Fura-2 fluorescence intensity, increased to 138±7 % (P<0.01) during ischemia and to 210±11 % (P<0.01) after reperfusion. The change of NCX current and the increase of [Ca2+]i during I/R can be blocked by SEA0400 in a dose-dependent manner with an EC50 value of 31 nM and 28 nM for the inward and outward NCX current, respectively. The results suggested that SEA0400 is a potent NCX inhibitor, which can protect mouse cardiac myocytes from Ca2+ overload during I/R injuries.
- MeSH
- aniliny terapeutické užití MeSH
- fenylethery terapeutické užití MeSH
- financování vládou MeSH
- ischemická choroba srdeční etiologie komplikace MeSH
- kardiomyocyty metabolismus MeSH
- metoda terčíkového zámku metody využití MeSH
- myši inbrední C57BL MeSH
- pumpa pro výměnu sodíku a vápníku antagonisté a inhibitory MeSH
- reperfuze myokardu MeSH
- Publikační typ
- srovnávací studie MeSH
