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Cytoplasmic Ca2+ Concentration (cytoplasmic + ca2+_concentration)
Selected AbstractsDihydropyridine- and voltage-sensitive Ca2+ entry in human parathyroid cellsEXPERIMENTAL PHYSIOLOGY, Issue 7 2009Keitaro Yokoyama Patch-clamp and fluorescence measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) were performed to directly detect extracellular Ca2+ entry into cultured parathyroid cells from patients with secondary hyperparathyroidism. Cells loaded with fluo-3 AM or fluo-4 AM showed a transient increase in fluorescence (Ca2+ transient) following 10 s exposure to 150 mm K+ solution in the presence of millimolar concentrations of external Ca2+. The Ca2+ transient was completely inactivated after 30,40 s exposure to the high-K+ solution, was reduced by dihydropyridine antagonists and was enhanced by FPL-64176, an L-type Ca2+ channel agonist. The electrophysiological and pharmacological properties of the whole-cell Ca2+ and Ba2+ currents were similar to those of L-type Ca2+ channels. The Ca2+ transients induced by 10 s exposure to 3.0 mm extracellular Ca2+ concentration ([Ca2+]o) were inhibited by dihydropyridine antagonists and were partly inactivated following 30,40 s exposure to the high-K+ solution. These results demonstrate, for the first time, that human parathyroid cells express L-type-like Ca2+ channels that are possibly involved in the [Ca2+]o -induced change in [Ca2+]i. This Ca2+ entry system might provide a compensatory pathway for the negative feedback regulation of parathyroid hormone secretion, especially in hyperplastic conditions in which the Ca2+ -sensing receptor is poorly expressed. [source] Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytesGLIA, Issue 8 2008Erik B. Malarkey Abstract Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+ -dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells. © 2008 Wiley-Liss, Inc. [source] Agonist-evoked Ca2+ wave progression requires Ca2+ and IP3JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010John G. McCarron Smooth muscle responds to IP3 -generating agonists by producing Ca2+ waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP3 and the caged Ca2+ buffer diazo-2. Waves, evoked by the IP3 -generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca2+ concentration ([Ca2+]c) over a single though substantial length (,30,µm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (,9,µm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP3 may explain the amplitude changes. Ca2+ was required for IP3 -mediated wave progression to occur. Increasing the Ca2+ buffer capacity in a small (2,µm) region immediately in front of a CCh-evoked Ca2+ wave halted progression at the site. However, the wave front does not progress by Ca2+ -dependent positive feedback alone. In support, colliding [Ca2+]c increases from locally released IP3 did not annihilate but approximately doubled in amplitude. This result suggests that local IP3 -evoked [Ca2+]c increases diffused passively. Failure of local increases in IP3 to evoke waves appears to arise from the restricted nature of the IP3 increase. When IP3 was elevated throughout the cell, a localized increase in Ca2+ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP3 and Ca2+ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334,344, 2010. © 2010 Wiley-Liss, Inc. [source] Regulation by FK506 and rapamycin of Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTORBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2009D MacMillan Background and purpose:, The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca2+ concentration ([Ca2+]cyto) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR). Experimental approach:, Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca2+]cyto measured using fluo-3. IP3Rs were activated by photolysis of caged IP3 and RyRs activated by hydrostatic application of caffeine. Key results:, FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca2+]cyto rise evoked by activation of either RyR or IP3R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP3 -evoked [Ca2+]cyto rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP3 -evoked [Ca2+]cyto release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP3R-mediated Ca2+ release. The mTOR inhibitor LY294002, like rapamycin, decreased IP3 -evoked Ca2+ release. Conclusions and implications:, Ca2+ release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP3R-mediated Ca2+ release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP3 -mediated Ca2+ release may be explained by mTOR inhibition. [source] Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca2+ responses in endothelial cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2008M Campos-Toimil Background and purpose: We previously reported that agonist-induced rises in cytoplasmic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short-term (2 min) pre-treatment with cAMP-elevating agents. The aim of this work was to study the effects of longer term (8 h) pre-treatment with dibutyryl-cAMP (db-cAMP) or rolipram, a specific inhibitor of phosphodiesterase-4 (PDE4), on [Ca2+]i, cAMP levels and PDE activity and expression in HUVEC. Experimental approach: [Ca2+]i changes were measured in isolated HUVEC by Fura-2 imaging. Intracellular cAMP levels and PDE4 activity were assessed by enzyme-immunoassay and radio-enzymatic assay, respectively. PDE expression was measured by northern and western blot analysis. Key results: Long-term pre-treatment of HUVEC with rolipram or db-cAMP significantly increased ATP-, histamine- and thrombin-induced [Ca2+]i rises. Short-term pre-treatment with rolipram was associated with an increase in cAMP, whereas long-term pre-treatment was associated with a decrease in cAMP. Long-term pre-treatment with rolipram or db-cAMP induced a significant increase in PDE4 activity and the expression of 74 kDa-PDE4A and 73 kDa-PDE4B was specifically enhanced. All these effects were suppressed by cycloheximide. Conclusions and implications: Our data suggest that sustained inhibition of PDE4 by rolipram induced an increase in PDE4 activity, possibly as a compensatory mechanism to accelerate cAMP degradation and that PDE4A and PDE4B were implicated in the regulation of [Ca2+]i. Thus, isozyme-specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca2+]i handling is altered, such as atherosclerosis, hypertension and tolerance to ,-adrenoceptor agonists. [source] Characteristics and function of cardiac mitochondrial nitric oxide synthaseTHE JOURNAL OF PHYSIOLOGY, Issue 4 2009Elena N. Dedkova We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H2DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations ([Ca2+]i= 1, 2 and 5 ,m) resulted in a dose-dependent increase of NO production by mitochondria when l -arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of l -arginine, mitochondrial NO production during stimulation of Ca2+ uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit l -arginine availability resulted in 50% inhibition of Ca2+ -induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)- N -(4-amino-5[aminoethyl]aminopentyl)- N,-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor l - N5 -(1-iminoethyl)ornithine (l -NIO) or iNOS inhibitor N -(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca2+ -induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of l -arginine, mitochondrial Ca2+ uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by l -arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH4; 100 ,m) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca2+ uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l -arginine and BH4. In conclusion, our data show the importance of l -arginine and BH4 for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS. [source] Small-Molecule Inhibitors of Store-Operated Calcium EntryCHEMMEDCHEM, Issue 5 2009Zachary Abstract Molecules that inhibit store-operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small-molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors. Controlled variation in intracellular calcium concentration is a key component of the immune response signaling pathway in lymphocytes. Store-operated calcium entry (SOCE) in these cells provides a prolonged increase in cytoplasmic Ca2+ concentrations and ultimately leads to the production of pro-inflammatory cytokines. Molecules that inhibit SOCE could therefore be useful immunomodulating agents for the treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and other conditions. Although the presence of the SOCE signaling pathway in lymphocytes and other cells involved in the immune response has been known for many years, key proteins involved in SOCE were identified only recently. The identification of these proteins may further enable the identification of agents that inhibit SOCE without affecting other cellular processes. This contribution documents representative examples of the small-molecule inhibitors of SOCE that have been reported to date. Where possible, methods that were used to characterize the mechanism of action of the inhibitors are also described. [source] | ||||||||||