Home About us Contact
|
Home About us Contact | ||
|
|
||
Mitochondrial Ca2+ (mitochondrial + ca2+)
Terms modified by Mitochondrial Ca2+ Selected AbstractsModulation of calcium signalling by intracellular organelles seen with targeted aequorinsACTA PHYSIOLOGICA, Issue 1 2009M. T. Alonso Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source] Effects of sulfonylureas on mitochondrial ATP-sensitive K+ channels in cardiac myocytes: implications for sulfonylurea controversyDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2006Toshiaki Sato Abstract Background Mitochondrial ATP-sensitive K+ (mitoKATP) channel plays a key role in cardioprotection. Hence, a sulfonylurea that does not block mitoKATP channels would be desirable to avoid damage to the heart. Accordingly, we examined the effects of sulfonylureas on the mitoKATP channel and mitochondrial Ca2+ overload. Methods Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoKATP channel activity. The mitochondrial Ca2+ concentration was measured by loading cells with rhod-2. Results The mitoKATP channel opener diazoxide (100 µM) reversibly increased flavoprotein oxidation to 31.8 ± 4.3% (n = 5) of the maximum value induced by 2,4-dinitrophenol. Glimepiride (10 µM) alone did not oxidize the flavoprotein, and the oxidative effect of diazoxide was unaffected by glimepiride (35.4 ± 3.2%, n = 5). Similarly, the diazoxide-induced flavoprotein oxidation was unaffected both by gliclazide (10 µM) and by tolbutamide (100 µM). Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca2+ overload, and the intensity of rhod-2 fluorescence increased to 197.4 ± 7.2% of baseline (n = 11). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca2+ overload (149.6 ± 5.1%, n = 11, p < 0.05 versus ouabain alone), and the effect was antagonized by the mitoKATP channel blocker 5-hydroxydecanoate (189.8 ± 27.8%, n = 5) and glibenclamide (193.1 ± 7.7%, n = 8). On the contrary, cardioprotective effect of diazoxide was not abolished by glimepiride (141.8 ± 7.8%, n = 6), gliclazide (139.0 ± 9.4%, n = 5), and tolbutamide (141.1 ± 4.5%, n = 7). Conclusions Our results indicate that glimepiride, gliclazide, and tolbutamide have no effect on mitoKATP channel, and do not abolish the cardioprotective effects of diazoxide. Therefore, these sulfonylureas, unlike glibenclamide, do not interfere with the cellular pathways that confer cardioprotection. Copyright © 2006 John Wiley & Sons, Ltd. [source] Walker tumor cells express larger amounts of the antiapoptotic protein Bcl-2 and presents higher resistance to toxic concentrations of Ca2+ than the tumor cells K 562DRUG DEVELOPMENT RESEARCH, Issue 4 2001Graziela Milani Abstract Ca2+ homeostasis was studied in two tumor cell lines (Walker 256 and K 562) previously shown to exhibit different mitochondrial Ca2+ accumulation capacity. When intact, both cells present cytosolic Ca2+ concentrations within the range expected for mammalian cells, as determined through fura-2 fluorescence ratios. In order to study intracellular Ca2+ distribution, digitonin was used to permeabilize the plasma membrane without affecting intracellular organelle structure, as assessed using electron microscopy. Digitonin-permeabilized Walker 256 cells incubated with Ca2+ presented uptake of the cation exclusively through mitochondrial activity. In addition, very large Ca2+ loads were necessary to promote a disruption of Walker 256 mitochondrial membrane potential. K 562 cells presented active Ca2+ uptake through both nonmitochondrial and mitochondrial compartments and suffered disruption of mitochondrial membrane potential at lower Ca2+ loads than Walker 256 mitochondria. The higher Ca2+ resistance in Walker 256 cells could be attributed to Bcl-2 overexpression, as evidenced by immunocytochemical staining. Thus, we correlate natural Bcl-2 overexpression, observed in Walker 256 cells, with higher resistance to mitochondrial Ca2+ overload, as was shown previously in mitochondria from cells transfected with the bcl-2 gene. Drug Dev. Res. 52:508,514, 2001. © 2001 Wiley-Liss, Inc. [source] Molecular characterization of mitocalcin, a novel mitochondrial Ca2+ -binding protein with EF-hand and coiled-coil domainsJOURNAL OF NEUROCHEMISTRY, Issue 1 2006Mitsutoshi Tominaga Abstract Here we have identified and characterized a novel mitochondrial Ca2+ -binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function. [source] Differential effects of the mitochondrial uncoupling agent, 2,4-dinitrophenol, or the nitroxide antioxidant, Tempol, on synaptic or nonsynaptic mitochondria after spinal cord injuryJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2009Samir P. Patel Abstract We recently documented the progressive nature of mitochondrial dysfunction over 24 hr after contusion spinal cord injury (SCI), but the underlying mechanism has not been elucidated. We investigated the effects of targeting two distinct possible mechanisms of mitochondrial dysfunction by using the mitochondrial uncoupler 2,4-dinitrophenol (2,4-DNP) or the nitroxide antioxidant Tempol after contusion SCI in rats. A novel aspect of this study was that all assessments were made in both synaptosomal (neuronal)- and nonsynaptosomal (glial and neuronal soma)-derived mitochondria 24 hr after injury. Mitochondrial uncouplers target Ca2+ cycling and subsequent reactive oxygen species production in mitochondria after injury. When 2,4-DNP was injected 15 and 30 min after injury, mitochondrial function was preserved in both populations compared with vehicle-treated rats, whereas 1 hr postinjury treatment was ineffective. Conversely, targeting peroxynitrite with Tempol failed to maintain normal bioenergetics in synaptic mitochondria, but was effective in nonsynaptic mitochondria when administered 15 min after injury. When administered at 15 and 30 min after injury, increased hydroxynonenal, 3-NT, and protein carbonyl levels were significantly reduced by 2,4-DNP, whereas Tempol only reduced 3-NT and protein carbonyls after SCI. Despite such antioxidant effects, only 2,4-DNP was effective in preventing mitochondrial dysfunction, indicating that mitochondrial Ca2+ overload may be the key mechanism involved in acute mitochondrial damage after SCI. Collectively, our observations demonstrate the significant role that mitochondrial dysfunction plays in SCI neuropathology. Moreover, they indicate that combinatorial therapeutic approaches targeting different populations of mitochondria holds great potential in fostering neuroprotection after acute SCI. © 2008 Wiley-Liss, Inc. [source] Diazoxide acts more as a PKC- , activator, and indirectly activates the mitochondrial KATP channel conferring cardioprotection against hypoxic injuryBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2006M-Y Kim Background and purpose: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoKATP) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoKATP channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoKATP channel in diazoxide-induced cardioprotection. Experimental approach: In H9c2 cells and neonatal rat cardiomyocytes, PKC-, activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca2+ and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. Key results: Diazoxide (100 ,M) induced translocation of PKC-, from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-, by either ,V1-2 or dominant negative mutant PKC-, (PKC-, KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either ,V1-2 or PKC-, KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-, induced by diazoxide. Transfection with wild type PKC-, mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by ,V1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by ,V1-2 or 5-HD. Conclusions and Implications: Diazoxide induced isoform-specific translocation of PKC-, as an upstream signaling molecule for the mitoKATP channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway. British Journal of Pharmacology (2006) 149, 1059,1070. doi:10.1038/sj.bjp.0706922 [source] | |||||||||||||