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Ca2+ Load (ca2+ + load)
Selected AbstractsNo evidence for calcium electrogenic exchanger in frog semicircular canal hair cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002M. Martini Abstract We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 µm) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+ - and K+ -free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+ -free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2,-{[1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy)}bis(acetate) and calcium 2,2,-{[1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)} bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+ -ATPases as the primary contributors to Ca2+ extrusion. [source] Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Stefan Kahlert Abstract Generation of reactive oxygen species (ROS) in brain tissue leads to neurodegeneration. The major source of ROS is the mitochondrial respiratory chain. We studied regulation of Ca2+ level, mitochondrial potential, and ROS generation in defined mixed hippocampal cell cultures exposed to glutamate (100 ,M). Recordings were made from individually identified astrocytes and neurons to compare the physiologic responses in both cell types. Neurons identified by synaptotagmin immunoreactivity were characterized functionally by the fast Ca2+ increase with K+ (50 mM) stimulation, and the astrocytes identified by glial fibrillary acidic protein (GFAP) staining had the functional characteristic of a transient Ca2+ peak in response to ATP (10 ,M) stimulation. We found that the glutamate-mediated Ca2+ response in neurons is due largely to influx of extracellular Ca2+. This is consistent with our finding that in cultured hippocampal neurons, stores depending on the activity of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump had a low Ca2+ content, regardless of whether the neurons were challenged or not with K+ before applying the SERCA inhibitor cyclopiazonic acid (CPA). Astrocytes displayed a large CPA-mediated Ca2+ response, indicating a high level of Ca2+ load in the stores in astrocytes. Importantly, the rise in ROS generation due to glutamate application was cell-type specific. In neurons, glutamate induced a marked rise in generation of ROS, but not in astrocytes. In both astrocytes and neurons, the mitochondrial potential was increased in response to glutamate challenge. We conclude that in neurons, Ca2+ influx accounts for the increased ROS generation in response to glutamate. This might explain the high vulnerability of neurons to glutamate challenge compared to the vulnerability of astrocytes. The high resistance of astrocytes is accompanied by an efficient downregulation of cytosolic Ca2+, which is not found in neurons. © 2004 Wiley-Liss, Inc. [source] Cardiac Overexpression of Alcohol Dehydrogenase Exacerbates Cardiac Contractile Dysfunction, Lipid Peroxidation, and Protein Damage After Chronic Ethanol IngestionALCOHOLISM, Issue 7 2003Kadon K. Hintz Background: Alcoholic cardiomyopathy is manifested as ventricular dysfunction, although its specific toxic mechanism remains obscure. This study was designed to examine the impact of enhanced acetaldehyde exposure on cardiac function via cardiac-specific overexpression of alcohol dehydrogenase (ADH) after alcohol intake. Methods: ADH transgenic and wild-type FVB mice were placed on a 4% alcohol or control diet for 8 weeks. Mechanical and intracellular Ca2+ properties were evaluated in cardiac myocytes. Levels of acetaldehyde, lipid peroxidation, and protein carbonyl formation were determined. Results: FVB and ADH mice consuming ethanol exhibited elevated blood ethanol/acetaldehyde, cardiac acetaldehyde, and cardiac hypertrophy compared with non-ethanol-consuming mice. However, the levels of cardiac acetaldehyde and hypertrophy were significantly greater in ADH ethanol-fed mice than FVB ethanol-fed mice. ADH transgene itself did not affect mechanical and intracellular Ca2+ properties with the exception of reduced resting intracellular Ca2+ and Ca2+ re-sequestration at low pace frequency. Myocytes from ethanol-fed mice showed significantly depressed peak shortening, velocity of shortening/relengthening, rise of intracellular Ca2+ transients, and sarco(endo)plasmic reticulum Ca2+ load associated with similar duration of shortening/relengthening compared with myocytes from control mice. Strikingly, the ethanol-induced mechanical and intracellular Ca2+ defects were exacerbated in ADH myocytes compared with the FVB group except velocity of shortening/relengthening. The lipid peroxidation end products malondialdehyde and protein carbonyl formation were significantly elevated in both livers and hearts after chronic ethanol consumption, with the cardiac lipid and protein damage being exaggerated by ADH transgene. Conclusion: These data suggest that increased cardiac acetaldehyde exposure due to ADH transgene may play an important role in cardiac contractile dysfunctions associated with lipid and protein damage after alcohol intake. [source] Evolution and modulation of intracellular calcium release during long-lasting, depleting depolarization in mouse muscleTHE JOURNAL OF PHYSIOLOGY, Issue 19 2008Leandro Royer Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that in many types of cells appears to be tightly controlled by changes in [Ca2+] within the store. In contrast with cardiac muscle, where depletion of Ca2+ in the sarcoplasmic reticulum is a crucial determinant of termination of Ca2+ release, in skeletal muscle there is no agreement regarding the sign, or even the existence of an effect of SR Ca2+ level on Ca2+ release. To address this issue we measured Ca2+ transients in mouse flexor digitorum brevis (FDB) skeletal muscle fibres under voltage clamp, using confocal microscopy and the Ca2+ monitor rhod-2. The evolution of Ca2+ release flux was quantified during long-lasting depolarizations that reduced severely the Ca2+ content of the SR. As in all previous determinations in mammals and non-mammals, release flux consisted of an early peak, relaxing to a lower level from which it continued to decay more slowly. Decay of flux in this second stage, which has been attributed largely to depletion of SR Ca2+, was studied in detail. A simple depletion mechanism without change in release permeability predicts an exponential decay with time. In contrast, flux decreased non-exponentially, to a finite, measurable level that could be maintained for the longest pulses applied (1.8 s). An algorithm on the flux record allowed us to define a quantitative index, the normalized flux rate of change (NFRC), which was shown to be proportional to the ratio of release permeability P and inversely proportional to Ca2+ buffering power B of the SR, thus quantifying the ,evacuability' or ability of the SR to empty its content. When P and B were constant, flux then decayed exponentially, and NFRC was equal to the exponential rate constant. Instead, in most cases NFRC increased during the pulse, from a minimum reached immediately after the early peak in flux, to a time between 200 and 250 ms, when the index was no longer defined. NFRC increased by 111% on average (in 27 images from 18 cells), reaching 300% in some cases. The increase may reflect an increase in P, a decrease in B, or both. On experimental and theoretical grounds, both changes are to be expected upon SR depletion. A variable evacuability helps maintain a constant Ca2+ output under conditions of diminishing store Ca2+ load. [source] Mitochondrial modulation of Ca2+ sparks and transient KCa currents in smooth muscle cells of rat cerebral arteriesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2004Serguei Y. Cheranov Mitochondria sequester and release calcium (Ca2+) and regulate intracellular Ca2+ concentration ([Ca2+]i) in eukaryotic cells. However, the regulation of different Ca2+ signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca2+ sparks, Ca2+ waves and global [Ca2+]i by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 ,m) and rotenone (an electron transport chain complex I inhibitor; 10 ,m) depolarized mitochondria, reduced Ca2+ spark and wave frequency, and elevated global [Ca2+]i in smooth muscle cells of intact arteries. In voltage-clamped (,40 mV) cells, mitochondrial depolarization elevated global [Ca2+]i, reduced Ca2+ spark amplitude, spatial spread and the effective coupling of sparks to large-conductance Ca2+ -activated potassium (KCa) channels, and decreased transient KCa current frequency and amplitude. Inhibition of Ca2+ sparks and transient KCa currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca2+ load, and occurred in the presence of diltiazem, a voltage-dependent Ca2+ channel blocker. Ru360 (10 ,m), a mitochondrial Ca2+ uptake blocker, and lonidamine (100 ,m), a permeability transition pore (PTP) opener, inhibited transient KCa currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 ,m), a mitochondrial Na+,Ca2+ exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient KCa currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage-independent elevation in global [Ca2+]i and Ca2+ spark and transient KCa current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca2+ sparks and transient KCa currents via PTP opening and a decrease in intramitochondrial [Ca2+]. [source] Modulation of excitation,contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2+ load and Ca2+ current triggerTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Kenneth S. Ginsburg Cardiac Ca2+ transients are enhanced by cAMP-dependent protein kinase (PKA). However, PKA-dependent modulation of ryanodine receptor (RyR) function in intact cells is difficult to measure, because PKA simultaneously increases Ca2+ current (ICa), SR Ca2+ uptake and SR Ca2+ loading (which independently increase SR Ca2+ release). We measured ICa and SR Ca2+ release ± 1 ,m isoproterenol (ISO; isoprenaline) in voltage-clamped ventricular myocytes of rabbits and transgenic mice (expressing only non-phosphorylatable phospholamban). This mouse model helps control for any effect of ISO-enhanced SR uptake on observed release, but the two species produced essentially identical results. SR Ca2+ load and ICa were adjusted by conditioning. We thus evaluated PKA effects on SR Ca2+ release at constant SR Ca2+ load and ICa trigger (with constant unitary ICa). The amount of SR Ca2+ release increased as a function of either ICa or SR Ca2+ load, but ISO did not alter the relationships (measured as gain or fractional release). This was true over a wide range of SR Ca2+ load and ICa. However, the maximal rate of SR Ca2+ release was ,50% faster with ISO (at most loads and ICa levels). We conclude that the isolated effect of PKA on SR Ca2+ release is an increase in maximal rate of release and faster turn-off of release (such that integrated SR Ca2+ release is unchanged). The increased amount of SR Ca2+ release normally seen with ISO depends primarily on increased ICa trigger and SR Ca2+ load, whereas faster release kinetics may be the main result of RyR phosphorylation. [source] Regulation of junctional and non-junctional sarcoplasmic reticulum calcium release in excitation-contraction coupling in cat atrial myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2003Katherine A. Sheehan We have characterized the dependence on membrane potential (Vm) and calcium current (ICa) of calcium-induced calcium release (CICR) from the junctional-SR (j-SR, in the subsarcolemmal (SS) space) and non-junctional-SR (nj-SR, in the central (CT) region of the cell) of cat atrial myocytes using whole-cell voltage-clamp together with spatially resolved laser-scanning confocal microscopy. Subsarcolemmal and central [Ca2+]i transient amplitudes and ICa had a bell-shaped dependence on Vm, but [Ca2+]i reached a maximum at more negative Vm (-10 to 0 mV) than ICa (+10 mV). Termination of ICa after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca2+]i transient, leaving the development of the CT [Ca2+]i transient unaffected. Block of SR function with 20 ,m ryanodine and 2 ,m thapsigargin, revealed that > 90 % of the control [Ca2+]i transient amplitude was attributable to active SR Ca2+ release through ryanodine receptors (RyRs). The gain of SR Ca2+ release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na+ -Ca2+ exchange resulted in prolonged and higher amplitude [Ca2+]i transients, elevated resting [Ca2+]i, accelerated propagation of CICR, decreased extrusion of Ca2+ and an increase in j-SR Ca2+ load. Increasing the cytosolic Ca2+ buffer capacity by internal perfusion with 1 mm EGTA limited SR Ca2+ release to the SS region, indicating that Ca2+ release from nj-SR is initiated by diffusion of Ca2+ from the cell periphery and propagating CICR. Junctional-SR Ca2+ release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation-contraction coupling in cat atrial myocytes, only Ca2+ release from the j-SR is directly activated by Ca2+ entering via ICa. Elevation of SS [Ca2+]i is required to provide the cytosolic Ca2+ gradient needed to initiate regenerative and propagating CICR from nj-SR. [source] EXCITATION,CONTRACTION COUPLING FROM THE 1950s INTO THE NEW MILLENNIUMCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 9 2006AF Dulhunty SUMMARY 1Excitation,contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca2+ release from the sarcoplasmic reticulum. 2We now know that excitation,contraction coupling depends on a macromolecular protein complex or ,calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a ,voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca2+ load within the calcium store. 4The molecular components of the ,calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation,contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca2+ through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years. [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] Ca2+ -induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex IJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Tatyana V. Votyakova Abstract Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 µm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+ -challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired. [source] | |||||||||||||