Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.     

Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3

Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.     

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.     

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Effect of p-chloroamphetamine on calcium movement and viability in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of p-chloroamphetamine, a neurotoxin that depletes intracellular serotonin, on intracellular Ca2+ concentration ([Ca2+]i) and viability was measured by using the Ca2+-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium. p-Chloroamphetamine (> or = 10 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. p-Chloroamphetamine-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. p-Chloroamphetamine-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which p-chloroamphetamine failed to increase [Ca2+]i; also, pretreatment with p-chloroamphetamine reduced 50% of thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not p-chloroamphetamine)-induced [Ca2+]i rise. Overnight incubation with 1-500 microM p-chloroamphetamine decreased cell viability. These findings suggest that p-chloroamphetamine evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Thrombin and ionomycin can raise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using fura-2

In the presence of 1 mM EGTA, the addition of the calcium ionophore ionomycin to human platelets loaded with 30 microM fura-2 could elevate [Ca2+]i from less than 100 nM to a maximum of greater than 3 microM, presumably by discharge of Ca2+ from internal stores. Under the same conditions thrombin could maximally increase [Ca2+]i to a peak of greater than 1 microM which then declined to near resting levels within 3-4 minutes; by contrast in platelets loaded with 1 mM quin2 thrombin could raise [Ca2+]i to only about 200 nM. In the presence of 1 mM Ca2+ the peak response to thrombin in fura-2-loaded platelets was higher (1.4 microM) than that observed in the presence of EGTA (1.1 microM) and the elevation in [Ca2+] was prolonged, presumably by Ca2+ influx. These results with fura-2-loaded platelets indicate that mobilisation of internal Ca2+ can contribute a substantial proportion of the early peak [Ca2+]i evoked by thrombin directly confirming the deductions from previous work with different loadings of quin2. Under natural conditions the major role of Ca2+ influx may be to prolong the [Ca2+]i rise rather than to make it larger.     

Ketoconazole-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC)

The effect of ketoconazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2+ levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 microM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The ketoconazole-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 50 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM ketoconazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2+]i rise.     

Prostaglandin F2alpha potentiates the calcium dependent activation of mitochondrial metabolism in luteal cells

Cytoplasmic Ca2+ signals are transferred to the mitochondria and activate the Krebs cycle. We have compared the efficiency of this process for two Ca2+ mobilising agonists, PGF2alpha and ATP (acting at metabotropic P2 receptors) in rat luteal cells. [Ca2+]c, [Ca2+]m and mitochondrial NAD(P)H were monitored by means of microspectrofluorimetry and confocal microscopy. While both agonists caused similar elevations of [Ca2+]c, changes in NAD(P)H were larger in response to PGF2alpha than to ATP. PGF2alpha more effectively increased NAD(P)H level also in mouse luteal cells. PGF2alpha caused a faster rate of rise of NAD(P)H fluorescence than ATP when reoxidation was prevented with rotenone, suggesting a faster rate of NAD(P)+ reduction. The NAD(P)H response to both agonists was dependent on the mobilisation of stored Ca2+. We found no difference in the efficacy of transmission of the [Ca2+]c signal to mitochondria in response to PGF2alpha and ATP. Raising [Ca2+]c with ionomycin increased the NAD(P)H signal, which was further raised by PGF2alpha but not by ATP. These data suggest that PGF2alpha potentiates the Ca2+-induced stimulation of mitochondrial metabolism by a Ca2+-independent mechanism and shows that agonists may modulate mitochondrial function differentially through a novel process beyond the simple transfer of Ca2+ from ER to mitochondria.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Effect of t-butyl hydroperoxide on Ca2+ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca2+ ([Ca2+]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca2+]i in a concentration-dependent fashion between 50-250 microM with an EC50 of 100 microM. The [Ca2+]i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca2+. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca2+]i increase. The 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase in Ca2+ medium was reduced by 42 +/- 5% by pretreatment with 0.1 microM nicardipine but not by 10 microM verapamil, nifedipine, nimodipine or diltiazem, or by 50 microM La3+ or Ni2+. Pretreatment with 10 microM t-butyl hydroperoxide for 40 min did not affect 10 microM ATP-induced [Ca2+]i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca2+]i increase in PC12 cells by causing store Ca2+ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ influx via a nicardipine-sensitive pathway.     

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Olvanil (5-100 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. Olvanil-induced [Ca2+]i rise was prevented by 70 and 90% by removal of extracellular Ca2+ and La3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of olvanil on [Ca2+]i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca2+]i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 microM) and a vanilloid receptor antagonist (capsazepine; 100 microM) did not alter olvanil (50 microM)-induced [Ca2+]i rise. These results suggest that olvanil rapidly increases [Ca2+]i in renal tubular cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.     

Effect of nortriptyline on intracellular Ca2+ handling and viability in canine renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of nortriptyline, an antidepressant, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Nortriptyline (> 10 microM) caused a rapid increase of [Ca2+]i in a concentration-dependent manner (EC50 = 75 microM). Nortriptyline-induced [Ca2+]i increase was prevented by 40% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i, increase, after which the increasing effect of nortriptyline on [Ca2+], was abolished; also, pretreatment with nortriptyline reduced a large portion of thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, abolished ATP (but not nortriptyline)-induced [Ca2+]i increase. Overnight incubation with 10 microM nortriptyline decreased cell viability by 16%, and 50 microM nortriptyline killed all cells. Prechelation of cytosolic Ca2+ with BAPTA did not alter nortriptyline-induced cell death. These findings suggest that nortriptyline rapidly increased [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and was cytotoxic at higher concentrations in a Ca(2+)-dissociated manner.