Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Metabolic requirements for maintenance of the chlortetracycline-labeled pool of membrane-bound calcium in human neutrophils

Human neutrophils labeled with chlortetracycline (CTC), commonly used as a probe of membrane-bound calcium, release lysosomal enzymes and exhibit a rapid decrease in fluorescence when exposed to the chemotactic peptide fMet-Leu-Phe or the lectin Con A. This decrease has been attributed to the release of calcium from a membrane-associated "trigger pool." The nature of this putative pool has been further characterized by examining the effects of various inhibitors on the CTC fluorescence response and lysosomal enzyme release from stimulated neutrophils. These agents included inhibitors of glycolysis (2-deoxyglucose and iodoacetate), an uncoupler of oxidative- phosphorylation (KCN), and a sulfhydryl inhibitor (N-ethylmaleimide). Resting neutrophils labelled with CTC demonstrated an enhanced decay of baseline fluorescence when exposed to 2-deoxyglucose or iodoacetate. This suggested that the pool of membrane-bound calcium labelled by this probe was maintained by glycolytic metabolism. Furthermore, 2-deoxyglucose and iodoacetate inhibited both the stimulated decrease in CTC fluorescence and lysosomal enzyme release induced by fMet-Leu-Phe and Con A in a time-dependent manner. KCN did not inhibit either response to stimulation, but did retard the recovery of CTC fluorescence observed when fMet-Leu-Phe was used as the stimulus. High concentrations of N-ethylmaleimide (100 microM) completely inhibited both the CTC fluorescence response and lysosomal enzyme release almost immediately; low concentrations of N-ethylmaleimide (30 microM) inhibited lysosomal enzyme release in a time-dependent manner without significantly affecting changes in CTC fluorescence. These results are consistent with the hypothesis that CTC serves as a probe of membrane-bound "trigger" calcium, the release of which is dependent upon intact glycolysis and is a requirement for lysosomal enzyme release.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.     

Phosphatidylinositol 4,5-bisphosphate may represent the site of release of plasma membrane-bound calcium upon stimulation of human platelets

Thrombin stimulation of human blood platelets caused an extensive (up to 45%) and rapid (5-10 s) decline in endogenous phosphatidylinositol 4,5-bisphosphate (PI-P2). Thrombin initiated an equally rapid loss of membrane-bound Ca, as indicated by the decrease in fluorescence of chlortetracycline (CTC)-loaded platelets. PI-P2 breakdown also correlated with decreased CTC fluorescence upon use of other platelet stimuli: Arachidonate caused moderate and slow decreases in both PI-P2 and CTC fluorescence, while ionophore only induced minimal changes. Thrombin-induced decreases in PI-P2 content could account for release of sufficient membrane-bound Ca to raise cytoplasmic free [Ca2+] to 1-2 microM, supporting the hypothesis that PI-P2 represents the Ca-binding site involved in the stimulus-dependent increase in cytoplasmic Ca2+ evoked by receptor-ligand interactions.     

Ca2+ influx pathways mediated by swelling or stores depletion in mouse thymocytes

We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of approximately 70 s, these swelling-activated [Ca2+]i (SWAC) transients reached approximately 650 nM from resting levels of approximately 100 nM and declined from a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 microM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG- treated thymocytes, [Ca2+]i rose steadily for approximately 100 s, peaked at approximately 900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 microM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes. TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Trypsin-induced increase in cyclic AMP concentration in rat thymocytes. An effect independent of calcium and calmodulin.

Trypsin produces a dose-related increase in cellular cyclic AMP concentration in rat thymocytes [Shneyour, Patt & Trainin (1976) J. Immunol. 117, 2143-2149; Segal & Ingbar (1983) Clin. Res. 31, 277A]. In the present study, I examined whether this effect of trypsin requires Ca2+ and whether it is modified by calmodulin. In fresh thymocytes suspended in standard medium (containing 1 mM-Ca2+), trypsin produced a concentration-dependent increase in cytoplasmic free Ca2+ concentration, which was evident at a concentration of 50 micrograms of trypsin/ml and reached maximal values at about 1 mg/ml. This effect of trypsin was very prompt in onset, almost immediate, and reached maximal values within 2-3 min. But in cells suspended in essentially Ca2+-free medium (6 nM free Ca2+), trypsin had no effect on cytoplasmic free Ca2+ concentration, which indicates that trypsin acted by increasing Ca2+ uptake rather than Ca2+ release from an intracellular pool. However, the increase in thymocyte cyclic AMP concentration produced by trypsin was independent of extracellular Ca2+ and was not influenced by calmodulin, because it was the same in the presence or absence of Ca2+ and was not changed by the calmodulin inhibitor trifluoperazine. I therefore suggest that in rat thymocytes the trypsin-induced increase in cyclic AMP concentration does not require Ca2+ and is not influenced by calmodulin.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.     

Calcium pools in saponin-permeabilized guinea pig hepatocytes

The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.     

Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca2+] is maintained around 20 nM and the cells contain membrane-bound Ca2+ pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca2+ ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca2+ both in the presence and in the absence of extracellular Ca2+. In addition to the ER, an acidic compartment appears to be involved in Ca2+ storage, as collapse of intracellular pHgradients by monensin, a Na+ -H+ exchanger, and nigericin, a K+ -H+ exchanger, induce the release of Ca2+ from internal pools. A vacuolar H+ pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca2+ pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

Inositol 1,4,5-trisphosphate and the endoplasmic reticulum Ca2+ cycle of a rat insulinoma cell line

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

The role of changes in the cytoplasmic concentration of Ca2+ in dexamethasone-induced thymocyte death]

The putative role of changes in cytosolic Ca2+ concentration ([Ca2+]i) in the dexamethasone (DM) induced thymocyte death was investigated. Incubation of rat thymocytes with 10(-7) M DM for different time intervals from 0.1 to 8 h did not change the basal [Ca2+]i level ca 100 nM as measured with Ca(2+)-fluorescent probe Quin-2. Ca2+ influx measured by the rate of 45Ca2+ uptake was also just the same in DM treated and control cells. At the same time a 6-8 h incubation of cell suspension with 10(-7) M DM results in significant increase in DNA fragmentation and pyknosis, and a 24 h incubation is associated with the decrease in the percentage of cells not staining with Trypan blue. Thus, the results obtained indicate that 10(-7) M DM induces thymocyte death without any significant and constant [Ca2+]i rise during the first 8 h after hormone application.     

Modelling of Mg2+, ATP-dependent mitochondrial Ca ions transport in smooth muscle cells using protonophore CCCP-sensitive fluorescent tetracycline

Mg2+, ATP-dependent Ca2+ accumulation in the rat myometrial mitochondria was investigated in complex experiment using Ca2+ isotope (45Ca2+) and Ca(2+)-sensitive label tetracycline. Monotonous increase of the fluorescence signal, insensitive to thapsigargin (100 nM) was observed with following establishing the stationary state of incubation at 2 min. which correlates with results obtained using isotope technique. Experiments with isotope label signify, that protonophore CCCP, ruthenium red and sodium azide, in concentration 1 microM, 10 microM and 10 mM respectively, totally inhibits the accumulation of the Ca ions in mitochondria. At the same time, in conditions of Mg2+, ATP-dependent Ca2+ accumulation modeling in these cellular structures, CCCP and sodium azide, used in the same concentration, diminished tetracycline fluorescence signal increase. In the same conditions, the introduction of the CCCP (1 mM) into the incubation medium at 75 sec. after initiation of the transport process induced reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP presence in the medium. According to data obtained in the experiment, using Ca2+ isotope, Ca(2+)-ionophore A-23187 induces both the reversible release of previously accumulated Ca ions, and cause reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP (1 mM) and sodium azide (10 mM) presence in the incubation medium. Conclusion was drawn that the thapsigargin-insensitive and CCCP, sodium azide and A-23187-sensitive tetracycline fluorescence increasing in case of modeling of Mg2+, ATP-dependent Ca2+ accumulation in myometrial mitochondria reflect the Ca2+ uniporter functioning in those subcellular structures.     

Chlorotetracycline fluorescence is a quantitative measure of the free internal Ca2+ concentration achieved by active transport. In situ calibration and application to bovine cardiac sarcolemmal vesicles

Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+.     

Effect of SH-reagents on Ca2+ level in lymphocyte cytoplasm

The effect of SH-reagents on cytoplasmic free Ca2+ concentration [( Ca2+]i) in rat thymocytes and B lymphoma Raji cells has been studied by means of fluorescent Ca2+ indicator quin-2. N-ethylmaleimide and ethylmercurythiosalicylate have been found to induce a dose-dependent increase of Ca2+ concentration from about 100 nM in the control cells up to 1000 nM. The effect is weakened with a decrease of the external Ca2+ concentration and is not observed already with Ca2+ concentration in the medium less than 0.2 mM. Reduction of the level of intracellular ATP does not suppress the Ca2+ response to SH-reagents. The effect of SH-reagents is weakened with a decrease of the temperature from 37 to 24 degrees C. Addition of 1 mM Mn2+ or Ca2+ into the standard medium containing 1 mM CaCl2 prevents Ca2+ concentration increase in the cytoplasm under the action of SH-reagents. The conclusion is made that in lymphocytes Ca2+ permeability is regulated by a protein(s) sensitive to the SH-reagent and that a high level of SH-group oxidation is necessary to maintain the low Ca2+ permeability of lymphocyte plasma membrane. Mechanisms of SH-reagents action on the Ca2+ level in the cell are discussed.