Effect of hyperglycemia on the changes of intracellular [Ca2+]i in heart myoblast

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.     

Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin.

The free intracellular calcium ion concentration ([Ca2+]i) was measured in single cells of a population containing 65-80% somatotrophs, using the fluorescent Ca(2+)-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca2+]i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca2+ oscillations were blocked by the Ca2+ channel blocker CoCl2 and were thus proposed to be the result of influx of Ca2+ into the cell, possibly as the result of spontaneous electrical activity. GHRH (10-100 nM) increased [Ca2+]i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca2+]i rose from 170 +/- 26 nM to 321 +/- 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca2+ oscillations in a number of cells, and some previously quiescent cells showed Ca2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca2+]i in 10 out of 14 cells tested. Somatostatin (SS) (10-80 nM) rapidly reduced basal [Ca2+]i, blocked Ca2+ oscillations, and blocked the [Ca2+]i response to GHRH. The Ca2+ channel blocker CoCl2 (4 mM) had similar actions on [Ca2+]i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca2+ entry into the cell through the cell membrane. The [Ca2+]i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca2+]i, which in turn, may be influenced by the frequency of [Ca2+]i oscillations in bovine somatotrophs.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment.

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.     

Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures.

To explore how to manage pH when calibrating Ca2+ probes at different temperatures, the dissociation constant (Kd) of indo-1 was determined both in pH-stat (pH is fixed despite the temperature) and in alpha-stat (pH changes with temperature as in cells). The results showed that the Kd was much more sensitive to temperature in pH-stat than in alpha-stat, demonstrating that alpha-stat calibration should be preferred when using a Ca2+ probe to measure intracellular free calcium ([Ca2+]i) at different temperatures. Based on the calibration in situ and in alpha-stat, we showed a striking increase of [Ca2+]i from 141+/-8 nM at 30 degrees C to 218+/-22 nM at 10 degrees C in indo-1 loaded rat ventricular cells, which supports that intracellular calcium overload takes place in cardiac myocytes of non-hibernating mammals during hypothermia.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.     

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.     

Maintenance of intracellular calcium in Escherichia coli

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.     

Increases in cytosolic calcium ion concentration can be dissociated from the killing of cultured hepatocytes by tert-butyl hydroperoxide

Digital imaging fluorescence microscopy was used to study the effect of tert-butyl hydroperoxide (TBHP) on the cytosolic free calcium concentration ([Ca2+]i) of single rat hepatocytes in primary culture. Within minutes of the addition of TBHP, individual hepatocytes displayed one or more peaks of increased [Ca2+]i that promptly returned to the prestimulation level. This was followed by a slower increase of [Ca2+]i that reached a plateau of 696 +/- 260 nM (basal 194 +/- nM) after 20 min. Another rise in [Ca2+]i, abrupt and much larger, preceded the death of the cells after about 45 min. Pretreatment of the hepatocytes with deferoxamine, a ferric iron chelator, or the addition of the antioxidants N,N'-diphenyl-p-phenylenediamine or catechol prevented the loss of viability. Neither the number of hepatocytes displaying the initial [Ca2+]i transients nor the magnitude of these oscillations was affected by deferoxamine, N,N'-diphenyl-p-phenyl-enediamine, or catechol. However, both the plateau phase and the abrupt rise in [Ca2+]i were prevented. Treatment of the hepatocytes with TBHP in a low calcium buffer (less than 2 microM Ca2+) reduced or abolished the initial [Ca2+]i transients and eliminated both the plateau phase and abrupt rise in [Ca2+]i. The onset of cell death was delayed by 10 min in the low calcium medium. Addition of 3.5 mM EGTA to the cultures lowered the basal calcium concentration, prevented both the initial [Ca2+]i spikes and the delayed changes, and further prolonged the onset of cell death. These data indicate that the killing of the cultured hepatocytes by TBHP can be dissociated from changes in intracellular calcium homeostasis. An influx of extracellular Ca2+ ions may aggravate somewhat the mechanisms of cell injury by an oxidative stress and accelerate the time of onset of cell death.     

Induction of calcium influx from extracellular fluid by beauvericin in human leukemia cells

Beauvericin, a cyclic hexadepsipeptide, is a mycotoxin that can induce cell death in human lymphoblastic leukemia CCRF-CEM cells. Our previous data have shown that beauvericin induces cell death in CCRF-CEM cells in a dose- and time-dependent manner, and that this beauvericin-induced cell death can be prevented by administration of intracellular calcium chelator-BAPTA. Therefore, the intracellular Ca2+ concentration ([Ca2+]i) may play an important role in beauvericin-induced cell death in CCRF-CEM cells. In this study, the effect of beauvericin on [Ca2+]i and the possible mechanism responsible for the changes of [Ca2+]i in CCRF-CEM cells were investigated. Beauvericin caused a rapid and sustained [Ca2+]i rise in a dose-dependent manner. Excess extracellular Ca2+ facilitated beauvericin-induced [Ca2+]i rise by adding 1 mM CaCl2 in the bathing medium. On the other hand, beauvericin-induced [Ca2+]i rise was prevented in Ca2+-free Tyrode's solution by 200 microM EGTA. In addition, beauvericin-induced [Ca2+]i rise was also attenuated by intracellular Ca2+ chelator-BAPTA/AM. It is worthy to note that neither the voltage-dependent Ca2+ channel blocker, nimodipine, nor depletion of intracellular Ca2+ with thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, has any effect on beauvericin-induced [Ca2+]i rise. The data from present study indicate that beauvericin acts as a potent Ca2+ mobilizer by stimulating extracellular Ca2+ influx CCRF-CEM cells.     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.