The V-Quick patch versus the standard 12-lead ECG system: time is the essence.

INTRODUCTION: The V-Quick patch template system is compared with the standard 12-lead electrocardiogram (ECG) acquisition technique in this paper. The objectives of the study were: (a) to study and compare the time taken to produce the printed 12-lead ECG and (b) to look at the level of agreement when the ECGs were compared by two blinded, independent assessors. METHODS: One hundred and fifty each of male and female volunteers signed an informed consent form to participate in the clinical study. Nurses were put through a 4-h training session to familiarise themselves with the V-Quick patch system. The timings were measured with a stopwatch with the specific start and end points defined. The final ECG printouts were then compared by two blinded, independent assessors for several set criteria. RESULTS: The V-Quick patch system was proved to be significantly faster than the standard 12-lead system in the acquisition of the ECG in both male and female volunteers. The time taken in male volunteers was also noted to be significantly faster than in female volunteers. CONCLUSION: The two assessors shared a 100% agreement level when comparing the ECGs acquired by both techniques in the same individual (intra-assessor agreement) and when each ECG was read by the two assessors separately (inter-assessor agreement).

Interactions of bismuth with human lactoferrin and recognition of the Bi(III)-lactoferrin complex by intestinal cells.

Several bismuth compounds are currently used as antiulcer drugs, but the mechanism of action still remains unclear. The antimicrobial activity of Bi(III) complexes toward Gram-negative bacteria is reported to be dependent on the iron uptake system [Domenico, P., et al. (1996) J. Antimicrob. Chemother. 38, 1031-1040]. Electronic absorption and 13C NMR spectroscopic data show that Bi(III) binds to human lactoferrin at the specific Fe(III) sites along with either carbonate or oxalate as the synergistic anion. The uptake of Bi(III) by apo-hLF was rapid [minutes in 10 mM Hepes buffer and 5 mM bicarbonate (pH 7.4)], and almost equal in both lobes. The presence of ATP facilitates the release of Bi(III) from the Bi2-hLF complex when the pH is lowered. The Bi2-hLF complex blocked the uptake of the radiolabeled 59Fe-hLF complex into rat IEC-6 cells. Surprisingly, apo-hLF (but not apotransferrin) was almost as effective in blocking 59Fe uptake as bismuth-loaded lactoferrin. These results suggest that Bi(III)-loaded hLF might be recognized by the lactoferrin receptor and be taken up into cells.

bcl-2 protects HL-60 cells from apoptosis by stabilizing their intracellular calcium pools.

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis induced under different circumstances. In this study we investigated the effects of bcl-2 overexpression on the homeostasis of subcellular organelles such as ER and mitochondria. In our study, HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Apoptosis was evaluated by both DNA fragmentation and flow cytometry qualitatively and quantitatively, and the intracellular calcium by Fura-2/AM. Thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump, and Br-A23187, a calcium ionophore, were used in this study. Our results showed that overexpression of bcl-2 significantly blocked TG- and Br-A23187-induced apoptosis in calcium containing buffer. Measurement of intracellular calcium showed that bcl-2 overexpression could reduce sustained elevation of cytosolic Ca2+ induced by these agents. However, in calcium-free medium, bcl-2 overexpression maintained Ca2+ uptake in ER of both TG- and Br-A23187-treated cells. Moreover, the depletion of Ca2+ by EGTA enhanced TG- and Br-A23187-induced apoptosis, and reduced the anti-apoptotic action of bcl-2, suggesting that cytosolic Ca2+ elevation may be required for optimal ER pool refilling. These findings suggest that bcl-2 facilitates and maintains the replenishment of Ca2+ in intracellular stores and, as a result, influences the intracellular calcium, thus protecting the cells from death. In addition, there were no cytochrome c release from mitochondria into the cytosol in TG- and Br-A23187- induced apoptosis, suggesting that cytochrome c release is not a universal phenomenon in the apoptotic process.

Telomerase activity is not related to apoptosis in leukemic cell lines.

Any deregulation of apoptosis or an escape from cellular senescence will drive the cells to neoplasia. It remains unclear whether there is a direct linkage between apoptosis and telomerase activity particularly in transformed cell lines. In the present study, we investigated the telomerase activities in three leukemic cell lines (HL-60, U937 and K562) after treating these cells with various doses of antitumor drugs, puromycin or actinomycin D (ActD). Our results showed that HL-60 cells underwent apoptosis rapidly when treated with either 20 microg/ml of puromycin or 5 microg/ml of Act D with more than 60% of the cells becoming apoptotic at 6 hrs and almost 100% at 12 hrs. But telomerase activity analyzed by TRAP assay in these apoptotic cells remained unchanged as compared with the untreated control cells suggesting that whether the cells were apoptotic or not, it had no effect on telomerase activity. However, if lower dosages of the drugs were used, that is, 0.5-1.5 microg/ml of puromycin or 0.01-0.5 microg/ml of Act D, a decrease in telomerase activity was observed at 24-48 hrs, and was completely undetectable at 72 hrs. This decrease in telomerase activity was dose- and time-dependent. The suppression of telomerase activity by low doses of these two drugs is probably due to the inhibitory effect of the drugs on protein translation or RNA transcription rather than direct inhibition of the telomerase activity. Flow cytometry analysis of the cell cycle of the drug-treated cells showed that these drugs unselectively induced apoptosis at all phases of the cell cycle and was unrelated to the changes in telomerase activity. Similar results were observed in U937 and K562 cells except that K562 cells underwent apoptosis more slowly than the former two cell lines.

Redistribution of cytochrome c is not an essential requirement in C2-ceramide induced apoptosis in HL-60 cells.

bcl-2 has been shown to enhance cell survival by inhibiting apoptosis. The present study investigates the potential role of bcl-2 on apoptosis in HL-60 cells induced by different agents. HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Staurosporine (STS) promoted DNA fragmentation dose-dependently in the 6 h exposure assay while C2-ceramide was relatively slow in the induction of apoptosis (approximately 40% after 24 h) and required higher concentrations (> 20 microM). Caspases inhibitors, Ac-YVAD-cmk (100 microM) and zVAD-fmk (20 microM) had no effect on DNA fragmentation themselves. However, they blocked C2-ceramide-induced caspase-3 cleavage and apoptosis, but not the release of cytochrome c from the mitochondria. In addition, we found that both Ac-YVAD-cmk and zVAD-fmk failed to protect STS-induced apoptosis in HL-60 cells. Overexpression of bcl-2 inhibited STS and C2-ceramide induced cytochrome c redistribution, caspase-3 activation and apoptosis. These results suggest a protective role of bcl-2 in the regulation of apoptosis and cytochrome c release is unlikely to be involved in the final common pathway in apoptosis.

Regulation of transferrin function and expression: review and update.

The cellular iron uptake is a precisely controlled process to fulfill the iron demand for the synthesis and functions of a variety of iron-containing proteins, and one of the main molecules involved is the transferrin receptor (TfR), which mediates the uptake process via the transferrin cycle. The TfR expression is tightly regulated by factors such as intracellular iron level, cell proliferation or erythropoiesis at levels of receptor recycling, transcriptional or posttranscriptional control. The iron-regulatory protein/iron-responsive element system has been widely used to explain changes in receptor expression during iron loading or depletion, oxidative stress and nitric oxide stimulation. On the other hand, transcriptional control of TfR expression appears to be more important in erythroid differentiation and general cell proliferation. There is also an increasing awareness of the clinical application and experimental therapeutics based on the TfR functioning and expression. In this review, we attempt to provide a concise account of the studies of TfR structure and function as well as those areas that have not been reviewed in depth, in particular, tissue-specific regulation of TfR, the molecular mechanisms of TfR expression, and the use of TfR as diagnostic and therapeutic tools. The regulation of TfR expression in various tissues is related to its specific cellular iron requirements. Hemoglobin-synthesizing cells exhibit distinct features of iron metabolism and TfR expression as compared to most non-erythroid cells which synthesize a much lower amount of heme. For most non-erythroid cells, iron can regulate the TfR expression in a reciprocal manner through modulating the stability of the receptor mRNA whereas in hemoglobin-synthesizing cells, the TfR expression is independent of the cellular iron loading. In spite of a wide heterogeneity in the way receptor redistribution is in response to various stimuli, regulation of the constitutive expression of TfR is one of the ways of regulating the cellular iron uptake. This expression operates on both transcriptional and posttranscriptional levels. In general, factors related to cell growth and differentiation operate on the gene transcription level, whereas iron regulates the fate of the mature mRNA.

Lactation increases prolactin receptor expression in spleen and thymus of rats.

Prolactin receptors (PRL-R) are widely expressed on cells of the immune system. During lactation, the increase in serum PRL levels may modify the gene expression of these receptors. Specific PRL binding sites and the expressions of both PRL-R-L and PRL-R-S forms in thymus and spleen of nulliparous control, 10-day postpartum lactating, and 10-day postpartum nonlactating rats were studied. A semi-quantitative RT-PCR technique was used to detect the PRL-R gene transcript in the tissues. Our results showed that specific PRL binding sites and PRL-R-L mRNA and PRL-R-S mRNA were present in the lymphoid tissues with the PRL-R-L mRNA predominant. Lactation markedly increased specific binding sites and PRL-R-L mRNA in both spleen and thymus and only PRL-R-S in spleen. No differences between nulliparous control and postpartum nonlactating rats were observed in any of the parameters measured. The parallel increase in specific PRL binding sites and PRL-R-L mRNA suggests that lactation up-regulates PRL-R expression in lymphoid tissues and may be beneficial to the maternal immune system.

Spontaneous Mn2+ entry is specifically inhibited by calyculin A in human leukemic HL-60 cells.

The effects of calyculin A and other agents which enhance protein Ser/Thr phosphorylation, on the cytosolic free Ca2+ concentration ([Ca2+]i) and spontaneous Mn2+ entry were investigated in fura-2-loaded human leukemic HL-60 cells. Calyculin A (30 nM), a specific inhibitor of protein Ser/Thr phosphatase (PP) 1 and 2A, significantly decreased [Ca2+]i. By contrast, another structurally unrelated inhibitor of PP1 and 2A, okadaic acid (1 microM), caused a slight elevation in [Ca2+]i. Forskolin (30 microM), which could enhance protein kinase A activity by raising cAMP concentration, also caused a rise in [Ca2+]i. Phorbol myristate acetate (PMA, 300 nM), an activator of protein kinase C, did not have a significant effect on [Ca2+]i. Spontaneous entry of Mn2+ (a surrogate ion for Ca2+) was strongly inhibited by calyculin A, but not okadaic acid, forskolin or phorbol myristate acetate. Such inhibition was not significantly affected by staurosporine (300 nM), a non-specific inhibitor of protein Ser/Thr kinases. Our results suggest that calyculin A inhibited a plasmalemmal leak pathway to Mn2+ (and Ca2+), probably leading to a decrease in [Ca2+]i. Inhibition of spontaneous Mn2+ entry by calyculin A may depend on a specific protein phosphorylation pattern induced by staurosporine-insensitive protein kinase(s).

Specific interaction between tetrandrine and Quillaja saponins in promoting permeabilization of plasma membrane in human leukemic HL-60 cells.

Spontaneous Ni2+ entry (leak), measured as fluorescence quench in fura-2-loaded HL-60 cells at the excitation wavelength of 360 nm, was strongly inhibited by tetrandrine (TET, 100 microM), a Ca2+ antagonist of Chinese herbal origin. Exposure of the cells for 5 min to saponins from Quillaja saponaria (QS, 30 microg/ml), surfactants well known to permeabilize the plasma membrane by complexing with cholesterol, promoted Ni2+ entry without causing fura-2 leak-out. Unexpectedly, TET caused an immediate (within 2.5 min) augmentation of QS-promoted Ni2+ entry; and a 5-min treatment with both TET and QS resulted not only in an enhanced Ni2+ entry, but also a fura-2 leak-out. Ginseng saponins (100 microg/ml) alone or together with TET did not cause such a permeabilization. Permeabilization induced by 1-3 microM digitonin, another cholesterol-complexing glycoside, could not be enhanced by TET. TET did not affect permeabilization induced by Triton X-100 (0.01%), a detergent which non-specifically disrupts the hydrophobic interaction at the plasma membrane. TET also did not enhance Ni2+ entry triggered by ionomycin (0.35 microM) or SK&F 96365 (20 microM). Further, it did not augment Ni2+ entry when the plasma membrane fluidity was modulated by changes of temperature (27-47 degrees C) or treatment with 5% ethanol. This QS-promoted Ni2+ entry could not be amplified by other lipophilic Ca2+ antagonists, such as diltiazem (100 microM) and verapamil (100 microM). The results hence indicate that TET enhanced Ni2+ entry (or permeabilization) elicited by QS treatment, but not other perturbations of the plasma membrane. We suggest that pore formation at the plasma membrane, a consequence of QS-cholesterol interaction, can be specifically enhanced by TET. Also, a comparative study of the effects of TET and its very close analogues, hernandezine and berbamine, reveals that the methoxyl group at the R2 position of TET appears to be crucial in enhancing QS-promoted Ni2+ entry.

Effects of tetrandrine and closely related bis-benzylisoquinoline derivatives on cytosolic Ca2+ in human leukaemic HL-60 cells: a structure-activity relationship study.

1. Previously it has been shown that tetrandrine (TET), a bis-benzylisoquinoline alkaloid, isolated from a Chinese herb Stephania tetrandra, can block non-voltage-operated Ca2+ entry activated by intracellular Ca2+ store depletion induced by thapsigargin (TSG) and can release intracellular Ca2+ in HL-60 cells. The present study attempted to identify the chemical group(s) of the TET molecule responsible for these dual effects. The effects of TET and its closely related analogues, hernandezine (HER) and berbamine (BER), on Ca2+ entry and Ca2+ release were compared in fura-2-loaded HL-60 cells. 2. Berbamine was much less potent (IC50 = 200 mumol/L) than TET and HER (both IC50 values = 25 mumol/L) in inhibiting Ca2+ entry activated by TSG. Furthermore, at 100 mumol/L, BER was much less effective than TET and HER in suppressing TSG-induced Mn2+ entry. At 30-100 mumol/L, BER was significantly less effective than both TET and HER in causing Ca2+ release from internal stores. However, only BER was able to cause store depletion-activated Ca2+ entry (or the so-called 'capacitative Ca2+ entry') upon Ca2+ readmission. 3. Taken together, the data from this structure-activity relationship study reveal that the -OCH3 group of one particular benzene ring of TET, which distinguishes TET from BER, in part produces the dual pharmacological actions of TET.

Differential effects of phorbol ester on apoptosis in HL-60 promyelocytic leukemia cells.

The role of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in apoptosis of HL-60 cells was investigated. PMA inhibited DNA fragmentation induced by thapsigargin (TG) and 4-bromo-calcium ionophore (Br-A23187). The inhibitory effect of PMA was concentration-related and was abolished by a specific PKC inhibitor, bisindolylmaleimide (GF109203X. In addition TG-induced apoptosis was decreased in cells in which PKC activity was down-regulated by long-term pretreatment with PMA. These results indicate that PKC activation by PMA inhibits HL-60 cell apoptosis induced by TG and Br-A23187, and that this inhibition is not influenced by the down-regulation of PKC. However, PMA did not inhibit DNA fragmentation induced by 1-beta-D-arabinofuranosylcytosine (Ara-C) and cycloheximide. PMA suppressed TG- or Br-A23187. Our results indicate that PKC participates in the regulation of apoptosis only by some pathways. Down-regulation of PKC is not responsible for the diverse effects of PKC activators on apoptosis. The effect of a PKC modulator on apoptosis is dependent upon interaction with individual apoptotic stimulus.

Dual effects of SK&F 96365 in human leukemic HL-60 cells. Inhibition of calcium entry and activation of a novel cation influx pathway.

The effects of the receptor-mediated Ca2+ entry blocker, SK&F 96365 on thapsigargin (TSG)-induced Ca2+ entry in fura-2-loaded HL-60 cells were studied. After Ca2+ release induced by 30 nM TSG, readmission of Ca2+ resulted in a sustained Ca2+ entry, which could be partially inhibited by 1-3 microM SK&F 96365. Surprisingly, SK&F 96365 at 30-100 microM, instead of causing a stronger inhibition, actually promoted Ca2+ entry. Furthermore, at 16-100 microM, this drug released intracellular Ca2+ on its own and induced Ca2+ entry upon readmission of Ca2+. This SK&F 96365-activated Ca2+ entry pathway was insensitive to nifedipine and, interestingly, accessible to Ni2+ and La3+. However, SK&F 96365 (30 microM) almost completely blocked (basal) Mn2+ entry and only caused 4.4% of the cells to be stained with trypan blue, strongly suggesting that the SK&F 96365-activated cation entry was not due to damage nor to a very nonselective permeabilization of the plasma membrane. These data indicate that low concentrations of SK&F 96365 inhibited Ca2+ entry and higher concentrations activated a novel cation entry pathway. Because these 2 opposing effects overlapped at an intermediate concentration (16 microM), which is within the range commonly used to block Ca2+ entry, cautious use of this Ca2+ antagonist appears to be warranted.

Transcriptional regulation of transferrin receptor expression during phorbol-ester-induced HL-60 cell differentiation. Evidence for a negative regulatory role of the phorbol-ester-responsive element-like sequence.

The mechanism involved in the regulation of transferrin receptor (TfR) expression during phorbol-ester-induced HL-60 cell differentiation was investigated. The mRNA of the TfR was constitutively expressed in proliferating HL-60 cells. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h resulted in a gradual decrease in the expression of the TfR mRNA. Nuclear run-on assays revealed that the transcription of the TfR gene was inhibited by prior treatment of cells with PMA. The effect of PMA on the binding of nuclear proteins to the TfR gene promoter region was then investigated. Based on sequence similarity and previous footprinting data, the promoter region of the TfR gene seems to contain a sequence like that of the phorbol-ester-responsive element (TRE). Our results showed that the binding of nuclear extracts to the TfR gene promoter region containing the TRE-like sequence was increased in PMA-treated cells. This binding activity could be abolished by prior incubation of the nuclear extracts with a synthetic oligonucleotide containing the consensus TRE sequence. In vitro transcription assays revealed that prior incubation of the nuclear extracts of PMA-treated cells with excess consensus TRE oligonucleotide enhanced the gene transcription driven by the TfR gene promoter. These findings suggest that the TRE-like element may play a role in the inhibition of TfR gene transcription.

Capacitative Ca2+ entry in HL-60 cells: tetrandrine and SK & F 96365 as probes.

Agonist-activated Ca2+ entry is important in many biological responses such as secretion and cell growth(1,2). In nonexcitable cells which have no voltage-operated Ca2+ channels (VOCC), agonist-receptor interaction can trigger Ca2+ entry across the plasmalemma via several entry pathways(1-3) (Fig 1): (A) channels which are intrinsic structures of the receptor (receptor-operated channels), (B) channels which are coupled to receptors via a G-protein (G-protein-operated channels), (C) channels which are activated by some second messengers (second-messenger-operated channels), and (D) channels which open upon intracellular nonmitochondrial Ca2+ store depletion (Ca2+ release-activated channels) resulting from inositol 1, 4, 5-trisphosphate-induced Ca2+ release or inhibition of Ca2+ re-uptake (see next section). Ca2+ entry via the 4th type of channel, also known as "capacitative Ca2+ entry" (CCE)[4], has aroused much interest in the past decade because of its intriguing nature as retrograde signalling. In this brief review, we present the evidence for and the possible biochemical processes involved in CCE. We also discuss the use of 2 novel Ca2+ entry blockers: tetrandrine and SK&F 96365. Emphasis will be put on the human leukemic HL-60 cell line, a popular cell system for intracellular Ca2+ homeostasis studies and also a model the signal transduction of which we have been investigating during the past few years.

Effects of Na+/H+ antiport and intracellular pH in the regulation of HL-60 cell apoptosis.

The roles of Na+/H+ antiport and intracellular pH in apoptosis of HL-60 cells were investigated here. We found that dimethyl amiloride, a specific Na+/H+ antiport inhibitor, induced intracellular acidification but not apoptosis; while sodium ionophore, monensin caused intracellular alkalinization as well as apoptosis in HL-60 cells. Br-A23187 and thapsigargin could induce a various degree of intracellular alkalinization through the stimulation of Na+/H+ antiport. Dimethyl amiloride blocked the intracellular alkalinization and inhibited apoptosis induced by Br-A23187 and thapsigargin. PMA also stimulated Na+/H+ antiport and induced intracellular alkalinization which was completely blocked by dimethyl amiloride and partially attenuated by PKC inhibitors. PMA could inhibit apoptosis in HL-60 cells. PMA-induced suppression of apoptosis was, however, not interfered by dimethyl amiloride, but could be abolished by PKC inhibitors. These results indicate that pHi alkalinization and/or the stimulation of Na+/H+ antiport, instead of intracellular acidification, are contributory to the induction of apoptosis. PMA-induced inhibition of apoptosis is not necessarily associated with intracellular alkalinization, but primarily due to activation of PKC. We suggest that stimulation of Na+/H+ antiport and pHi alkalinization act as facilitating factors in the induction of apoptosis.

The effect of nicotine and its interaction with carbon tetrachloride in the rat liver.

In order to study the effects of nicotine on liver, groups of rats were given nicotine doses that simulated those seen in chronic smoking (54 and 108 mumol/l of nicotine) for 10 days. A subgroup was also given a single subcutaneous injection of 6 g/kg of carbon tetrachloride (CCl4) shortly before the animals of the group were killed. Histology demonstrated a significant hepatotoxic effect in the group receiving 108 mumol/l of nicotine when compared with the control group in the form of fatty change, focal or confluent necrosis and dark-cell change. The effects in pregnant rats were less severe. Carbon tetrachloride alone induced significant fatty change and focal necrosis in non-pregnant rats but not in pregnant rats. Nicotine also aggravated the CCl4 induced pathological changes in livers of both non-pregnant and pregnant animals. Thus nicotine alone, when given at a concentration of 108 mumol/l, exerted hepatotoxic effects; the alkaloid also aggravated the hepatotoxicity of CCl4. Pregnant rats were more resistant to the hepatotoxic effects produced by nicotine and CCl4.

Effects of protein kinase modulators on transferrin receptor expression in human leukaemic HL-60 cells.

The mRNA of transferrin receptor (TfR) is constitutively expressed in proliferating human leukaemic HL-60 cells. Treatment of HL-60 cells with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, or dibutyryl-cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, resulted in a 90% decrease in the level of TfR mRNA. Inhibition of TfR mRNA expression induced by 10 nM PMA and 100 microM dbcAMP was abolished by prior incubation of cells with 0.1-1.0 microM GF109203X, a PKC-specific inhibitor, and 1-10 microM H-89, a PKA-specific inhibitor, respectively. The blocking effects of GF109203X and H-89 were dose-dependent and complete at the highest concentrations of the inhibitors used. Although treatment of cells with GF109203X or H-89 alone did not alter the constitutive expression of TfR mRNA, incubation of cells with 30-100 nM staurosporine, a wide-spectrum protein kinase inhibitor, resulted in suppression of the constitutive expression of TfR mRNA in a dose-dependent manner. These results suggest that (i) the down-regulation of TfR mRNA expression during the differentiation of HL-60 cells can be mediated by activation of either PKC or PKA; (ii) the constitutive expression of TfR mRNA in proliferating HL-60 cells is staurosporine-sensitive and is probably maintained by protein kinase(s) other than PKC and PKA.

Roles of calcium in the regulation of apoptosis in HL-60 promyelocytic leukemia cells.

Increase in intracellular calcium concentrations ([Ca2+]i) is critical for the initiation of apoptosis in cells such as thymocytes and in other cells, calcium chelators may promote apoptosis. However, calcium modulators, such as calcium ionophore 4-bromo-calcium ionophore (Br-A23187) and thapsigargin (TG), induce apoptosis in different cells, including HL-60 cells in which the induction of apoptosis seems a calcium-independent process. These observations imply that the disturbance of calcium homeostasis is probably the most important factor in the regulation of apoptosis. In this article, reagents with different potencies of modulating calcium homeostasis were used to study the possible role of [Ca2+]i and the status of intracellular calcium stores in the causation of HL-60 cell apoptosis. We found that an increase in [Ca2+]i alone did not result in apoptosis, while the depletion of TG-sensitive calcium stores in the endoplasmic reticulum was closely related with the induction of apoptosis. In HL-60 cells, extracellular and intracellular calcium chelators promoted apoptosis. Calmodulin antagonist did not attenuate apoptosis induced by other reagents. Our results suggest that the depletion of Ca2+ stores is an important mean to modulate calcium homeostasis and that the mobilization of calcium (Ca2+) from intracellular stores, rather than an increase in [Ca2+]i, provides the signal for the induction of apoptosis in HL-60 cells.

Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL-60 cells: intracellular calcium release and calcium entry blockade.

1. Tetrandrine (TET, a Ca2+ antagonist of Chinese herbal origin) and thapsigargin (TSG, an endoplasmic reticulum Ca2+ pump inhibitor) concentration-dependently mobilized Ca2+ from intracellular stores of HL-60 cells, with EC50 values of 20 microM and 0.8 nM, respectively. After intracellular Ca2+ release by 30 nM TSG, there was no more discharge of Ca2+ by TET (100 microM), and vice versa. 2. Pretreatments with 100 nM rauwolscine (alpha 2-adrenoceptor antagonist), 100 nM prazosin (alpha 1-adrenoceptor antagonist), 10 nM phorbol myristate acetate (PMA, a protein kinase C activator) or 100 nM staurosporine (a protein kinase C inhibitor) had no effect on 100 microM TET-induced intracellular Ca2+ release. 3. After intracellular Ca2+ release by 30 nM TSG in Ca(2+)-free medium, readmission of Ca2+ caused a substantial and sustained extracellular Ca2+ entry. The latter was almost completely inhibited by 100 microM TET (IC50 of 20 microM) added just before Ca2+ readmission. In Ca(2+)-containing medium, 30 nM TSG caused a sustained phase of cytosolic Ca2+ elevation, which could be abolished by 100 microM TET. TET was also demonstrated to retard basal entry of extracellular Mn2+ and completely inhibit TSG-stimulated extracellular Mn2+ entry. 4. TSG-induced extracellular Ca2+ entry was insensitive to the L-type Ca2+ channel blocker, nifedipine (1 microM), but was completely inhibited by the non-selective Ca2+ channel blocker La3+ (300 microM). Depolarization with 100 mM KCl did not raise the cytosolic Ca2+ level. 5. These data suggest that (a) TET and TSG mobilized the same Ca2+ pool and TET-induced intracellular Ca2+ release was independent of protein kinase C activity and ox-adrenoceptor activation,and (b) TET blocked the voltage-insensitive Ca2+ entry pathway activated by TSG. These dual effects on HL-60 cells were also observed with hernandezine (HER), a TET-like compound and in another cell type, murine B lymphoma M12.4 cells.

Cytosolic calcium pre-elevation amplifies agonist-induced calcium release in human leukaemic HL-60 cells.

Histamine, ATP, and two microsomal Ca(2+)-pump inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), were able to release intracellular Ca2+ in human leukaemic HL-60 cells. The relationships between the agonist-, TG- and CPA-sensitive Ca2+ pools were investigated with optimal concentrations of these agents in Ca(2+)-free medium. CPA failed to release Ca2+ after the Ca2+ stores of the cells had been discharged by TG, and vice versa, suggesting that the TG- and CPA-sensitive pools exactly overlap. Using this protocol, it was further demonstrated that (a) histamine and ATP utilized the same agonist-sensitive pool, and (b) the CPA- or TG-sensitive pool was much larger than, and encompassed, the agonist-sensitive pool. Although optimal (30 microM) CPA treatment for 5 min totally emptied the agonist-sensitive pool, a brief exposure (1.5 min) to a sub-optimal concentration (3 microM) of CPA, which only slightly raised cytosolic free Ca2+ concentration ([Ca2+]i), substantially enhanced subsequent agonist-induced Ca2+ release. Brief pretreatments with sub-optimal concentrations of TG or ionomycin, which caused moderate [Ca2+]i elevation, also caused such enhancement. However, sub-optimal CPA pretreatment had no prominent effect on Ca2+ release, which was InsP3-independent: it did not enhance TG-induced Ca2+ release, and only relatively weakly augmented ionomycin-induced Ca2+ release. Our results represent a novel observation showing that low concentrations of CPA, TG and ionomycin can potentiate subsequent agonist-induced Ca2+ release, and suggest that a 'priming' moderate [Ca2+]i elevation can amplify subsequent InsP3-dependent Ca2+ release in HL-60 cells.