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N Demaurex

Publications and source records attributed to N Demaurex.

At least 37 records · Page 2Linked to original sources
PubMed1997

Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease.

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+ indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+ release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.

Animals↗
PubMed1997

Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion.

Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation. It has been shown that calreticulin associates with the cytoplasmic domains of integrin alpha-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibroblasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.

Animals↗
PubMed1997

ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids.

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.

Adenosine Triphosphate↗
PubMed1996

Intracellular pH regulation during spreading of human neutrophils.

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.

Actin Cytoskeleton↗
PubMed1995

[Characterization of Ca2+ influx in human neutrophils using the patch clamp technique].

In neutrophils, Ca2+ influx across the plasma membrane occurs in response to a variety of agonists. It plays an important role in the regulation of neutrophil function, in particular exocytosis and release of inflammatory mediators. Using the combination of patch clamp and microfluorimetry techniques, we have found the following characteristics of neutrophil Ca2+ influx: (a) neutrophils do not possess voltage-dependent Ca2+ channels; (b) Ca2+ influx is activated by depletion of intracellular Ca2+ stores, either by Ins(1,4,5)P3, or by Ca(2+)-ATPase inhibitors; (c) Ca2+ influx is not activated by [Ca2+]i elevations and not associated with the activation of non-selective cation channels; (d) Ca2+ influx is associated with the activation of a small, voltage-independent current through a highly Ca(2+)-selective conductance. The current was only seen upon depletion of intracellular Ca2+ stores and showed a pharmacological profile similar to the fluorimetrically detected Ca2+ influx.

Calcium↗
PubMed1995

Highly cooperative Ca2+ elevations in response to Ins(1,4,5)P3 microperfusion through a patch-clamp pipette.

To study the initial kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations with a high time resolution and to avoid the problem of cell-to-cell heterogeneity, we have used the combined patch-clamp/microfluorimetry technique. The mathematical description of the microperfusion of Ins(1,4,5)P3 and the subsequent Ca2+ release consists of a monoexponential decay (cytosolic Ins(1,4,5)P3 concentration) and a Hill equation (Ins(1,4,5)P3 dose-response curve). Two additional Hill equations and an integration were necessary to include a putative dependence of Ins(1,4,5)P3-induced Ca2+ release on [Ca2+]i. Best-fitting analysis assuming [Ca2+]i-independent Ca2+ release yielded Hill coefficients between 4 and 12. The high cooperativity was also observed with the poorly metabolizable analog Ins(2,4,5)P3 and was independent of extracellular [Ca2+]. Best-fitting analysis including a positive [Ca2+]i feedback suggested a cooperativity on the level of Ins(1,4,5)P3-induced channel opening (n = 2) and an enhancement of Ins(1,4,5)P3-induced Ca2+ release by [Ca2+]i. In summary, the onset kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations in single HL-60 granulocytes showed a very high cooperativity, presumably because of a cooperativity on the level of channel opening and a positive Ca2+ feedback, but not because of Ca2+ influx or Ins(1,4,5)P3 metabolism. This high cooperativity, acting in concert with negative feedback mechanisms, might play an important role in the fine-tuning of the cellular Ca2+ signal.

Calcium↗
PubMed1995

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

Animals↗
PubMed1995

Ion transport and the function of phagocytic cells.

Inorganic ions constitute the predominant osmolytes of blood cells and are therefore the main determinant of the cellular volume. In leukocytes, maintenance of constant cell size is accomplished by modulating the ionic permeability of the membrane. In addition, the plasmalemmal ionic permeability dictates the transmembrane potential, which influences a variety of cellular responses. Inorganic ions are also essential for the regulation of the intracellular pH and play an active role in signal transduction during phagocyte activation. This chapter reviews recent progress in the area of ion transport in phagocytic leukocytes, with special reference to the physiologic implications to the activation process.

Animals↗
PubMed1994

Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol trisphosphate-dependent mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) that appear to be independent of cAMP. To study the mechanisms involved in this response, we used the patch-clamp technique to microperfuse various agents into single rat gonadotrophs while monitoring [Ca2+]i with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (> 1 microM) oscillations in [Ca2+]i, which were blocked by intracellular application of GDP beta S (guanosine 5'-O-2-thiodiphosphate), indicating the involvement of a G-protein. To identify the intracellular messenger(s) involved, we microperfused gonadotrophs with cAMP, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of cAMP (100 microM) had no significant effect on basal [Ca2+]i and did not alter the PACAP-stimulated Ca2+ response. Heparin, but not its inactive isoform, completely blocked the PACAP-stimulated increase in [Ca2+]i, while Ins(1,4,5)P3 stimulated oscillations in [Ca2+]i very similar to those observed in response to PACAP. These results strongly suggest that PACAP mobilizes Ca2+ through an Ins(1,4,5)P3-dependent mechanism. The fact that PACAP stimulates two signaling pathways in pituitary cells could substantially enhance the signaling potential of this hypothalamic peptide.

Animals↗
PubMed1994

Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils.

1. It is not known to what extent the emptying of intracellular Ca2+ stores participates in the mediation of chemoattractant-induced Ca2+ influx in human neutrophils. To study this question, we compared the properties of bivalent-cation influx in response to the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenyl-alanine (f-MLP) and to the microsomal Ca(2+)-ATPase inhibitor thapsigargin. 2. The influx pathway activated by f-MLP and thapsigargin had identical properties of permeation. Mn2+ influx became saturated at around 1 mM extracellular Mn2+, whereas Ca2+ influx did not become saturated up to concentrations of 10 mM. 3. The influx of the two bivalent cations, Mn2+ and Ca2+, was activated to a similar extent and with identical kinetics of activation. 4. The Mn2+ influx activated by f-MLP and thapsigargin was blocked, with identical dose-inhibition curves, by four imidazole analogues. 5. The same relationship between the emptying of Ca2+ stores and bivalent-cation influx was observed for f-MLP and thapsigargin, with a half-maximal activation of the influx at 40% emptying of intracellular stores. 6. In conclusion, neutrophils possess a single type of Ca(2+)-influx pathway that is activated by receptor agonists and by store depletion. Receptor agonists activate this influx pathway to a large extent, if not completely, through the depletion of intracellular Ca2+ stores.

Biological Transport↗
PubMed1994

Na+/H+ antiport: modulation by ATP and role in cell volume regulation.

Na+/H+ antiport is a major determinant of intracellular pH (pHi) and also plays an important role in the maintenance of cellular volume. Na+/H+ exchange through NHE-1, the ubiquitous isoform of the antiporter, is accelerated by cytosolic acidification and also by osmotically induced cell shrinking, thereby promoting recovery of the physiological pHi and volume, respectively. Although hydrolysis of ATP is not required for transport of ions through the antiporter, metabolic depletion exerts a marked inhibitory effect. Depletion of ATP also prevents osmotic activation and volume regulation. Contrary to earlier suggestions, however, changes in the phosphorylation state of the antiporter itself are not involved in the effects of either metabolic depletion or osmotic stimulation. Nevertheless, the cytosolic carboxy-terminal segment of the antiporter, which contains the major phosphorylation sites, is essential for the ATP dependence as well as for osmotic activation. It is conceivable that this domain interacts with ancillary phosphorylated or nucleotide-binding proteins, with the cytoskeleton and/or with specific phospholipids, which modulate the rate of transport. Nucleotide dependence and osmotic sensitivity have been compared in three different isoforms of the antiporter, heterologously expressed in fibroblastic cells. Like NHE-1, NHE-2 and NHE-3 were severely inhibited by depletion of ATP. In contrast, whereas NHE-2 was stimulated by osmotic shrinkage, NHE-3 was inhibited. The possible physiological significance of the ATP-dependence and osmotic responsiveness of the antiporter isoforms is discussed.

Adenosine Triphosphate↗
PubMed1993

Proton channels, plasma membrane potential, and respiratory burst in human neutrophils.

When confronted with invading microorganisms, neutrophils undergo a number of nearly synchronous reactions including the generation of microbicidal reactive oxygen intermediates by the NADPH oxidase. These reactions are accompanied by a slow depolarization, from resting values of-60 mV to levels probably exceeding 0 mV. The depolarization is transient, indicating that a compensatory charge transport mechanism is activated. Activation of the oxidase system causes a massive burst of metabolic acid generation that would, if uncompensated, lower the intracellular pH of neutrophils by over 5 units, to lethal levels (pH = 2). Neutrophils must therefore possess particularly effective regulatory systems to avoid excessive cytosolic acidification. The recently described H+ conductance of neutrophils may counteract both the acidification and the depolarization. Activation of the H+ conductance occurs at depolarizing voltages and is promoted by cytosolic acidification, a combination that takes place during the respiratory burst. The NADPH oxidase of neutrophils is thus associated to an unusual, particularly efficient mechanism of H+ export and charge compensation. The sequential activation of these two systems causes neutrophils to depolarize through the activation of an electron transport chain, and to repolarize through the activation of a H+ conductance.

Cell Membrane↗
PubMed1993

Highly co-operative Ca2+ activation of intermediate-conductance K+ channels in granulocytes from a human cell line.

1. To study Ca(2+)-activated K+ currents in dimethyl sulphoxide (DMSO)-differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-L-phenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2+]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca(2+)-activated current was a function of the extracellular K+ concentration (56.1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to +100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nM [Ca2+]i, and a highly co-operative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca(2+)-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca(2+)-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS). 8. In summary, we describe a charybdotoxin-sensitive, intermediate-conductance Ca(2+)-activated K+ channel in HL-60 granulocytes. The characteristics of the Ca2+ activation of this current (i.e. sensitivity to submicromolar [Ca2+]i, high co-operativity and voltage independence) are similar to the Ca2+ activation of the apamin-sensitive small-conductance K+ channel. Our results also suggest that [Ca2+]i elevations are the predominant, if not the only, activators of this channel during physiological stimulation of HL-60 granulocytes.

Apamin↗
PubMed1993

Ion channels and receptor-mediated Ca2+ influx in neutrophil granulocytes.

Although generally classified as non-excitable cells, human neutrophils possess a variety of ion channels that play a crucial role in the regulation of cellular activity. The mechanism of receptor-mediated Ca2+ influx in neutrophils is complex. Receptor agonists empty intracellular Ca2+ stores via generation of Ins(1,4,5)P3. The emptying of intracellular Ca2+ stores leads by an hitherto not understood mechanism to the activation of Ca2+ influx across the plasma membrane. Neutrophils possess at least 2 types of K+ channels. Voltage-activated K+ channels, important for the maintenance of the resting potential, and Ca2+ activated K+ channels, important for the repolarization after cellular activation. Neutrophils also possess voltage- and pH activated H+ channels that serve to extrude protons, generated by the neutrophil respiratory burst. Neutrophils depolarize in response to activation by agonists. The mechanism of neutrophil depolarization involves electron transport by the respiratory burst oxidase. Neutrophil depolarization serves as a negative feed-back mechanism, it also activates the H+ channels and thereby stimulates extrusion of protons.

Calcium↗
PubMed1993

Proton currents in human granulocytes: regulation by membrane potential and intracellular pH.

1. To determine whether conductive pathways contribute to the H+ efflux from granulocytes, we used the whole-cell patch-clamp technique combined with microfluorimetric determinations of cytosolic pH (pHi) in single, dimethylsulphoxide-differentiated HL-60 cells. 2. In voltage-clamp mode, depolarization of the cell from the resting potential (around -60 mV) to +60 mV caused an increase in pHi that was accompanied by a sizeable outward current. 3. Ion substitution experiments and analysis of the reversal potential of tail currents indicated that the outward current is carried largely by H+ ions. 4. Full activation of the H+ current occurred within 1-2s after depolarization and deactivation within 100-200 ms upon repolarization. 5. This H+ conductance was strongly dependent on pHi, being larger at acidic pH. In addition, at low pHi the threshold for voltage activation of the H+ conductance was shifted to more negative values. 6. Addition of millimolar concentrations of Cd2+ and Zn2+ to the bath solution reduced the maximum H+ conductance and shifted the voltage dependence of the H+ conductance to more positive potentials. The effects were reversible. 7. In conclusion, our results demonstrate that granulocytic HL-60 cells possess a voltage-gated and pHi-sensitive H+ conductance. Because both a depolarization and a cytosolic acidification occur during the activation of granulocytes, this conductance may play a role in pHi homeostasis of granulocytes during microbial killing.

Cations, Divalent↗
PubMed1992

Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells.

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.

Biological Transport↗