Histamine increases cytosolic Ca2+ in HL-60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist profile and induces functional differentiation.

Histamine H1 receptors mediate activation of phospholipase C, with subsequent increases in cytosolic Ca2+ concentration ([Ca2+]i), and H2 receptors mediate accumulation of cAMP. HL-60 promyelocytes possess H2 receptors, but it is not known whether these cells also possess H1 receptors. We studied the effects of histamine on [Ca2+]i and the functional importance of histamine receptors in HL-60 promyelocytes. In these cells, histamine and dimaprit increased [Ca2+]i with EC50 values of 15 microM and 30 microM, respectively. Diphenhydramine inhibited the effect of histamine (100 microM) on [Ca2+]i up to 40%, with an IC50 of 100 nM. Famotidine and cimetidine diminished the effect of histamine (100 microM) up to 75%, with IC50 values of 85 nM and 300 nM, respectively. Diphenhydramine plus famotidine abolished histamine-induced rises in [Ca2+]i. Impromidine, with an IC50 of 100 nM, abolished the effect of histamine (100 microM) on [Ca2+]i. Diphenhydramine, famotidine, cimetidine, and impromidine showed marked noncompetitive antagonism with histamine. Histamine-induced increases in [Ca2+]i were largely due to influx of Ca2+ from the extracellular space. Ca2+ influx was inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). Histamine activated phospholipase C. Histamine induced expression of formyl peptide receptors, which effect was abolished by famotidine. In U-937 promonocytes and in the human erythroleukemia cell lines HEL and K-562, histamine did not induce rises in [Ca2+]i. Our data suggest the following. (i) In HL-60 promyelocytes, histamine increases [Ca2+]i predominantly via H2 receptors and to a lesser extent via H1 receptors. (ii) The agonist/antagonist profile of the H2 receptor-mediated increases in [Ca2+]i differs markedly from that for cAMP accumulation, suggesting the involvement of different H2 receptor subtypes. (iii) In HL-60 promyelocytes, histamine activates nonselective cation channels and induces functional differentiation via H2 receptors.     

Histamine inhibits activation of human neutrophils and HL-60 leukemic cells via H2-receptors

Summary The effects of prostaglandin E₁ (PGE₁) and histamine on activation of superoxide (O_22/su–) formation, exocytosis of -glucuronidase and aggregation in human neutrophils and HL-60 leukemic cells were studied. PGE₁, histamine and impromidine, a potent H₂-agonist, inhibited O₂ ^– formation in neutrophils induced by the chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe) with IC₅₀ values of 0.5 µM, 8 µM and 2 µM, respectively. The full H₁-agonist and weak partial H₂-agonist, betahistine, was much less potent and effective than histamine. Dibutyryl cyclic AMP and forskolin mimicked the effects of histamine and PGE₁, on O₂ ^– formation. The H2-antagonist, famotidine, competitively reversed histamine-induced inhibition of O₂ ^– formation with a pA₂ value of 7.5. Histamine inhibited O₂ ^– formation when added prior to or after fMet-Leu-Phe. fMet-Leu-Phe-induced aggregation and release of -glucuronidase in neutrophils were less sensitive to inhibition by PGE₁, histamine, dibutyryl cyclic AMP and forskolin than_OZ formation. The inhibitor of cyclic AMP-specific phosphodiesterase, rac-4-(3-butoxy4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), additively enhanced the inhibitory effects of histamine and PGE₁, on the above cell functions. In HL-60 cells differentiated by dimethyl sulfoxide or dibutyryl cyclic AMP, histamine, impromidine and PGE₁, but not betahistine inhibited fMet-Leu-Phe-induced O₂ ^– formation as well. Our data suggest that histamine inhibits activation of neutrophils and HL-60 cells via H2-receptors through activation of adenylyl cyclase and increased formation of cyclic AMP. As stimulated basophils and mast cells release high quantities of histamine, this intercellular signal molecule may play an inhibitory role in the activation of cytotoxic functions of myeloid cells. Send offprint requests to R. Seifert at the above address     

Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores

The results of binding studies suggest the presence of histamine H₁-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and superoxide anion (O₂ ^– ) formation in HL-60 cells differentiated towards monocytes with 1,25-dihydroxychole-calciferol. In HL-60 monocytes, HA increased [Ca²⁺]_i with a half-maximal effect at 8 M and a maximum at 30–100 M. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca²⁺]_i. Betahistine, a weak partial H₁-receptor agonist, also increased [Ca²⁺]_i, whereas H₂- and H₃-receptor agonists were ineffective. H₁- but not H₂- and H₃-receptor antagonists inhibited HA induced rises in [Ca²⁺]_i. HA-induced rises in [Ca²⁺]_i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4\-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-{\-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), to induce rises in [Ca²⁺]_i. Unlike fMLP, HA did not activate O₂ ^– formation. Our data indicate that HL-60 monocytes possess H₁-receptors coupled to heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) of the G_i-family and PTX-insensitive G-proteins which mediate activation of NSC channels without concomitant activation of Ca²⁺ mobilization from intracellular stores, that homologous desensitization of HA-induced Ca²⁺ influx is independent of protein kinase C and that the stimulatory effect of HA on Ca²⁺ influx is too small to result in activation of O_{2 –} formation. Correspondence to: R. Seifert at the above address     

Zn2+ increases resting cytosolic Ca2+ levels and abolishes capacitative Ca2+ entry induced by ATP in MDCK cells

The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.     

STIM1 but not STIM2 is an essential regulator of Ca influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

Extracellular Ca²⁺ entry, primarily mediated through store-operated Ca²⁺ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca²⁺ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca²⁺ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca²⁺ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca²⁺ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca²⁺ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca²⁺ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.     

Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells.

1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.     

Stimulation of histamine H2- (and H1)-receptors activates Ca2+ influx in all-atrans-retinoic acid-differentiated HL-60 cells independently of phospholipase C or adenylyl cyclase

In human neutrophils, histamine H₂-receptors mediate activation of adenylyl cyclase (AC) and inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced superoxide anion (0 _inf2 ^sup– ) formation, and in HL-60 promyelocytes, H₂-receptors mediate parallel activation of AC, phospholipase C (PLC) and non-selective cation (NSC) channels. As all-trans-retinoic acid (RA) is successfully used in the differentiation therapy of acute promyelocytic leukaemia, we studied signal transduction in RA-differentiated HL-60 cells. Histamine and the H₂-receptor agonist, impromidine, induced both rises in cAMP levels and cytosolic Ca²⁺ ([Ca²⁺]_i). Substances acting at post-receptor sites to increase cAMP did not increase [Ca²⁺]_i. H₂-but not H₁-receptor antagonists inhibited histamine-induced cAMP accumulation and rises in [Ca²⁺]_i were more effectively inhibited by H₂- than by H₁-receptor antagonists. Histamine-induced rises in [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were abolished by the blocker of NSC channels, Gd³⁺, but were resistant to inhibition by pertussis toxin. Unlike FMLP, histamine did not activate PLC. The effects of FMLP on [Ca²⁺]_i were less sensitive to blockade by Gd³⁺ than those of histamine, and there was no cross-desensitization between the two stimuli. FMLP, but not histamine, inhibited transiently thapsigargin-induced rises in [Ca²⁺]₁. Taken together, our results show that histamine activates AC-mediated cAMP accumulation in RA-differentiated HL-60 cells via H₂-receptors and NSC channel-mediated Ca²⁺ influx via H₂- (and H₁)-receptors. Histamine-induced NSC channel activation is not the consequence of AC- or PLC stimulation and occurs, directly or indirectly, via pertussis toxin-insensitive guanine nucleotide-binding proteins. FMLP and histamine activate Ca²⁺ influx by different mechanisms. There are similarities in H₂-receptor-mediated signal transduction between RA-differentiated HL-60 cells and HL-60 promyelocytes and differences between the former cells and neutrophils, indicating that RA-differentiated HL-60 cells must be considered as partially immature.     

Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

Alpha 1-adrenoceptor stimulation increases intracellular pH and Ca2+ in cardiomyocytes through Na+/H+ and Na+/Ca2+ exchange

The effects of alpha 1-adrenergic stimulation on intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were investigated in isolated rat cardiomyocytes with fluorescence dyes, BCECF and fura-2, respectively. In the presence of 5 or 25 mM HCO3- norepinephrine (NE) increased pHi in a dose-dependent manner. Intracellular alkalinization was inhibited by prazosin and phentolamine but not by yohimbine. NE-induced alkalinization was inhibited in the presence of a Na+/H+ exchange inhibitor (5-(N,N-hexamethylene) amiloride (HMA)), a C kinase inhibitor (H-7) or a calmodulin inhibitor (W-7), or in the absence of extracellular Na+. NE also increased [Ca2+]i following the pHi increase, which was abolished in the absence of extracellular Na+ or Ca2+. This Ca2+ influx was inhibited by HMA but not by diltiazem (10(-5) M). Thus, we conclude that alpha 1-adrenergic stimulation enhances Na+/H+ exchange by activation of C kinase, thereby allowing intracellular alkalinization, and that subsequent activation of Na+/Ca2+ exchange increases Ca2+ influx.     

Safrole-induced cellular Ca2+ increases and death in human osteosarcoma cells.

The effect of the carcinogen safrole on intracellular Ca2+ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca2+ handling and viability in MG63 human osteosarcoma cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 130 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 450 microM. The Ca2+ signal was reduced by 30% by removing extracellular Ca2+. Addition of Ca2+ after safrole had depleted intracellular Ca2+ induced Ca2+ influx, suggesting that safrole caused Ca2+ entry. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca2+; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca2+]i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca2+ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca2+]i increase. Trypan exclusion assays revealed that incubation with 65 microM safrole for 30 min did not kill cells, but incubation with 650 microM safrole for 10-30 min nearly killed all cells. Flow cytometry demonstrated that safrole evoked apoptosis in a concentration-dependent manner. Safrole-induced cytotoxicity was not reversed by chelation of Ca2+ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca2+ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca2+-independent manner.     

Thimerosal-induced cytosolic Ca2+ elevation and subsequent cell death in human osteosarcoma cells.

The effect of the oxidizing agent thimerosal on cytosolic free Ca(2+) concentration ([Ca(2+)]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether thimerosal alters Ca(2+) levels and causes cell death in MG63 human osteosarcoma cells. [Ca(2+)]i and cell death were measured using the fluorescent dyes fura-2 and WST-1, respectively. Thimerosal at concentrations above 5 microM increased [Ca(2+)]i in a concentration-dependent manner. The Ca(2+) signal was reduced by 80% by removing extracellular Ca(2+). The thimerosal-induced Ca(2+) influx was sensitive to blockade of La(3+), and dithiothreitol (50 microM) but was insensitive to nickel and several L-type Ca(2+) channel blockers. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), thimerosal failed to induce [Ca(2+)]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change thimerosal-induced [Ca(2+)]i rises. At concentrations of 5, 10 and 20 microM thimerosal killed 33, 55 and 100% cells, respectively. The cytotoxic effect of 5 microM thimerosal was reversed by 54% by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in MG63 cells, thimerosal induced a [Ca(2+)]i rise by causing Ca(2+) release from endoplasmic reticulum stores and Ca(2+) influx from extracellular space. Furthermore, thimerosal can cause Ca(2+)-related cytotoxicity in a concentration-dependent manner.     

Competitive inhibition of cytosolic Ca2+-dependent phospholipase A2 by acteoside in RBL-2H3 cells

The aim of this study was to investigate whether acteoside isolated from Clerodendron trichotomum Thunberg may act as a selective inhibitor of phospholipase A(2) in RBL-2H3 cells. Acteoside dose-dependently inhibited 0.5 μM melittin-induced release of [(3)H]arachidonic acid, which was due to the inhibition of cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)) rather than secretory PLA(2) (sPLA(2)). In Dixon plots, the apparent K ( i ) value of acteoside on cPLA(2) was 5.9 μM and the inhibitory pattern appeared to be a competitive inhibitor. The above data, suggests that acteoside acts as a competitive inhibitor of cPLA(2) in RBL-2H3 cells.     

Histamine regulates growth of malignant melanoma implants via H2 receptors in mice

The present study examined the effect of histamine H₂-receptor antagonists and exogenous histamine on growth of malignant melanoma implant in mice. Drugs were administered to B16BL6 malignant-melanoma-implanted syngeneic mice, and the tumor volume was measured throughout the experiments. Cell proliferation was assessed by MTT assay and mRNA expression was determined by RT-PCR. Both roxatidine and cimetidine significantly suppressed growth of B16BL6 implant compared with vehicle. On the other hand, systemically administered histamine significantly stimulated growth of B16BL6 implants. In addition, the histamine-stimulated B16BL6 implant growth was markedly suppressed by co-administration of cimetidine in a dose-dependent manner. H₂-receptor antagonists, however, failed to affect in vitro proliferation of B16BL6 cells. H₂-receptor mRNA was detected in B16BL6 implants but not in the cell line. These results indicated that both endogenous and exogenous histamine have ability to stimulate growth of malignant melanoma implants via H₂ receptors expressed in host cells.     

Agonistic activities of histamine-albumin conjugates at histamine H2 receptors on human HL-60 promyelocytic leukemia cells

We previously identified functional histamine H2 receptors on human HL-60 promyelocytic leukemia cells [J. Biol. Chem. 264: 18356-18362 (1989)]. In the present study, we have compared the action of histamine-albumin conjugates on H2 receptor activation with that of histamine alone. Both histamine and conjugates increased intracellular levels of Ca2+ in an H2 receptor-specific manner. However, binding of fluoresceinated histamine-albumin conjugates to HL-60 cells was not dissociated by 10(-4) M unlabeled histamine, although this concentration of histamine significantly desensitized conjugate responses. These data suggest that histamine-albumin conjugates not only activate H2 receptors but also bind to HL-60 cells nonspecifically or in an H2 receptor-unrelated manner. Moreover, histamine-induced Ca2+ mobilization was transient, whereas conjugate-induced Ca2+ mobilization was sustained for more than 10 min, as a result of the influx of extracellular Ca2+. Therefore, the functional difference between histamine and conjugates may provide a good model for the further understanding of the activation mechanisms of receptor-operated Ca2+ influx.     

Ca2+ entry but not Ca2+ release is necessary for desensitization of ETA receptors in airway epithelial cells

Ca2+ transients evoked by endothelin-1 (ET-1) were measured in single cells of a human tracheal epithelial cell line using the fluorescent Ca2+ indicator fura-2. In line with a previous study, a single exposure to ET-1 (10 nM) for 10-20 s resulted in a long-lasting desensitization to a subsequent challenge by the peptide, without affecting sensitivity to agonists for other Ca2+-mobilizing receptors such as P2y or H1, respectively. In the absence of extracellular Ca2+ ET-1 elicited a Ca2+ signal of comparable amplitude as in the presence of extracellular Ca2+ but of shorter duration. Exposure to ET-1 in the absence of Ca2+ caused significantly less desensitization. Inhibition of the Ca2+ entry component of the Ca2+ transient by means of SK&F 96365, an inhibitor of Ca2+ entry, had effects comparable to Ca2+ removal. The Ca2+ transient was shortened but not significantly reduced in amplitude, and desensitization was reduced in the presence of the compound. These data demonstrate that desensitization of ET(A) receptors (ET(A)R) is promoted by transmembrane Ca2+ entry but not by Ca2+ release.     

Histamine stimulates glycogen breakdown and increases 45Ca2+ permeability in rat astrocytes in primary culture

In astrocyte-enriched cultures from rat brain hemispheres prelabeled with [3H]glucose, histamine stimulates [3H]glycogen breakdown in a concentration-dependent manner, with an EC50 of 0.6 microM. This effect can be induced by activation of both H1 and H2 receptors independently. Thus, neither 1 microM promethazine, an H1 antagonist, or 100 microM metiamide, an H2 antagonist, inhibited the glycogenolytic response to histamine unless they were present together. In addition, the maximal effect of histamine (55% decrease in [3H]glycogen) was also elicited by 300 microM 2-thiazolylethylamine, an H1 agonist, and by 1 mM dimaprit, an H2 agonist. These agonist effects were inhibited by promethazine and metiamide, respectively, and were not additive, indicating that the same glycogen pool was affected. Histamine was more potent in eliciting glycogenolysis through H1 (EC50 of 0.4 microM in the presence of 100 microM metiamide) than through H2 (EC50 of 3.3 microM in the presence of 1 microM promethazine) receptors, as also shown previously for the H1-mediated phosphoinositide hydrolysis compared with the H2-mediated cAMP formation in the same cells. Both dibutyryl cyclic AMP and the Ca2+ ionophore A23187 could independently mimic the glycogenolytic effect of histamine, whereas the absence of extracellular Ca2+ abolished the H1 component of the response. Histamine also stimulated rapid transmembrane 45Ca2+ influx (maximum, 48% of basal at 15 sec) and efflux (maximum, 25% of basal at 1 min) in astrocytes by activation of H1 receptors. This histamine-increased 45Ca2+ entry was abolished by the nonspecific Ca2+ channel blocker lanthanum but not by the voltage-operated Ca2+ channel inhibitor nifedipine. The enhanced 45Ca2+ release was more a consequence of the histamine-increased Ca2+ permeability than intracellular Ca2+ mobilization, because it was largely diminished when Ca2+ entry was prevented and was little affected by pretreatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate. Thus, the histamine-induced glycogen breakdown in astrocytes may involve increases in cAMP formation and in intracellular Ca2+ levels, this latter resulting mainly from H1-mediated extracellular Ca2+ uptake.     

Chronic Li+ attenuates agonist- and phorbol ester-mediated Na+/H+ antiporter activity in HL-60 cells

The effects of Li+ on signal transduction in dibutyryl cAMP-differentiated HL-60 cells were studied. Upon differentiation, these human promyelocytic leukemia cells express a chemotactic formyl peptide receptor, which is coupled through a guanine nucleotide-binding protein to phospholipase C. Stimulation with fMet-Leu-Phe results in changes in intracellular pH which are thought to be mediated by protein kinase C regulation of Na+/H+ antiporter function. Acute LiCl treatment (10 mM) was without any effect on Na+/H+ activity. However, pretreatment of HL-60 cells with 1 or 10 mM LiCl for at least 5 days resulted in a marked attenuation of fMet-Leu-Phe effects on Na+/H+ activity. In undifferentiated HL-60 cells, which lack fMet-Leu-Phe receptors, intracellular acidification induced by the proton ionophore nigericin generates an alkalinization response. Chronic (but not acute) Li+ treatment also resulted in an inhibition of the nigericin-mediated response. Furthermore, stimulation of the Na+/H+ antiporter by the phorbol ester, phorbol-12-myristate-13-acetate, was also markedly attenuated by chronic LiCl treatment, suggesting an impairment of protein kinase C activity. In contrast, fMet-Leu-Phe-induced increases in intracellular Ca2+ and phospho-inositide breakdown were unchanged in cells treated with Li+ for 5 days. These results indicate that chronic but not acute Li+ treatment alters intracellular pH regulation possibly at a site distal to the fMet-Leu-Phe receptor.