2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

The signal transduction mechanism involved in kazinol B-stimulated superoxide anion generation in rat neutrophils

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated.
2. Kazinol B concentration-dependently stimulated the superoxide anion (O₂[dot over 2]⁻) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2±1.4 nmol O₂[dot over 2]⁻ 10 min⁻¹ per 10⁶ cells) was observed at 10 μM kazinol B.
3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O₂[dot over 2]⁻ generation following the subsequent stimulation of cells with kazinol B.
4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O₂[dot over 2]⁻ generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response.
5. Kazinol B significantly stimulated [Ca²⁺]_i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP₂ and IP₃) formation with a 1 min lag time.
6. The membrane-associated PKC-α and PKC-θ but not PKC-ι were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-θ than PKC-α by kazinol B. Kazinol B partially inhibited the [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to the neutrophil cytosolic PKC.
7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O₂[dot over 2]⁻ generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein.
8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca²⁺]_i elevation in rat neutrophils.

     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca²⁺ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca²⁺]i elevation of neutrophils suspended in Ca²⁺-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC₅₀ values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC₅₀ value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca²⁺ mobilization of neutrophils suspended in Ca ²⁺-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC₅₀ values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca²⁺]_i elevation of neutrophils challenged by fMLP in Ca²⁺-containing medium, but not in Ca²⁺-free medium, with IC₅₀ value 22.30 ± 1.61 µM. In Mn²⁺-quench studies, U-73122 suppressed the Mn²⁺ influx in CPA-activated neutrophils (IC₅₀ value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC₅₀ value was 6.72 ± 0.30 M). U-73343 also suppressed the Mn²⁺ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca²⁺]i of activated neutrophils is attributed partly to the suppression of Ca²⁺ release from the intracellular Ca²⁺ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca²⁺ entry from the extracellular space through PLC-independent processes.     

Heparin specifically inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned rat aortic smooth muscle cells in primary culture

Summary By measuring the ⁴⁵Ca²⁺ movement in saponin-skinned primary cultured rat aortic smooth muscle cells, we examined the specificity of the inhibitory effect of heparin on the IP₃-induced Ca²⁺ release. IP₃ (100 mol/l) markedly (98%) decreased the MgATP-dependent ⁴⁵Ca²⁺ content in the non-mitochondrial Ca²⁺ stores in the presence of 1 mol/l free Ca²⁺. Heparin (1–100 g/ml) dose-dependently inhibited this Ca²⁺ release by IP₃. In Ca²⁺-free solution, heparin (100 g/ml) inhibited the increases in ⁴⁵Ca²⁺ efflux rate evoked by 10 mol/l IP₃. De-N-sulfated heparin did not inhibit the IP₃-induced Ca²⁺ release. Hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C and 2,6-disulfated d-glucosamine had no inhibitory effects on the IP₃-induced Ca²⁺ release. High concentrations (over 1 mg/ml) of heparin inhibited the ⁴⁵Ca²⁺ influx and decreased the Ca²⁺ content in skinned cells. These results suggest that heparin (1–100 g/ml) specifically inhibits the IP₃-induced increase in Ca²⁺ permeability of Ca²⁺ stores and that three sulfate groups at different locations on the molecule of heparin, two at the d-glucosamine and one at the iduronic acid, may be important for this action, in skinned vascular smooth muscle cells, in culture. Send offprint requests to H. Kanaide at the above address     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Effects of timolol on Ca2+ handling and viability in human prostate cancer cells

Timolol is a medication used widely to treat glaucoma. Regarding Ca²⁺ signaling, timolol was shown to modulate Ca²⁺-related physiology in various cell types, however, the effect of timolol on Ca²⁺ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100–1000?μM induced [Ca²⁺]_i rises. The Ca²⁺ signal in Ca²⁺-containing medium was reduced by removal of extracellular Ca²⁺ by approximately 75%. Timolol (1000?μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Timolol-induced Ca²⁺ entry was partially inhibited by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished timolol-evoked [Ca²⁺]_i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca²⁺]_i rises. Timolol at concentrations between 200 and 600?μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca²⁺]_i rises by evoking Ca²⁺release from the endoplasmic reticulum in a PLC-dependent manner, and Ca²⁺ influx via PKC-regulated store-operated Ca²⁺ entry. Timolol also caused cell death that was not linked to preceding [Ca²⁺]_i rises.     

Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166

Summary The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3, 4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-trimethoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1, 4-benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl^– but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC₅₀-values of 70 nM (5–11 times less potent than nifedipine, 2–16 times more potent than verapamil), the respective S-(–)-enantiomer being 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(–)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4–15 times more potent inhibitor of reversible (+)-[³H]PN200-110, (–)-[³H]desmethoxyverapamil and d-cis [³H]diltiazem binding compared to its pharmacologically less active (S)-(–)-enantiomer, with IC₅₀ values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca²⁺-antagonistic effect by binding to a Ca²⁺-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca²⁺ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.Send offprint requests to H. Glossmann     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg²⁺]_o to 0.1 mM can evoke repetitive [Ca²⁺]_i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg²⁺]_o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca²⁺ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg²⁺]_o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca²⁺ channel antagonist nimodipine, which blocked 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca²⁺]_i spikes. The intracellular Ca²⁺ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca²⁺]_i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca²⁺]_i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg²⁺]_o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca²⁺]_i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus

The effects of honokiol and magnolol extracted from the Magnolia officinalis on muscular contractile responses and intracellular Ca²⁺ mobilization were investigated in the non-pregnant rat uterus. Honokiol and magnolol (1–100 mol/l) were observed to inhibit spontaneous and uterotonic agonists (carbachol, PGF₂, and oxytocin)-, high K⁺-, and Ca²⁺ channel activator (Bay K 8644)-induced uterine contractions in a concentration-dependent manner. The inhibition rate of honokiol on spontaneous contractions appeared to be slower than that of magnolol-induced response. The time periods that were required for honokiol and magnolol, at 100 mol/l, to abolish 50% spontaneous contractions were approximately 6 min. Furthermore, honokiol and magnolol at 10 mol/l also blocked the Ca²⁺-dependent oscillatory contractions. Consistently, the increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by PGF₂ and high K⁺ were suppressed by both honokiol and magnolol at 10 mol/l. After washout of these treatments, the rise in [Ca²⁺]_i induced by PGF₂ and high K⁺ was still partially abolished. In conclusion, the inhibitory effects of honokiol and magnolol on uterine contraction may be mediated by blockade of external Ca²⁺ influx, leading to a decrease in [Ca²⁺]_i. Honokiol and magnolol may be considered as putative Ca²⁺ channel blockers and be of potential value in the treatment of gynecological dysfunctions associated with uterine muscular spasm and dysmenorrhea.     

N,N-dimethyl-D-erythro-sphingosine increases intracellular Ca2+ concentration via Na+-Ca2+-exchanger in HCT116 human colon cancer cells

N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca²⁺ increase concentration ([Ca²⁺]_i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca²⁺]_i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca²⁺]_i in colon cancer cells is composed of Ca²⁺ release from intracellular Ca²⁺ stores and subsequent Ca²⁺ influx. The Ca²⁺ release is not related to modulation of inositol 1,4,5-trisphosphate (IP₃) receptor or ryanodine receptor. On the other hand, the Ca²⁺ influx is mediated largely through Ca²⁺ channels sensitive to verapamil, nifedipine, Ga³⁺, and La³⁺. Furthermore, we found that the response is inhibited by bepridil and Ni²⁺, specific inhibitors of Na⁺-Ca²⁺-exchanger, suggesting involvement of Na⁺-Ca²⁺ exchanger in the DMS-induced [Ca²⁺]_i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.