Dynamic regulation of [Ca2+]i by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca(2+)-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]i elicited by activation of capacitative Ca2+ entry (CCE) served to characterize kinetic parameters of these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca(2+)- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]i has been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+ extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system. The contribution by the low-affinity NCX to Ca2+ clearance became apparent at [Ca2+]i > approximately 150 nM under conditions of submaximal activation of the PMCA.     

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.     

Heterogeneous changes in [Ca2+]i induced by glucose, tolbutamide and K+ in single rat pancreatic B cells

The effects of glucose, tolbutamide and K+ on cytosolic free Ca2+ ([Ca2+]i) in single rat pancreatic B cells were examined using Fura-2 and dual wavelength microfluorimetry. At basal glucose concentration (2.8 mM), about half of the cells were found to display spontaneous Ca2+ oscillations. Glucose (greater than or equal to 11.1 mM), tolbutamide (greater than or equal to 50 microM) and K+ (50 mM) induced rises in [Ca2+]i that could be inhibited by the Ca2+ channel blocker D600. The pattern of response and the sensitivity to the secretagogues were characterized by a marked heterogeneity. The majority of the cells responded to glucose and tolbutamide by a progressive increase in [Ca2+]i onto which sinusoidal oscillations were superimposed. The periodicity of these oscillations was about 2.5/min. Occasionally, some cells displayed slow and major waves in Ca2+ levels (about 0.2/min). None of the cells responded to glucose by displaying an initial decrease in [Ca2+]i. Likewise, the sugar failed to decrease [Ca2+]i in the absence of extracellular Ca2+. The present study shows that, despite a large heterogeneity of the response, the majority of the pancreatic B cells respond to different secretagogues by displaying fast [Ca2+]i oscillations that are reminiscent of the bursts of electrical activity recorded in B cells.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca²⁺]_i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca²⁺]_i depend on the intensity of external electric field. In the presence of Ca²⁺ chelant EGTA or Ca²⁺ channels blocker La³⁺ in the pulsation solutions, the increase of [Ca²⁺]_i was still observable. It was also observed that [Ca²⁺]_i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca²⁺]_i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system.The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field.The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field.In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions,the increase of [Ca2+]i was still observable.It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

Effects of hypercapnia and hypocapnia on [Ca2+]i mobilization in human pulmonary artery endothelial cells.

The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI2) through a process depending on intracellular Ca2+ concentration ([Ca2+]i). We studied the influence of CO2-related pH changes on [Ca2+]i and PGI2 production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca2+]i from 112 +/- 24 to 157 +/- 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca2+]i comparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca2+]i was unchanged by the removal of Ca2+ from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca2+ stores. Hypercapnic acidosis may thus release Ca2+ from pH-sensitive but thapsigargin-insensitive intracellular Ca2+ stores. Hypocapnic alkalosis caused a fivefold increase in [Ca2+]i compared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca2+]i. The hypocapnia-evoked increase in [Ca2+]i was decreased from 242 +/- 56 to 50 +/- 32 nmol/l by the removal of extracellular Ca2+. The main mechanism affecting the hypocapnia-dependent [Ca2+]i increase was thought to be the augmented influx of extracellular Ca2+ mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI2 production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca2+]i in HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.     

Sulfur dioxide derivatives modulate Na/Ca exchange currents and cytosolic [Ca2+]i in rat myocytes

We have recently shown that sulfur dioxide (SO(2)) derivatives (bisulfite and sulfite, 1:3 M/M) modulated L-type calcium, sodium, and potassium channels in rat myocytes. The aim of this study was to investigate whether SO(2) derivatives could alter Na/Ca exchanger current and the intracellular free [Ca(2+)]. The nickel-sensitive Na/Ca exchanger current was measured in rat myocytes exposed to ramp pulses in Tyrode's solution containing ouabain, nifedipine, and +/-Ni (5 mmol/l). Myocytes were loaded with the fluorescent Ca(2+) indicator Fura-2/AM to estimate intracellular Ca(2+) concentration. SO(2) derivatives significantly inhibited both outward and inward Ni-sensitive Na/Ca exchanger currents without a shift in the reversal potential. The intracellular free [Ca(2+)] was raised by SO(2) derivatives in several concentrations. SO(2) derivatives increased [Ca(2+)](i) in rat myocytes and its mechanism might involve SO(2) derivatives significantly inhibiting Na/Ca exchanger current and enhancing L-type calcium channel.     

卵细胞受精相关胞质钙离子波、钙离子震荡信号特征及其形成机制

胞质[Ca2 ]i震荡的动力学变化在哺乳动物早期胚胎发育中发挥重要作用。卵母细胞的成熟伴随间断的、快速的[Ca2 ]i震荡的时空表达;在受精过程中精子因子诱导的反复[Ca2 ]i震荡的振幅和持续时间是卵细胞最有效的激活信号,这种信号形成自然连续的受精[Ca2 ]i波,并以长时持续[Ca2 ]i震荡形式在受精卵空间传递并持续数小时,直至受精完成;受精卵内源性的Ca2 释放所引起的[Ca2 ]i震荡形成第一次卵裂信号,启动早期胚胎的发育。精子PLCζ和cPKCs是形成受精卵[Ca2 ]波、[Ca2 ]震荡的重要因素。     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.