Changes In Intracellular Free Calcium Research Articles

ORAI Calcium Channels: Regulation, Function, Pharmacology, and Therapeutic Targets.

The changes in intracellular free calcium (Ca2+) levels are one of the most widely regulators of cell function; therefore, calcium as a universal intracellular mediator is involved in very important human diseases and disorders. In many cells, Ca2+ inflow is mediated by store-operated calcium channels, and it is recognized that the store-operated calcium entry (SOCE) is mediated by the two partners: the pore-forming proteins Orai (Orai1-3) and the calcium store sensor, stromal interaction molecule (STIM1-2). Importantly, the Orai/STIM channels are involved in crucial cell signalling processes such as growth factors, neurotransmitters, and cytokines via interaction with protein tyrosine kinase coupled receptors and G protein-coupled receptors. Therefore, in recent years, the issue of Orai/STIM channels as a drug target in human diseases has received considerable attention. This review summarizes and highlights our current knowledge of the Orai/STIM channels in human diseases and disorders, including immunodeficiency, myopathy, tubular aggregate, Stormorken syndrome, York platelet syndrome, cardiovascular and metabolic disorders, and cancers, as well as suggesting these channels as drug targets for pharmacological therapeutic intervention. Moreover, this work will also focus on the pharmacological modulators of Orai/STIM channel complexes. Together, our thoughtful of the biology and physiology of the Orai/STIM channels have grown remarkably during the past three decades, and the next important milestone in the field of store-operated calcium entry will be to identify potent and selective small molecules as a therapeutic agent with the purpose to target human diseases and disorders for patient benefit.

Revisiting the Large-Conductance Calcium-Activated Potassium (BKCa) Channels in the Pulmonary Circulation.

Potassium ion concentrations, controlled by ion pumps and potassium channels, predominantly govern a cell′s membrane potential and the tone in the vessels. Calcium-activated potassium channels respond to two different stimuli-changes in voltage and/or changes in intracellular free calcium. Large conductance calcium-activated potassium (BKCa) channels assemble from pore forming and various modulatory and auxiliary subunits. They are of vital significance due to their very high unitary conductance and hence their ability to rapidly cause extreme changes in the membrane potential. The pathophysiology of lung diseases in general and pulmonary hypertension, in particular, show the implication of either decreased expression and partial inactivation of BKCa channel and its subunits or mutations in the genes encoding different subunits of the channel. Signaling molecules, circulating humoral molecules, vasorelaxant agents, etc., have an influence on the open probability of the channel in pulmonary arterial vascular cells. BKCa channel is a possible therapeutic target, aimed to cause vasodilation in constricted or chronically stiffened vessels, as shown in various animal models. This review is a comprehensive collation of studies on BKCa channels in the pulmonary circulation under hypoxia (hypoxic pulmonary vasoconstriction; HPV), lung pathology, and fetal to neonatal transition, emphasising pharmacological interventions as viable therapeutic options.

Qibai Pingfei capsule medicated serum inhibits the proliferation of hypoxia-induced pulmonary arterial smooth muscle cells via the Ca2+/calcineurin/nuclear factor of activated T-cells 3 pathway

ObjectiveTo observe the effect of Qibai Pingfei capsule (QBPF) medicated serum on the proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions and to investigate its key molecular effects on the Ca2+/calcineurin/nuclear factor of activated T-cells 3 (NFATc3) signaling pathway. MethodsQBPF was provided to rats via continuous gavage for 10 days. Primary rat PASMCs were cultured using the direct adherent culture method. Methyl thiazolyl tetrazolium assay was used to evaluate the effect of QBPF on PASMCs proliferation under hypoxia conditions. Laser scanning confocal microscopy was used to detect changes in intracellular free calcium ([Ca2+]i) in PASMC-loaded Fluo-3-AM. Real-time quantitative polymerase chain reaction and western blot were used to detect the transcription and protein expression levels of calcineurin and NFATc3 genes in PASMCs. ResultsCompared with normoxia conditions, PASMCs proliferated at 12, 24, 48, and 72 h under hypoxia conditions. QBPF at concentrations of 5%, 10%, and 20% could inhibit hypoxia-induced PASMC proliferation to different degrees. The inhibitory effect was most significant in the 20% QBPF group under 24 h hypoxia conditions. The concentration of [Ca2+]i in PASMCs under hypoxia was increased and [Ca2+]i was significantly decreased when co-incubated with QBPF at 5%, 10%, and 20%. Compared with normoxia conditions, the mRNA and protein expression levels of calcineurin and NFATc3 in PASMCs induced by hypoxia were up-regulated. QBPF application significantly down-regulated mRNA and protein expression levels of calcineurin and NFATc3 in PASMCs. ConclusionQBPF can effectively inhibit hypoxia-induced proliferation of PASMCs through down-regulation of key molecular expression via the Ca2+/calcineurin/NFATc3 pathway.

The effects and mechanism of islet amyloid polypeptide on insulin secretion in INS-1 cells stimulated by glibenclamide

To observe the effects, and study the mechanism of islet amyloid polypeptide (IAPP) on insulin secretion in INS-1 cells stimulated by glibenclamide. Whole cell patch clamp technique was employed to study the influences of short exposure to IAPP on electrophysiological characteristics of ATP-sensitive K+ channel (K(ATP) channel) upon sulfonylurea stimulation. Intracellular free calcium changes in this process was observed by laser scanning confocal microscope. Insulin was measured by enzyme-linked immunoassay. (1) Insulin secretion stimulated by 1 micomol/L glibenclamide was significantly decreased from (11.43 +/- 1.22) microg/L to (9.40 +/- 0.87) microg/L and to (7.11 +/- 1.85) microg/L after 1 micromol/L and 10 micromol/L IAPP incubation, respectively. (2) Glibenclamide-stimulated calcium influx was dose dependently inhibited by IAPP from 1 micromol/L to 10 micromol/L, with the AUC of fluorescence intensity-time reduced from 427.78 +/- 2.32 to 380.59 +/- 1.49, and to 246.53 +/- 8.41, respectively. (3) Compared with that in control cells (14.59 +/- 0.69) mV, the half activation voltage of KA, channel in response to glibenclamide was significantly increased to (28.75 +/- 0.77) mV and to (46.95 +/- 1.81) mV in cells pretreated with 1 micromol/L and 10 micromol/L IAPP, implicating an inhibitory effect of IAPP on activation of K(ATP) channel. Short-term exposure to high concentration of IAPP inhibited glibenclamide-induced closure of K(ATP) channels and decreased calcium influx, which may ultimately lead to the reduction of insulin secretion in INS-1 cells

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation.

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll-like receptors (TLR) and protease-activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet-bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg-gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1-agonist Pam3 CSK4 , which evoked a time-dependent release of the chemokine. Interestingly, the TLR2/1-evoked response was abolished by a following addition of viable P. gingivalis wild-types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam3 CSK4 -stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor-1, and that also these responses were antagonized by P. gingivalis. These results supports immune-modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.

PTK2b function during fertilization of the mouse oocyte

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ

A change in intracellular free calcium (Ca2+) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca2+ signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca2+-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca2+ overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca2+ overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca2+-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca2+ overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

Potassium Depolarization and Raised Calcium Induces α-Synuclein Aggregates

α-Synuclein is the key aggregating protein in Parkinson's disease (PD), which is characterized by cytoplasmic protein inclusion bodies, termed Lewy bodies, thought to increase longevity of the host neuron by sequestering toxic soluble α-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca(2+) buffering protein, calbindin, and recent cell culture and in vitro studies have shown that α-synuclein aggregation can be induced by Ca(2+). We hypothesized that depolarization with potassium resulting in raised Ca(2+) in a PD cell culture model will lead to the formation of α-synuclein protein aggregates and that the intracellular Ca(2+) buffer, BAPTA-AM, may suppress their formation. Live cell fluorescence microscopy was performed to monitor changes in intracellular free calcium in HEK293T, SH-SY5Y neuroblastoma or stably transfected HEK293T/α-synuclein cells. Raised intracellular free Ca(2+) was consistently observed in cells treated with KCl, but not controls. Immunohistochemistry analysis on cells 48-72h after K(+) treatment revealed two subsets of cells with either large (>2μm), perinuclear α-synuclein aggregates or multiple smaller (<2μm), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca(2+) transient following KCl treatment and no α-synuclein aggregates at TMO concentrations >5μM. Quantitative analysis revealed a significant increase in the number of cells bearing α-synuclein cytoplasmic inclusions in both HEK293T/α-synuclein and SHSY-5Y cells when transient intracellular raised Ca(2+) was induced (p=0.001). BAPTA-AM pre-loading significantly suppressed α-synuclein aggregates (p=0.001) and the intracellular free Ca(2+) transient. This study indicates that raised intracellular Ca(2+) mediated by K(+) depolarization can lead to α-synuclein aggregation.

Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc &lt;em&gt;Aplysia&lt;/em&gt;

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Role of large conductance calcium-activated potassium channels in neuronal Ca2+ overload following traumatic brain injury

Objective To investigate the role of large conductance calcium-activated potassium channels (BK) in neuronal Ca2+ overload following traumatic brain injury (TBI). Methods Neuronal cells of C57BL/6 mouse cortex were collected and cultured. Patch-clamp technique was applied to investigate the changes of intracellular free calcium [Ca2+] i and firing frequency of neuronal action potentials in rest condition or evoked by 100 pA electric current lasting 500 ms after perfusion of Iberiotoxin ( 100 nmmol/L), a BK specific blocker. The cells were divided randomly into experimental group ( plus Iberiotoxin) and control group. Extracellular solution of cultured neurons was further perfused with KCl (20 mmol/L) to induce elevation of [Ca2 +]i and influence of Iberiotoxin ( 100 nmol/L) on amplitude of [Ca2 +] i elevation was determined. Results No significant changes of neuronal spontaneous action potential frequency and [Ca2 +]i were observed in rest condition after perfusion of Iberiotoxin (P＞0.05).However, when evoked by electric current, the frequency of action potential was (10.4 ± 3.0) Hz,which was increased to ( 13.8 ± 3.7 ) Hz after perfusion of Iberiotoxin, with statistical difference (P＜0.05 ). The [Ca2 +] i level was ( 14.21 ± 16.98 ) nmol/Lbefore perfusion with Iberiotoxin but was increased to (44.07 ± 34. 4) nmol/L after perfusion of Iberiotoxin (P ＜ 0.05 ). Extracellular high concentration of KCl increased [Ca2 +] i of neurons, while perfudion of Iberiotoxin further elevated [Ca2 +]i (P ＜ 0.05).Conclusion BK may play an important role in the regulation of neuronal [Ca2+] i and in neuronal Ca2+overload following TBI. Key words: Brain injuries; Neuron; Calcium; Large conductance calcium-activated potassium channel

Intracellular calcium as a second messenger following growth stimulation of human keratinocytes.

The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF-deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium-free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing.

Flavonoid‐induced calcium signalling in Rhizobium leguminosarum bv. viciae

• Legume-rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals - flavonoids - before infection. Host plants sense counterpart rhizobial signalling molecules - Nod factors - through transient changes in intracellular free-calcium. Here we investigate the potential involvement of Ca(2+) in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae. • By using aequorin-expressing rhizobial strains, we monitored intracellular Ca(2+) dynamics and the Ca(2+) dependence of nod gene transcriptional activation. • Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca(2+) that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca(2+) variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca(2+) signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca(2+) signalling event. • These findings demonstrate a key role played by Ca(2+) in sensing and transducing plant-specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid-NodD paradigm of nod gene regulation.

Role of ATP‐sensitive K+ channels in glucose sensing by immortalized adrenomedullary chromaffin cells

Adrenomedullary chromaffin cells exhibit a critical Ca2+‐dependent catecholamine secretion when directly exposed to perinatal stressors (e.g. hypoxia). Using an immortalized chromaffin cell line (MAH), we investigated their potential sensitivity to low glucose (hypoglycemia) by monitoring changes in intracellular free calcium ([Ca2+]i) with the fluorescent probe Fura‐2. The majority of MAH cells (86%; n=398) were unresponsive on switching from 10 to 0 mM glucose. Interestingly, 0 glucose markedly inhibited (~75%) the rise in [Ca2+]i induced by the depolarizing stimulus high K+ (10 mM). To test the possibility that this was due to KATP channel activation, the KATP channel blocker, glibenclamide (50 μM), was used. In normal K+, glibenclamide increased the proportion of 0 glucose‐responsive cells from 14% to 65%, and the mean Δ[Ca2+]i response increased fourfold. RT‐PCR and Western blot analysis confirmed the presence of both the inward rectifying (Kir6.2) and sulfonylurea (Sur1) subunits of KATP channels. These findings suggest that hypoglycemia‐induced activation of KATP channels may limit [Ca2+]i increases and catecholamine secretion in chromaffin cells. Furthermore, the increased proportion of 0 glucose‐responsive cells in the presence of glibenclamide suggests that hypoglycemia causes a separate excitatory pathway in MAH cells.Supported by NSERC and the HSFO.

Detailed confocal fluorescent visualization of Ca 2+ and membrane potential changes in pulmonary neuroepithelial bodies (NEBs) in a murine lung slice model

Conclusive physiological data on potential stimuli for pulmonary NEBs are very scarce because of the lack of a reliable in vitro model. We recently developed a method for selective staining of NEBs by 4-Di-2-ASP in vibratome slices of live mouse lungs. Aim of the study was optimizing the model for live cell imaging, allowing detailed confocal visualization of changes in intracellular free calcium ([Ca2+]i) and membrane potential, using the fluorescent indicators Fluo-4 and DiBac4 (3), respectively. In all NEBs, experimental short-term elevation of extracellular potassium ([K+]o) was seen to evoke a reproducible rise in cytoplasmic fluorescence for both Fluo-4, corresponding to an increase in [Ca2+]i, and DiBac4 (3), indicative for membrane depolarization. So-called Clara-like cells, known to almost completely shield NEBs from the airway lumen, appeared to exhibit a slightly delayed increase in Fluo-4 fluorescence after high [K+]o, while DiBac4 (3) did not reveal changes. The Ca2+ response in NEBs and Clara-like cells was abolished in Ca2+ free solution. This study provides the first evidence that physiological activity of pulmonary NEBs, and surrounding tissues, can be visualized in live lung slices using fluorescent indicators. The delayed increase in [Ca2+]i in Clara-like cells might be explained by stimulation of Clara-like cells via depolarization-induced neurotransmitter release from NEB cells. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)

Calcium gradients and the Golgi

Changes in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function. Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate (IP 3) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis.

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca 2+ plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca 2+] i) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca 2+ dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca 2+] i in U937-derived macrophages in both acute and chronic treatments ( P < 0.01). In particular, the increases of the induced [Ca 2+] i were different in the presence or absence of extracellular Ca 2+ under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24 h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca 2+] i induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.

How adhesion, migration, and cytoplasmic calcium transients influence interleukin-1beta mRNA stabilization in human monocytes.

We investigated the mechanisms by which primary human monocyte migration and the production of important cytokines are co-regulated. Motile monocytes underwent cyclic morphologic and adhesive changes that were associated with intracellular free calcium changes; in such cells, cytokine transcripts were unstable and translationally repressed. Agents that activate monocytes, including lipopolysacharrides (LPS), cytomegalovirus (CMV), and tumor necrosis factor (TNFalpha), have been shown to de-repress translation and these agents stabilize adhesion-induced transcripts for IL-lbeta and IL-8 and markedly diminish cell migration in the presence of autologous serum. LPS suppressed Rho A activity and either this agent or C3 transferase elevated intracellular free calcium, stabilized transcripts, and, in tandem, inhibited cell migration by preventing tail retraction, a prerequisite for cell translocation. These results, therefore, suggest that monocyte activating agents inhibit the RhoA pathway and continuously elevate intracellular calcium leading to a concomitant decrease in monocyte migration and stabilization of cytokine transcripts prior to translation.

In vivo monitoring of intracellular free calcium changes during uterine activation by prostaglandin f(2alpha) and oxytocin.

It has been well established that oxytocin (OXT) increases intracellular free calcium ([Ca(2+)](i)) by targeting both intracellular and extracellular stores, but the mechanisms involved in the increase through activation with prostaglandin F(2alpha) (PGF(2alpha)) are still incompletely understood. This study was designed to elucidate the source(s) of increased [Ca(2+)](i) in response to PGF(2alpha) (10(-6) M) or OXT (10(-8) M) administration in the near-term rat myometrium. The animals were divided into an in vitro group (n= 8), where the developed tension of uterine strips was assessed, and an in vivo group (n= 5), where a lobe of the uterus with intact innervation and circulation was loaded with the fluorescent indicator Indo-1 AM to assess [Ca(2+)](i). PGF(2alpha) and OXT induced a 30.1% and 35.9%, respectively, increase in developed tension in the potassium chloride-depolarized myometrial strips. Nifedipine reduced the PGF(2alpha) and OXT increased tension by 65.8% and 49.4%, respectively. In vivo, both PGF(2alpha) and OXT increased [Ca(2+)](i) in the potassium chloride-depolarized uterine muscle by 35.7% and 44.6%, respectively, increases similar to the rises in tension in vitro. Nifedipine reduced these effects of PGF(2alpha) and OXT by 45.3% and 39.6%. These findings indicate that in near-term myometrium the source of increased [Ca(2+)](i) after administration of PGF(2alpha), similar to OXT, is both extracellular and intracellular.

In Vivo Monitoring of Intracellular Free Calcium Changes During Uterine Activation By Prostaglandin F2α and Oxytocin

In Vivo Monitoring of Intracellular Free Calcium Changes During Uterine Activation By Prostaglandin F2α and Oxytocin