Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Domain-dependent modulation of insulin-induced AS160 phosphorylation and glucose uptake by Ca2+/calmodulin-dependent protein kinase II in L6 myotubes

In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca²⁺ mobilisation and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100 nM] for 15 min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca²⁺ (Ca_i²⁺) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160 kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Ca_i²⁺ and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Ca_i²⁺ or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Ca_i²⁺ mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Ca_i²⁺ and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca²⁺ and CaMKII are components of the insulin signalling pathway in L6 myotubes.     

Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca²⁺ homeostasis plays a key role in cardiac function, we investigated how Ca²⁺ channels and Ca²⁺ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca²⁺ imaging experiments proved the functional expression of the L-type Ca²⁺ current (I_Ca) starting from day 7 of EB development. On day 7, using 10 mM Ca²⁺ as charge carrier, I_Ca was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of I_Ca proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, I_Ca was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP₃-sensitive intracellular Ca²⁺ stores and Ca²⁺-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca²⁺ stores, detected by caffeine-induced Ca²⁺ release, appeared in most GFP-positive cells 1–2 d after I_Ca. Coexpression of both I_Ca and ryanodine-sensitive Ca²⁺ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.Thus, the functional expression of voltage-dependent L-type Ca²⁺ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP₃ receptors and sarcoplasmic Ca²⁺-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.     

Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardium

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca²⁺ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa²⁺/Mg²⁺-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca²⁺ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca²⁺-stimulated Mg²⁺-ATPase and in the pCa₅₀ of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-³²P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the³² P incorporation counted by the Molecular Imager. Ca²⁺/phosphatidylserine/sn-1,2 diolein-dependent³² P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the pCa₅₀ from 6.03 ± 0.04 to 6.44 ± 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg²⁺-ATPase activity. [+]EMD 60263 (3 µM) produced a leftward shift of the myofibrillar pCa²⁺/Mg²⁺-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca²⁺ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca²⁺-stimulated Mg²⁺-ATPase, the pCa₅₀ and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Crosstalk between cellular morphology and calcium oscillation patterns Insights from a stochastic computer model

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca²⁺]₁) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca²⁺ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such ‘Ca ²⁺ fingerprints’ can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca ²⁺ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca²⁺-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca²⁺ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca²⁺ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells

This work investigated the role of Ca²⁺ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca²⁺ chelator BAPTA-AM, but not by the Ca²⁺ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca²⁺. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca²⁺ mobilization and extracellular Ca²⁺ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca²⁺ mobilization and heterotrimeric G protein activation.     

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca²⁺ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca²⁺ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca²⁺ concentration, which was completely prevented by the antioxidants. An intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca²⁺ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca²⁺ increase and induction of apoptosis were significantly inhibited by an extracellular Ca²⁺ chelator or Na⁺/Ca²⁺ exchanger blockers (bepridil and benzamil), whereas neither Ca²⁺ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca²⁺ through the activation of reverse mode of Na⁺/Ca²⁺ exchanger. However, tBOOH decreased intracellular Na⁺ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca²⁺ concentration by direct activation of the reverse mode of Na⁺/Ca>²⁺ exchanger, rather than indirect elevation of intracellular Na⁺ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca²⁺ may mediate this action of tBOOH. These results further suggest that Na⁺/Ca²⁺ exchanger may be a target for the management of oxidative hepatotoxicity.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.