Peroxynitrite affects Ca2+ influx through voltage-dependent calcium channels

The effect of peroxynitrite (OONO-) on voltage-dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO- time- and dose-dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4-benzoic acid) porphyrin, a scavenger for OONO-. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO(-)-induced [45Ca2+] influx. OONO-, as well as 30 mm KCl, significantly increased fluorescence intensity of cell-associated bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose-dependently suppressed OONO(-)-induced [45Ca2+] influx. Although each of 1 microM nifedipine and 1 microM omega-agatoxin VIA (omega-ATX) significantly inhibited the OONO(-)-induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 microM omega-conotoxin GVIA (omega-CTX) showed no effect on the influx. On the other hand, OONO- itself reduced 30 mM KCl-induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO- alone, and the magnitude of this reduction was as same as that of KCl-induced [45Ca2+] influx by omega-CTX. These results indicate that OONO- increases [45Ca2+] influx into the neurones through opening P/Q- and L-type VDCCs subsequent to depolarization, and inhibits the influx through N-type VDCCs.     

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.     

Involvement of STIM1 in the proteinase‐activated receptor 1‐mediated Ca2+ influx in vascular endothelial cells

Thrombin increases the cytosolic Ca²⁺ concentrations and induces NO production by activating proteinase‐activated receptor 1 (PAR₁) in vascular endothelial cells. The store‐operated Ca²⁺ influx is a major Ca²⁺ influx pathway in non‐excitable cells including endothelial cells and it has been reported to play a role in the thrombin‐induced Ca²⁺ signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca²⁺ content, thereby regulating the store‐operated Ca²⁺ influx. However, the functional role of STIM1 in the thrombin‐induced Ca²⁺ influx and NO production in endothelial cells still remains to be elucidated. Fura‐2 and diaminorhodamine‐4M fluorometry was utilized to evaluate the thrombin‐induced changes in cytosolic Ca²⁺ concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1‐targeted siRNA suppressed the STIM1 expression and the thapsigargin‐induced Ca²⁺ influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca²⁺ influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca²⁺ influx and a partial reduction of the NO production induced by thrombin. The thrombin‐induced Ca²⁺ influx exhibited the similar sensitivity toward the Ca²⁺ influx inhibitors to that seen with the thapsigargin‐induced Ca²⁺ influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR₁‐mediated Ca²⁺ influx and Ca²⁺‐dependent component of the NO production in endothelial cells. J. Cell. Biochem. 108: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

Capacitative calcium influx in human epithelial breast cancer and non-tumorigenic cells occurs through Ca2+ entry pathways with different permeabilities to divalent cations

The operation of capacitative Ca(2+) entry (CCE) in human breast cancer (SKBR3) and non-tumorigenic (HBL100) cell lines was investigated as an alternative Ca(2+) entry route in these cells. Ca(2+) readdition after thapsigargin-induced store depletion showed activation of CCE in both cell lines. SKBR3 cells exhibited retarded store depletion and CCE decay kinetics compared to the non-tumorigenic HBL100 cells, suggesting alterations in Ca(2+) homeostasis. CCE was also highly permeable to Mn(2+) and to a lesser extent to Sr(2+), but not to Ba(2+). In HBL100 cells, CCE is contributed (30%) by a Ca(2+)/Mn(2+) permeable route insensitive to low (1 microM) Gd(3+) and a Ca(2+)/Sr(2+)/Mn(2+) permeable non-selective pathway (70%) sensitive to 1 microM Gd(3+). In SKBR3 cells, the relative contribution to CCE of both routes was opposite to that in non-tumorigenic cells.     

Arachidonic acid and docosahexaenoic acid suppress thrombin-evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid liberation

Arachidonic (AA) and docosahexaenoic acid (DHA) are the major polyunsaturated fatty acids (PUFAs) in the brain. However, their influence on intracellular Ca2+ signalling is still widely unknown. In astrocytes, the amplitude of thrombin- induced Ca2+ response was time-dependently diminished by AA and DHA, or by the AA tetraynoic analogue ETYA, but not by eicosapentaenoic acid (EPA). Thrombin-elicited Ca2+ response was reduced (20-30%) by 1-min exposure to AA or DHA. Additionally, 1-min application of AA or DHA together with thrombin in Ca2+-free medium blocked Ca2+ influx, which followed after readdition of extracellular Ca2+. EPA and ETYA, however, were ineffective. Long-term treatment of astrocytes with AA and DHA, but not EPA reduced the amplitude of the thrombin-induced Ca2+ response by up to 80%. AA and DHA caused a comparable decrease in intracellular Ca2+ store content. Only DHA and AA, but not EPA or ETYA, caused liberation of endogenous AA by cytosolic phospholipase A2 (cPLA2). Therefore, we reasoned that the suppression of Ca2+ response to thrombin by AA and DHA could be due to release of endogenous AA. Possible participation of AA metabolites, however, was excluded by the finding that specific inhibitors of the different oxidative metabolic pathways of AA were not able to abrogate the inhibitory AA effect. In addition, thrombin evoked AA release via activation of cPLA2. From our data we propose a novel model of positive/negative-feed-back in which agonist-induced release of AA from membrane phospholipids promotes further AA release and then suppresses agonist-induced Ca2+ responses.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation

The physical interaction of the human growth factor receptor‐bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser³⁵⁸, Ser³⁶², and Ser³⁶⁶ in hGrb14 by glycogen synthase kinase‐3 repressed hGrb14–IR complex formation. In this study, we investigated phosphatase‐mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14‐derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser³⁵⁸ and Ser³⁶². Furthermore, coimmunoprecipitation experiments suggested that insulin‐induced hGrb14–IR complex formation was repressed by the substitution of Ser³⁵⁸ or Ser³⁶² with glutamic acid. These findings suggested that phosphate groups on Ser³⁵⁸ and Ser³⁶² in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14–IR complex formation.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

Induced Ca2+ uptake and callose synthesis in suspension-cultured cells of Catharanthus roseus are decreased by the protein phosphatase inhibitor okadaic acid

Subtoxic concentrations of the saponin digitonin. the polyene antibiotic amphotericin B and the bacterial phytotoxin syringomycin induce increased uptake of ⁴⁵Ca²⁺ into suspension-cultured plant cells and a rapid Ca²⁺-dependent defense response, callose synthesis. Both reactions were inhibited by preincubation of the cells with okadaic acid, a specific inhibitor of type 1 and type 2A protein phosphatases. These results suggest that Ca²⁺ uptake induced by the above agents does not occur due to unspecific perturbation of plasma membrane permeability but involves transport proteins which are controlled by protein phosphorylation/dephosphorylation. Phosphoproteins appear also to be involved in the regulation of callose synthesis, although it remains open whether this control is effected at the level of Ca²⁺ transport or at the 1,3-ß-glucan synthase involved in deposition of the polymer.     

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.     

L-type voltage-operated Ca(+2) channels modulate transient Ca(+2) influx triggered by activation of Sertoli cell surface L-selectin

Near the base of mammalian seminiferous epithelium, Sertoli cells are joined by tight junctions, which constitute the blood-testis barrier. Differentiating germ cells are completely enveloped by Sertoli cells and must traverse the tight junctions during spermatogenic cycle. Following the specific ligand activation of L-selectin, the up-regulated Rho family small G-proteins have been implicated as important modulators of tight junctional dynamics. Although the activation of L-selectin transmits subsequent intracellular signals in a Ca(+2)-dependent fashion in various cell types, little is understood regarding the signaling pathways utilized by L-selectin in Sertoli cells. Therefore, we have examined the possible resultant calcium influx triggered by specific ligand-activation of cell surface L-selectin receptors or by cross-linking of L-selectin with anti-L-selectin. Spectrofluorimetric studies demonstrate increase of intracellular Ca(+2) levels immediately after the treatment of the L-selectin ligands, fucoidan and sialyl Lewis-a, or after treatment with anti-L-selectin antibody. We then determined the mechanism of Ca(+2) influx by investigating L- and T-type voltage-operated Ca(+2) channels, which have been suggested to present in the membranes of Sertoli cells. Data demonstrate that Sertoli cells treated with L-type voltage-operated Ca(+2) channel antagonists, nifedipine, diltiazem, or verapamil, lead to dose-dependent blockage of L-selectin-induced Ca(+2) influx. Cells treated with mibedradil, a T-type voltage-operated Ca(+2) channel antagonist, results in little or no blocking effect. Therefore, we conclude that activation of Sertoli cell L-selectin induces Ca(+2) influx, which is at least partially regulated by L-type voltage-operated Ca(+2) channels.     

Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.     

Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10–100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca²⁺ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca²⁺-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils. © 1996 Wiley-Liss, Inc.     

Sigma-1 receptor as regulator of neuronal intracellular Ca2+: clinical and therapeutic relevance

Preserving brain function and cognitive faculties during aging and psychiatric diseases (e.g. psychotic, anxiety and affective disorders, dementia) is essential for the self-reliance and quality of life of patients. Cognitive loss involves not only memory, but also motor function. The decrease of catecholaminergic and excitatory neurotransmissions, as well as of protein phosphorylation, have currently been identified as prominent biological markers of the above-mentioned diseases. Such deleterious biological events are well known to occur downstream of a progressive decline of intracellular Ca2+ signalling. This latter constitutes a key target for the neuronal plasticity that has also been reported during aging and psychiatric disorders. Most of the medicines used in psychiatry are active on the sigma-1 receptor. This membrane bound receptor is widely distributed in memory-associated cortical and motor-related brainstem areas, prompting the hypothesis that it might contribute to the pathophysiology of these behavioural brain diseases. The sigma-1 receptor is characterized by a unique mode of action by regulating both Ca2+ entry at the plasma membrane level (i.e. via potassium channels, voltage-sensitive Ca2+ channels) and Ca2+ mobilization from endoplasmic stores [i.e. via Ins(1,4,5)P3 receptors]. This review presents recent data supporting the notion that drugs acting via the endoplasmic reticulum-coupled sigma-1 receptor might reverse these deleterious events by restoring both extra- and intra-cellular Ca(2+)-dependent neuronal responses.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid

Protein phosphorylation and cytosolic-free [Ca2+] ([Ca2+]i) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2, 1.3 +/- 0.7 mm) but not the non-hydrolyzable analog ATPgammaS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca2+ current and [Ca2+]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+]i increases in the guard cells.