Angiotensin-2-Mediated Ca2+ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca²⁺inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca²⁺response. RPE cells from Atrap^−/− mice showed smaller AngII-evoked Ca²⁺peak (by 22%) and loss of sustained Ca²⁺elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca²⁺-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca²⁺-rise. Application of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca²⁺transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca²⁺transients in the RPE by releasing Ca²⁺from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca²⁺elevation.     

Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca²⁺ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Moreover, Jujuboside B increased intracellular Ca²⁺ concentration dose-dependently, which was inhibited by EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Besides, Jujuboside B induced a rapid Ca²⁺ influx instantaneously after depleting intracellular Ca²⁺ store, which was significantly inhibited by {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca²⁺ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved endothelium-dependent hyperpolarizaiton through endothelial potassium channels. Jujuboside B is a natural compound with new pharmacological effects on improving endothelial dysfunction and treating vascular diseases.     

The P2Y2 Nucleotide Receptor Mediates the Proliferation and Migration of Human Hepatocellular Carcinoma Cells Induced by ATP

ATP is an abundant biochemical component of the tumor microenvironment and a physiologic ligand for the P2Y₂ nucleotide receptor (P2Y₂R). In this study, we investigated the effect of ATP on the cellular behavior of human hepatocellular carcinoma (HCC) cells and the role of P2Y₂R in ATP action and aimed to find a new therapeutic target against HCC. The experiments were performed in native isolated human HCC cells, normal hepatocytes, human HCC cell lines, and nude mice. We found that the mRNA and protein expression levels of P2Y₂R in native human HCC cells and the human HCC cell lines HepG2 and BEL-7404 were enhanced markedly compared with human normal hepatocytes and the normal hepatocyte line LO2, respectively. ATP induced intracellular Ca²⁺ increases in HCC cells and promoted the proliferation and migration of HCC cells and the growth of HCC in nude mice. The P2Y receptor antagonist suramin, P2Y₂R-specific shRNA, the store-operated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and 1-(β-3-(4-methoxy-phenyl) propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride ({"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365), and stromal interaction molecule (STIM1)-specific shRNA inhibited the action of ATP on HCC cells. In conclusion, P2Y₂R mediated the action of ATP on the cellular behavior of HCC cells through store-operated calcium channel-mediated Ca²⁺ signaling, and targeting P2Y₂R may be a promising therapeutic strategy against human HCC.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.     

Fusion-Activated Ca2+ Entry: An “Active Zone” of Elevated Ca2+ during the Postfusion Stage of Lamellar Body Exocytosis in Rat Type II Pneumocytes

Background

Ca²⁺ is essential for vesicle fusion with the plasma membrane in virtually all types of regulated exocytoses. However, in contrast to the well-known effects of a high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) in the prefusion phase, the occurrence and significance of Ca²⁺ signals in the postfusion phase have not been described before.

Methodology/Principal Findings

We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, Ca²⁺- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca²⁺]_c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t_1/2 of decay = 3.2 s) rise of localized [Ca²⁺]_c originating at the site of lamellar body fusion. [Ca²⁺]_c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca²⁺ from, or addition of Ni²⁺ to the extracellular solution, strongly inhibited these [Ca²⁺]_c transients, whereas Ca²⁺ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca²⁺]_c. Both effects were reduced by the non-specific Ca²⁺ channel blocker {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365.

Conclusions/Significance

Fusion-activated Ca²⁺ entry (FACE) is a new mechanism that leads to [Ca²⁺]_c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca²⁺ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions.     

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca²⁺ entry (SOCE) with Orai1 as principal Ca²⁺ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca²⁺ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1^−/− and Orai1^−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca²⁺ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2^−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca²⁺ signals of Stim1^−/− and Orai1^−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1^−/− and Orai1^−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, a blocker of (receptor-operated) Ca²⁺ entry, inhibited Ca²⁺ and procoagulant responses even in Stim1^−/− and Orai1^−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca²⁺ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca²⁺ entry and PS exposure, only one relying on STIM1-Orai1 interaction.     

A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry,release, and retrograde signaling to the DHPR

Bidirectional signaling between the sarcolemmal L-type Ca²⁺ channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca²⁺ release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation–contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K⁺ depolarization. The new findings are: (a) For all three genotypes, Ca²⁺ transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca²⁺ or the addition of Ca²⁺ entry blockers (nifedipine, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, and Ni²⁺) enhanced the rate of decay in all genotypes. (d) When Ca²⁺ entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K⁺ depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor.     

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

SKF83959, an Agonist of Phosphatidylinositol-Linked D1-Like Receptors,Promotes ERK1/2 Activation and Cell Migration in Cultured Rat Astrocytes

Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the mitogen-activated protein kinase family. It can mediate cell migration. Classical dopamine receptor-mediated ERK1/2 phosphorylation is widely studied in neurons. Here, we report that ERK1/2 phosphorylation is also modulated by putative phosphatidylinositol-linked D₁-like receptors in cultured rat astrocytes. 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine ({"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959), an agonist of the putative phosphatidylinositol-linked D₁-like receptors, was found to enhance ERK1/2 phosphorylation, which then promoted the migration of cultured astrocytes. The {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced ERK1/2 phosphorylation was found to be Ca²⁺-independent based on the following observations: i. chelating intracellular Ca²⁺ did not inhibit ERK1/2 phosphorylation and astrocyte migration; ii. blockage of the release of intracellular Ca²⁺ from the endoplasmic reticulum by an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor did not attenuate ERK1/2 phosphorylation. However, inhibition of phospholipase C (PLC), the upstream molecule of internal Ca²⁺ release, disabled {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959’s ability to elevate the level of ERK1/2 phosphorylation. Both non-selective protein kinase C (PKC) inhibitor and PKCδ selective inhibitor prevented ERK1/2 phosphorylation increase and astrocyte migration, but PKCα inhibitor did not. This suggests that Ca²⁺-independent and diacylglycerol-dependent PKCδ acts downstream of putative phosphatidylinositol-linked D₁-like receptor activation and mediates {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced elevation of ERK1/2 phosphorylation in order to modulate astrocyte migration. In conclusion, our results demonstrate that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-induced increases in ERK1/2 phosphorylation and astrocyte migration are dependent on PLC-PKCδ signals. This might help us to further understand the functions of the putative phosphatidylinositol-linked D₁-like receptors in the nervous system.     

Arylbenzazepines Are Potent Modulators for the Delayed Rectifier K+ Channel: A Potential Mechanism for Their Neuroprotective Effects

(±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D₁-like dopamine receptor agonistic activity. The precise mechanism for the (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-mediated neuroprotection remains elusive. We report here that (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is a potent blocker for delayed rectifier K⁺ channel. (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 inhibited the delayed rectifier K⁺ current (I _K) dose-dependently in rat hippocampal neurons. The IC ₅₀ value for inhibition of I _K was 41.9±2.3 µM (Hill coefficient = 1.81±0.13, n = 6), whereas that for inhibition of I _A was 307.9±38.5 µM (Hill coefficient = 1.37±0.08, n = 6). Thus, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is 7.3-fold more potent in suppressing I _K than I _A. Moreover, the inhibition of I _K by (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 with the K⁺ channel. The intracellular application of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 had no effects of on I _K, indicating that the compound most likely acts at the outer mouth of the pore of K⁺ channel. We also tested the enantiomers of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, R-(+) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-201), and S-(−) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-202), as well as {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393; all these compounds inhibited I _K. However, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I _K , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959.     

Muscarinic Agonists Activate Ca<Superscript>2+</Superscript> Store-operated and -independent Ionic Currents in Insulin-secreting HIT-T15 Cells and Mouse Pancreatic β-Cells

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic -cells by depolarizing the membrane to enhance voltage-gated Ca²⁺ influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal -cell line (HIT-T15) and mouse pancreatic -cells. In whole-cell recordings, the muscarinic agonist carbachol (CCh) dose-dependently and reversibly activated a voltage-independent, nonselective current (whole-cell conductance 24 pS/pF, reversal potential ~-15 mV). The current, which we refer to as I_musc, was blocked by atropine, a muscarinic receptor antagonist, and SKF 96365, a nonspecific ion channel blocker. The magnitude of the current decreased by 52% when extracellular Na⁺ was removed, but was not affected by changes in extracellular Ca²⁺, confirming that I_musc is a nonselective current. To determine if I_musc activates following release of Ca²⁺ from an intracellular store, we blocked intracellular IP₃ receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca²⁺ store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential (~0 mV) than the current activated by CCh under control conditions. This result indicates that heparin had blocked a component of I_musc, which likely activates following release of stored Ca²⁺. Depleting IP₃-sensitive calcium stores with thapsigargin also activated a non-selective, SKF 96365-blockable current in HIT cells. The properties of this putative store-operated current were similar to the component of I_musc that was blocked by heparin, being voltage-independent and reversing near –30 mV. We conclude that I_musc consists of store-operated and store-independent components, both of which may contribute to the depolarizing action of muscarinic agonists on pancreatic -cells.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Role of Endogenous ENaC and TRP Channels in the Myogenic Response of Rat Posterior Cerebral Arteries

Aims

Mechanogated ion channels are predicted to mediate pressure-induced myogenic vasoconstriction in small resistance arteries. Recent findings have indicated that transient receptor potential (TRP) channels and epithelial sodium channels (ENaC) are involved in mechanotransduction. The purpose of this study was to investigate the role of TRP channels and ENaC in the myogenic response. Our previous study suggested that ENaC could be a component of the mechanosensitive ion channels in rat posterior cerebral arteries (PCA). However, the specific ion channel proteins mediating myogenic constriction are unknown. Here we found, for the first time, that ENaC interacted with TRPM4 but not with TRPC6 using immunoprecipitation and confocal microscopy.

Methods and Results

Treatment with a specific βENaC inhibitor, amiloride, a specific TRPM4 inhibitor, 9-phenanthrol, and a TRPC6 inhibitor, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, resulted in inhibition of the pressure-induced myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with small interfering RNA of βENaC, TRPM4, and TRPC6. Co-treatment with amiloride and 9-phenanthrol showed a similar inhibitory effect on myogenic contraction compared to single treatment with amiloride or 9-phenanthrol. The myogenic response was not affected by 9-phenanthrol or amiloride treatment in PCA transfected with βENaC or TRPM4 siRNA, respectively. However, pressure-induced myogenic response was fully inhibited by co-treatment with amiloride, 9-phenanthrol, and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, and by treatment with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 in PCA transfected with βENaC siRNA.

Conclusion

Our results suggest that ENaC, TRPM4, and TRPC6 play important roles in the pressure-induced myogenic response, and that ENaC and TRPM4 interact in rat PCA.     

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.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca²⁺ ATP pump inhibitor, thapsigargin (10 μM) produced a time-dependent increase in the V_max for d-[³H]aspartate transport that reached a maximum at 15 min (143% of control; P < 0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the V_max of sodium-dependent d-[³H]aspartate transport (190% of control; P < 0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in d-[³H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 μM) or SKF 96365 (10 μM) caused a net inhibition of d-[³H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in d-[³H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.     

Intracellular ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana

In Arabidopsis thaliana cell suspension,abscisic acid (aBa) induces changes in cytosolic calcium concentration ([Ca²⁺]_cyt) which are the trigger for aBa-induced plasma membrane anion current activation, H⁺-aTPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca²⁺ stores in aBa signal transduction through electrophysiological current measurements, cytosolic Ca²⁺ activity measurements with the apoaequorin Ca²⁺ reporter protein and external pH measurement. Intracellular Ca²⁺ stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (1) Ca²⁺ release in the cytosol, (2) anion channel activation and H⁺-ATPase inhibition leading to plasma membrane depolarization and (3) stomatal closure. Intracellular Ca²⁺ release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.