Addition Of Extracellular Ca2 Research Articles

How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP

BackgroundFor many cell types, directional locomotion depends on their maintaining filopodia at the leading edge. Filopodia lack any Ca2+-binding structural protein but respond to store-operated Ca2+ entry (SOCE).MethodsSOCE was induced by first replacing the medium with Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+ in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+ influx restored by addition of extracellular Ca2+. Intracellular Ca2+ levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing.ResultsDepletion of the ER Ca2+ increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+ increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+ altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis.ConclusionsBecause the media were devoid of ligands that activate receptors during depletion and Ca2+ replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+ influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell’s ability to orient itself in response to attractive or repulsive cues.AnZD29f44bXN87L4SDxgRWVideo

Enhanced transient receptor potential channel-mediated Ca2+ influx in the cells with phospholipase C-δ1 overexpression: its possible role in coronary artery spasm.

We reported that coronary spasm was induced in the transgenic mice with the increased phospholipase C (PLC)-δ1 activity. We investigated the effect of enhanced PLC-δ1 on Ca2+ influx and its underlying mechanisms. We used human embryonic kidney (HEK)-293 and coronary arteries smooth muscle cells (CASMC). Intracellular free Ca2+ concentration ([Ca2+ ]i ; nm) was measured by fura-2, and Ca2+ influx was evaluated by the increase in [Ca2+ ]i after addition of extracellular Ca2+ . Acetylcholine (ACh) was used to induce Ca2+ influx. ACh-induced peak Ca2+ influx was 19 ± 3 in control HEK-293 cells and 71 ± 8 in the cells with PLC-δ1 overexpression (P < 0.05 between two groups). Nifedipine partially suppressed this Ca2+ influx, whereas either 2-APB or knockdown of classical transient receptor potential channel 6 (TRPC6) blocked this Ca2+ influx. In the human CASMC, ACh-induced peak Ca2+ influx was 29 ± 6 in the control and was increased to 45 ± 16 by PLC-δ1 overexpression (P < 0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or knockdown of TRPC6 blocked it. ACh-induced Ca2+ influx was enhanced by PLC-δ1 overexpression, and was blocked partially by nifedipine and completely by 2-APB. TRPC-mediated Ca2+ influx may be related to the enhanced Ca2+ influx in PLC-δ1 overexpression.

Abstract 16460: Enhanced Trpc Channel-Mediated Ca2+ Influx in the Cells With Phospholipase C-δ1 Overexpression

Background: We recently showed that ergometrine induces coronary spasm in the mice with enhanced phospholipase C (PLC)-δ1 activity, which was detected in patients with coronary spasm (Circulation 2012), and PLC-mediated Ca2+ entry is involved in the genesis of coronary spams. We examined the role of enhanced PLC-δ1 in extracellular Ca2+ entry and its underlying mechanisms in human embryonic kidney (HEK)-293 cells and human coronary arteries smooth muscle cells (CASMC). Methods and Results: The cells were stimulated with acetylcholine (ACh) in a extracellular Ca2+-free condition, and the increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of extracellular Ca2+ was defined as Ca2+ influx. [Ca2+]i was measured by fura-2. In HEK-293 cells, ACh-induced Ca2+ influx (nM ) was 21±2 in the control and 52±6 in the cells with PLC-δ1 overexpression (p&lt;0.05). ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by nifedipine in a dose-dependent manner but was partially by 36±13% at 10-5M (p&lt;0.05), thereby even after treatment with nifedipine at 10-5M, ACh-induced Ca2+ influx was increased by 2.9±0.1 times by enhanced PLC-δ1 compared with the control (p&lt;0.05). To clarify the role of diacylglycerol (DAG) in Ca2+ influx, the effect of blockers for DAG-activated transient receptor potential (TRPC) channel was examined. ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by 2-APB, an inhibitor of non-selective cation channel TRPC, in a dose-dependent manner, and was almost completely blocked by 89±12% at 10-4M (p&lt;0.05). While TRPC3 siRNA did not affect ACh-induced Ca2+ influx (59±27 vs 75±23 nM, p=ns), TRPC6 siRNA suppressed Ca2+ influx to 37±28 nM (p&lt;0.05). In human CASMC, ACh-induced Ca2+ influx was 41±11 in the control and 64±15 in the cells with PLC-δ1 overexpression (p&lt;0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or TRPC6 siRNA almost completely blocked it. Conclusion: ACh-induced Ca2+ influx, mediated by both voltage-gated Ca2+ channels and non-selective cation channels TRPC6, is enhanced by PLC-δ1 overexpression. Inhibition of TRPC may be effective in enhanced PLC-δ1-mediated coronary spasm.

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein

Ca2+ dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca2+ homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca2+ homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca2+ upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca2+ in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca2+ store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca2+ release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca2+ homeostasis affect APP protein interactions. Our results suggest that Ca2+ and Homers play a significant role in the development of Alzheimer's disease pathology.

Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl)-1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O2−) production, elastase release, and CD11b expression in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O2− production. The peak cytosolic calcium concentration ([Ca2+]i) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca2+]i was significantly shortened. In a calcium-free solution, changes in [Ca2+]i caused by the addition of extracellular Ca2+ were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca2+]i changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE.

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation.

BackgroundAgonist stimulation of airway smooth muscle (ASM) results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells.MethodsThe expression profile of NCX (which encodes the Na+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.ResultsNCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways.ConclusionsTaken together, these data demonstrate a potentially important role for NCX1 in control of Ca2+ homeostasis and link store depletion via STIM1 directly with NCX activation.

478 ZO-1 C-Terminal Domains Are Essential for Tight Junction Assembly and Maintenance

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

479 Myosin Light Chain Kinase Inhibition Decreases the Incidence of Severe Bowel Injury in a Neonatal Mouse Model of Necrotizing Enterocolitis

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

482 The Human Milk Factor Erythropoietin Protects Against Necrotizing Enterocolitis (NEC) in Preterm Rats By Maintaining Barrier Function Through Upregulation of Tight Junction Protein Expression

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

481 The RNA-Binding Protein HuR Regulates Occludin Translation Through Chk2-Dependent HuR Phosphorylation Modulating Intestinal Epithelial Barrier Function

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Role of symmetric dimethylarginine in vascular damage by increasing ROS via store-operated calcium influx in monocytes

The guanidines asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction, and its counterpart symmetric dimethylarginine (SDMA), considered inert, are accumulated in chronic kidney disease (CKD). The present study evaluates their effect on monocyte function, since previous data demonstrated leukocyte activation by other guanidino compounds. The effect of ADMA and SDMA on reactive oxygen species (ROS) production in human whole blood at baseline and after N-formyl-methionine-leucine-phenylalanine (fMLP) stimulation was evaluated. By using the fluorescent probe Fluo3-AM, the role of changes in monocytic cytoplasmic calcium ([Ca2+]i) was studied. Thapsigargin, and removal followed by addition of extracellular Ca2+ (Ca2+(ex)), was used to investigate the contribution of store-operated Ca2+-channels (SOCs). SKF96365 was used as a selective inhibitor of the SOCs. A pharmacologic intervention with captopril, known to affect Ca2+ influx, was tested. SDMA enhanced ROS production in fMLP-stimulated monocytes using heparinized blood, and this effect was abolished in EDTA-anticoagulated blood. In the presence of SDMA, an increased Ca2+ entry from the extracellular milieu resulted in an elevated amplitude of the peak [Ca2+]i change triggered by fMLP. None of these effects were seen with ADMA. Depletion of the intracellular stores with thapsigargin in the absence of Ca2+(ex), followed by re-addition of Ca2+(ex) triggered a significantly larger Ca2+ entry after SDMA treatment versus saline. This effect was prevented with SKF96365, as was the SDMA-enhanced oxidative burst after fMLP. Pre-incubation with captopril also reduced the increased ROS production seen with SDMA. SDMA, a uraemic retention solute considered inert, stimulates ROS production of monocytes by acting on Ca2+ entry via SOCs. This pro-inflammatory effect may trigger vascular pathology and may be involved in altering the prevalence of cardiovascular disease in CKD.

Evidence for the Role of Calcineurin in Morphogenesis and Calcium Homeostasis during Mycelium-to-Yeast Dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25 degrees C to 37 degrees C and is critical for pathogenicity. Intracellular Ca(2+) levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca(2+) with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca(2+) accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca(2+) homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca(2+) channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca(2+)/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca(2+) homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis.

Calcium‐sensing receptor mediates phenylalanine‐induced cholecystokinin secretion in enteroendocrine STC‐1 cells

Intraluminal L-phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells.

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Following smooth muscle excitation and contraction, depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE) to replenish stores and sustain cytoplasmic Ca(2+) (Ca(2+)(i)) elevations. The objectives of the present study were to characterize CCE and the Ca(2+)(i) dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca(2+)-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca(2+)(i) as well as activation of nonselective cation currents. To deplete Ca(2+)(i) stores, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca(2+)-free bathing solution. Under these conditions, addition of extracellular Ca(2+) (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 microM) and lanthanum (100 microM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca(2+)-imaging techniques, the CCE-evoked rise of Ca(2+)(i) was associated with inward currents carrying Ca(2+) that were inhibited by SKF-96365. Regional specializations in Ca(2+) influx and handling during CCE were observed. Distinct "hotspot" regions of Ca(2+) rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca(2+) entry occurs in hotspots contributing to localized Ca(2+) elevations in human colonic smooth muscle.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

Effects of calmodulin and protein kinase C modulators on transient Ca 2+ increase and capacitative Ca 2+ entry in human platelets: Relevant to pathophysiology of bipolar disorder

Disturbed intracellular calcium (Ca 2+) homeostasis has been implicated in bipolar disorder, which mechanisms may be involved in the dysregulation of protein kinase C (PKC) and calmodulin systems. In this study, we investigated a transient intracellular Ca 2+ increase induced by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca 2+-ATPase pump (SERCA), and a capacitative Ca 2+ entry followed by addition of extracellular Ca 2+, in the presence or absence of PKC/calmodulin modulators in the platelets of healthy subjects in order to elucidate the role of SERCA in Ca 2+ homeostasis and to assess how both PKC and calmodulin systems regulate the two Ca 2+ responses. Moreover, we also examined the thapsigargin-elicited transient Ca 2+ increase and capacitative Ca 2+ entry in patients with mood disorders. PKC and calmodulin systems have opposite regulatory effects on the transient Ca 2+ increase and capacitative Ca 2+ entry in the platelets of normal subjects. The inhibitory effect of PKC activation on capacitative Ca 2+ entry is significantly increased and the stimulatory effect of PKC inhibition is significantly decreased in bipolar disorder compared to major depressive disorder and normal controls. These results suggest the possibility that increased PKC activity may activate the inhibitory effect of capacitative Ca 2+ entry in bipolar disorder. However, this is a preliminary study using a small sample, thus further studies are needed to examine the PKC and calmodulin modulators on the capacitative Ca 2+ entry in a larger sample.

Retrograde regulation of store-operated calcium channels by the ryanodine receptor-associated protein triadin 95 in rat skeletal myotubes

The 95 kDa triadin (or T95), the main skeletal muscle triadin isoform, negatively regulates the mechanism of excitation–contraction coupling. T95 is a ryanodine receptor (RyR)-interacting protein but it also possesses a calsequestrin-interacting domain. RyR and calsequestrin are involved in Ca 2+ signalling and, for instance, influence the activity of store-dependent Ca 2+ channels (SOC). This work was undertaken to determine whether T95 was able to modulate the entry of Ca 2+ through SOC. The experiments were carried out on differentiated rat myotubes over-expressing T95 or DsRed (control cells) by means of an adenovirus infection. Intracellular Ca 2+ signals were analyzed using the Ca 2+ indicator Fluo-4. The sarco–endoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca 2+ stores. When applied in the presence of a Ca 2+-free medium, thapsigargin elicited transient but long-lasting Fluo-4 responses by elevating the cytoplasmic concentration of Ca 2+ ([Ca 2+] i). The over-expression of T95 reduced the thapsigargin-dependent [Ca 2+] i increase, with respect to control myotubes. Addition of extracellular Ca 2+ after the depletion of this Ca 2+ pool was accompanied by a [Ca 2+] i increase that was sensitive to the SOC blockers 2-APB, SKF-96365 and La 3+. The over-expression of T95 reduced this Ca 2+ influx, without changing its pharmacological properties, showing that T95 over-expression did not alter the properties of the SOC. In conclusion, the RyR-interacting molecule T95, recently shown to inhibit the excitation–contraction coupling, has also the ability to interfere with the skeletal muscle Ca 2+ signalling by depressing thapsigargin-dependent Ca 2+ release and influx.

Effects of Intracellular and Extracellular Concentrations of Ca 2+, K +, and Cl − on the Na +-Dependent Mg 2+ Efflux in Rat Ventricular Myocytes

Intracellular Mg 2+ concentration ([Mg 2+] i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25°C). After the myocytes were loaded with Mg 2+, the initial rate of decrease in [Mg 2+] i (initial Δ[Mg 2+] i/Δ t) was estimated upon introduction of extracellular Na +, as an index of the rate of Na +-dependent Mg 2+ efflux. The initial Δ[Mg 2+] i/Δ t values with 140 mM [Na +] o were essentially unchanged by the addition of extracellular Ca 2+ up to 1 mM (107.3 ± 8.7% of the control value measured at 0 mM [Ca 2+] o in the presence of 0.1 mM EGTA, n = 5). Intracellular loading of a Ca 2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 μM for 3.5 h) did not significantly change the initial Δ[Mg 2+] i/Δ t: 115.2 ± 7.5% (seven BAPTA-loaded cells) and 109.5 ± 10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K + and Cl − were modified under constant [Na +] o (70 mM), [Ca 2+] o (0 mM with 0.1 mM EGTA), and membrane potential (–13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Δ[Mg 2+] i/Δ t: 1), changes in [K +] o between 0 mM and 75 mM (65.6 ± 5.0% ( n = 11) and 79.0 ± 6.0% ( n = 8), respectively, of the control values measured at 140 mM [Na +] o without any modification of extracellular and intracellular K + and Cl −); 2), intracellular perfusion with K +-free (Cs +-substituted) solution from the patch pipette in combination with removal of extracellular K + (77.7 ± 8.2%, n = 8); and 3), extracellular and intracellular perfusion with K +-free and Cl −-free solutions (71.6 ± 5.1%, n = 5). These results suggest that Mg 2+ is transported in exchange with Na +, but not with Ca 2+, K +, or Cl −, in cardiac myocytes.

Inhibition of the renal betaine transporter by calcium ions

Chronic upregulation of the renal betaine/GABA transporter (BGT1) by hypertonic stress has been well documented, but it is not known whether BGT1 can be regulated acutely after insertion in the basolateral plasma membrane. Related transporters, such as the rat brain GABA transporter, can be rapidly removed from the plasma membrane through activation of G protein-coupled receptors. The goal of the present study was to determine whether acute changes in extracellular and/or intracellular Ca(2+) will regulate BGT1 transport activity at the plasma membrane level in Madin-Darby canine kidney cells subjected to 24-h hypertonic stress. After brief pretreatment with a Ca(2+)-free solution, the addition of extracellular Ca(2+) in the transport assay produced dose-dependent inhibition of Na(+)-GABA cotransport. Maximum inhibition was 49% at 2 mM Ca(2+) (P < 0.05). Fura 2 imaging confirmed that addition of 2 mM Ca(2+) produced a transient increase in intracellular Ca(2+) that preceded transport inhibition. Acute inhibition of Na(+)-GABA cotransport was reproduced by addition of thapsigargin (5 microM) and ionomycin (10 microM). Amino acid transport system A, assayed as a control, was not inhibited. Brief treatment with phorbol esters reproduced the specific inhibition of Na(+)-GABA cotransport, and the inhibition was blocked by staurosporine. Surface biotinylation confirmed that the response to phorbol esters was accompanied by loss of BGT1 protein from the plasma membrane, and immunohistochemistry showed a shift to an intracellular distribution. We conclude that BGT1 can be inhibited acutely by extracellular Ca(2+) through a mechanism involving BGT1 protein internalization, and protein kinase C may play a role.