Calcium Channel Subunit α2δ-1 as a Potential Biomarker Reflecting Illness Severity and Neuroinflammation in Patients with Acute Ischemic Stroke.

BACKGROUND: Voltage-gated calcium channels (VGCCs) dysfunction is involved in the development of acute ischemic stroke (AIS). As a subunit of VGCC complexes, we detected the levels of α2δ-1 subunit in serum and cerebrospinal fluid (CSF) specimens from AIS patients. METHODS: The study included 105 patients with first-ever AIS, who were admitted within 48 hours after stroke onset. The serum and CSF levels of α2δ-1 were measured with ELISA and the severity of AIS patients was evaluated according to the National Institutes of Health Stroke Scale (NIHSS) score. The cerebral infarct volume was calculated through the Pullicino formula based on the cranial CT or MRI scan. C-reactive protein (CRP) and serum amyloid A (SAA) were measured using the latex-enhanced immunoturbidimetric assay. RESULTS: Compared to the control subjects, the serum α2δ-1 level was significantly increased in AIS patients with large infarct volume and in severe AIS cases with high NIHSS score, which correlated positively with the inflammatory markers CRP and SAA. Furthermore, the concentration of α2δ-1 in CSF was elevated with the infarct volume, which was higher in severe AIS patients. CONCLUSION: Our study suggests that the increased α2δ-1 levels in serum and CSF specimens may be used as a potential marker for reflecting VGCCs dysfunction, illness severity and neuroinflammation in AIS patients.

TRPV6 Is Associated with Prognosis of ST-Elevation Acute Myocardial Infarction.

OBJECTIVE: To investigate the relationship between Transient Receptor Potential Vanilloid 6 (TRPV6) and ST-elevation acute myocardial infarction (STEMI) patients. METHODS: This observational research included a total of 221 patients with STEMI admitted during January 2017~August 2019. Additionally, 50 cases of non-ST-elevation acute myocardial infarction (NSTEMI) patients and 50 healthy individuals were enrolled as the control. Serum levels of TRPV6 were detected by ELISA method. The relationship between TRPV6, clinical characteristics, laboratory indices of CK-MB, TnI, NT-pro-B-type natriuretic peptide (NT-pro-BNP), C-reactive protein (CRP), and the left ventricular ejection fraction (LVEF%) was analyzed by statistical methods. K-M curve was performed for survival time. RESULTS: Serum levels of TRPV6 were remarkably lower in STEMI and NSTEMI patients compared with the healthy control. Levels of NT-pro-BNP and CK-MB were significantly higher and serum levels of TRPV6 were dramatically lower in deceased STEMI patients in comparison with the surviving patients. The levels of TRPV6 were negatively correlated with CK-MB and NT-pro-BNP. Meanwhile, TRPV6 was negatively expressed in tissues of STEMI patients and positively expressed in normal tissues. Patients with lower TRPV6 levels had remarkably lower LVEF ratio, higher GRACE scores, higher CK-MB and NT-pro-BNP levels, as well as higher ratios of cardiovascular death, malignant arrhythmia, cumulative MACE, and shorter survival time than patients with higher TRPV6. CONCLUSION: The lower expression of TRPV6 was associated with poor clinical outcomes and prognosis of STEMI patients.

Predictors of Renal Function and Calcifications in Primary Hyperparathyroidism: A Nested Case-Control Study.

Context: Some patients with primary hyperparathyroidism (PHPT) develop renal calcifications. Investigation of urinary and nonurinary risk factors are essential. Objective: We aimed to study the prevalence and potential biochemical predictors of renal calcifications. Design: Nested case-control study. Setting: University hospital. Participants: We identified 792 patients with PHPT from 2005 to 2015. We used biochemical data to validate the diagnosis of PHPT. Main Outcome Measures: The prevalence of renal calcifications defined as nephrolithiasis or nephrocalcinosis assessed by a routine CT scan at the time of diagnosis. Results: A total of 792 patients with PHPT were identified among whom 617 patients (78%) had a CT scan preformed. We found a prevalence of renal calcifications of 23%, equally frequent between sexes. A total of 76 patients (12%) had nephrolithiasis and 75 patients (12%) had nephrocalcinosis where 7 patients (1%) had both nephrolithiasis and nephrocalcinosis. Compared with patients without renal calcifications, patients with renal calcifications had significantly higher levels of ionized calcium, parathyroid hormone, and 24-hour calcium excretion (Pall < 0.01). Patients with nephrocalcinosis had higher plasma levels of phosphate and a higher calcium-phosphate product compared with patients with nephrolithiasis (Pall < 0.05). Impaired renal function (estimated glomerular filtration rate <60 mL/min) was observed in 12% of patients. However, no differences in renal function were observed between those with and without renal calcifications. Conclusion: Renal calcifications are frequent in patients with PHPT and are associated with the severity of the disease. Impaired renal function is also common in PHPT, but renal function was not associated with renal calcifications.

Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients.

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19(+) B cells, CD56(bright) and CD56(dim) cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56(bright) TRPM3 35.72 % ± 7.37; CD56(dim) 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19(+) B cells (1.56 ± 0.191) and CD56(bright) NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19(+) B lymphocytes. CD56(bright) NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

Correlation Between DNA Methylation of TRPA1 and Chronic Pain States in Human Whole Blood Cells.

OBJECTIVES: Neuro-immune interactions with functional changes in the peripheral blood cells including changes in the transient receptor potential ankyrin 1 (TRPA1) appear to play a pivotal role in the development of chronic pain in humans. The aim of this study was to examine the association between TRPA1 DNA methylation in whole blood cells and the pain states in chronic pain patients. METHODS: After collecting blood samples from 12 chronic pain patients, the authors measured DNA methylation levels in whole blood cells. Significant associations between the patient's demographic data and the chronic pain states were determined by a multiple linear regression analysis that used age, body mass index, pain duration, depression, anxiety, cognitive impairment, activities of daily living, neuropathic pain, and pain states as the dependent variables, and the TRPA1 DNA methylation levels as the independent variables. RESULTS: Multiple regression analysis revealed a significant correlation between increases of the methylation levels of the CpG island in the TRPA1 gene and increases in the number of neuropathic pain symptoms, which were evaluated using the Douleur Neuropathique 4 (DN4) questionnaire. Decreases in the TRPA1 mRNA expression were also significantly related to increases in the DN4 score. The presence of a burning sensation, which is one of pain symptoms in the DN4 questionnaire, was significantly correlated with the increase in DNA methylation level of TRPA1. CONCLUSIONS: TRPA1 DNA methylation levels in whole blood cells appear to be associated with pain symptoms in chronic pain patients.

Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19+ B cells, CD56bnght and CD56dim cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56bright TRPM3 35.72 % ± 7.37; CD56dim 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19+ B cells (1.56 ± 0.191) and CD56bright NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19+ B lymphocytes. CD56bright NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+ release.

Platelet dense granules (PDGs) are acidic calcium stores essential for normal hemostasis. They develop from late endosomal compartments upon receiving PDG-specific proteins through vesicular trafficking, but their maturation process is not well understood. Here we show that two-pore channel 2 (TPC2) is a component of the PDG membrane that regulates PDG luminal pH and the pool of releasable Ca(2+). Using a genetically encoded Ca(2+) biosensor and a pore mutant TPC2, we establish the function of TPC2 in Ca(2+) release from PDGs and the formation of perigranular Ca(2+) nanodomains. For the first time, Ca(2+) spikes around PDGs--or any organelle of the endolysosome family--are visualized in real time and revealed to precisely mark organelle "kiss-and-run" events. Further, the presence of membranous tubules transiently connecting PDGs is revealed and shown to be dramatically enhanced by TPC2 in a mechanism that requires ion flux through TPC2. "Kiss-and-run" events and tubule connections mediate transfer of membrane proteins and luminal content between PDGs. The results show that PDGs use previously unknown mechanisms of membrane dynamics and content exchange that are regulated by TPC2.

Non-paraneoplastic voltage-gated calcium channels antibody-mediated cerebellar ataxia responsive to IVIG treatment.

Non-paraneoplastic cerebellar ataxia associated with voltage-gated calcium channel (VGCC) antibodies is a rare entity with only few cases reported in literature. We describe a 60 year-old man with subacute cerebellar ataxia and subclinical Lambert-Eaton myasthenic syndrome (LEMS) in whom VGCC antibodies were detected at high titer in serum and cerebrospinal fluid. Screening for underlying malignancies was negative. Intravenous immunoglobulin treatment led to the improvement of clinical picture and reduction of serum antibody titer over a 13-month follow-up period. We emphasize that VGCC antibodies should be included in the diagnostic work-up of patients with subacute cerebellar ataxia and that treatment with IVIG can improve the clinical picture and prevent disability.

A functional interaction between TRPC/NCKX induced by DAG plays a role in determining calcium influx independently from PKC activation.

Ca(2+)influx might occur through K(+)-dependent Na(+)/Ca(2+) exchanger operating in reverse mode (rNCKX). In a cellular model different from platelets, an interaction between canonical transient receptor potential cation (TRPC) channels and NCX has been found. The aim of this study was to verify whether the TRPC/NCKX interaction operates in human platelets. Our results showed that the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced rNCKX-mediated Ca(2+) influx through TRPC-mediated Na(+) influx. DAG-induced activation of TRPC/NCKX occurs independently of protein kinase C (PKC) activation, as PKC inhibitor did not modify OAG-mediated Ca(2+) influx. Moreover, as both rNCKX and TRPC inhibitors reduced OAG-induced platelet aggregation which, conversely, was increased by flufenamic acid, known to develop TRPC activity, it could be suggested that the TRPC/NCKX interaction has a role in OAG-dependent platelet aggregation.

Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation.

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca(2+). Entry of Ca(2+) through the Ca(2+) release-activated Ca(2+) (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient (sgk1(-/-)) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1(-/-) MCs. Confirming previous results, release of ß-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1(-/-) MCs compared with sgk1(+/+) MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 µM), MC degranulation was strongly decreased in both sgk1(+/+) and sgk1(-/-) MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1(+/+) MCs and sgk1(-/-) MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1(+/+) MCs but not in sgk1(-/-) MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1(+/+) MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca(2+) entry in mast cells as a novel mechanism regulating actin cytoskeleton.

[The functional characteristics oF TRPV5 and TRPV6 channels in normal and transformed human blood lymphocytes].

Calcium signaling and Ca(2+)-conducting channels are involved in the development of immune response, cell proliferation, growth and differentiation of lymphocytes. In this paper the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of T cell line Jurkat and normal human blood lymphocytes. The channels were activated by removing Ca2+ and Mg2+ from surrounding solution, characterized by inward rectification, and were inactivated by the effective blocker of TRPV5 and TRPV6, ruthenium red. Channel activity was significantly higher in Jurkat cells, than in normal human lymphocytes. Quantitative PCR analysis revealed higher levels of mRNA genes encoding channels TRPV5 and TRPV6 in the proliferation cells compared with resting lymphocytes. In general these data showed that TRPV5/TRPV6 in human lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets.

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca2+ signalling. Two distinct Ca2+ entry pathways amplify and sustain platelet Ca2+ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca2+ entry (SOCE) and non-capacitative Ca2+ entry (NCCE) in thrombin-induced Ca2+ signalling and PS exposure by using two Ca2+ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca2+ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca2+ signalling at higher thrombin concentrations. However, despite being involved in Ca2+ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca2+ entry is an important regulator of thrombin-induced PS exposure in platelets.

STIM and Orai in hemostasis and thrombosis.

At sites of vascular injury, platelets rapidly adhere to the exposed subendothelial extracellular matrix, become activated and, together with the coagulation system, form a plug that seals the lesion. This process is essential to prevent blood loss, however, under pathological conditions it may lead to vessel occlusion. Agonist-induced elevation of intracellular Ca(2+) levels is essential for platelet activation. It occurs through two different mechanisms: Ca(2+) release from internal stores, involving phospholipase C (PLC)-dependent generation of inositol-1,4,5-trisphosphate (IP3) and activation of IP3 sensitive channels in the store membrane, and Ca(2+) influx across the plasma membrane. Store operated Ca(2+) entry (SOCE), triggered by store depletion, is the main influx pathway for extracellular Ca(2+) in platelets, but the molecular mechanism underlying this pathway has long remained elusive. In the last years, however, the Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the channel protein Orai1 emerged as the key players in platelet SOCE. This review summarizes the current knowledge about the role of these proteins in platelet physiology and thrombus formation and discusses their suitability as antithrombotic targets.

Lack of association between ORAI1/CRACM1 gene polymorphisms and Kawasaki disease in the Taiwanese children.

OBJECTIVE: Kawasaki disease (KD) is characterized by systemic vasculitis of an unknown cause. A previous study has indicated that a polymorphism of the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene is involved in the susceptibility to KD. ORAI (also known as CRACM1) is one of the components of store-operated calcium channels involved in regulating immune and inflammatory reactions. This study was conducted to investigate if polymorphisms in ORAI1/CRACM1, a gene downstream from ITPKC, are associated with KD susceptibility and clinical outcomes. MATERIALS AND METHODS: A total of 1,056 subjects (341 KD patients and 715 controls) were investigated to identify five tagging single nucleotide polymorphisms (tSNPs) in ORAI1/CRACM1 (rs12313273, rs6486795, rs7135617, rs12320939, and rs712853) by using the TaqMan Allelic Discrimination assay. RESULTS: No significant associations between genotype and allele frequency of the five ORAI1/CRACM1 tSNPs were observed in the KD patients and controls. In KD patients, no significant associations between ORAI1/CRACM1 polymorphisms and coronary artery lesion (CAL) formation or intravenous immunoglobulin (IVIG) treatment response were observed. The results from haplotype analysis were insignificant. CONCLUSIONS: This study showed for the first time that ORAI1/CRACM1 polymorphisms are not associated with KD susceptibility, CAL formation, or IVIG treatment response in the Taiwanese population.

The levels of Pdx1/insulin, Cacna1c and Cacna1d, and ß-cell mass in a rat model of intrauterine undernutrition.

OBJECTIVE: To elucidate the underlying mechanisms responsible for ß-cell mass and function in response to intrauterine growth restriction (IUGR). METHODS: Offspring from undernourished mother rats with birth weight < -2 SD (IUGR group) and from standard-nourished rats with birth weight between mean +/- 1SD normal birth weight (NBW group) were examined. Levels of fasting glucose, serum insulin, and insulin-like growth factor-1 (IGF-1) were analyzed. Entire pancreas or islet-like cell clusters (ICCs) were collected to evaluate relative ß-cell mass and determine the genetic and protein profiles of pancreatic and duodenal homeobox 1 (Pdx1), Cacna1c and Cacna1d using real-time PCR, immunohistochemical staining, and western blotting at day (d) of birth and at d21 of age. RESULTS: Fasting serum insulin and IGF-1 concentrations were significantly lower in the IUGR group than in the NBW group at d0 and d21. The levels of Pdx1 and insulin mRNAs in IUGR pancreas were also decreased. At birth, the ratios of ß-cell mass to body weight were not significantly different between the two groups. However, by d21 the relative ß-cell mass in IUGR had not grown to compensate for the increase in body weight, as compared to the NBW group (p < 0.05). The Cacna1c and Cacna1d proteins were significantly higher in the NBW group than that in the IUGR group at birth, but there were no statistical differences at d21. CONCLUSION: Decreased Pdx1 levels and IGF-1 concentration restrain ß-cell mass and insulin expression in rat offspring from undernourished mothers. However, Cacna1c and Cacna1d expression were able to reach normal levels as the IUGR rats were aged, indicating that these factors were not responsible for the IUGR rat phenotype of insulin resistance and ß-cell dysfunction in later life.

Src family tyrosine kinases activate thrombin-induced non-capacitative cation entry in human platelets.

Platelet activation is critically regulated by an increase in intracellular calcium concentration ([Ca2+](i)). Although Ca2+ release from intracellular Ca2+ stores and subsequent store-operated Ca2+ entry are often thought to be the major contributors to increases in [Ca2+](i) evoked by most agonists, high concentrations of thrombin activate a Ca2+ entry pathway that is independent of Ca2+ store depletion (known as 'non-capacitative cation entry'-NCCE). The channel that conducts NCCE has not previously been clearly identified, and the mechanisms that regulate its activation are also unknown. Here we have investigated NCCE using fura-2-loaded human platelets. To investigate NCCE independently of other Ca2+ signaling pathways, the intracellular Ca2+ stores were first rapidly depleted in the absence of extracellular Ca2+. Sr2+ was then added to monitor maximal store-operated cation influx. Thrombin was then added to stimulate NCCE. Flufenamic acid, which inhibits Ca2+ entry through most TRPC isoforms, but potentiates entry through TRPC6, was found to block store-operated cation entry. In contrast, thrombin-induced NCCE was increased, suggesting the possible involvement of TRPC6. Since TRPC6 is regulated by Src family tyrosine kinases in some cells, we investigated the possible role of this kinase family in NCCE. PP2, a Src family tyrosine kinase inhibitor, completely abolished thrombin-induced NCCE. Furthermore, NCCE was enhanced by phenylarsine oxide and could be directly induced by vanadyl hydroperoxide, both tyrosine phosphatase inhibitors. These data indicate that Src family tyrosine kinase activation is a required step in NCCE activation. In conclusion NCCE may be an important regulator of platelet activation when local thrombin concentrations are high.

STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets.

Ca(2+) entry, particularly store-operated Ca(2+) entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca(2+) content of the stores, where the intraluminal Ca(2+) sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca(2+) stores to the store-operated Ca(2+)-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25-139) antibody, directed towards the Ca(2+)-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288-301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557-571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca(2+) entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

Store-operated Ca2+ entry is sensitive to the extracellular Ca2+ concentration through plasma membrane STIM1.

Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) influx in platelets and other cells activated by a reduction in Ca(2+) concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca(2+), although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca(2+). Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn(2+) entry, which was inhibited by extracellular Ca(2+) in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25-139 of PM-STIM1 that contains the Ca(2+)-binding domain, prevented the inactivation of Ca(2+) entry induced by extracellular Ca(2+). TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca(2+)-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca(2+), an event that is likely to be mediated by interaction with Orai1.

L-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes.

In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1), which transports L-arginine, L-lysine, L-ornithine, and L-histidine. The expression level of human CAT1 (hCAT1) mRNA in mononuclear cells (MNCs) fractionated according to lineage-selective markers was examined by reverse transcriptase-polymerase chain reaction. The expression of CAT1 in glycophorin A-positive erythroid cells was 8 times higher than in nonfractionated MNC (control) cells. Characteristics of L-arginine uptake by K562 cells, an established leukemic cell line used as an erythroid model, were similar to those of CAT1 in regards to saturation kinetics, sodium independence, and substantial inhibition of L-arginine uptake by N-ethylmaleimide, which is a specific inhibitor of system y+ amino acid transporter. Removal of L-arginine from the culture medium prevented both proliferation and differentiation of K562 cells, while removal of L-lysine or L-histidine had little effect on differentiation, though proliferation was blocked. Hematopoietic stem cells obtained from human cord blood failed to develop into erythroid cells in the absence of L-arginine in the culture medium. These findings indicate that hCAT1 is involved in erythroid hematopoiesis through its role in importing L-arginine, which appears to be essential for the differentiation of red blood cells.