Elevated Intracellular Calcium Levels Research Articles

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte-mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1(G93A) mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1(G93A) mice as well as human induced pluripotent stem cell (iPSC)-derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co-culture. Increased Cx43 expression led to important functional consequences when tested in SOD1(G93A) astrocytes when compared to control astrocytes over-expressing wild-type SOD1 (SOD1(WT) ). We observed SOD1(G93A) astrocytes exhibited enhanced gap junction coupling, increased hemichannel-mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1(G93A) astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS-related motor neuron loss. GLIA 2016;64:1154-1169.

Rhein triggers apoptosis via induction of endoplasmic reticulum stress, caspase-4 and intracellular calcium in primary human hepatic HL-7702 cells

Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells.

Altered cellular localization and hemichannel activities of KID syndrome associated connexin26 I30N and D50Y mutations.

BackgroundGap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells.ResultsImmunostaining experiments showed that Cx26I30N and D50Y failed to form gap junction plaques at cell-cell contact sites. Further, these mutations resulted in the retention of Cx26 protein in the Golgi apparatus. Examination of hemichannel function by fluorescent dye uptake assays revealed that cells with Cx26I30N and D50Y mutations had increased dye uptake compared to Cx26WT (wild-type) containing cells, indicating abnormal hemichannel activities. Cells with mutant proteins had elevated intracellular calcium levels compared to Cx26WT transfected cells, which were abolished by a hemichannel blocker, carbenoxolone (CBX), as measured by Fluo-3 AM loading and flow cytometry.ConclusionsHere, we demonstrated that Cx26I30N and D50Y mutations resulted in the formation of aberrant hemichannels that might result in elevated intracellular calcium levels, a process which may contribute to the hyperproliferative epidermal phenotypes of KID syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0081-0) contains supplementary material, which is available to authorized users.

Immunomodulatory Protein from Ganoderma microsporum Induces Pro-Death Autophagy through Akt-mTOR-p70S6K Pathway Inhibition in Multidrug Resistant Lung Cancer Cells.

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Hemeoxygenase-1 maintains bone mass via attenuating a redox imbalance in osteoclast

Heme oxygenase-1 (HO-1) has long been considered to be an endogenous antioxidant. However, the role of HO-1 is highly controversial in developing metabolic diseases. We hypothesized that HO-1 plays a role in maintaining bone mass by alleviating a redox imbalance. We investigated its role in bone remodeling. The absence of HO-1 in mice led to decreased bone mass with elevated activity and number of OCs, as well as higher serum levels of reactive oxygen species (ROS). HO-1, which is constitutively expressed at a high level in osteoclast (OC) precursors, was down-regulated during OC differentiation. HO-1 deficiency in bone marrow macrophages (BMM) in vitro resulted in increased numbers and activity of OCs due to enhanced receptor activator of nuclear factor-κB ligand (RANKL) signaling. This was associated with increased activation of nuclear factor-κB and of nuclear factor of activated T-cells, cytoplasmic 1 along with elevated levels of intracellular calcium and ROS. Decreased bone mass in the absence of HO-1 appears to be mainly due to increased osteoclastogenesis and bone resorption resulting from elevated RANKL signaling in OCs. Our data highlight the potential role of HO-1 in maintaining bone mass by negatively regulating OCs.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

Abstract 29: Calcium/calmodulin-dependent Protein Kinase Kinase β (CaMKK β) is Neuroprotective in Stroke in Aged Mice

Background: CaMKK β is a major kinase activated by elevated levels of intracellular calcium. Our previous data has suggested that CaMKK β is neuroprotective after stroke in young mice as inhibition of this kinase aggravated stroke outcome. As aging is an important determinant of stroke outcomes, here we evaluated the functional role of CaMKK β in stroke in aged mice. Methods: Aged wild type (WT) males received intracerebral injections of lentiviral vectors carrying either CaMKK β (LV-CaMKK β) or GFP (LV-GFP) 7 days before middle cerebral artery occlusion (MCAO, 90 minutes). Acute infarcts and neurological deficits scores were then analyzed at 72 hours post MCAO. In chronic survival studies, aged male CaMKK β knockout (KO) and WT mice were subjected to 60 minutes MCAO. Following stroke, long-term behavioral assessments were continuously performed for 3 weeks in KO and WT mice until the sacrifice for tissue loss assessments. Results: Baseline levels of CaMKK β in aged brain were significantly lower when compared to that in young mice measured by Western Blots (Aged WT 0.66±0.06 vs. Young WT 1.00±0.05, n=3 p/g, p<0.05). Administration of LV-CaMKK β to WT mice increased levels of this protein in brain (LV-CaMKK β 1.65±0.11 vs. LV-GFP control 1.07±0.05, n=3 p/g, p<0.05). Further, LV-CaMKK β treatment reduced infarcts assessed 3 days after stroke (total: LV-CaMKK β 16.76± 3.16% vs. LV-GFP control 26.43±4.30%, n=7 p/g, p<0.05) and neurological deficits [LV-CaMKK β 1(1) vs. LV-GFP 2(1), median (interquartile range), n=7 p/g, p<0.05]. In chronic survival experiments, CaMKK β KO mice showed increased tissue loss in ipsilateral hemispheres measured at 3 weeks after stroke (tissue loss: Aged KO 20.59±1.90% vs. Aged WT 11.89±1.00%, n=6 KO, 7 WT, p<0.05). Additionally, KO mice had worsened functional recovery when compared to WT controls during 3 weeks survival measured by rotarod test, locomotor activity in an open field, and in the novel object recognition test. Conclusions: Our studies demonstrated that CaMKK β is neuroprotective in stroke in the aged. As the elderly population has worse functional recovery after stroke, our data suggested that this protein may be a potential target for reducing long term disability of stroke patients.

Elevated Calcium after Acute Ischemic Stroke: Association with a Poor Short-Term Outcome and Long-Term Mortality

Background and PurposeAn elevated intracellular calcium level is known to be a major initiator and activator of ischemic cell death pathway; however, in recent studies, elevated serum calcium levels have been associated with better clinical outcomes and smaller cerebral infarct volumes. The pathophysiological role played by calcium in ischemic stroke is largely unknown.MethodsAcute stroke patients from a prospective stroke registry, consecutively admitted during October 2002-September 2008, were included. Significant associations between the modified Rankin scale distribution at discharge and serum calcium or albumin-corrected calcium were identified using ordinal logistic regression analysis. Cox proportional hazard models were used for survival analysis.ResultsMean serum calcium and albumin-corrected calcium levels of the 1,915 participants on admission were 8.97±0.58 mg/dL and 9.07±0.49 mg/dL, respectively. Second [adjusted odds ratio 1.32 (95% confidence interval 1.07-1.61)] and third [1.24 (1.01-1.53)] tertiles of serum calcium level and the third [1.24 (1.01-1.53)] tertile of albumin-corrected calcium level were found to be independent risk factors for a poor discharge outcome. Significant relationships were observed with serum calcium [1.19 (1.03-1.38)] and albumin-corrected calcium [1.21(1.01-1.44)] as linear variables. However, only albumin-corrected calcium was associated with long-term mortality, third tertile [adjusted hazard ratio 1.40 (1.07-1.83)], and increase by 1 mg/dL [1.46 (1.16-1.84)].ConclusionsElevated albumin-corrected serum calcium levels are associated with a poorer short-term outcome and greater risk of long-term mortality after acute ischemic stroke.

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma.

BackgroundHuman multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.MethodsThe proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.ResultsTreatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.ConclusionsOur results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.

Intracellular calcium regulates nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons (PTCs) and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent an important target for therapeutic intervention. Here we have developed a novel multicolored, bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides including ouabain and digoxin as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the Na+/K+-ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has important implications for exploiting NMD in the treatment of disease.

Myocardial protection using del nido cardioplegia solution in adult reoperative aortic valve surgery.

The immediate postischemic period is marked by elevated intracellular calcium levels, which can lead to irreversible myocyte injury. Del Nido cardioplegia was developed for use in the pediatric population to address the inability of immature myocardium to tolerate high levels of intracellular calcium following cardiac surgery. Our aim in this study is to determine if this solution can be used safely and effectively in an adult, reoperative population. All patients undergoing isolated reoperative aortic valve replacement at our institution from 2010 to 2012 were retrospectively reviewed. Demographics, comorbidities, operative variables, postoperative complications, and patient outcomes were collected. Patients were divided into two groups based on cardioplegia strategy used: whole blood cardioplegia (WB, n = 61) and del Nido cardioplegia (DN, n = 52). Mean age in the study was 73.4 ± 14.3 years and 86 patients were male (76.1%). Eighty-four patients had undergone prior coronary artery bypass graft (CABG) (74.3%). Patients in the DN group required significantly lower total volume of cardioplegia (1147.6 ± 447.2 mL DN vs. 1985.4 ± 691.1 mL WB, p < 0.001) and retrograde cardioplegia dose (279.3 ± 445.1 mL DN vs. 1341.2 ± 690.8 mL WB, p < 0.001). There were no differences in cross-clamp time, bypass time, postoperative complication rate, or patient outcomes between groups. Del Nido cardioplegia use in an adult, reoperative aortic valve population offers equivalent postoperative outcomes when compared with whole blood cardioplegia. In addition, use of del Nido solution requires lower total and retrograde cardioplegia volumes in order to achieve adequate myocardial protection.

Overview of the genetic determinants of primary aldosteronism

Primary aldosteronism is the most common cause of secondary hypertension. The syndrome accounts for 10% of all cases of hypertension and is primarily caused by bilateral adrenal hyperplasia or aldosterone-producing adenoma. Over the last few years, the use of exome sequencing has significantly improved our understanding of this syndrome. Somatic mutations in the KCNJ5, ATP1A1, ATP2B3 or CACNA1D genes are present in more than half of all cases of aldosterone-producing adenoma (~40%, ~6%, ~1% and ~8%, respectively). Germline gain-of-function mutations in KCNJ5 are now known to cause familial hyperaldosteronism type III, and an additional form of genetic hyperaldosteronism has been reported in patients with germline mutations in CACNA1D. These genes code for channels that control ion homeostasis across the plasma membrane of zona glomerulosa cells. Moreover, all these mutations modulate the same pathway, in which elevated intracellular calcium levels lead to aldosterone hyperproduction and (in some cases) adrenal cell proliferation. From a clinical standpoint, the discovery of these mutations has potential implications for patient management. The mutated channels could be targeted by drugs, in order to control hormonal and overgrowth-related manifestations. Furthermore, some of these mutations are associated with high cell turnover and may be amenable to diagnosis via the sequencing of cell-free (circulating) DNA. However, genotype-phenotype correlations in patients harboring these mutations have yet to be characterized. Despite this recent progress, much remains to be done to elucidate the yet unknown mechanisms underlying sporadic bilateral adrenal hyperplasia.

Aryl hydrocarbon receptor (AhR) agonists increase airway epithelial matrix metalloproteinase activity

The aryl hydrocarbon receptor (AhR) agonists may upregulate matrix metalloproteinases (MMPs) and contribute to many airway diseases, such as asthma and chronic obstructive pulmonary disease. Elucidation of the detailed molecular mechanisms regulating MMPs may provide the scientific basis for diagnostic and therapeutic opportunities to improve the care of various pulmonary diseases, especially those related to xenobiotic agents. In this study, we investigated the detailed mechanisms of how AhR agonists modulated the expressions and activities of MMPs in bronchial epithelial cells. Treating the cells (Beas-2B or HBE135-E6E7) with 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester or 2,3,7,8-tetrachlorodibenzo-p-dioxin, we found these AhR agonists increased the expression and activity of MMP-1 via a noncanonical AhR pathway and increased the activity of MMP-2 and MMP-9 in an MMP-1-dependent manner. AhR agonists increased the expression of MMP-1 via the activation of mitogen-activated protein kinase (MAPK) pathways by increased cytosolic calcium level and activated calcium/calmodulin-dependent protein kinase II (CaMKII). The activated MAPK pathways phosphorylated c-Jun, c-Fos, and ATF-2, resulting in their nuclear translocation and binding to the activator protein-1 (AP-1) elements of the MMP-1 promoter region. These findings correlated clinically to the significantly higher plasma/serum MMP-1 level in asthmatic patients. In conclusion, the present study demonstrated a novel signaling pathway by which AhR agonists elevated intracellular calcium levels, which activated CaMKII, leading to increased MMP-1 expression through MAPK pathways in bronchial epithelial cell lines. This novel regulatory pathway may serve as a potential target for the treatment of airway remodeling of many pulmonary diseases, such as asthma. AhR agonists increase MMP-1 expression in bronchial epithelial cells. The underlying AhR pathway involves CaMKII, MAPKs, and AP-1 elements. The upregulated MMP-1 further activated MMP-2 and MMP-9. Asthmatic patients have higher serum MMP-1 level. This novel regulatory pathway is a potential target for treating asthma.

The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.

Functional characterization of zebrafish K2P18.1 (TRESK) two-pore-domain K+ channels

The human KCNK18 gene is predominantly expressed in brain, spinal cord, and dorsal root ganglion neurons. Encoded K2P18.1K(+) channels are functionally implicated in migraine, pain and anesthesia. Data delineating the in vivo significance of K2P18.1 are still limited owing to a lack of model systems allowing for rapid, whole organism phenotypic analyses. We hypothesized that zebrafish (Danio rerio) might close this scientific gap. This work was designed to characterize the zebrafish ortholog of K2P18.1 in comparison to human K2P18.1 channels. The complete coding sequence of zKCNK18 was amplified from zebrafish cDNA. Zebrafish KCNK18 expression was assessed by in situ hybridization. Human and zebrafish K2P18.1 currents were functionally analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. KCNK18 mRNA is expressed in zebrafish brain and eyes. Human and zebrafish K2P18.1 proteins share 32% identity. Zebrafish K2P18.1 channels mediate K(+)-selective background currents that stabilize the negative resting membrane potential. Functional similarities between human and zK2P18.1 currents include open rectification properties, inhibition by barium, and regulation by signaling molecules protein kinase (PK)C, PKA, and phospholipase C. In contrast to the human ortholog, zK2P18.1 exhibited reduced sensitivity to elevation of intracellular calcium levels by ionomycin and was virtually insensitive to inhibition by quinidine. Zebrafish and human K2P18.1 channels share functional and regulatory properties, indicating that the zebrafish may serve as model to assess K2P18.1 function in vivo. However, distinct differences in K2P18.1 current regulation require careful consideration when zebrafish data are extrapolated to human physiology.

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS.

Real-time in vivo analysis of T cell migration and activation in the periphery and in the central nervous system using a genetically encoded calcium indicator (P4103)

Abstract Interaction of T cells with cells of the surrounding milieu may result in activation of one or both partners. In particular, T cell activation following recognition of antigen presented by antigen presenting cells (APCs) triggers a swift rise of intracellular calcium levels within the T cell. Synthetic calcium indicators have been successfully used to monitor the activation status of T cells in vitro. However, these chemical dyes do not persist intracellularly for a sustained period which creates a major challenge for calcium imaging in the living animal. We now present a newly developed fluorescence resonance energy transfer (FRET)-based, genetically encoded calcium indicator (GECI) expressed in (auto)antigen-specific CD4+ T cells to study T cell activation in vivo at real time. Using two-photon microscopy, we explored the responses of retrovirally transduced, GECI-expressing T cells to antigen. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around APCs concomitant with an instant rise of cytosolic calcium levels. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium levels while still meandering through the space. This approach enables us for the first time to follow the migration and activation patterns of T cells in vivo during the course of the disease.

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.

Transcriptional response of zebrafish embryos exposed to neurotoxic compounds reveals a muscle activity dependent hspb11 expression.

Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sopfixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.

Elevated BCR signaling and decreased survival of Lyn‐deficient transitional and follicular B cells

The Src-family tyrosine kinase Lyn negatively regulates BCR signaling and also myeloid cell activity. Mice deficient in Lyn have substantially decreased numbers of peripheral B cells, despite spontaneously producing IgG anti-DNA antibodies. Here, we examine the mechanism underlying the B-cell depletion in these mice. Lyn-deficient B cells were out-competed by WT B cells in mixed BM chimeras at two steps, at the T1 to T2 transitional maturation stage in the spleen and again between the T2 or T3 stage and the mature follicular B-cell population. Lyn-deficient T2 and follicular B cells expressed elevated levels of the pro-apoptotic factor Bim and deletion of Bim restored splenic B cells of Lyn-deficient mice to close to WT numbers. Lyn-deficient T2 and later stage B cells also had changes in cell surface phenotype consistent with increased in vivo BCR signaling. Similarly, an increased proportion of T2 and follicular B cells had elevated basal intracellular free calcium levels. Overall, these observations suggest that increased BCR signaling is responsible for increased death of weakly self-reactive Lyn-deficient B cells both at the T2 stage and additionally as these cells mature to follicular B cells.