By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Objective

Computational models of calcium (Ca²⁺) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²⁺ signaling model for RPE based on our experimental data of mechanically induced Ca²⁺ wave in the in vitro model of RPE, the ARPE-19 monolayer.

Methods

We combined six essential Ca²⁺ signaling components into a model: stretch-sensitive Ca²⁺ channels (SSCCs), P₂Y₂ receptors, IP₃ receptors, ryanodine receptors, Ca²⁺ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²⁺ waves reproduced our control experimental data and data where gap junctions were blocked.

Results

Our model was able to explain Ca²⁺ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²⁺ through plasma membrane SSCCs and gap junctions conduct the Ca²⁺ and IP³ between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²⁺ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP₃ receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca²⁺ signal in the distance.

Conclusions

The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P₂Y₂ receptors or accelerate P₂Y₂ receptor phosphorylation, which may partially be the reason for Ca²⁺ wave attenuation by suramin. Being the first RPE Ca²⁺ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.     

Anti-Epileptic Effect of Ganoderma Lucidum Polysaccharides by Inhibition of Intracellular Calcium Accumulation and Stimulation of Expression of CaMKII α in Epileptic Hippocampal Neurons

Purpose

To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated.

Method

Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg²⁺ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg²⁺ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg²⁺ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg²⁺ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca²⁺ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca²⁺ turnover was observed under a laser scanning confocal microscope.

Results

The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca^{2 +} fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes.

Conclusion

GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.     

Regulation of TRPM7 Function by IL-6 through the JAK2-STAT3 Signaling Pathway

Aims

Previous studies have demonstrated that expression of the TRPM7 channel, which may induce delayed cell death by mediating calcium influx, is precisely regulated. However, functional regulation of TRPM7 channels by endogenous molecules has not been elucidated. The proinflammatory cytokine IL-6 contributes to regulation of Ca²⁺ influx in cerebral ischemia, but the role of IL-6 in regulating TRPM7 functioning is unknown. Thus, we here investigated the interaction between IL-6 and TRPM7 channels and the relevant mechanisms.

Materials and Methods

Using whole-cell patch-clamping, we first investigated the effect of IL-6 on TRPM7-like currents in primary cultured cortical neurons. Next, TRPM7-overexpressing HEK293 cells were used to confirm the effect of IL-6/sIL-6R on TRPM7. Finally, we used specific signaling pathway inhibitors to investigate the signaling pathways involved.

Results

IL-6 or IL-6/sIL-6R dose-dependently inhibited inward TRPM7 currents, in both primary cultured neurons and HEK293 cells overexpressing TRPM7. In intracellular Mg²⁺-free conditions, extracellular Ca²⁺ or the α-kinase domain of TRPM7 did not participate in this regulation. The inhibitory effect of IL-6 on TRPM7 could be blocked by specific inhibitors of the JAK2−STAT3 pathway, but not of the PI3K, ERK1/2, or PLC pathways.

Conclusions

IL-6 inhibits the inward TRPM7 current via the JAK2−STAT3 signaling pathway.     

Role of Sarcoplasmic Reticulum Calcium in Development of Secondary Calcium Rise and Early Afterdepolarizations in Long QT Syndrome Rabbit Model

Background

L-type calcium current reactivation plays an important role in development of early afterdepolarizations (EADs) and torsades de pointes (TdP). Secondary intracellular calcium (Ca_i) rise is associated with initiation of EADs.

Objective

To test whether inhibition of sarcoplasmic reticulum (SR) Ca²⁺ cycling suppresses secondary Ca_i rise and genesis of EADs.

Methods

Langendorff perfusion and dual voltage and Ca_i optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB) was created by radiofrequency ablation. After baseline studies, E4031, SR Ca²⁺ cycling inhibitors (ryanodine plus thapsigargin) and nifedipine were then administrated subsequently, and the protocols were repeated.

Results

At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD) was significantly prolonged and the amplitude of secondary Ca_i rise was enhanced, and 7 (70%) rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced (2 hearts, 20%, p = 0.03). Although APD was significantly prolonged (from 298 ± 30 ms to 457 ± 75 ms at pacing cycle length of 1000 m, p = 0.007) by ryanodine plus thapsigargin, the secondary Ca_i rise was suppressed (from 8.8 ± 2.6% to 1.2 ± 0.9%, p = 0.02). Nifedipine inhibited TdP inducibility in all rabbit hearts.

Conclusion

In this AVB and long QT rabbit model, inhibition of SR Ca²⁺ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Ca_i rise and genesis of EADs.     

Store-Operated Ca2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

Aims

Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and Results

We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca²⁺ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions

These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca²⁺ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

Background

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias.

Methods

Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca²⁺ was measured using Indo-1AM and Ca²⁺ alternans (Ca-ALT) was induced with a standard ramp pacing protocol.

Results

As predicted, SR Ca²⁺ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca²⁺ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20).

Conclusions

Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.     

The Relationship between Zinc Status and Inflammatory Marker Levels in Rural Korean Adults Aged 40 and Older

Background

Serum cytokines and C-reactive protein (CRP) are known as one of the major risk factors in atherosclerosis. The antioxidant and anti-inflammatory properties of zinc have been suggested, but few data are available on the relationship between zinc status and inflammatory markers in epidemiological studies.

Objective

The present study aims to investigate the cross-sectional relationships of serum cytokines and CRP with dietary zinc intake and serum zinc levels in healthy men and women aged 40 and older in rural areas of South Korea.

Materials and Methods

A group of 1,055 subjects (404 men, 651 women) was included in dietary zinc analysis while another group of 695 subjects (263 men, 432 women) was included in serum zinc analysis. Serum IL-6, TNF-α, and CRP were measured as inflammatory markers.

Results

There was no significant inverse relationship between dietary zinc intake and inflammatory markers. We found a significant inverse relationship between serum zinc levels and all three inflammatory markers in women (P for trend = 0.0236 for IL-6; P for trend = 0.0017 for TNF-α; P for trend = 0.0301 for CRP) and between serum zinc levels and a single inflammatory marker (IL-6) in men (P for trend = 0.0191), although all R2 values by regression were less than 10%.

Conclusion

In conclusion, serum zinc levels may be inversely related to inflammatory markers (IL-6, TNF-α, and CRP), particularly in women.     

Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Background

Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca²⁺ homeostasis and tTG activity.

Methods/Principal Findings

We studied Ca²⁺ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca²⁺ from intracellular stores. Specifically, peptide 31–43 mobilized Ca²⁺ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca²⁺ only from mitochondria. We also found that gliadin peptide-induced Ca²⁺ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes.

Conclusions

By inducing Ca²⁺ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells.     

Post-Prandial Protein Handling: You Are What You Just Ate

Background

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective

To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design

12 healthy young males ingested 20 g intrinsically [1-¹³C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-²H₅]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[1-¹³C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results

55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min^-1·100 mL leg volume^-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h^-1 based upon the muscle protein bound L-[ring-²H₅]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-¹³C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion

Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration

trialregister.nl 3638     

K+ Channel Inhibition Differentially Regulates Migration of Intestinal Epithelial Cells in Inflamed vs. Non-Inflamed Conditions in a PI3K/Akt-Mediated Manner

Background

Potassium channels have been shown to determine wound healing in different tissues, but their role in intestinal epithelial restitution–the rapid closure of superficial wounds by intestinal epithelial cells (IEC)–remains unclear.

Methods

In this study, the regulation of IEC migration by potassium channel modulation was explored with and without additional epidermal growth factor (EGF) under baseline and interferon-γ (IFN-γ)-pretreated conditions in scratch assays and Boyden chamber assays using the intestinal epithelial cell lines IEC-18 and HT-29. To identify possibly involved subcellular pathways, Western Blot (WB)-analysis of ERK and Akt phosphorylation was conducted and PI3K and ERK inhibitors were used in scratch assays. Furthermore, mRNA-levels of the potassium channel KCNN4 were determined in IEC from patients suffering from inflammatory bowel diseases (IBD).

Results

Inhibition of Ca²⁺-dependent potassium channels significantly increased intestinal epithelial restitution, which could not be further promoted by additional EGF. In contrast, inhibition of KCNN4 after pretreatment with IFN-γ led to decreased or unaffected migration. This effect was abolished by EGF. Changes in Akt, but not in ERK phosphorylation strongly correlated with these findings and PI3K but not ERK inhibition abrogated the effect of KCNN4 inhibition. Levels of KCNN4 mRNA were higher in samples from IBD patients compared with controls.

Conclusions

Taken together, we demonstrate that inhibition of KCNN4 differentially regulates IEC migration in IFN-γ-pretreated vs. non pretreated conditions. Moreover, our data propose that the PI3K signaling cascade is responsible for this differential regulation. Therefore, we present a cellular model that contributes new aspects to epithelial barrier dysfunction in chronic intestinal inflammation, resulting in propagation of inflammation and symptoms like ulcers or diarrhea.     

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.     

Gastrointestinal transport of Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> during the digestion of a single meal in the freshwater rainbow trout

A diet containing an inert marker (ballotini beads, quantified by X-radiography) was used to quantify the transport of two essential minerals, Ca²⁺ and Mg²⁺ from the diet during the digestion and absorption of a single meal of commercial trout food (3% ration). Initially, net uptake of Ca²⁺ was observed in the stomach followed by subsequent Ca²⁺ fluxes along the intestine which were variable, but for the most part secretory. This indicated a net secretion of Ca²⁺ along the intestinal tract resulting in a net assimilation of dietary Ca²⁺ of 28%. Similar handling of Ca²⁺ and Mg²⁺ was observed along the gastrointestinal tract (GI), although net assimilation differed substantially between the cations, with Mg²⁺ assimilation being close to 60%, mostly a result of greater uptake by the stomach. The stomach displayed the highest net uptake rates for both cations (1.5 and 1.3 mmol kg⁻¹ fish body mass for Ca²⁺ and Mg²⁺, respectively), occurring within 2 h following ingestion of the meal. Substantial secretions of both Ca²⁺ and Mg²⁺ were observed in the anterior intestine, which were attributed to bile and other intestinal secretions, while fluxes in the mid and posterior intestine were small and variable. The overall patterns of Ca²⁺ and Mg²⁺ handling in the GI tract were similar to those observed for Na⁺ and K⁺ (but not Cl⁻) in a previous study. Overall, these results emphasize the importance of dietary electrolytes in ionoregulatory homeostasis.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

Background

It has recently been suggested that RhoA plays an important role in the enhancement of the Ca²⁺ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca²⁺ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.

Methods

Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.

Results

Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K⁺-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca²⁺ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca²⁺ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca²⁺ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.

Conclusion

These findings suggest that the augmentation of Ca²⁺ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.