The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.

AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

Thymol improves salinity tolerance of tobacco by increasing the sodium ion efflux and enhancing the content of nitric oxide and glutathione.

BACKGROUND AND OBJECTIVE: Salt stress is one of the most important abiotic stresses affecting the yield and quality of tobacco (Nicotiana tabacum). Thymol (a natural medicine) has been widely used in medical research because of its antibacterial and anti-inflammatory activities. However, the influence of thymol on the root growth of tobacco is not fully elucidated. In this study, the regulatory effects of different concentrations of thymol were investigated. METHODOLOGY: Here, histochemical staining and biochemical methods, non-invasive micro-test technology (NMT), and qPCR assay were performed to investigate the effect of thymol and mechanism of it improving salinity tolerance in tobacco seedlings. RESULTS: In this study, our results showed that thymol rescued root growth from salt stress by ameliorating ROS accumulation, lipid peroxidation, and cell death. Furthermore, thymol enhanced contents of NO and GSH to repress ROS accumulation, further protecting the stability of the cell membrane. And, thymol improved Na+ efflux and the expression of SOS1, HKT1, and NHX1, thus protecting the stability of Na+ and K+. CONCLUSION: Our study confirmed the protecting effect of thymol in tobacco under salt stress, and we also identified the mechanism of it, involving dynamic regulation of antioxidant system and the maintenance of Na+ homeostasis. It can be a new method to improve salinity tolerance in plants.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Tissue and salinity specific Na+/Cl- cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus).

Two orthologues of the gene encoding the Na+-Cl- cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl- cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

N-Methyl-d-aspartic Acid (NMDA) Receptor Is Involved in the Inhibitory Effect of Ketamine on Human Sperm Functions.

Ketamine, which used to be widely applied in human and animal medicine as a dissociative anesthetic, has become a popular recreational drug because of its hallucinogenic effect. Our previous study preliminarily proved that ketamine could inhibit human sperm function by affecting intracellular calcium concentration ([Ca2+]i). However, the specific signaling pathway of [Ca2+]i induced by ketamine in human sperm is still not clear yet. Here, the N-methyl-d-aspartic acid (NMDA) receptor was detected in the tail region of human sperm. Its physiological ligand, NMDA (50 µM), could reverse ketamine's inhibitory effect on human sperm function, and its antagonist, MK801 (100 µM), could restrain the effect of NMDA. The inhibitory effect caused by 4 mM ketamine or 100 µM MK801 on [Ca2+]i, which is a central factor in the regulation of human sperm function, could also be recovered by 50 µM NMDA. The results suggest that the NMDA receptor is probably involved in the inhibitory effect of ketamine on human sperm functions.

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action.

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 µM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 µM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 µM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 µM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

cAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

Hepcidin induces intestinal calcium uptake while suppressing iron uptake in Caco-2 cells.

Abnormal calcium absorption and iron overload from iron hyperabsorption can contribute to osteoporosis as found in several diseases, including hemochromatosis and thalassemia. Previous studies in thalassemic mice showed the positive effects of the iron uptake suppressor, hepcidin, on calcium transport. However, whether this effect could be replicated in other conditions is not known. Therefore, this study aimed to investigate the effects of hepcidin on iron and calcium uptake ability under physiological, iron uptake stimulation and calcium uptake suppression. To investigate the potential mechanism, effects of hepcidin on the expression of iron and calcium transporter and transport-associated protein in Caco-2 cells were also determined. Our results showed that intestinal cell iron uptake was significantly increased by ascorbic acid together with ferric ammonium citrate (FAC), but this phenomenon was suppressed by hepcidin. Interestingly, hepcidin significantly increased calcium uptake under physiological condition but not under iron uptake stimulation. While hepcidin significantly suppressed the expression of iron transporter, it had no effect on calcium transporter expression. This indicated that hepcidin-induced intestinal cell calcium uptake did not occur through the stimulation of calcium transporter expression. On the other hand, 1,25(OH)2D3 effectively induced intestinal cell calcium uptake, but it did not affect intestinal cell iron uptake or iron transporter expression. The 1,25(OH)2D3-induced intestinal cell calcium uptake was abolished by 12 mM CaCl2; however, hepcidin could not rescue intestinal cell calcium uptake suppression by CaCl2. Taken together, our results showed that hepcidin could effectively and concurrently induce intestinal cell calcium uptake while reducing intestinal cell iron uptake under physiological and iron uptake stimulation conditions, suggesting its therapeutic potential for inactive calcium absorption, particularly in thalassemic patients or patients who did not adequately respond to 1,25(OH)2D3.

Resveratrol improves the iron deficiency adaptation of Malus baccata seedlings by regulating iron absorption.

BACKGROUND: Resveratrol (Res), a phytoalexin, has been widely reported to participate in plant resistance to fungal infections. However, little information is available on its role in abiotic stress, especially in iron deficiency stress. Malus baccata is widely used as apple rootstock in China, but it is sensitive to iron deficiency. RESULTS: In this study, we investigated the role of exogenous Res in M. baccata seedings under iron deficiency stress. Results showed that applying 100 µM exogenous Res could alleviate iron deficiency stress. The seedlings treated with Res had a lower etiolation rate and higher chlorophyll content and photosynthetic rate compared with the apple seedlings without Res treatment. Exogenous Res increased the iron content in the roots and leaves by inducing the expression of MbAHA genes and improving the H+-ATPase activity. As a result, the rhizosphere pH decreased, iron solubility increased, the expression of MbFRO2 and MbIRT1 was induced, and the ferric-chelated reductase activity was enhanced to absorb large amounts of Fe2+ into the root cells under iron deficiency conditions. Moreover, exogenous Res application increased the contents of IAA, ABA, and GA3 and decreased the contents of DHZR and BL for responding to iron deficiency stress indirectly. In addition, Res functioned as an antioxidant that strengthened the activities of antioxidant enzymes and thus eliminated reactive oxygen species production induced by iron deficiency stress. CONCLUSION: Resveratrol improves the iron deficiency adaptation of M. baccata seedlings mainly by regulating iron absorption.

Sex difference in kidney electrolyte transport III: Impact of low K intake on thiazide-sensitive cation excretion in male and female mice.

We compared the regulation of the NaCl cotransporter (NCC) in adaptation to a low-K (LK) diet in male and female mice. We measured hydrochlorothiazide (HCTZ)-induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK), and fractional (FENa, FEK) excretion in male and female mice on control-K (CK, 1% KCl) and LK (0.1% KCl) diets for 7 days. With CK, NCC-dependent ENa and FENa were larger in females than males as observed previously. However, with LK, HCTZ-induced ENa and FENa increased in males but not in females, abolishing the sex differences in NCC function as observed in CK group. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K-secretory apparatus in the distal nephron is strongly suppressed under these conditions. We also examined LK-induced changes in Na transport protein expression by Western blotting. Under CK conditions females expressed more NCC protein, as previously reported. LK doubled both total (tNCC) and phosphorylated NCC (pNCC) abundance in males but had more modest effects in females. The larger effect in males abolished the sex-dependence of NCC expression, consistent with the measurements of function by renal clearance. LK intake did not change NHE3, NHE2, or NKCC2 expression, but reduced the amount of the cleaved (presumably active) form of γENaC. LK reduced plasma K to lower levels in females than males. These results indicated that males had a stronger NCC-mediated adaptation to LK intake than females.

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) with Lidocaine Provokes Pyroptosis of Glioblastoma Cells.

The study examines the problem whether pyroptosis of U87-MG glioblastoma cells can result from activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by a local anesthetic. Glioblastoma cells exposed to various concentrations of typical local anesthetic lidocaine demonstrated augmented cytosolic flux of Ca2+, while suppression of CaMKII expression with the corresponding siRNA significantly inhibited this effect in cells treated with 2 mM lidocaine. Lidocaine up-regulated the expression of mRNA caspase-3 and gasdermin GSDME proteins, whereas silencing of CaMKII gene with siRNA significantly moderated this effect. In addition, lidocaine inhibited proliferation of U87-MG cells, and this effect was prevented by silencing CaMKII gene. Thus, lidocaine activated protein kinase CaMKII, which phosphorylated TRPV1 ion channels and induced calcium overload of U87-MG glioblastoma cells, thereby provoking their pyroptosis.

Ticagrelor alleviates high-carbohydrate intake induced altered electrical activity of ventricular cardiomyocytes by regulating sarcoplasmic reticulum-mitochondria miscommunication.

Metabolic syndrome (MetS) is associated with additional cardiovascular risk in mammalians while there are relationships between hyperglycemia-associated cardiovascular dysfunction and increased platelet P2Y12 receptor activation. Although P2Y12 receptor antagonist ticagrelor (Tica) plays roles in reduction of cardiovascular events, its beneficial mechanism remains poorly understood. Therefore, we aimed to clarify whether Tica can exert a direct protective effect in ventricular cardiomyocytes from high-carbohydrate diet-induced MetS rats, at least, through affecting sarcoplasmic reticulum (SR)-mitochondria (Mit) miscommunication. Tica treatment of MetS rats (150 mg/kg/day for 15 days) significantly reversed the altered parameters of action potentials by reversing sarcolemmal ionic currents carried by voltage-dependent Na+ and K+ channels, and Na+/Ca2+-exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca2+ level and depressed SR Ca2+ load were also reversed in Tica-treated cells, at most, though recoveries in the phosphorylation levels of ryanodine receptors and phospholamban. Moreover, there were marked recoveries in Mit structure and function (including increases in both autophagosomes and fragmentations) together with recoveries in Mit proteins and the factors associated with Ca2+ transfer between SR-Mit. There were further significant recoveries in markers of both ER stress and oxidative stress. Taken into consideration the Tica-induced prevention of ER stress and mitochondrial dysfunction, our data provided an important document on the pleiotropic effects of Tica in the electrical activity of the cardiomyocytes from MetS rats. This protective effect seems through recoveries in SR-Mit miscommunication besides modulation of different sarcolemmal ion-channel activities, independent of P2Y12 receptor antagonism.

Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Effect of a chemical dispersant (Corexit 9500A) on the structure and ion transport function of blue crab (Callinectes sapidus) gills.

Chemical dispersants are commercially available mixtures of surfactants and solvents that have become important tools in the remediation of spilled oil. Given the importance of oil to the world economy, the recurring nature of spills, and the prevalence of dispersant use in remediation, there is a critical need to understand potential toxic impacts of dispersants on invertebrate and vertebrate animals. Blue crabs (Callinectes sapidus) play ecologically important roles in the environments they inhabit and support economically important fisheries along the Atlantic Coast and in the Gulf of Mexico. In studies reported here, we assessed the impact of a chemical dispersant, Corexit 9500A, on the structure and ion transport function of blue crab gills. Exposure of blue crabs to Corexit 9500A for 24 h (0-300 ppm in artificial seawater under static conditions) revealed a 24-h lethal concentration 50 (LC50) estimate of 210 ppm. A histological analysis of gills from crabs exposed for 24 h to a sub-lethal concentration of Corexit 9500A (125 ppm) revealed evidence of loss or disruption of cuticle, and an increase in stained amorphous material in the hemolymph spaces of gill lamellae. Quantitative image analysis of stained gill sections revealed the area/length ratio of gill lamellae in crabs exposed to Corexit 9500A (24 h, 125 ppm), was greater than that in gill lamellae from control crabs; the results are consistent with the presence of edematous swelling in gill lamellae from dispersant-exposed crabs. Quantitative PCR was used to measure the relative abundance of transcripts encoding three ion transport proteins (Na+/K+ ATPase, plasma membrane Ca2+ ATPase (PMCA), and sarcoplasmic reticulum/endoplasmic reticulum Ca2+ ATPase (SERCA)) in gills from Corexit-exposed and control crabs. In general, the abundance of transcripts encoding each ion transport protein was lower in gills from dispersant-exposed crabs than in gills from control crabs. The combined results are consistent with the hypothesis that 24-h exposure of blue crabs to a sublethal concentration of Corexit 9500A impacts both the structure and ion transport function of gills.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Ivacaftor partially corrects airway inflammation in a humanized G551D rat.

Animal models have been highly informative for understanding the pathogenesis and progression of cystic fibrosis (CF) lung disease. In particular, the CF rat models recently developed have addressed mechanistic causes of the airway mucus defect characteristic of CF, and how these may change when cystic fibrosis transmembrane conductance regulator (CFTR) activity is restored using new modulator therapies. We hypothesized that inflammatory changes to the airway would develop spontaneously and progressively, and that these changes would be resolved with modulator therapy. To test this, we used a humanized-CFTR rat expressing the G551D variant that responds to the CFTR modulator ivacaftor. Markers typically found in the CF lung were assessed, including neutrophil influx, small airway histopathology, and inflammatory cytokine concentration. Young hG551D rats did not express inflammatory cytokines at baseline but did upregulate these in response to inflammatory trigger. As the hG551D rats aged, histopathology worsened, accompanied by neutrophil influx into the airway and increasing concentrations of TNF-α, IL-1α, and IL-6 in the airways. Ivacaftor administration reduced concentrations of these cytokines when administered to the rats at baseline but was less effective in the rats that had also received inflammatory stimulus. Therefore, we conclude that administration of ivacaftor resulted in an incomplete resolution of inflammation when rats received an external trigger, suggesting that CFTR activation may not be enough to resolve inflammation in the lungs of patients with CF.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts.

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

Resveratrol is an inhibitor of sodium-dependent inorganic phosphate transport in triple-negative MDA-MB-231 breast cancer cells.

Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non-flavonoid polyphenol. Resveratrol inhibits metastatic processes, such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumor progression has been demonstrated. The metastatic process in breast cancer is associated with Na+ -dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na+ -dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na+ -dependent Pi transport non-competitively. Resveratrol also inhibits adhesion/migration in MDA-MB-231 cells, likely related to inhibition of the Na+ -dependent Pi transporter.