Basal Intracellular Ca2 Research Articles

Activation of Pannexin-1 channels causes cell dysfunction and damage in mesangial cells derived from angiotensin II-exposed mice.

Chronic kidney disease (CKD) is a prevalent health concern associated with various pathological conditions, including hypertensive nephropathy. Mesangial cells are crucial in maintaining glomerular function, yet their involvement in CKD pathogenesis remains poorly understood. Recent evidence indicates that overactivation of Pannexin-1 (Panx1) channels could contribute to the pathogenesis and progression of various diseases. Although Panx1 is expressed in the kidney, its contribution to the dysfunction of renal cells during pathological conditions remains to be elucidated. This study aimed to investigate the impact of Panx1 channels on mesangial cell function in the context of hypertensive nephropathy. Using an Ang II-infused mouse model and primary mesangial cell cultures, we demonstrated that in vivo exposure to Ang II sensitizes cultured mesangial cells to show increased alterations when they are subjected to subsequent in vitro exposure to Ang II. Particularly, mesangial cell cultures treated with Ang II showed elevated activity of Panx1 channels and increased release of ATP. The latter was associated with enhanced basal intracellular Ca2+ ([Ca2+]i) and increased ATP-mediated [Ca2+]i responses. These effects were accompanied by increased lipid peroxidation and reduced cell viability. Crucially, all the adverse impacts evoked by Ang II were prevented by the blockade of Panx1 channels, underscoring their critical role in mediating cellular dysfunction in mesangial cells. By elucidating the mechanisms by which Ang II negatively impacts mesangial cell function, this study provides valuable insights into the pathogenesis of renal damage in hypertensive nephropathy.

Methylmercury Decreases AMPA Receptor Subunit GluA2 Levels in Cultured Rat Cortical Neurons.

Methylmercury (MeHg) is a well-known environmental pollutant that has harmful effects on the central nervous systems of humans and animals. The molecular mechanisms of MeHg-induced neurotoxicity at low concentrations are not fully understood. Here, we investigated the effects of low-concentration MeHg on the cell viability, Ca2+ homeostasis, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels, which determine Ca2+ permeability of AMPA receptors, in rat primary cortical neurons. Exposure of cortical neurons to 100 and 300 nM MeHg for 7 d resulted in a decrease in GluA2 levels, an increase in basal intracellular Ca2+ concentration, increased phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 and p38, and decreased cell viability. Moreover, glutamate stimulation exacerbated the decrease in cell viability and increased intracellular Ca2+ levels in MeHg-treated neurons compared to control neurons. MeHg-induced neuronal cell death was ameliorated by 1-naphthyl acetyl spermine, a specific antagonist of Ca2+-permeable, GluA2-lacking AMPA receptors. Our findings raise the possibility that decreased neuronal GluA2 levels and the subsequent increase in intracellular Ca2+ concentration may contribute to MeHg-induced neurotoxicity.

Biphasic regulation of CFTR expression by ENaC in epithelial cells: The involvement of Ca2+-modulated cAMP production

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl− transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

Vasorin plays a critical role in vascular smooth muscle cells and arterial functions.

Within the cardiovascular system, the protein vasorin (Vasn) is predominantly expressed by vascular smooth muscle cells (VSMCs) in the coronary arteries and the aorta. Vasn knockout (Vasn-/- ) mice die within 3 weeks of birth. In the present study, we investigated the role of vascular Vasn expression on vascular function. We used inducible Vasn knockout mice (VasnCRE-ERT KO and VasnSMMHC-CRE-ERT2 KO , in which respectively all cells or SMCs only are targeted) to analyze the consequences of total or selective Vasn loss on vascular function. Furthermore, in vivo effects were investigated in vitro using human VSMCs. The death of VasnCRE-ERT KO mice 21 days after tamoxifen injection was concomitant with decreases in blood pressure, angiotensin II levels, and vessel contractibility to phenylephrine. The VasnSMMHC-CRE-ERT2 KO mice displayed concomitant changes in vessel contractibility in response to phenylephrine and angiotensin II levels. In vitro, VASN deficiency was associated with a shift toward the SMC contractile phenotype, an increase in basal intracellular Ca2+ levels, and a decrease in the SMCs' ability to generate a calcium signal in response to carbachol or phenylephrine. Additionally, impaired endothelium-dependent relaxation (due to changes in nitric oxide signaling) was observed in all Vasn knockout mice models. Our present findings highlight the role played by Vasn SMC expression in the maintenance of vascular functions. The mechanistic experiments suggested that these effects are mediated by SMC phenotype switching and changes in intracellular calcium homeostasis, angiotensin II levels, and NO signaling.

Involvement of Ca2+ and ROS signals in nickel-impaired human sperm function

As one of the main environmental pollutants and occupational hazards, nickel has been reported to have mutagenic, carcinogenic, and teratogenic properties, as well as reproductive toxicity. However, how nickel affects human reproduction is still unclear. In this study, the toxicity of nickel on human sperm and the underlying mechanisms were evaluated in vitro. We found that NiCl2 (10, 50, and 250 μM) impaired sperm total motility and progressive motility in a dose- and time-dependent manner. In addition, sperm hyperactivation and the ability of human sperm to penetrate a viscous medium were found to be compromised after nickel exposure. Mechanically, NiCl2 significantly inhibited the basal intracellular Ca2+ signaling. Besides, reactive oxygen species (ROS), superoxide, and malondialdehyde levels were increased in human sperm after exposure to different concentrations of NiCl2. Consistently, eliminating excess ROS by N-acetyl-L-cysteine or tocopherol significantly alleviated nickel-impaired sperm motility. Taken together, these results revealed that nickel could compromise sperm functions by interfering with Ca2+ signaling and inducing excessive oxidative stress. These findings suggest that, in the high and occupational nickel exposure environments, the contribution of nickel toxicity to the males who wish to preserve their fertility is worthy of careful evaluation.

Presence of TRPA1 Modifies CD4+/CD8+ T Lymphocyte Ratio and Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) has been reported to influence neuroinflammation and lymphocyte function. We analysed the immune phenotype and activation characteristics of TRPA1-deficient mice (knockout—KO) generated by targeted deletion of the pore-loop domain of the ion channel. We compared TRPA1 mRNA and protein expression in monocyte and lymphocyte subpopulations isolated from primary and secondary lymphatic organs of wild type (WT) and KO mice. qRT-PCR and flow cytometric studies indicated a higher level of TRPA1 in monocytes than in lymphocytes, but both were orders of magnitude lower than in sensory neurons. We found lower CD4+/CD8+ thymocyte ratios, diminished CD4/CD8 rates, and B cell numbers in the KO mice. Early activation marker CD69 was lower in CD4+ T cells of KO, while the level of CD8+/CD25+ cells was higher. In vitro TcR-mediated activation did not result in significant differences in CD69 level between WT and KO splenocytes, but lower cytokine (IL-1β, IL-6, TNF-α, IL-17A, IL-22, and RANTES) secretion was observed in KO splenocytes. Basal intracellular Ca2+ level and TcR-induced Ca2+ signal in T lymphocytes did not differ significantly, but interestingly, imiquimod-induced Ca2+ level in KO thymocytes was higher. Our results support the role of TRPA1 in the regulation of activation, cytokine production, and T and B lymphocytes composition in mice.

Connexin 43 hyper-phosphorylation at serine 282 triggers apoptosis in rat cardiomyocytes via activation of mitochondrial apoptotic pathway.

Cx43 is the major connexin in ventricular gap junctions, and plays a pivotal role in control of electrical and metabolic communication among adjacent cardiomyocytes. We previously found that Cx43 dephosphorylation at serine 282 (pS282) caused cardiomyocyte apoptosis, which is involved in cardiac ischemia/reperfusion injury. In this study we investigated whether Cx43-S282 hyper-phosphorylation could protect cardiomyocytes against apoptosis. Adenovirus carrying rat full length Cx43 gene (Cx43-wt) or a mutant gene at S282 substituted with aspartic acid (S282D) were transfected into neonatal rat ventricular myocytes (NRVMs) or injected into rat ventricular wall. Rat abdominal aorta constriction model (AAC) was used to assess Cx43-S282 phosphorylation status. We showed that Cx43 phosphorylation at S282 was increased over 2-times compared to Cx43-wt cells at 24 h after transfection, while pS262 and pS368 were unaltered. S282D-transfected cells displayed enhanced gap junctional communication, and increased basal intracellular Ca2+ concentration and spontaneous Ca2+ transients compared to Cx43-wt cells. However, spontaneous apoptosis appeared in NRVMs transfected with S282D for 34 h. Rat ventricular myocardium transfected with S282D in vivo also exhibited apoptotic responses, including increased Bax/Bcl-xL ratio, cytochrome c release as well as caspase-3 and caspase-9 activities, while factor-associated suicide (Fas)/Fas-associated death domain expression and caspase-8 activity remained unaltered. In addition, AAC-induced hypertrophic ventricles had apoptotic injury with Cx43-S282 hyper-phosphorylation compared with Sham ventricles. In conclusion, Cx43 hyper-phosphorylation at S282, as dephosphorylation, also triggers cardiomyocyte apoptosis, but through activation of mitochondrial apoptosis pathway, providing a fine-tuned Cx43-S282 phosphorylation range required for the maintenance of cardiomyocyte function and survival.

Excessive Accumulation of Intracellular Ca2+ After Acute Exercise Potentiated Impairment of T-cell Function.

Ca2+ is an important intracellular second messenger known to regulate several cellular functions. This research aimed to investigate the mechanisms of exercise-induced immunosuppression by measuring intracellular calcium levels, Ca2+-regulating gene expression, and agonist-evoked proliferation of murine splenic T lymphocytes. Mice were randomly assigned to the control, sedentary group (C), and three experimental groups, which performed a single bout of intensive and exhaustive treadmill exercise. Murine splenic lymphocytes were separated by density-gradient centrifugation immediately (E0), 3h (E3), and 24h after exercise (E24). Fura-2/AM was used to monitor cytoplasmic free Ca2+ concentration in living cells. The combined method of carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry was used for the detection of T cell proliferation. The transcriptional level of Ca2+-regulating genes was quantified by using qPCR. Both basal intracellular Ca2+ levels and agonist (ConA, OKT3, or thapsigargin)-induced Ca2+ transients were significantly elevated at E3 group (p<0.05 vs. control). However, mitogen-induced cell proliferation was significantly decreased at E3 group (p<0.05 vs. control). In parallel, the transcriptional level of plasma membrane Ca2+-ATPases (PMCA), sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), TRPC1, and P2X7 was significantly downregulated, and the transcriptional level of IP3R2 and RyR2 was significantly upregulated in E3 (p<0.01 vs. control). In summary, this study demonstrated that acute exercise affected intracellular calcium homeostasis, most likely by enhancing transmembrane Ca2+ influx into cells and by reducing expression of Ca2+-ATPases such as PMCA and SERCA. However, altered Ca2+ signals were not transduced into an enhanced T cell proliferation suggesting other pathways to be responsible for the transient exercise-associated immunosuppression.

Atrial Fibrillation in Heart Failure Is Associated with High Levels of Circulating microRNA-199a-5p and 22-5p and a Defective Regulation of Intracellular Calcium and Cell-to-Cell Communication.

MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promotion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca2+ channel, NCX and connexin-40, leading to lower basal intracellular Ca2+ levels, fewer inward currents, a moderate reduction in Ca2+ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF patients. Cells exposed to both miRNAs exhibited altered Ca2+ handling and defective cell-to-cell communication, both findings being potential arrhythmogenic mechanisms.

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells.

Regulation of the Ca2+-activated Cl− channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl− channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca2+ Signals.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl− channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1−/−, was larger in male when compared to female Pkd1−/− cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1−/− males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.

Effects of oral calcium bolus supplementation on intracellular polymorphonuclear leukocyte calcium levels and functionality in primiparous and multiparous dairy cows

The objectives of this study were (1) to characterize Ca levels and polymorphonuclear leukocyte (PMN) function in primiparous and multiparous animals following oral Ca bolus supplementation, and (2) to determine differential responses of boluses containing a lower dose of Ca than traditionally used in primiparous animals on Ca levels and PMN function. Jersey × Holstein crossbred animals (n = 104) were enrolled within 24 h of parturition. All animals were blocked by time relative to calving and randomly assigned to treatment. The Ca boluses were composed of a mixture of Ca chloride, Ca sulfate, and Ca propionate. For objective 1, animals were assigned to control (CON; no Ca supplementation), or a series of 2 Ca boluses given 24 h apart for a total of 50 g of Ca. Objective 2 treatments included control (CON; no Ca supplementation), a series of 2 Ca boluses given 24 h apart containing 50 g of Ca, or a series of 2 Ca boluses given 24 h apart containing 25 g of Ca. Blood samples were collected on d 1 (<24 h), 2, 3, 5, and 7 relative to parturition. Total serum Ca, serum haptoglobin, PMN intracellular Ca, PMN intracellular Ca after stimulation with an environmental Escherichia coli, PMN L-selectin surface expression, and PMN phagocytic and oxidative burst activities were analyzed. For objective 1 a tendency was detected for a treatment difference on basal intracellular PMN Ca and a treatment difference on E. coli-stimulated intracellular PMN Ca. We detected a parity × DIM effect for PMN oxidative burst intensity. However, no other interactions or parity effects on other functional PMN variables were detectable. In primiparous animals, we found a treatment difference for E. coli-stimulated intracellular PMN Ca among animals given 50 g of Ca but no treatment difference on basal intracellular PMN Ca. The 50 g of Ca treatment increased both PMN phagocytosis and oxidative burst intensities. Supplementing animals with 50 g of oral Ca increased intracellular PMN Ca and influenced PMN function.

Pentachlorophenol inhibits CatSper function to compromise progesterone’s action on human sperm

Pentachlorophenol (PCP), a highly toxic contaminant of chlorophenols, is common in a variety of environments and presents serious risks to animal and human health. However, the reproductive toxicity and potential actions of PCP have not been investigated thoroughly, especially in humans. Here, human spermatozoa were used to evaluate the effect of PCP on cell function and to explore the underlying mechanisms. PCP had no substantive effects on sperm viability or motility, nor on the ability to penetrate viscous medium, sperm hyperactivation or spontaneous acrosome reactions. However, PCP significantly inhibited these properties induced by progesterone (P4). Consistent with the functional observations, although PCP itself did not affect the basal intracellular Ca2+ concentrations and CatSper current, PCP dose-dependently inhibited increases of intracellular Ca2+ concentrations caused by P4. In addition, the activation of CatSper induced by P4 was largely suppressed by PCP. This is the first report showing that PCP may serves as an antagonist of the P4 membrane receptor to interfere with Ca2+ signaling by compromising the action of P4 on regulating sperm function. These findings suggest that the reproductive toxicity of PCP should also be a matter of concern as a mammalian health risk.

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl- Channel TMEM16A.

TMEM16A is a Ca2+ activated Cl− channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl− secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A.

TMEM16A drives renal cyst growth by augmenting Ca2+ signaling in M1 cells

Polycystic kidney disease (PKD) leads to continuous decline of renal function by growth of renal cysts. Enhanced proliferation and transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated TMEM16A Cl− channels is thought to cause an increase in cyst volume. Recent work shows the pro-proliferative role of the Ca2+ activated Cl− channel TMEM16A (anoctamin 1), and demonstrates the essential contribution of TMEM16A to CFTR-dependent Cl− secretion. The present data demonstrate an increase in intracellular Ca2+ ([Ca2+]i) signals and Cl− secretion by TMEM16A, in renal collecting duct principle cells from dog (MDCK) and mouse (M1) as well as primary tubular epithelial cells from PKD1−/− knockout mice. M1 organoids proliferated, increased expression of TMEM16A, and secreted Cl− upon knockdown of endogenous polycystin 1 or 2 (PKD1,2), by retroviral transfection with shPKD1 and shPKD2, respectively. Knockdown of PKD1 or PKD2 increased basal intracellular Ca2+ levels and enhanced purinergic Ca2+ release from endoplasmic reticulum. In contrast, ryanodine receptors were found not to be expressed in mouse renal epithelial cells and caffeine had no effects on [Ca2+]i. Ca2+ signals, proliferation, and Cl− secretion were largely reduced by knockdown or blockade of TMEM16A. TMEM16A may be therefore important for enhanced Ca2+ release from IP3-sensitive Ca2+ stores in polycystic kidney disease.Key messages• ADPKD leads to continuous decline of renal function by growth of renal cysts.• Knockdown of PKD1 or PKD2 increases TMEM16A expression.• TMEM16A enhanced intracellular Ca2+ signals, Cl− secretion, and proliferation.• TMEM16A contributes to cyst growth in ADPKD.

Role of TG2-Mediated SERCA2 Serotonylation on Hypoxic Pulmonary Vein Remodeling.

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pumps take up Ca2+ from the cytoplasm to maintain the balance of intracellular Ca2+. A decline in expression or activity of SERCA results in persistent store-operated calcium entry (SOCE). In cardiomyocytes as well as vascular smooth muscle cells (SMCs), SERCA2 acts as an important regulator of calcium cycling. The purpose of this study is to identify and better understand the role of transglutaminases2 (TG2) as a key factor involved in SERCA2 serotonination (s-SERCA2) and to elucidate the underlying mechanism of action. Human pulmonary venous smooth muscle cell in normal pulmonary lobe were isolated and cultured in vitro. Establishment of hypoxic pulmonary hypertension model in wild type and TG2 knockout mice. SERCA2 serotonylation was analyzed by co-(immunoprecipitation) IP when the TG2 gene silenced or overexpressed under normoxia and hypoxia in vivo and in vitro. Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia. Real-time (RT)-PCR and Western blot analyzed expression of TG2, TRPC1, and TRPC6 under normoxia and hypoxia. Bioactivity of cells were analyzed by using Cell Counting Kit (CCK)-8, flow cytometry, wound healing, RT-PCR, and Western blot under PST-2744 and cyclopiazonic acid. We confirmed that 1) hypoxia enhanced the expression and activity of TG2, and 2) hypoxia increased the basal intracellular Ca2+ concentration ([Ca2+]i) and SOCE through activating TRPC6 on human pulmonary vein smooth muscle cells (hPVSMC). Then, we investigated the effects of overexpression and downregulation of the TG2 gene on the activity of SERCA2, s-SERCA2, basal [Ca2+]i, and SOCE under normoxia and hypoxia in vitro, and investigated the activity of SERCA2 and s-SERCA2 in vivo, respectively. We confirmed that SERCA2 serotonylation inhibited the activity of SERCA2 and increased the Ca2+ influx, and that hypoxia induced TG2-mediated SERCA2 serotonylation both in vivo and in vitro. Furthermore, we investigated the effect of TG2 activity on the biological behavior of hPVSMC by using an inhibitor and agonist of SERCA2, respectively. Finally, we confirmed that chronic hypoxia cannot increase vessel wall thickness, the right ventricular systolic pressure (RVSP), and right ventricular hypertrophy index (RVHI) of vascular smooth muscle-specific Tgm2−/− mice. These results indicated that hypoxia promoted TG2-mediated SERCA2 serotonylation, thereby leading to inhibition of SERCA2 activity, which further increased the calcium influx through the TRPC6 channel. Furthermore, tissue-specific conditional TG2 knockout mice prevents the development of pulmonary hypertension caused by hypoxia. In summary, we uncovered a new target (TG2) for treatment of chronic hypoxic pulmonary hypertension (CHPH).

Calcium signalling in mammalian cell lines expressing wild type and mutant human \u03b11-Antitrypsin

A possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca2+-signalling, which may contribute to the pathology associated with another serpinopathy, α1-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca2+, its responses to thapsigargin (TG), an ER Ca2+-ATPase blocker, and store-operated Ca2+-entry (SOCE). Our fura2 based Ca2+ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.

The ellagitannin metabolite urolithin C is a glucose-dependent regulator of insulin secretion through activation of L-type calcium channels.

The pharmacology of polyphenol metabolites on beta-cell function is largely undetermined. We sought to identify polyphenol metabolites that enhance the insulin-secreting function of beta-cells and to explore the underlying mechanisms. INS-1 beta-cells and rat isolated islets of Langerhans or perfused pancreas preparations were used for insulin secretion experiments. Molecular modelling, intracellular Ca2+ monitoring, and whole-cell patch-clamp recordings were used for mechanistic studies. Among a set of polyphenol metabolites, we found that exposure of INS-1 beta-cells to urolithins A and C enhanced glucose-stimulated insulin secretion. We further characterized the activity of urolithin C and its pharmacological mechanism. Urolithin C glucose-dependently enhanced insulin secretion in isolated islets of Langerhans and perfused pancreas preparations. In the latter, enhancement was reversible when glucose was lowered from a stimulating to a non-stimulating concentration. Molecular modelling suggested that urolithin C could dock into the Cav 1.2 L-type Ca2+ channel. Calcium monitoring indicated that urolithin C had no effect on basal intracellular Ca2+ but enhanced depolarization-induced increase in intracellular Ca2+ in INS-1 cells and dispersed cells isolated from islets. Electrophysiology studies indicated that urolithin C dose-dependently enhanced the L-type Ca2+ current for levels of depolarization above threshold and shifted its voltage-dependent activation towards more negative potentials in INS-1 cells. Urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca2+ channel opening and Ca2+ influx into pancreatic beta-cells. Our work paves the way for the design of polyphenol metabolite-inspired compounds aimed at ameliorating beta-cell function.

De novo expression of functional connexins 43 and 45 hemichannels increases sarcolemmal permeability of skeletal myofibers during endotoxemia

Endotoxemia caused by bacterial lipopolysaccharides (LPSs) leads to severe skeletal muscular deterioration, starting with higher membrane permeability and decline in resting membrane potential (RMP). However, the molecular mechanism of such changes remains unclear. Here, we evaluated the possible involvement of connexin43- and connexin45-based hemichannels (Cx43 and Cx45 HCs, respectively) as putative mediators of sarcolemmal dysfunctions induced by LPS in control (Cx43fl/flCx45fl/fl) and Cx43/Cx45 expression-deficient (Cx43fl/flCx45fl/fl:Myo-Cre) skeletal mice myofibers. At 5 h of endotoxemia, control myofibers presented Cx43 and Cx45 proteins forming functional HCs. Additionally, myofibers from endotoxic control mice showed dye uptake in vivo, which was inhibited by carbenoxolone, a Cx HC blocker. A similar increase in membrane permeability was observed in myofibers freshly isolated from skeletal muscle of mice treated for 5 h with LPS, which was blocked by the Cx HC blocker and was absent in myofibers from mice simultaneously treated with LPS and boldine, which is a Cx HC blocker. The increase in sarcolemmal permeability was mimicked by isolated myofibers treated with pro-inflammatory cytokines (TNF-α and IL-1β) and occurred at 5 h after treatment. Endotoxemia also induced a significant increase in basal intracellular Ca2+ signal and a drop in RMP in control myofibers. These two changes were not elicited by myofibers deficient in Cx43/Cx45 expression. Therefore, sarcolemmal dysfunction characterizing endotoxemia is largely explained by the expression of functional Cx43 and Cx45 HCs. Hence, current therapy options for individuals suffering from endotoxic shock could be greatly improved with selective Cx HC inhibitors avoiding the underlying skeletal muscle dysfunction.