Changes In Extracellular Ca2 Research Articles

Molecular Pathophysiology of Parathyroid Tumorigenesis-The Lesson from a Rare Disease: The "MEN1 Model".

Primary hyperparathyroidism represents the third most prevalent endocrine disease in the general population, consisting of an excessive secretion of parathyroid hormone from one or, more frequently, more of the parathyroid glands, leading to a dysregulation of calcium homeostasis. Schematically, its development occurs primarily by pathophysiological events with genetic mutation, at the germline and/or somatic level, that favor the neoplastic transformation of parathyroid cells and promote their aberrant proliferation, and mutations determining the shift in the PTH "set-point", thus interfering with the normal pathways of PTH secretion and leading to a "resetting" of Ca2+-dependent PTH secretion or to a secretion of PTH insensitive to changes in extracellular Ca2+ levels. Familial syndromic and non-syndromic forms of primary hyperparathyroidism are responsible for approximately 2-5% of primary hyperparathyroidism cases and most of them are inherited forms. The history of the genetic/molecular studies of parathyroid tumorigenesis associated with multiple endocrine neoplasia type 1 syndrome (MEN1) represents an interesting model to understand genetic-epigenetic-molecular aspects underlying the pathophysiology of primary hyperparathyroidism, both in relation to syndromic and non-syndromic forms. This minireview aims to take a quick and simplified look at the MEN1-associated parathyroid tumorigenesis, focusing on the molecular underlying mechanisms. Clinical, epidemiological, and observational studies, as well as specific guidelines, molecular genetics studies, and reviews, have been considered. Only studies submitted to PubMed in the English language were included, without time constraints.

A light-addressable potentiometric sensor-based extracellular calcium dynamic monitoring and imaging platform for cellular calcium channel drug evaluation

Disruption and dysregulation of cellular calcium channel function can lead to diseases such as ischemic stroke, heart failure, and arrhythmias. Corresponding calcium channel drugs typically require preliminary efficacy evaluations using in vitro models such as cells and simulated tissues before clinical testing. However, traditional detection and evaluation methods often encounter challenges in long-term continuous monitoring and lack calcium specificity. In this study, a dynamic monitoring system based on ion-sensitive membranes for light-addressable potentiometric sensor (LAPS) was developed to meet the demand for monitoring changes in extracellular calcium ion (Ca2+) concentration in live cells. The effects of Ca2+ channel agonists and blockers on 2D and 3D HL-1 cells were investigated, with changes in extracellular Ca2+ concentration reflecting cellular calcium metabolism, facilitating drug evaluation. Additionally, calcium imaging technology with optical addressing capability complemented the LAPS system's ability to perceive 3D cell morphology, enhancing its drug evaluation capabilities. This work provides a novel, label-free, specific, and stable technique for monitoring cellular calcium metabolism. It achieves both continuous monitoring at single points and custom sensing area calcium imaging, holding significant implications for drug screening and disease treatment related to human calcium homeostasis.

The A986S calcium sensing receptor variant supports the adaptation of breast cancer cells to high calcium — a novel paradigm in breast cancer progression

Breast cancer and other solid tumors that metastasize to bone are frequently accompanied by a progressive increase in circulating calcium as the disease becomes more malignant. The increase in circulating calcium not only disrupts systemic calcium homeostasis but also alters the ability of cells to sense slight changes in extracellular Ca2+ via the ubiquitous calcium sensing receptor (CaSR). Of the CASR polymorphisms at rs1801725 (p.A986S), rs1801726 (p.Q1011E), and rs1042636 (p.R990G) in exon 7 of the receptor, previous studies have shown that only the A986S CaSR variant is associated with calcium in breast, prostate and skin cancers. In this study, we assessed if exon 7 inactivating A986S (AS-CaSR) and Q1011E (QE-CaSR) variants of the receptor influence the survival of breast tumor cells at higher Ca2+. We generated isogenic HEK293T and BT-549 cells expressing these CaSR variants and carried out bulk RNA sequencing of high Ca2+ adapted control and breast cancer cells. CaSR expression was detected by western blotting using anti-flag M2 (Sigma) antibody. By using phospho-ERK1/2 as the readout, the activation of AS-CaSR required higher concentrations of Ca2+ (EC50 ~5.28 mM) compared to the QE-CaSR (EC50 of ~3.81 mM) and the wild type (WT-CaSR; EC50 ~3.69 mM) receptor. When the CaSR variants transfected cells initially cultured at 7.5 mM for 6 days, and then sub-cultured in medium containing 7.5 to 20 mM Ca2+, the viability of empty vector control and WT-CaSR transfected cells strongly decreased at Ca2+ levels beyond 7.5 mM and these cells were mostly necrotic at ≥15 mM Ca2+. The QE-CaSR variant transfected cells on the other hand exhibited limited cell death beyond 10 mM Ca2+, while the surviving colonies of the AS-CaSR variant were viable at up to 15 mM Ca2+ with invasive tendencies. Analysis of differentially expressed genes in high Ca2+ adapted mesenchymal-like BT-549 and epithelial MDA-MB-468 breast cancer cells compared to MCF10A control cells revealed a small set of differentially expressed genes that were significantly associated with relapse free survival of basal-like breast cancer patients. Among these genes are ligands and negative regulators of the WNT/FZD pathway. Our data suggest that the A986S-CaSR variant with reduced sensitivity to Ca2+ supported adaptation of TNBC cells at high Ca2+, and that high Ca2+ adaptation of breast cancer cells may be mediated via a panel of high Ca2+ inducible genes with implications in bone metastasis. This study is supported by NIH SC1GM139814, T32AI007281, T32GM144927, T32HL007737; and the American Cancer Society. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oral expressions and functional analyses of the extracellular calcium-sensing receptor (CaSR) in chicken

In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca2+. CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.

The significance of calcium-sensing receptor in sustaining photosynthesis and ameliorating stress responses in plants.

Calcium ions (Ca2+) regulate plant growth and development during exposure to multiple biotic and abiotic stresses as the second signaling messenger in cells. The extracellular calcium-sensing receptor (CAS) is a specific protein spatially located on the thylakoid membrane. It regulates the intracellular Ca2+ responses by sensing changes in extracellular Ca2+ concentration, thereby affecting a series of downstream signal transduction processes and making plants more resilient to respond to stresses. Here, we summarized the discovery process, structure, and location of CAS in plants and the effects of Ca2+ and CAS on stomatal functionality, photosynthesis, and various environmental adaptations. Under changing environmental conditions and global climate, our study enhances the mechanistic understanding of calcium-sensing receptors in sustaining photosynthesis and mediating abiotic stress responses in plants. A better understanding of the fundamental mechanisms of Ca2+ and CAS in regulating stress responses in plants may provide novel mitigation strategies for improving crop yield in a world facing more extreme climate-changed linked weather events with multiple stresses during cultivation.

Development of FRET-based indicators for visualizing homophilic trans interaction of a clustered protocadherin

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

Rare Case of Severe Hypocalcemia Due to PTH Resistance Related to Diet Pill

Introduction: The production of parathyroid hormone (PTH) is essential for calcium the maintenance of normal mineral metabolism. Parathyroid cells have cell-surface calciumsensing receptors, even small changes in extracellular Ca induce rapid changes in PTH secretion. Hypocalcaemia is a well-recognized manifestation of magnesium deficiency. We present rare case of severe hypocalcemia due to PTH resistance caused by hypomagnesemia related to diet pill. Case Report: 61 year old Hispanic obese female with chronic gastritis on omeprazole 20 mg daily, no other significant past medical and surgical history, not taking any prescribed medications presents to ED with complaints of tingling and numbness around the mouth, abdominal cramps, chest pain, shortness of breath and anxiety. On arrival calcium was found to be 6.0mg/dl(8.2–10.2mg/dl) with ionized calcium of 0.60mmol/l(1.13-1.32mmol/l) and EGFR>90ml/min, Albumin-4.0g/l, Magnesium-1.1mg/dl(1.6–2.3mg/dl), Phosphorus-6.1mg/dl(2.4–4.5mg/dl),rest of the electrolytes were normal. Patient was given IV calcium gluconate 2g and magnesium which helped improving her symptoms. PTH was 1700pg/ml(23-73pg/ml), low Vitamin D 25-hydroxy 20ng/dl(30-100ng/dl). Urinary calcium was <1mg/dl(2.0–17.5mg/dl). Vitamin D1,25- dihydroxy 34pg/ml(18-78pg/ml), PTH-like peptide levels 0.6pmol/l(<4.2pmol/l). EKG was normal no QT interval changes. For 4 weekspatient was taking weight loss medication was given to her by her brother called nucific-bio-x4. Patient had lost 4 pounds while taking the medication and had suppression of appetite. Physical features of pseudohypoparathyroidism were not seen. Patient had poor dietary intake of calcium, denied taking vitamin D supplements. Patient was given calcium acetate 2001 mg TID with meals, calcitriol 0.5mcg daily and calcium gluconate 2g IV intermittently was given. That improved her calcium levels to 6.8mg/dl with ionized calcium-0.90mmol/l. Magnesium was replaced IV and discharged on magnesium oxide 400mg BID to maintain magnesium the normal range. The patient was given loading dose of ergocalciferol 50,000 IUfor 8 weeks. At the time of discharge repeat PTH levels were 1600 pg/ml and calcium levels were 8.2mg/dl. 3D CT of the neck did not parathyroid mass. After discharge patient continued on oral calcium and magnesium to maintain calcium levels between 8.4–10.2mg/dl. After discharge calcium levels were 8.6mg/dl, vitamin D 25-hydroxy levels were 32ng/dl and maintenance dose of vitamin D3 2000 IU daily also continued. Nucific-bio-x4 pill was discontinued. Conclusion: In conclusion, severe life threatening hypocalcemia can occur with unsupervised weight lossmedications due to malabsorption of magnesium in patient with likely mutation in magnesium receptors causing PTH resistance. Vitamin D deficiency worsens hypocalcemia resulting into secondary hyperparathyroidism.

Repurposing calcium-sensing receptor agonist cinacalcet for treatment of CFTR-mediated secretory diarrheas.

Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures, such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca2+. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl– secretion by greater than 80%. Cinacalcet also reduced Cl– secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) Cl– secretion following activation of CaSR and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed loop models of cholera and traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3–mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas, including cholera, traveler’s diarrhea, and VIPoma.

The calcium sensing receptor modulates H+-ATPase activity in intercalated cells.

Previous studies found that calcium sensing receptor (CaSR) it's expressed in intercalated cells of the collecting duct and that its activation by calcium in the luminal membrane promotes acidification of urine. Therefore, the aim of the study was to analyze the effects of CaSR stimulus on the biochemical activity of the vacuolar H+-ATPase in a cellular model of intercalated cells, MDCK-C11 cells. Biochemical activity of H+-ATPase was performed using cell homogenates and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium ([Ca2+]i) were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300 μM), neomycin (200 μM) and by the calcimimetic R-568 (1 μM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ concentration ([Ca2+]o) between 10-2 and 2 mM. The calciolytic NPS 2143 (150 nM) significantly reduced the vacuolar H+-ATPase activity observed with 2 mM [Ca2+]o. Inhibition of phospholipase C (PLC) activity with U73122 (5 x 10-7 M) reversed the increase in pump activity observed in the presence of Gd3+. Activation of CaSR by the specific CaSR agonist R-568 produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of thapsigargin. In summary, CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of MDCK-C11 cells, an effect that involves an increase in [Ca2+]i and require PLC activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially reducing the formation of calcium phosphate stones.

Extracellular Ca2+-Sensing Fluorescent Protein Biosensor Based on a Collagen-Binding Domain.

The importance of extracellular gradients of biomolecules is increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, the dynamics of extracellular calcium concentration is rarely studied. Here, we present a low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable scaffold-based biosensing of Ca2+ in the proximity of cells in live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of the biosensor displays response to [Ca2+] over a range of ∼2-2.5 ns, making it attractive for multiplexed FLIM assays. To evaluate the performance of this biosensor in physiological measurements, we applied it to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+ upon chelation with EGTA. When combined with spectrally resolved FLIM of lipid droplets using Nile red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+ depletion, suggesting that the intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate Twitch-2B as a prospective Ca2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment.

Role of calcium-sensing receptor in cadmium-induced apoptosis of rat primary osteoblasts in vitro

Calcium is essential to various physiological and pathophysiological cellular processes. Calcium-sensing receptor (CasR), a seven-transmembrane-spanning protein that responds to changes in extracellular Ca2+, partly modulates calcium homeostasis, thereby influencing bone metabolism. In this study, we aimed to elucidate the role of CasR in Cd-induced calcium homeostasis disruption and OB apoptosis, and the underlying mechanisms. Cd treatment dramatically increased the protein expression of CasR and elevated the intracellular calcium concentration. Meanwhile, OBs apoptosis rate and caspase-dependent apoptosis protein levels, including cleaved caspase 3, cleaved caspase 9 and the ratio of Bax/Bcl-2 were increased. However, downregulation of CasR by CasR siRNA effectively suppressed the effects of Cd on theses phenomena. At the same time, we illustrated that CasR siRNA pretreatment blocked Cd-inhibited the phosphorylation of PKC and decreased Cd-induced the phosphorylation of PI3K/AKT.Our results suggested that CasR-mediated PKC and PI3K/AKT signaling pathways involve in calcium oscillation and apoptosis in OB caused by Cd maybe responsible for the bone homeostasis.

Rare earth elements induce cytoskeleton-dependent and PI4P-associated rearrangement of SYT1/SYT5 endoplasmic reticulum-plasma membrane contact site complexes in Arabidopsis.

In plant cells, environmental stressors promote changes in connectivity between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM). Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in interorganelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+- and lipid-binding protein 1 (CLB1/SYT7). This complex is enriched at ER-PM contact sites (EPCSs), has slow responses to changes in extracellular Ca2+, and displays severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositide species at the PM.

Biochemical features of blood vessel extracellular calcium-sensing receptor and their physiological mechanism of action in the regulation of vascular tone and blood pressure

The extracellular calcium-sensing receptor (CaR) is a G protein coupled receptor superfamily that has ability to sense very small changes in extracellular Ca2+ with primary physiological function of maintenance of constant blood Ca2+ levels. In addition to being expressed in tissues involved in [Ca2+]o homeostasis, it is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions such as control vasomotor tone and blood pressure. However, the exact cellular mechanisms involved in these responses remain unclear. Stimulation of CaRs on perivascular nerves is proposed to evoke synthesis and release of nitric oxide (NO) from pre-synaptic terminals, which activate large conductance Ca2+-activated K+ channels in adjacent VSMCs that induce membrane hyperpolarisation and subsequent vasorelaxation. Plus, activation of CaRs on endothelial cells is reported to generate NO or vasoactive lipids or induce activation of intermediate conductance Ca2+-activated K+ channels to induce vasorelaxations. In contrast, CaR-induced activation of Gq and MAPK kinase-mediated pathways in VSMCs are linked to vasoconstriction and cell proliferation respectively. Taken together, these findings indicate that stimulation of vascular CaRs is reported to induce both constrictions and relaxations. In the endothelium intact artery, CaR mediated vasorelaxations are the dominant physiological phenomenon.

Calcium-sensing receptor deletion in the mouse esophagus alters barrier function.

Calcium-sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca2+ concentrations. CaSR has been reported to play a role in glandular and fluid secretion in the gastrointestinal tract and to regulate differentiation and proliferation of skin keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating keratin 5 CreER;CaSRFlox+/+compound mutants, in which loxP sites flank exon 7 of CaSR gene. Recombination was initiated with multiple tamoxifen injections, and we demonstrated exon 7 deletion by PCR analysis of genomic DNA. Quantitative real-time PCR and Western blot analyses showed a significant reduction in CaSR mRNA and protein expression in the knockout mice (EsoCaSR-/-) as compared with control mice. Microscopic examination of EsoCaSR-/- esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and bacterial buildup on the luminal epithelial surface. Western analysis revealed a significant reduction in levels of adherens junction proteins E-cadherin and β catenin and tight junction protein claudin-1, 4, and 5. Levels of small GTPase proteins Rac/Cdc42, involved in actin remodeling, were also reduced. Ussing chamber experiments showed a significantly lower transepithelial resistance in knockout (KO) tissues. In addition, luminal-to-serosal-fluorescein dextran (4 kDa) flux was higher in KO tissues. Our data indicate that CaSR plays a role in regulating keratinization and cell-cell junctional complexes and is therefore important for the maintenance of the barrier function of the esophagus.NEW & NOTEWORTHY The esophageal stratified squamous epithelium maintains its integrity by continuous proliferation and differentiation of the basal cells. Here, we demonstrate that deletion of the calcium-sensing receptor, a G protein-coupled receptor, from the basal cells disrupts the structure and barrier properties of the epithelium.

Methylation determines the extracellular calcium sensitivity of the leak channel NALCN in hippocampal dentate granule cells

The sodium leak channel NALCN is a key player in establishing the resting membrane potential (RMP) in neurons and transduces changes in extracellular Ca2+ concentration ([Ca2+]e) into increased neuronal excitability as the downstream effector of calcium-sensing receptor (CaSR). Gain-of-function mutations in the human NALCN gene cause encephalopathy and severe intellectual disability. Thus, understanding the regulatory mechanisms of NALCN is important for both basic and translational research. This study reveals a novel mechanism for NALCN regulation by arginine methylation. Hippocampal dentate granule cells in protein arginine methyltransferase 7 (PRMT7)-deficient mice display a depolarization of the RMP, decreased threshold currents, and increased excitability compared to wild-type neurons. Electrophysiological studies combined with molecular analysis indicate that enhanced NALCN activities contribute to hyperexcitability in PRMT7−/− neurons. PRMT7 depletion in HEK293T cells increases NALCN activity by shifting the dose-response curve of NALCN inhibition by [Ca2+]e without affecting NALCN protein levels. In vitro methylation studies show that PRMT7 methylates a highly conserved Arg1653 of the NALCN gene located in the carboxy-terminal region that is implicated in CaSR-mediated regulation. A kinase-specific phosphorylation site prediction program shows that the adjacent Ser1652 is a potential phosphorylation site. Consistently, our data from site-specific mutants and PKC inhibitors suggest that Arg1653 methylation might modulate Ser1652 phosphorylation mediated by CaSR/PKC-delta, leading to [Ca2+]e-mediated NALCN suppression. Collectively, these data suggest that PRMT7 deficiency decreases NALCN methylation at Arg1653, which, in turn, decreases CaSR/PKC-mediated Ser1652 phosphorylation, lifting NALCN inhibition, thereby enhancing neuronal excitability. Thus, PRMT7-mediated NALCN inhibition provides a potential target for the development of therapeutic tools for neurological diseases.

Calcium-sensing receptor (CaSR) modulates vacuolar H+-ATPase activity in a cell model of proximal tubule.

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H+-ATPase in a cellular model of proximal tubule cells, OKP cells. Biochemical activity of H+-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca2+]i were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300µM) and neomycin (200µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ (Ca2+o) between 10-4 and 2mM. Gd3+ and neomycin produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of 0.1µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5 × 10-8M) reduced the increase in [Ca2+]i induced by neomycin. CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of OKP cells, an effect that involves an increase in [Ca2+]i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

Desmoglein 3 Order and Dynamics in Desmosomes Determined by Fluorescence Polarization Microscopy

Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca2+ dependent, and reduction of extracellular Ca2+ leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca2+ concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.

TRPC1 stimulates calcium‑sensing receptor‑induced store‑operated Ca2+ entry and nitric oxide production in endothelial cells.

Store-operated Ca2+ entry (SOCE) via store-operated Ca2+ channels (SOCC), encoded by transient receptor potential canonical (TRPC) channel proteins, is an important underlying mechanism regulating intracellular Ca2+ concentration ([Ca2+]i) and various intracellular functions in endothelial cells (ECs). TRPC1, the probable candidate for SOCC, is expressed in ECs. Ca2+-sensing receptor (CaSR) is functionally expressed in vascular endothelium and is important in Ca2+ mobilization and cardiovascular functions. To date, there have been no reports demonstrating an association between CaSR and TRPC1 in ECs. The present study investigated the effects of TRPC1 on CaSR-induced Ca2+ influx and nitric oxide (NO) production in human umbilical vein ECs (HUVECs). TRPC1 and CaSR proteins in HUVECs were measured by immunostaining and western blot analysis. [Ca2+]i levels were measured using the Fura-2-acetoxymethyl ester method. The indicator 3-amino, 4-aminomethyl-2, 7-difluorescein diacetate was used to measure NO production in HUVECs. The expression of TRPC1 protein in HUVECs was silenced by transfecting HUVECs with small interfering RNA (siRNA) against TRPC1. Although changes in extracellular Ca2+ failed to alter [Ca2+]i in HUVECs, the CaSR agonist spermine increased [Ca2+]i and NO production in HUVECs. NO production in HUVECs was diminished in Ca2+-free medium or following treatment with a CaSR negative allosteric modulator (Calhex231), SOCC inhibitor (MRS1845) or TRPC inhibitor (SKF96365). The spermine-induced increases in [Ca2+]i and NO production were reduced in HUVECs transfected with TRPC1 siRNA. These results suggested that TRPC1 is a primary candidate in forming SOCC that stimulates CaSR-induced SOCE and NO production in HUVECs and is a potential therapeutic target for vascular diseases.

Molecular Physiology of the Hypocalcemic Action of Fibroblast Growth Factor 23 in Zebrafish (Danio rerio)

Fibroblast growth factor 23 (FGF23), a hormone required for phosphorus metabolism, was recently proposed to act on Ca2+ uptake; however, the available evidence of how FGF23 controls the body fluid Ca2+ homeostasis needs to be further clarified. The use of zebrafish as a model system revealed that FGF23 is specifically expressed in the corpuscles of Stannius (CS), an organ involved in Ca2+ homeostasis in fish, and that its expression is stimulated by ambient water with a high Ca2+ level. The overexpression of FGF23 inhibited Ca2+ uptake by downregulating the messenger RNA (mRNA) expression of epithelium calcium channel. Calcium-sensing receptor (CaSR), which senses changes in extracellular Ca2+ levels and modulates calciotropic hormones in organs controlling Ca2+ homeostasis in vertebrates, was found to be coexpressed with FGF23 in the CS. In addition, upregulated expression of FGF23 mRNA was detected in morphants of stanniocalcin 1 (stc1, another hypocalcemic factor synthesized in the CS), and knockdown of CaSR suppressed such upregulation and enhanced Ca2+ uptake. Taken together, our data indicate that FGF23 functions as a hypocalcemic hormone in zebrafish and that the CaSR/STC1-FGF23 axis is involved in body fluid Ca2+ homeostasis in vertebrates.

Ca2+ dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca2+ calmodulin kinases

The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca2+ regulation of NET and found that Ca2+ influenced both Vmax and Km for NE transport into cortical synaptosomes. Changes in extracellular Ca2+ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28–47 in the NET NH2-terminus abolished the Ca2+ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca2+- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca2+-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K+ increased NET surface expression in a process that required external Ca2+ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca2+ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.