Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells

Background Impaired mineral ion metabolism is a hallmark of CKD–metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. Methods To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells (SM22αCaSRΔflox/Δflox). Results Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. Conclusions These results suggest that, in addition to CaSR’s established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics

The calcium-sensing receptor: one of a kind

New Findings What is the topic of this review? The extracellular calcium-sensing receptor, CaSR, ensures whole-body Ca2+ homeostasis. Recent developments highlight the importance of the CaSR beyond mineral ion metabolism. This review focuses on novel roles and the use of CaSR-based therapeutics within the vasculature, the gut and the lung. What advances does it highlight? The ability of the CaSR to act as a multimodal chemosensor has led to the identification of signalling pathways that are ligand and cellular context dependent. Development of cell-specific CaSR modulators is now being harnessed to rescue aberrant CaSR function outside the extracellular Ca2+ homeostatic system. The extracellular calcium-sensing receptor, CaSR, is the first G protein-coupled receptor found to have an inorganic ion, calcium (Ca2+), as its physiological agonist. It is highly expressed in all organs involved in the regulation of mineral ion metabolism, namely the parathyroid gland, the kidney and bone. The CaSR is the master controller of extracellular Ca2+ concentration, as highlighted by the evidence that both inherited and acquired mutations in the CASR gene cause disturbances in mineral ion metabolism. CaSR positive allosteric modulators have been successfully employed in the clinic for over a decade to restore CaSR function, which is reduced in hyperparathyroidism secondary to kidney failure, while negative allosteric modulators are currently being tested in patients with hypocalcaemia with hypercalciuria due to gain-of-function CASR mutations. In addition to its expression within the bone–kidney–parathyroid axis, the CaSR can be found in other tissues, including but not limited to the gut, the vasculature and the lung. Here, the CaSR acts as a chemosensor, integrating signals deriving from nutrient availability, salinity, acidification and the presence of ubiquitous polyamines. Knowledge of what these stimuli are and of the cell-specific signalling responses they evoke is crucial to our understanding of the non-calciotropic roles of the CaSR in physiology and how these are affected in disease states

Topical therapy with negative allosteric modulators of the calcium-sensing receptor (calcilytics) for the management of asthma: the beginning of a new era?

In this review article we present the evidence to date supporting the role of the calcium-sensing receptor (CaSR) as a key, pluripotential molecular trigger for asthma and speculate on the likely benefits of topical therapy of asthma with negative allosteric modulators of the CaSR: calcilytics

The calcium-sensing receptor: one of a kind

New Findings What is the topic of this review? The extracellular calcium-sensing receptor, CaSR, ensures whole-body Ca2+ homeostasis. Recent developments highlight the importance of the CaSR beyond mineral ion metabolism. This review focuses on novel roles and the use of CaSR-based therapeutics within the vasculature, the gut and the lung. What advances does it highlight? The ability of the CaSR to act as a multimodal chemosensor has led to the identification of signalling pathways that are ligand and cellular context dependent. Development of cell-specific CaSR modulators is now being harnessed to rescue aberrant CaSR function outside the extracellular Ca2+ homeostatic system. The extracellular calcium-sensing receptor, CaSR, is the first G protein-coupled receptor found to have an inorganic ion, calcium (Ca2+), as its physiological agonist. It is highly expressed in all organs involved in the regulation of mineral ion metabolism, namely the parathyroid gland, the kidney and bone. The CaSR is the master controller of extracellular Ca2+ concentration, as highlighted by the evidence that both inherited and acquired mutations in the CASR gene cause disturbances in mineral ion metabolism. CaSR positive allosteric modulators have been successfully employed in the clinic for over a decade to restore CaSR function, which is reduced in hyperparathyroidism secondary to kidney failure, while negative allosteric modulators are currently being tested in patients with hypocalcaemia with hypercalciuria due to gain-of-function CASR mutations. In addition to its expression within the bone–kidney–parathyroid axis, the CaSR can be found in other tissues, including but not limited to the gut, the vasculature and the lung. Here, the CaSR acts as a chemosensor, integrating signals deriving from nutrient availability, salinity, acidification and the presence of ubiquitous polyamines. Knowledge of what these stimuli are and of the cell-specific signalling responses they evoke is crucial to our understanding of the non-calciotropic roles of the CaSR in physiology and how these are affected in disease states

Characterisation of negative allosteric modulators of the calcium-sensing receptor, CaSR, for repurposing as a treatment for asthma

Asthma is still an incurable disease and there is a recognised need for novel small molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor negative allosteric modulators (CaSR NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, originally developed for oral therapy for osteoporosis and previously tested in the clinic, as a novel, single and comprehensive topical anti-asthma therapy. We address the hypotheses, using murine asthma surrogates, that topically-delivered CaSR NAMs (i) abolish AHR; (ii) are unlikely to cause unwanted systemic effects; (iii) are suitable for topical application; (iv) inhibit airways inflammation to the same degree as the current standard of care, inhaled corticosteroid (ICS), and furthermore inhibit airways remodelling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice, and suppressed both AHR and airways inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, PK/PD, formulation and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation and abrogating some features of airways remodelling

Forced cell-cycle exit and modulation of GABAA, CREB and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal sub-types and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time-courses of functional maturation which are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to post-synaptic receptor maturation. Here, we describe a simple protocol which employs the sequential addition of just two supplemented media which have been formulated to separate the two key phases of neural differentiation - the neurogenesis and synaptogenesis - each characterized by different signaling requirements. Employing these media, this new protocol synchronised neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials, moreover they exhibited augmented: i) spontaneous electrical activity; ii) regenerative induced action potential train activity; iii) Na+ current availability, and; iv) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype which was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multi-electro array recordings. Furthermore, since this protocol can exploit expanded and frozen pre-patterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens

Single Protein Molecule Mapping with Magnetic Atomic Force Microscopy

Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to “see” magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space