Intracellular Calcium Activity Research Articles

Scar matrix drives Piezo1 mediated stromal inflammation leading to placenta accreta spectrum.

Scar tissue formation is a hallmark of wound repair in adults and can chronically affect tissue architecture and function. To understand the general phenomena, we sought to explore scar-driven imbalance in tissue homeostasis caused by a common, and standardized surgical procedure, the uterine scar due to cesarean surgery. Deep uterine scar is associated with a rapidly increasing condition in pregnant women, placenta accreta spectrum (PAS), characterized by aggressive trophoblast invasion into the uterus, frequently necessitating hysterectomy at parturition. We created a model of uterine scar, recapitulating PAS-like invasive phenotype, showing that scar matrix activates mechanosensitive ion channel, Piezo1, through glycolysis-fueled cellular contraction. Piezo1 activation increases intracellular calcium activity and Protein kinase C activation, leading to NF-κB nuclear translocation, and MafG stabilization. This inflammatory transformation of decidua leads to production of IL-8 and G-CSF, chemotactically recruiting invading trophoblasts towards scar, initiating PAS. Our study demonstrates aberrant mechanics of scar disturbs stroma-epithelia homeostasis in placentation, with implications in cancer dissemination.

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Therapeutic Effects of Capsaicin on Pain Management

Pain is an undesirable sensory experience that occurs from the noisome stimulus, it becomes a warning sign of an upcoming disease or existing disease thereby signaling immediate attention. There exists a variety of pharmacological and non-pharmacological methods to alleviate pain, with their own effects and impacts on health. The natural herb capsicum, used in most families as a spice is also found to have the therapeutic effects of for treating cardiovascular disorders, respiratory issues, gastrointestinal problems, urological disorders, cancer, diabetic peripheral neuropathy, obesity and pain reduction, especially in osteoarthritis and rheumatoid arthritis. It acts as a counter-irritant for various conditions like rheumatism, lumbago and neuralgia. It reduces pain by aiding in the depletion of substance-P and increases the activation of intracellular calcium by activating the calcium channel. The stimulation of transient receptor potential vanilloid 1 (TRPV1) causes effects like analgaesia, anti-infectious, anti-carcinogenic and antioxidant. It is permissible to use capsaicin in low concentrations such as in creams, lotions, gel patches and nasal sprays. High-concentration capsaicin is available in the form of oral formulations, intradermal, subcutaneous and intravenous. Capsaicin is mostly available in topical form, in many concentrations such as 0.025%, 0.0355%, 0.075% and 0.10%. It has been permitted to use capsaicin to the maximum concentration of 8%. Capsaicin is safe to use even in pregnancy, lactation, children and adolescents but is contraindicated for children less than 2 years old as well as for people with bleeding disorders..

Reducing the injury of hippocampal vascular endothelial cells after stroke via targeting SIRT1 by butylphthalide

Butylphthalide (NBP) can inhibit various pathological processes of ischemic stroke. This experiment explored the mechanism of NBP and SIRT1 on damage of hippocampal vascular endothelial cells after stroke. The neurons in the hippocampus of rats were stained with HE, and morphology and density of neurons were observed. Flow cytometry, commercial kits and Western blotting detected apoptosis of endothelial cells, levels of antioxidant enzymes and apoptotic proteins, intracellular calcium level and activity of Ca2+-ATPase. The damage to rat nerve cells was alleviated by butylphthalide to varying degrees, and the lost parts of rat nerve cells were recovered with decreased Bax and cleaved caspase-3 expression after butylphthalide treatment, and increased Bcl-2 (P <0.05), as well as decreased serum malondialdehyde (MDA) content and activity of catalase (CAT) decreased and elevated Superoxide dismutase (SOD) activity (P <0.05). The concentration of calcium ion also decreased but activity of Ca2+-ATPase increased (P <0.05) and mitochondria in the model group appeared with severe vacuolation and swelling. The vacuolation and swelling of mitochondria in the treatment group were improved. Additionally, mitochondrial membrane fluidity, potential and rat hippocampal ATPase activity in butylphthalide group were also increased. Compared to normal control group, model group, SIRT1 inhibitor group and butylphthalide+SIRT1 inhibitor group had lower levels of SIRT1 and higher p-NF-kB p65/p-IkBα levels. Butylphthalide has a protective effect on hippocampal neurons in stroke rats and can alleviate the damage degree of rat nerve cells.

Calcium-activated chloride channels in pericytes shape capillary electrical signaling

The brain vasculature, composed of penetrating arterioles (PAs) giving rise to the dense capillary network, facilitates continuous energy substrate delivery to neurons and glia by tightly controlling blood flow. The capillaries are covered by perivascular cells known as pericytes, which exhibit varying morphological and physiological properties based on their location in relation to the terminal arteriole, and these contribute to blood flow control within the capillary network in distinct ways. Thin-strand pericytes —the focus of this study—are located in the deep capillary bed. Serendipitously, we have discovered a robust chloride current during perforated patch clamp experiments on these thin-strand pericytes. We hypothesized that this current is mediated by calcium-activated chloride channels (CaCCs), which may influence electrical signaling and thereby regulate blood flow. Our data reveal that this robust current is dependent on external calcium concentration and can be blocked by voltage-gated calcium channel blockers, depletion of endoplasmic reticulum calcium stores, and calcium-activated chloride channel blockers. This suggests a mechanism wherein calcium entry into the cytosol via voltage-gated channels and intracellular calcium release govern the activity of CaCCs in the thin-strand pericyte membrane. Using two-photon microscopy, we further observed that blocking CaCCs led to PA dilation and hyperemia. Together, our data indicate a novel role for CaCCs in thin-strand pericytes, where they modulate electrical signaling across the capillary bed in response to intracellular calcium activity. By providing a depolarizing signal countering hyperpolarizing electrical signaling, these channels contribute to the intricate regulation of blood flow and vessel dilation. This work was supported by the NIH (grants 1R01AG066645, 5R01NS115401, 1DP2NS121347-01), and the American Heart Association (19IPLOI34660108). 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.

Association between Intracellular Calcium Signaling and Tumor Recurrence in Human Non-Functioning Pituitary Adenomas.

Clinically non-functioning pituitary adenomas (CNFPAs) are the second most frequent sellar tumor among studies on community-dwelling adults. They are characterized by the absence of hormonal hypersecretion syndrome, and patients present with compressive symptoms, such as a headache and visual field defects. Immunohistochemically, most CNFPAs are of gonadotrope differentiation, with only a few of them being truly null cell adenomas. Although these tumors express receptors for one or more hypothalamic releasing hormones, to what extent this has an impact on the biological and clinical behavior of these neoplasms remains to be defined. In this research, we evaluated the basal and hypothalamic secretagogue-stimulated intracellular calcium mobilization in 13 CNFPAs, trying to correlate this response to the phenotypic features of the patients. Our results indicate that the recurrence of a CNFPA correlates positively with cellular responsiveness, as measured by spontaneous intracellular calcium activity and the ability to respond to multiple hypothalamic secretagogues. We conclude that this finding may be a useful tool for predicting the clinicopathologic behavior of CNFPAs, by testing the variation of cellular responsiveness to hypothalamic secretagogues.

MicroRNA-9 promotes axon regeneration of mauthner-cell in zebrafish via her6/ calcium activity pathway

MicroRNA (miRNA), functioning as a post-transcriptional regulatory element, plays a significant role in numerous regulatory mechanisms and serves as a crucial intrinsic factor influencing axon regeneration. Prior investigations have elucidated the involvement of miRNA-9 in various processes, however, its specific contribution to axon regeneration in the central nervous system (CNS) remains uncertain. Hence, the zebrafish Mauthner axon regeneration model was employed to manipulate the expression of miRNA-9 in single cells, revealing that upregulation of miRNA-9 facilitated axon regeneration. Additionally, her6, a downstream target gene of miRNA-9, was identified as a novel gene associated with axon regeneration. Suppression of her6 resulted in enhanced Mauthner axon regeneration, as evidenced by the significantly improved regenerative capacity observed in her6 knockout zebrafish. In addition, modulation of her6 expression affects intracellular calcium levels in neurons and promoting her6 expression leads to a decrease in calcium levels in vivo using the new NEMOf calcium indicator. Moreover, the administration of the neural activity activator, pentylenetetrazol (PTZ) partially compensated for the inhibitory effect of her6 overexpression on the calcium level and promoted axon regeneration. Taken together, our study revealed a role for miRNA-9 in the process of axon regeneration in the CNS, which improved intracellular calcium activity and promoted axon regeneration by inhibiting the expression of downstream target gene her6. In our study, miRNA-9 emerged as a novel and intriguing target in the intricate regulation of axon regeneration and offered compelling evidence for the intricate relationship between calcium activity and the facilitation of axon regeneration.Graphical miRNA-9 can promote intracellular calcium activity in neurons by inhibiting the expression of its downstream target gene her6, which in turn promotes axonal regeneration.

Intracellular Calcium Overload and Activation of CaMKK/AMPK Signaling Are Related to the Acceleration of Muscle Glycolysis of Broiler Chickens Subjected to Acute Stress.

The current study investigated the effect of preslaughter transport on stress response and meat quality of broilers and explored the underlying mechanisms involved in the regulation of muscle glycolysis through calcium/calmodulin-dependent protein kinase kinase (CaMKK)/AMP-activated protein kinase (AMPK) signaling. Results suggested that transport induced stress responses of broilers and caused PSE-like syndrome of pectoralis major muscle. Preslaughter transport enhanced the mRNA expressions of glycogen phosphorylase and glucose transporters, as well as the activities of glycolytic enzymes, which accelerated the breakdown of glycolytic substrates and the accumulation of lactic acid. In addition, acute stress induced abnormal intracellular calcium homeostasis by disrupting calcium channels on the cell membrane and sarcoplasmic reticulum, which led to the activation of CaMKK and promoted AMPK phosphorylation. This study provides evidence that the intracellular calcium overload and the enhancement of CaMKK/AMPK signaling are related to the accelerated muscle glycolysis of broiler chickens subjected to acute stress.

S-22-1: GENETIC MECHANISMS OF HYPERTENSION IN DIFFERENT FORMS OF PRIMARY ALDOSTERONISM

Primary aldosteronism is the most common form of secondary arterial hypertension, affecting up to 10% of hypertensive patients. It is responsible for treatment resistance and increased risk of cardiovascular complications. Due to the difficulty of its diagnosis, PA is frequently missed, and as a result, treatment of the condition is either delayed by many years after the onset of hypertension, or is not initiated at all. Over the past 10 years, important discoveries have been made regarding the genetic basis of aldosterone producing adenoma and familial forms of primary aldosteronism. In most cases, genetic abnormalities are found in genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H, CLCN2) as well as ion pumps (ATP1A1, ATP2B3). They occur as somatic mutations in aldosterone producing adenoma and as germline mutations in familial forms of the disease. Mutations in these genes affect intracellular ion homeostasis and/or cell membrane potential, leading to increased intracellular calcium concentrations and activation of calcium signalling, which is the main regulator of aldosterone biosynthesis. In addition, double mutations in CTNNB1 and GNAQ/GNA11 have been identified in aldosterone producing adenoma presenting in puberty, pregnancy and menopause. Genetic studies have opened interesting diagnostic and treatment opportunities. While surrogate biomarkers of the mutation status may facilitate diagnosis and subtype identification in primary aldosteronism, innovative therapies are currently studied targeting mutated proteins. Finally, recent evidence suggests a continuum between unilateral and bilateral forms of primary aldosteronism, with somatic mutations being found in aldosterone producing cell clusters and aldosterone producing adenoma in patients with bilateral forms. We have recently identified genetic risk loci for primary aldosteronism through genome wide association studies, which may explain shared susceptibility and reveal new mechanisms involved in adrenal gland function and the development of aldosterone producing adenoma and bilateral adrenal hyperplasia.

Altered Calcium Permeability of AMPA Receptor Drives NMDA Receptor Inhibition in the Hippocampus of Murine Obesity Models.

Evidence has accumulated that higher consumption of high-fat diets (HFDs) during the juvenile/adolescent period induces altered hippocampal function and morphology; however, the mechanism behind this phenomenon remains elusive. Using high-resolution structural imaging combined with molecular and functional interrogation, a murine model of obesity treated with HFDs for 12weeks after weaning mice was shown to change in the glutamate-mediated intracellular calcium signaling and activity, including further selective reduction of gray matter volume in the hippocampus associated with memory recall disturbance. Dysregulation of intracellular calcium concentrations was restored by a non-competitive α-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonist, followed by normalization of hippocampal volume and memory recall ability, indicating that AMPARs may serve as an attractive therapeutic target for obesity-associated cognitive decline.

Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons

AimsNeurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs. Main methodshDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1–50 μM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and βIII-tubulin, respectively. Ten μM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity. Key findingsOur findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 μM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and βIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632. SignificanceAt the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.

Regulation of Cr(VI)-Induced Premature Senescence in L02 Hepatocytes by ROS-Ca2+-NF-κB Signaling.

Stress-induced premature senescence may be involved in the pathogeneses of acute liver injury. Hexavalent chromium [Cr(VI)], a common environmental pollutant related to liver injury, likely leads to premature senescence in L02 hepatocytes. However, the underlying mechanisms regarding hepatocyte premature senility in Cr(VI) exposure remain poorly understood. In this study, we found that chronic exposure of L02 hepatocytes to Cr(VI) led to premature senescence characterized by increased β-galactosidase activity, senescence-associated heterochromatin foci, G1 phase arrest, and decreased cell proliferation. Additionally, Cr(VI)-induced senescent L02 hepatocytes showed upregulated inflammation-related factors, such as IL-6 and fibroblast growth factor 23 (FGF23), which also exhibited reactive oxygen species (ROS) accumulation derived from mitochondria accompanied with increased concentration of intracellular calcium ions (Ca2+) and activity of nuclear factor kappa B (NF-κB). Of note is that ROS inhibition by N-acetyl-Lcysteine pretreatment not only alleviated Cr(VI)-induced premature senescence but also reduced the elevated intracellular Ca2+, activated NF-κB, and secretion of IL-6/FGF23. Intriguingly, the toxic effect of Cr(VI) upon premature senescence of L02 hepatocytes and increased levels of IL-6/FGF23 could be partially reversed by the intracellular Ca2+ chelator BAPTA-AM pretreatment. Furthermore, by utilizing the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), we confirmed that NF-κB mediated IL-6/FGF23 to regulate the Cr(VI)-induced L02 hepatocyte premature senescence, whilst the concentration of intracellular Ca2+ was not influenced by PDTC. To the best of our knowledge, our data reports for the first time the role of ROS-Ca2+-NF-κB signaling pathway in Cr(VI)-induced premature senescence. Our results collectively shed light on further exploration of innovative intervention strategies and treatment targeting Cr(VI)-induced chronic liver damage related to premature senescence.

Handling a mature calcium signature through optogenetics improves the differentiation of primary murine myotubes

Calcium takes part in numerous cellular processes such as proliferation, migration, differentiation, or cell death and plays a particular role in myogenesis of skeletal muscle. Indeed, intracellular calcium signaling participates, in a non-negligeable manner, to the “on” signal of muscle differentiation from undifferentiated cells to differentiated myotubes. Therefore, this differentiation can be modulated by controlling calcium activity with electrical or optogenetic stimulation approaches. In this study, we used the optogenetic tool channelrhodopsin 2 (ChR2) to control calcium activity and to modulate skeletal muscle differentiation. Using primary cultures of mouse myotubes, we showed that ChR2 stimulation was well-adapted to control intracellular calcium activity at the single cell or whole culture scale. To modulate the calcium-dependent myotube differentiation, we used an optical stimulation protocol based on GCAMP6s-decoded spontaneous calcium activity patterns of differentiated myotubes. The optical training of myotubes increased the fusion index and their contractile ability. This study demonstrates that handling a mature calcium signature with such optogenetic tool improves the differentiation of primary murine myotubes.

Design, synthesis and biological evaluation of novel 1,3,4,9-tetrahydropyrano[3,4-b]indoles as potential treatment of triple negative breast cancer by suppressing PI3K/AKT/mTOR pathway

Triple-negative breast cancer (TNBC) represents a subset of breast cancer characterized by high aggressiveness and poor prognosis. Currently, there is no curative therapeutic regimen for TNBC patients. In this study, molecular hybridization strategy is adopted by combining benzopyran and indole pharmacophores together, and a library of structurally simple 1,3,4,9-tetrahydropyrano[3,4-b]indoles was rapidly constructed. The structure-activity relationship studies indicated that compound 23 exhibited the most potent effect against the MDA-MB-231 cells with IC50 value of 2.29 μM. Mechanistic studies revealed that compound 23 inhibited cell proliferation via arresting cell cycle at G0/G1 phase. It induced cell apoptosis by disruption of mitochondrial membrane potential (MMP), accumulation of reactive oxygen species (ROS), reduction of glutathione (GSH), elevation of intracellular calcium ion (Ca2+) and activation of caspase cascade. Furthermore, compound 23 significantly inhibited the regulators of PI3K/AKT/mTOR pathway in MDA-MB-231 cells, suggesting that PI3K/AKT/mTOR pathway was involved in the 23-mediated apoptosis. To our knowledge, this is the first example of the anti-cancer activity study of indole-fused pyrans through suppressing PI3K/AKT/mTOR pathway. Overall, the current study suggested that compound 23 would serve as a promising lead compound for TNBC treatment.

Atrial Fibrillation Induced by Anticancer Drugs and Underling Mechanisms.

Cancer therapy has made major progress in the past several decades, but treatments are often accompanied by significant side effects. Arrhythmias are a widespread complication of some antineoplastic drugs, with atrial fibrillation (AF) being the most often encountered drug-associated arrhythmia. Preexisting AF risk factors are commonly present in cancer patients who develop drug-associated AF, and active cancer itself may cause or promote AF. Although anticancer drugs may induce AF in cancer patients without AF risk factors, it appears that most drug-associated AF develop when cancer drugs add or aggravate precancer-existing and/or cancer-related pro-AF factors/alterations, additively or synergistically producing AF. Abnormalities in intracellular calcium activity seem to be involved in the generation of anticancer drug-induced AF. In cancer survivors with cancer therapy-induced cardiomyopathy, AF often occurs, with most of the arrhythmias likely to develop secondary to the cardiomyopathy. AF may lead to modification or even cessation of cancer therapy. The management of AF in patients with cancer is currently conducted largely based on pragmatic assumptions. This review briefly discusses AF caused by anticancer drugs and the underlying mechanisms.

Experimental-numerical analysis of cell adhesion-mediated electromechanical stimulation on piezoelectric nanofiber scaffolds

Electrospun nanofibers exhibiting piezoelectricity are a specific class of smart materials which could provide electric stimulation to cells in a noninvasive way and contribute to tissue regeneration. During cell-material interaction, the materials display electromechanical behavior by transforming cell adhesion force into surface charge. In the process, how the cell adhesion states and the electromechanical properties of scaffolds determine the actual piezoelectric potential implemented on a cell is still unclear. Herein, we fabricated piezoelectric poly(vinylidene fluoride) (PVDF) nanofiber scaffolds with different topographies, and investigated their influences on cell morphology and cell adhesion-mediated electromechanical stimulation of mesenchymal stem cell (MSC). Our results demonstrated that MSC seeded on aligned piezoelectric nanofibers exhibited elongated morphology combined with higher intracellular calcium activity than those adhered on random nanofibers with rounded shape. The underlying mechanism was further quantitatively analyzed using a three-dimensional (3D) finite element method with respect to cell adhesion states and architecture parameters of nanofiber scaffolds. The results suggested that cell morphology and cell adhesion force influenced the piezoelectric output through modulating the location and magnification of force implemented on the scaffolds. In addition, the change of alignment, pore size and diameter of the nanofiber network could alter the mechanical property of the scaffolds, and then bias the actual piezoelectric output experienced by a cell. These findings provide new insights for probing the mechanism of cell self-stimulation on piezoelectric scaffolds, and pave the way for rational design of piezoelectric scaffolds for cell regulation and tissue regeneration.

Multiple ligand recognition sites in free fatty acid receptor 2 (FFA2R) direct distinct neutrophil activation patterns

The allosteric modulating free fatty acid receptor 2 ligands Cmp58 and AZ1729, increased the activity induced by orthosteric receptor agonists mediating a rise in intracellular calcium ions and activation of the neutrophil NADPH-oxidase. Together, the two modulators triggered an orthosteric-agonist-independent activation of the oxidase without any rise in the concentration of intracellular calcium ions. In this study, structurally diverse compounds presumed to be ligands for free fatty acid receptor 2 were used to gain additional insights into receptor-modulation/signaling. We identified two molecules that activate neutrophils on their own and we classified one as allosteric agonist and the other as orthosteric agonist. Ten compounds were classified as allosteric FFA2R modulators. Of these, one activated neutrophils when combined with AZ1729; the nine remaining compounds activated neutrophils solely when combined with Cmp58. The activation signals were primarily biased when stimulated by two allosteric modulators interacting with different binding sites, such that two complementary modulators together triggered an activation of the NADPH-oxidase but no increase in the intracellular concentration of calcium ions. No neutrophil activation was induced when allosteric receptor modulators suggested to be recognized by the same binding site were combined, results in agreement with our proposed model for activation, in which the receptor has two different sites that selectively bind allosteric modulators. The down-stream signaling mediated by cross-sensitizing allosteric receptor modulators, occurring independent of any orthosteric agonist, represent a new mechanism for activation of the neutrophil NADPH oxidase.

Modulation of Calcium Signaling by Chemogenetic Tools to Elucidate the Pathogenesis of Primary Aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote increased intracellular calcium concentrations and activation of calcium signaling, the main trigger for aldosterone biosynthesis.The aim of our study was to elucidate the mechanisms underlying the development of PA by modulating calcium signaling using chemogenetic tools.We have generated two different adrenocortical H295R_S2 cell lines stably expressing different chimeric ion channels generated by fusing the mutated extracellular ligand binding domain of the α7 nicotinic acetylcholine receptor to the ion pore domain of large Cys-loop receptor ion channel family; these receptors constitute PSAM (Pharmacologically Selective Actuator Modules). The mutations introduced in the ligand-binding domain allow to use synthetic ligands, PSEM (Pharmacologically Selective Effector Molecules) to activate the PSAM. We used two different PSAM: the chimeric receptor α7-5HT3 or a mutated acetylcholine receptor nAchR, allowing respectively modulation of sodium or calcium entry into the cells in response to the specific PSEM: Varenicline for α7-5HT3 or Compound 9S for mutated nAChR. The cells lines were characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production and steroidogenic gene expression.Cells expressing α7-5HT3 treated for 24h with increasing concentrations of Varenicline (10–9 to 10-5M) showed increased intracellular calcium concentrations and an increase in expression of steroidogenic genes such as StAR CYP17A1, CYP21A2 and CYP11B2. Cell proliferation was not affected. Calcium entry into cells expressing the mutated nAChR receptor treated for 24h with increasing concentrations of Compound 9S (10–9 to 10-5M) induced an increase in expression of steroidogenic genes such as StAR, CYP21A2 and HSD3B2, but not CYP11B2. Similarly to the results obtained in cells expressing α7-5HT3, cell proliferation was unaffected in response to Compound 9S.These cell lines, in which we can modulate the intracellular calcium concentration « on demand », are a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Upregulation of transient receptor potential melastatin 4 (TRPM4) in ventricular fibroblasts from heart failure patients.

The transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective monovalent cation channel belonging to the TRP channel superfamily. TRPM4 is widely expressed in various tissues and most abundantly expressed in the heart. TRPM4 plays a critical role in cardiac conduction. Patients carrying a gain-of-function or loss-of-function mutation of TRPM4 display impaired cardiac conduction. Knockout or over-expression of TRPM4 in mice recapitulates conduction defects in patients. Moreover, recent studies have indicated that TRPM4 plays a role in hypertrophy and heart failure. Whereas the role of TRPM4 mediated by cardiac myocytes has been well investigated, little is known about TRPM4 and its role in cardiac fibroblasts. Here we show that in human left ventricular fibroblasts, TRPM4 exhibits typical Ca2+-activation characteristics, linear current-voltage (I-V) relation, and monovalent permeability. TRPM4 currents recorded in fibroblasts from heart failure patients (HF) are more than 2-fold bigger than those from control individuals (CTL). The enhanced functional TRPM4 in HF is not resulted from changed channel properties, as TRPM4 currents from both HF and CTL fibroblasts demonstrate similar sensitivity to intracellular calcium activation and extracellular 9-phenanthrol (9-phen) blockade. Consistent with enhanced TRPM4 activity, the protein level of TRPM4 is about 2-fold higher in HF than that of CTL hearts. Moreover, TRPM4 current in CTL fibroblasts is increased after 24 hours of TGFβ1 treatment, implying that TRPM4 in vivo may be upregulated by fibrogenesis promotor TGFβ1. The upregulated TRPM4 in HF fibroblasts suggests that TRPM4 may play a role in cardiac fibrogenesis under various pathological conditions.

Decorin regulates cartilage pericellular matrix micromechanobiology

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2–4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.