Changes In Intracellular Ca2 Research Articles

Dual effects of the small-conductance Ca2+-activated K+ current on human atrial electrophysiology and Ca2+-driven arrhythmogenesis: an in silico study.

By sensing changes in intracellular Ca2+, small-conductance Ca2+-activated K+ (SK) channels dynamically regulate the dynamics of the cardiac action potential (AP) on a beat-to-beat basis. Given their predominance in atria versus ventricles, SK channels are considered a promising atrial-selective pharmacological target against atrial fibrillation (AF), the most common cardiac arrhythmia. However, the precise contribution of SK current (ISK) to atrial arrhythmogenesis is poorly understood, and may potentially involve different mechanisms that depend on species, heart rates, and degree of AF-induced atrial remodeling. Both reduced and enhanced ISK have been linked to AF. Similarly, both SK channel up- and downregulation have been reported in chronic AF (cAF) versus normal sinus rhythm (nSR) patient samples. Here, we use our multiscale modeling framework to obtain mechanistic insights into the contribution of ISK in human atrial cardiomyocyte electrophysiology. We simulate several protocols to quantify how ISK modulation affects the regulation of AP duration (APD), Ca2+ transient, refractoriness, and occurrence of alternans and delayed afterdepolarizations (DADs). Our simulations show that ISK activation shortens the APD and atrial effective refractory period, limits Ca2+ cycling, and slightly increases the propensity for alternans in both nSR and cAF conditions. We also show that increasing ISK counteracts DAD development by enhancing the repolarization force that opposes the Ca2+-dependent depolarization. Taken together, our results suggest that increasing ISK in human atrial cardiomyocytes could promote reentry while protecting against triggered activity. Depending on the leading arrhythmogenic mechanism, ISK inhibition may thus be a beneficial or detrimental anti-AF strategy.NEW & NOTEWORTHY Using our established framework for human atrial myocyte simulations, we investigated the role of the small-conductance Ca2+-activated K+ current (ISK) in the regulation of cell function and the development of Ca2+-driven arrhythmias. We found that ISK inhibition, a promising atrial-selective pharmacological strategy against atrial fibrillation, counteracts the reentry-promoting abbreviation of atrial refractoriness, but renders human atrial myocytes more vulnerable to delayed afterdepolarizations, thus potentially increasing the propensity for ectopic (triggered) activity.

In Vivo Immune Adjuvant Effects of CaCO3 Nanoparticles through Intracellular Ca2+ Concentration Regulation.

Calcium (Ca) is a vital component of the human body and plays a crucial role in intracellular signaling and regulation as a second messenger. Recent studies have shown that changes in intracellular Ca2+ concentration can influence immune cell function. In this study, we developed calcium carbonate nanoparticles (CaNPs) of various sizes using a Nanosystem Platform to modulate intracellular Ca2+ concentration in vitro and in vivo. Our findings demonstrate that intravenous administration of CaNPs led to changes in the number and ratio of immune cells in the spleen and stimulated the activation of dendritic cells (DCs) and macrophages. Notably, CaNPs exhibited strong adjuvant properties in the absence of antigenic stimuli. These results indicate that CaNPs have the potential to regulate immune cell function by modulating Ca2+ concentrations, offering a novel approach for disease prevention and treatment in combination with antigens or drugs. Overall, our study emphasizes the importance of modulating intracellular Ca2+ concentration as a means of regulating immune cell function.

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect.

Cytosolic Ca2+ signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca2+ concentration ([Ca2+]i) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+]i into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+]i to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14-16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer's disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system.

LIGHT ADAPTATION OF RETINAL ROD BIPOLAR CELLS.

The sensitivity of retinal cells is altered in background light to optimize the detection of contrast. For scotopic (rod) vision, substantial adaptation occurs in the first two cells, the rods and rod bipolar cells (RBCs), through sensitivity adjustments in rods and postsynaptic modulation of the transduction cascade in RBCs. To study the mechanisms mediating these components of adaptation, we made whole-cell, voltage-clamp recordings from retinal slices of mice from both sexes. Adaptation was assessed by fitting the Hill equation to response-intensity relationships with the parameters of half-maximal response (I1/2 ), Hill coefficient (n), and maximum response amplitude (Rmax ). We show that rod sensitivity decreases in backgrounds according to the Weber-Fechner relation with an I1/2 of about 50 R* s-1 The sensitivity of RBCs follows a near-identical function, indicating that changes in RBC sensitivity in backgrounds bright enough to adapt the rods are mostly derived from the rods themselves. Backgrounds too dim to adapt the rods can however alter n, relieving a synaptic nonlinearity likely through entry of Ca2+ into the RBCs. There is also a surprising decrease of Rmax , indicating that a step in RBC synaptic transduction is desensitized or that the transduction channels became reluctant to open. This effect is greatly reduced after dialysis of BAPTA at a membrane potential of +50 mV to impede Ca2+ entry. Thus the effects of background illumination in RBCs are in part the result of processes intrinsic to the photoreceptors and in part derive from additional Ca2+-dependent processes at the first synapse of vision.SIGNIFICANCE STATEMENT:Light adaptation adjusts the sensitivity of vision as ambient illumination changes. Adaptation for scotopic (rod) vision is known to occur partly in the rods and partly in the rest of the retina from presynaptic and postsynaptic mechanisms. We recorded light responses of rods and rod bipolar cells to identify different components of adaptation and study their mechanisms. We show that bipolar-cell sensitivity largely follows adaptation of the rods, but that light too dim to adapt the rods produces a linearization of the bipolar-cell response and a surprising decrease in maximum response amplitude, both mediated by a change in intracellular Ca2+ These findings provide a new understanding of how the retina responds to changing illumination.

Dysregulation of miR-146a: a causative factor in epilepsy pathogenesis, diagnosis, and prognosis.

miR-146a is an NF-κB-dependent miRNA that acts as an anti-inflammatory miRNA via the Toll-like receptor (TLR) pathway. miR-146a targets multiple genes and has been identified to directly or indirectly regulate processes other than inflammation, including intracellular Ca changes, apoptosis, oxidative stress, and neurodegeneration. miR-146a is an important regulator of gene expression in epilepsy development and progression. Furthermore, miR-146a-related single nucleotide polymorphisms (SNPs) and single nucleotide variants (SNVs) contribute to the genetic susceptibility to drug resistance and seizure severity in epilepsy patients. This study summarizes the abnormal expression patterns of miR-146a in different types and stages of epilepsy and its potential molecular regulation mechanism, indicating that miR-146a can be used as a novel biomarker for epilepsy diagnosis, prognosis, and treatment.

What makes the sinoatrial node tick? A question not for the faint of heart.

Even before the sinoatrial node (SAN) was discovered, cardiovascular science was engaged in an active investigation of when and why the heart would beat. After the electrochemical theory of bioelectric membrane potentials was formulated and the first action potentials were measured in contracting muscle cells, the field became divided: some investigators studied electrophysiology and ion channels, others studied muscle contraction. It later became known that changes in intracellular Ca2+ cause contraction. The pacemaking field was reunited by the coupled-clock theory of pacemaker cell function, which integrated intracellular Ca2+ cycling and transmembrane voltage into one rhythmogenic system. In this review, we will discuss recent discoveries that contextualize the coupled-clock system, first described in isolated SAN cells, into the complex world of SAN tissue: heterogeneous local Ca2+ releases, generated within SAN pacemaker cells and regulated by the other cell types within the SAN cytoarchitecture, variably co-localize and synchronize to give rise to relatively rhythmic impulses that emanate from the SAN to excite the heart. We will ultimately conceptualize the SAN as a brain-like structure, composed of intercommunicating meshworks of multiple types of pacemaker cells and interstitial cells, intertwined networks of nerves and glial cells and more. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intra- and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation, and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.

Protease-activated receptor-1 mediated calcium oscillations in renal mesangial cells

INTRODUCTION. Mesangial cells provide structural support to the glomerular tuft and modulate the glomerular capillary flow via their contractile properties. Mesangial cells undergo phenotypic changes into myofibroblast-like cells, such as proliferation, mesangial expansion, abnormal glomerular tuft formation, and reduced numbers of capillary loops, are present in several glomerular diseases, including diabetic nephropathy and glomerulonephritis. In addition, thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling and mechanisms in mesangial cells were not examined. This study aims to investigate protease-activated mechanisms regulating mesangial cell contraction and glomerular capillary flow. METHODS. We used primary human renal mesangial cells (HRMC) to determine the signaling mechanisms mediated by PAR1 thrombin-activated receptors. Confocal fluorescent microscopy was utilized to detect changes in intracellular Ca 2+ response to specific PAR1 modulators. Pharmacology and patch clamp electrophysiology was further applied to reveal downstream signaling mechanisms responsible for intracellular Ca 2+ oscillations. RESULTS. PAR1-mediated Ca 2+ response displayed high sensitivity to specific agonist (TFLLR-NH 2 ) with EC 50 values of 3 and 6.3 nM for male and female-derived cultured cells, respectively (the competition of a ligand for receptor binding fit converged; adj. R 2 =0.98). The response to PAR1 activation promoted initial cytosolic Ca 2+ increase followed by synchronized, damped Ca 2+ oscillations with a lag of 6.74±0.84 min between peaks. The pre-application of a specific inhibitor (RWJ56110) eliminated PAR1-mediated response, and oscillations were blocked by the changes of an extracellular solution to zero Ca 2+ . The specific inhibitors for store-operated calcium (SOCs) (Pyr6) and TRPC3 (GSK 2833503A) channels strongly attenuated oscillation behavior (up to 40% when added separately and up to 65% when both were applied; two-way ANOVA, * p<0.0001). In addition, the effect of a specific inhibitor of TRPC6 channels (BI-749327) had a minimal impact on Ca 2+ flux. Further single-channel electrophysiology experiments in HRMC cells confirmed the involvement of SOC and TRPC3 channels in PAR1-mediated GPCR activation. CONCLUSION. Our results indicate that coagulation proteases like thrombin may strongly regulate mesangial cell contraction and corresponding glomerular capillary flow by PAR1 GPCRs-related activation. The contraction mechanism is mediated presumably through SOCs entry and TRPC3 channels. Since high thrombin levels are linked to poor diabetic control, the described signaling may play a crucial role in the development of diabetic glomerular disease. DK126720 (to OP), DK129227 (to AS and OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 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.

Nuciferine relieves type 2 diabetes mellitus via enhancing GLUT4 expression and translocation

Nuciferine contained in lotus leaves have been confirmed to have the effect of ameliorating hyperlipemia and hyperglycemia. A laser scanning confocal microscope was used to track the translocation of glucose transporter 4 (GLUT4) in L6 cells and the changes in intracellular Ca2+ levels in real time, and related protease inhibitors combined with western blotting were used to explore the mechanism of nuciferine. Meanwhile, KK-Ay mice, the spontaneous type 2 diabetic mice, were used to evaluate the in vivo activity of nuciferine. In this study, the in vitro studies indicated that nuciferine-induced GLUT4 translocation was regulated by G protein-PLC-PKC and AMPK pathways and nuciferine-enhanced intracellular Ca2+ was mediated by G protein-PLC-IP3-IP3R pathway, the increase in intracellular Ca2+ caused by nuciferine was not directly related to GLUT4 translocation, but both promote glucose uptake. The in vivo results suggested that nuciferine ameliorated weight gain induced by high-fat diet, abnormal lipid metabolism and the symptoms of insulin resistance in KK-Ay diabetic mice. Western blot results suggested that nuciferine increased AMPK and PKC phosphorylation levels in skeletal muscle and liver, and enhanced GLUT4 expression in skeletal muscle. Taken together, this research showed that nuciferine has the non-negligible potential in the treatment of type 2 diabetes mellitus.

Ca2+- and Voltage-Activated K+ (BK) Channels in the Nervous System: One Gene, a Myriad of Physiological Functions.

BK channels are large conductance potassium channels characterized by four pore-forming α subunits, often co-assembled with auxiliary β and γ subunits to regulate Ca2+ sensitivity, voltage dependence and gating properties. BK channels are abundantly expressed throughout the brain and in different compartments within a single neuron, including axons, synaptic terminals, dendritic arbors, and spines. Their activation produces a massive efflux of K+ ions that hyperpolarizes the cellular membrane. Together with their ability to detect changes in intracellular Ca2+ concentration, BK channels control neuronal excitability and synaptic communication through diverse mechanisms. Moreover, increasing evidence indicates that dysfunction of BK channel-mediated effects on neuronal excitability and synaptic function has been implicated in several neurological disorders, including epilepsy, fragile X syndrome, mental retardation, and autism, as well as in motor and cognitive behavior. Here, we discuss current evidence highlighting the physiological importance of this ubiquitous channel in regulating brain function and its role in the pathophysiology of different neurological disorders.

Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling.

Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.

The Curcumin Derivative GT863 Protects Cell Membranes in Cytotoxicity by Aβ Oligomers

In Alzheimer's disease (AD), accumulation of amyloid β-protein (Aβ) is one of the major mechanisms causing neuronal cell damage. Disruption of cell membranes by Aβ has been hypothesized to be the important event associated with neurotoxicity in AD. Curcumin has been shown to reduce Aβ-induced toxicity; however, due to its low bioavailability, clinical trials showed no remarkable effect on cognitive function. As a result, GT863, a derivative of curcumin with higher bioavailability, was synthesized. The purpose of this study is to clarify the mechanism of the protective action of GT863 against the neurotoxicity of highly toxic Aβ oligomers (Aβo), which include high-molecular-weight (HMW) Aβo, mainly composed of protofibrils in human neuroblastoma SH-SY5Y cells, focusing on the cell membrane. The effect of GT863 (1 μM) on Aβo-induced membrane damage was assessed by phospholipid peroxidation of the membrane, membrane fluidity, membrane phase state, membrane potential, membrane resistance, and changes in intracellular Ca2+ ([Ca2+]i). GT863 inhibited the Aβo-induced increase in plasma-membrane phospholipid peroxidation, decreased membrane fluidity and resistance, and decreased excessive [Ca2+]i influx, showing cytoprotective effects. The effects of GT863 on cell membranes may contribute in part to its neuroprotective effects against Aβo-induced toxicity. GT863 may be developed as a prophylactic agent for AD by targeting inhibition of membrane disruption caused by Aβo exposure.

Methods for stable multimeric molecular dynamics simulation and modeling of cardiac SK2 channel function.

Small conductance Ca2+-activated K+ (SK) channels are unique since they are gated solely by changes in intracellular Ca2+ and are known to be upregulated in heart failure. SK channels are regulated via the ubiquitous Ca2+ sensing protein, calmodulin (CaM). Currently there are no structures for SK1-3 channels bound to CaM, thus, significant need for a method to generate stable SK channel models to assess conduction and membrane interactions exists. Recent cryo-EM structures with nearly complete tetrameric SK4-CaM complex (PDB: 6CNM, 6CNN, 6CNO) allows the generation of nearly full-length SK2-CaM homology models (SK2-CaM). Molecular dynamic simulations (MD) performed on SK2-CaM models generated with established methods, however, critical loops and subunits were unstable due to multimeric and size of complex. We have developed a method for each state of SK4-CaM to be used as an input with output being 1μs+ stable MD simulation ofSK2-CaM complex. This process includes many novel approaches using Rosetta, NAMD, and Amber along with specific regional restraints during specific steps; the procedure can differ for various states. MD designed to assess stability and conduction of the relevant SK4-CaM PDBs listed above and comparing results to the similar MD simulations of the final SK2-CaM models, we propose potential mechanisms for ion conduction in SK2 and SK4. In addition, basic residue clustering was detected near the lower leaflet headgroup of the membrane, implying anionic lipid binding locations. SK2-CaM was also stable in all 3 tested states with POPC membrane and varying concentrations of anionic lipids, allowing for the exploration of binding/interacting mechanisms. Using our expertise on the SK2-CaM complex as a template, we propose a new protocol that can be used to create stable MD simulations of homology models for SK1-3 using SK4 as an input.

Calcium inhibits the funny current (If)in sinoatrial myocytes.

Hyperpolarization-activated cyclic nucleotide sensitive (HCN4) channels are the molecular correlate of the funny current (If), which is required for spontaneous action potentials in cardiac pacemaker cells. Activation of β-adrenergic receptors on pacemaker cells increases cAMP, which shifts HCN4 activation to more depolarized potentials. The resulting increase in current, along with activation of Ca2+ cycling proteins, increases action potential firing rate and, thus, heart rate. Here we show that Mg2+ and Ca2+ regulate the effects of cAMP on If and HCN4. We found that physiological concentrations of intracellular Ca2+ inhibited cAMP-dependent potentiation of native If channels in excised membrane patches from sinoatrial node myocytes (IC50 ≈ 9 μM). This inhibition was recapitulated in heterologously expressed HCN4 channels, where intracellular Ca2+ inhibited the channels with a similar IC50 (≈ 5 μM) and limited the cAMP-dependent shift in channel activation (4 mV in 100 µM Ca2+ vs. 12 mV in zero Ca2+). Interestingly, Mg2+ had opposing effects to Ca2+. Mg2+ has been shown to interact with cAMP-bound HCN4 channels at a metal coordination site composed of two residues in the C-linker and two residues in the S4-S5 linker. We found that Mg2+ is also required to functionally couple the inhibitory, unbound CNBD to the pore in the absence of cAMP — removal of Mg2+ mimicked the effects of cAMP-dependent disinhibition. Although the metal coordination site is conserved among mammalian HCN channel isoforms, Mg2+ and Ca2+ regulation appear to be specific for HCN4 — cAMP regulation of HCN2 was insensitive to divalents. These results suggest that divalent cations directly regulate HCN4, raising the possibility that If in pacemaker cells is dynamically regulated in response to beat-to-beat changes in intracellular Ca2+.

The Novel Cardiac Myosin Activator Danicamtiv Improves Cardiac Systolic Function at the Expense of Diastolic Dysfunction In Vitro and In Vivo: Implications for Clinical Applications.

Recent cardiotropic drug developments have focused on cardiac myofilaments. Danicamtiv, the second direct myosin activator, has achieved encouraging results in preclinical and clinical studies, thus implicating its potential applicability in the treatment of heart failure with reduced ejection fraction (HFrEF). Here, we analyzed the inotropic effects of danicamtiv in detail. To this end, changes in sarcomere length and intracellular Ca2+ levels were monitored in parallel, in enzymatically isolated canine cardiomyocytes, and detailed echocardiographic examinations were performed in anesthetized rats in the absence or presence of danicamtiv. The systolic and diastolic sarcomere lengths decreased; contraction and relaxation kinetics slowed down with increasing danicamtiv concentrations without changes in intracellular Ca2+ transients in vitro. Danicamtiv evoked remarkable increases in left ventricular ejection fraction and fractional shortening, also reflected by changes in systolic strain. Nevertheless, the systolic ejection time was significantly prolonged, the ratio of diastolic to systolic duration was reduced, and signs of diastolic dysfunction were also observed upon danicamtiv treatment in vivo. Taken together, danicamtiv improves cardiac systolic function, but it can also limit diastolic performance, especially at high drug concentrations.

Glial regulators of ions and solutes required for specific chemosensory functions in Caenorhabditis elegans.

Glia and accessory cells regulate the microenvironment around neurons and primary sensory cells. However, the impact of specific glial regulators of ions and solutes on functionally diverse primary cells is poorly understood. Here, we systemically investigate the requirement of ion channels and transporters enriched in Caenorhabditis elegans Amsh glia for the function of chemosensory neurons. Although Amsh glia ablated worms show reduced function of ASH, AWC, AWA, and ASE neurons, we show that the loss of glial enriched ion channels and transporters impacts these neurons differently, with nociceptor ASH being the most affected. Furthermore, our analysis underscores the importance of K+, Cl-, and nucleoside homeostasis in the Amphid sensory organ and uncovers the contribution of glial genes implicated in neurological disorders. Our findings build a unique fingerprint of each glial enriched ion channel and transporter and may provide insights into the function of supporting cells of mammalian sensory organs.

Isorhamnetin protects zearalenone-induced damage via the PI3K/Akt signaling pathway in porcine ovarian granulosa cells.

Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin has extensive pharmacological activity. However, the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear. This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism. Two experiments were conducted, where a minimum of 3 biological replicates were used for each treatment. In Exp. 1, ovarian granulosa cells were treated with different concentrations of isorhamnetin (1, 5, 10, 20 and 30μmol/L) and ZEA (0, 10, 30, 60, 90 and 120μmol/L) for 24h. Our results indicated that 60μmol/L ZEA (half-maximal inhibitory concentration value) and 20μmol/L isorhamnetin (the most effective concentration against ZEA-induced cytotoxicity) were optimum concentrations. In Exp. 2, ovarian granulosa cells were treated with isorhamnetin (20μmol/L) for 2h, before treatment with ZEA (60μmol/L) for 24h. Apoptosis, endoplasmic reticulum stress, oxidative stress, proliferation and hormone secretion of ovarian granulosa cells were detected. Our findings showed that isorhamnetin suppressed (P<0.05) ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins (B-cell lymphoma-2 [Bcl-2], Bcl2-associated x [Bax] and cleaved caspase-3 [C-Casp3]). Changes in intracellular Ca2+ levels and C/EBP homologous protein (CHOP), recombinant activating transcription factor 6 (ATF6), glucose regulated protein78 kD (GRP78) indicated that isorhamnetin rescued (P<0.05) ZEA-induced endoplasmic reticulum stress. Furthermore, isorhamnetin prevented (P<0.05) ZEA-induced oxidative stress via the mitogen-activated protein kinase (P38) signaling pathway. Mechanistically, isorhamnetin stimulated (P<0.05) the expression of proliferating cell nuclear antigen (PCNA) and cyclin D, thereby increasing the ratio of S phase cells in response to ZEA-induced apoptosis via phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. Isorhamnetin also recovered (P<0.05) ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (follicle-stimulating hormone receptor [FSHR] and cytochrome P450 family 19 subfamily a member 1 [CYP19A1]). Collectively, these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage, which promotes proliferation, alleviates apoptosis, endoplasmic reticulum stress, oxidative stress, and steroidogenesis disorder.

Establishment of a CaCC-based Cell Model and Method for High-throughput Screening of M3 Receptor Drugs.

Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, most M3 receptor drugs also have high affinity for other subtypes of muscarinic acetylcholine receptors (mAChRs) and produce the risk of side effects. Therefore, in order to find M3 receptor drugs with high specificity, high activity and low side effects, we established a cell model and method for efficient and sensitive screening of M3 receptor based on calcium-activated chloride channels (CaCCs), and this method is also suitable for the screening of other GPCR drugs. This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We verified that the model can sensitively detect changes in intracellular Ca2+ concentration using fluorescence quenching kinetics experiments, confirmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity.

Obesity is an important health problem, which can be prevented through appetite control. Ghrelin is an appetite-stimulating hormone considered to promote obesity. Thus, we examined whether gastric stretching affects ghrelin secretion. We investigated the role of transient receptor potential vanilloid 4 (TRPV4) in gastric glands in the regulation of ghrelin secretion. TRPV4 immunostaining was performed in tissue samples from 57 patients who underwent gastrectomy. TRPV4 expression was compared between patients with (body mass index (BMI) ≥ 30) and without (BMI <30) obesity. For in vitro experiments, we used MGN3-1 cells, a ghrelin-producing cell line derived from mice. To investigate the bioactivity of TRPV4, MGN3-1 cells were treated with TRPV4 agonists and antagonists, and changes in intracellular Ca2+ concentration were confirmed. The concentration of ghrelin in the cell supernatant was measured using the ELISA with and without 120% stretch stimulation. TRPV4 expression was significantly higher in patients with obesity than in those without at all sites, except the fornix. Immunostaining confirmed the expression of TRPV4 in MGN3-1 cells. TRPV4 agonist administration increased intracellular Ca2+ concentration and ghrelin secretion in MGN3-1 cells, whereas the administration of the agonist combined with the antagonist decreased intracellular Ca2+ concentration and ghrelin secretion. Ghrelin secretion significantly increased in response to a 120% stretch in MGN3-1 cells. However, secretion was not increased by stretch when cells were treated with a TRPV4 antagonist. TRPV4 regulates ghrelin secretion in response to stretch in the stomach, which may affect body weight.