Cytoplasmic Calcium Levels Research Articles

Cyclic nucleotide-gated ion channel 6 is involved in extracellular ATP signaling and plant immunity.

Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)-localized receptor P2K1 (LecRK-I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca2+ ]cyt ), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca2+ ]cyt are unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca2+ ]cyt response to eATP. Positional cloning revealed that the mutation resided in the cngc6 gene, which encodes cyclic nucleotide-gated ion channel 6 (CNGC6). Mutation of the CNGC6 gene led to a notable decrease in the PM inward Ca2+ current in response to eATP. eATP-induced mitogen-activated protein kinase activation and gene expression were also significantly lower in cngc6 mutant plants. In addition, cngc6 mutant plants were also more susceptible to the bacterial pathogen Pseudomonas syringae. Taken together, our results indicate that CNGC6 plays a crucial role in mediating eATP-induced [Ca2+ ]cyt signaling, as well as plant immunity.

Plant Immune Memory in Systemic Tissue Does Not Involve Changes in Rapid Calcium Signaling.

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses.

Interleukin-37 inhibits inflammation activation and disease severity of PM2.5-induced airway hyperresponsiveness

We have shown in the past studies that fine particulate matter (PM2.5) exposure increases airway hyperresponsiveness and leads to lung inflammation damage. Interleukin (IL)-37 plays a inhibitory role in inflammation activation and maintenance. However, the function of IL-37 in the above processes keep unclear. We aim to explore the role of IL-37 in PM2.5-induced airway hyperresponsiveness in this study. A nose-only PM2.5 online concentration, enrichment and exposure instrument was also applied to generate mice model of airway hyperresponsiveness. A transgenic mice strain using a CMV promoter to express human IL-37b (hIL-37tg) was obtained. PM2.5 exposure was shown to increase airway resistance, followed by lung inflammation and IL-1β, TNFα, and IL-6 release, which was inhibited by IL-37tg mice and mice administrated recombinant human IL-37 intranasally (i.n). Moreover, expression of the proliferation-related protein PCNA and migration-related proteins MMP-2, MMP-9, and Vimentin was reduced in lung tissues of IL-37tg mice and mice given recombinant human IL-37 i.n. Abnormal cell contraction, proliferation, and migration of human airway smooth muscle cells (hASMCs) incubated with PM2.5 were also decreased by IL-37 treatment. In addition, IL-37 intervention of hASMCs before PM2.5 incubation decreased cytoplasmic calcium level and expression of PCNA, MMP-2, MMP-9 and Vimentin. Finally, knockdown of the IL-37 receptor IL-1R8 gene eliminated the protective effects of IL-37 in the above responses. We conclude that IL-37 inhibits inflammation activation and disease severity of airway hyperreactivity by PM2.5 induction.

Cardioprotective mechanisms of sodium-glucose cotransporter 2 inhibitors

The findings of large-scale cardiovascular outcome trials have been demonstrated that sodium-glucose cotransporter 2 ­inhibitors (iSGLT-2) have shown beneficial cardiovascular effects. In this review proposed mechanisms underlying iSGLT-2-associated cardiovascular benefits have been discussed: haemodynamic and intracellular effects, including metabolic effects and electrolyte changes; and also, the effect on markers of cardiovascular disease (CVD). The hemodynamic effects of SGLT-2 are characterized by reduction of cardiac preload and afterload as a result of osmotic diuresis, a decrease in blood pressure and arterial stiffness. The metabolic effects of this medicine are accompanied by an increase the production of ketone bodies, followed by improving ATP production and myocardial energetics. Also, iSGLT-2 modulate ion transporters (NHE1 and NHE3). A reduction of cytoplasmic sodium and calcium levels and increasing mitochondrial calcium levels in the cardiomyocytes enhances the synthesis of ATP and increases cell viability. Effect of iSGLT-2 on CVD markers showed a decrease in the levels of the N-terminal pro-B-type natriuretic peptide and highly sensitive troponin I in elderly patients with type 2 diabetes mellitus (T2DM). Thus, this class of agents has a multifactorial effect on the functioning of cardiovascular system. Further studies will help to explain the all possible cardioprotective effects of iSGLT-2 in individuals with and without T2DM.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Oxidative Stress and Male Fertility: Role of Antioxidants and Inositols.

Infertility is defined as a couple’s inability to conceive after at least one year of regular unprotected intercourse. This condition has become a global health problem affecting approximately 187 million couples worldwide and about half of the cases are attributable to male factors. Oxidative stress is a common reason for several conditions associated with male infertility. High levels of reactive oxygen species (ROS) impair sperm quality by decreasing motility and increasing the oxidation of DNA, of protein and of lipids. Multi-antioxidant supplementation is considered effective for male fertility parameters due to the synergistic effects of antioxidants. Most of them act by decreasing ROS concentration, thus improving sperm quality. In addition, other natural molecules, myo-inositol (MI) and d-chiro–inositol (DCI), ameliorate sperm quality. In sperm cells, MI is involved in many transduction mechanisms that regulate cytoplasmic calcium levels, capacitation and mitochondrial function. On the other hand, DCI is involved in the downregulation of steroidogenic enzyme aromatase, which produces testosterone. In this review, we analyze the processes involving oxidative stress in male fertility and the mechanisms of action of different molecules.

Mibefradil reduces hepatic glucose output in HepG2 cells via Ca2+/calmodulin-dependent protein kinase II-dependent Akt/forkhead box O1signaling

The effects and underlying mechanisms of mibefradil on gluconeogenesis and glycogenesis were investigated using insulin-resistant HepG2 human hepatocellular carcinoma cells and a mouse model of type 2 diabetes mellitus (T2DM). HepG2 cells were divided into one of four groups: control, palmitate (PA)-induced insulin-resistance (0.25 mM), low-concentration mibefradil (0.025 μM), or high-concentration mibefradil (0.05 μM). Glycogen synthesis and glucose consumption were evaluated in these HepG2 cells, and quantitative polymerase chain reaction (qPCR) and western blotting techniques were used to detect expression of forkhead box O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase). Intracellular calcium concentrations were determined using Fluo-4 AM, and phosphorylation levels of calmodulin-dependent protein kinase II (CaMKII), protein kinase B (Akt) and FoxO1were detected by western blotting. Immunofluorescence was used for the localization and quantification of FoxO1.In vitro results were verified using a mouse model of T2DM. In HepG2 cells and mouse liver tissues, mibefradil decreased PA-induced cytoplasmic calcium levels and CaMKII phosphorylation, but increased the phosphorylation of Akt and FoxO1, thereby contributing to the cytoplasmic localization of FoxO1. Additionally, mibefradil alleviated PA-induced glucose output and insulin resistance through increased glucose consumption and glycogen synthesis, while decreasing the expression of key gluconeogenesis enzymes, including PEPCK and G6Pase. Mibefradil may help to control blood sugar levels by reducing glucose output and insulin resistance, and the mechanism of action may involve the Ca2+-CaMKII-dependent Akt/FoxO1 signaling pathway.

КАРДИОПРОТЕКТИВНЫЕ МЕХАНИЗМЫ ИНГИБИТОРОВ НАТРИЙ-ГЛЮКОЗНОГО КОТРАНСПОРТЕРА 2 ТИПА

The findings of large-scale cardiovascular outcome trials have been demonstrated that sodium-glucose cotransporter 2 ­inhibitors (iSGLT-2) have shown beneficial cardiovascular effects. In this review proposed mechanisms underlying iSGLT-2-associated cardiovascular benefits have been discussed: haemodynamic and intracellular effects, including metabolic effects and electrolyte changes; and also, the effect on markers of cardiovascular disease (CVD). The hemodynamic effects of SGLT-2 are characterized by reduction of cardiac preload and afterload as a result of osmotic diuresis, a decrease in blood pressure and arterial stiffness. The metabolic effects of this medicine are accompanied by an increase the production of ketone bodies, followed by improving ATP production and myocardial energetics. Also, iSGLT-2 modulate ion transporters (NHE1 and NHE3). A reduction of cytoplasmic sodium and calcium levels and increasing mitochondrial calcium levels in the cardiomyocytes enhances the synthesis of ATP and increases cell viability. Effect of iSGLT-2 on CVD markers showed a decrease in the levels of the N-terminal pro-B-type natriuretic peptide and highly sensitive troponin I in elderly patients with type 2 diabetes mellitus (T2DM). Thus, this class of agents has a multifactorial effect on the functioning of cardiovascular system. Further studies will help to explain the all possible cardioprotective effects of iSGLT-2 in individuals with and without T2DM.

Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.

Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.

Nicorandil attenuates high glucose-induced insulin resistance by suppressing oxidative stress-mediated ER stress PERK signaling pathway

IntroductionGlucose-induced insulin resistance is a typical character of diabetes. Nicorandil is now widely used in ischemic heart disease. Nicorandil shows protective effects against oxidative and endoplasmic reticulum (ER) stress, which...

Cortisol rapidly stimulates calcium waves in the developing trunk muscle of zebrafish

Glucocorticoids (GCs) play a role in stress coping by activating the glucocorticoid receptor (GR), a ligand-bound transcription factor. GCs also exert rapid effects that are nongenomic by modulating second messenger signaling, including Ca2+. However, the mechanism of action of GCs in modulating cytoplasmic free calcium level ([Ca2+]i) is unclear. We hypothesized that cortisol increases ([Ca2+]i) in zebrafish (Danio rerio) muscle, and this is independent of GR activation. Indeed, cortisol rapidly stimulated ([Ca2+]i) rise in the developing trunk muscle (DTM), and this response was not abolished in the GR knockout zebrafish. The rapid cortisol-induced ([Ca2+]i) rise was reduced with EGTA, and completely abolished by the pharmacological inhibition of the calcium release-activated calcium channel (CRACC). Also, cortisol stimulation rapidly increased the expression of Orai1, the pore forming protein subunit of CRACC, in the DTM. Altogether, rapid nongenomic action of cortisol on muscle function may involve Ca2+ signaling by CRACC gating in zebrafish.

Nicorandil Attenuates High Glucose-Induced Insulin Resistance by Suppressing Oxidative Stress-Mediated Endoplasmic Reticulum Stress Protein Kinase RNA-Like Endoplasmic Reticulum Kinase Singling Pathway

Background: Glucose- induced insulin resistance (IR) is a typical character of diabetes. Nicorandil is widely used in ischemic heart disease. Nicorandil shows protective effects against oxidative and endoplasmic reticulum (ER) stress which are involved in insulin resistance. Here we investigated mechanisms of nicorandil’s novel pharmacological activity on IR in diabetes. Methods: Nicorandil was administrated to diabetic animals and high-glucose exposed skeletal muscle cells. IR and glucose tolerance were evaluated. Molecular mechanisms concerning oxidative stress, ER stress and glucose uptake were assessed. Findings: Nicorandil attenuated high- glucose induced IR without affecting fasting blood glucose and glucose tolerance in whole body and skeletal muscle in diabetic rats. Nicorandil treatment suppressed protein kinase C(PKC)/ nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) system activities by reducing cytoplasmic free calcium level in skeletal muscle cells exposed to high glucose. As a result, the oxidative stress- mediated ER stress protein kinase RNA- like endoplasmic reticulum kinase (PERK)/ eukaryotic initiation factor (EIF) 2α/ activating transcription factor (ATF)4/CEBP homologous protein (CHOP)/tribbles homolog (TRB)3 signaling pathway activation was inhibited. Nicorandil down-regulated expression of TRB3 and thus facilitated Akt phosphorylation in response to insulin stimulation, leading to glucose transporter (GLUT)4 plasma membrane translocation which promoted glucose uptake capability of skeletal muscle cells. Interpretation: By reducing cytoplasmic calcium, nicorandil alleviated high-glucose induced IR by inhibiting oxidative stress- mediated ER stress PERK pathway. This study indicated nicorandil as a promising agent in treatment of patients with ischemic heart disease complicated with diabetes. Funding Statement: This study was supported by Innovative Talents Promotion Project of Shaanxi Province (2019KJXX-019); Fundamental Scientific Research Foundation of Xi’an Jiaotong University (xzy012019131); Health Scientific Foundation of Shaanxi Province (2018E11); Natural Science Basic Research Foundation of Shaanxi Province (2020JQ-941). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The experimental protocols were reviewed and approved by Institutional Animal Care and Use Committee of Medical Research Institute of Northwestern Polytechnical University.

Calmodulin Binding to Connexin 35: Specializations to Function as an Electrical Synapse.

Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium.

2094-P: Effects of Irisin on Human Pancreatic Islets from Type 2 Diabetic Subjects

Irisin is a hormone secreted by skeletal muscle following physical activity and excess of saturated fatty acids, able to improve metabolic homeostasis and promote energy expenditure. In a previous study, we have demonstrated that, in beta-cells, irisin promotes proliferation and glucose-stimulated insulin secretion (GSIS), increases insulin content levels, and reduces lipotoxicity-induced apoptosis. The aim of this study was to evaluate the effects of irisin on the function and survival of pancreatic islets isolated from patients with type 2 diabetes mellitus (T2DM), characterized by reduced ability to secrete insulin and high levels of apoptosis, and to investigate the molecular mechanisms underlying this action. Pancreatic islets isolated from T2DM patients (n=7) and nondiabetic subjects (ND) (n=3), as well as INS-1E cells, were exposed to 100 nM irisin for different times. GSIS, insulin content, apoptosis and cytoplasmic calcium levels were measured by ELISA assays; the activation of the main mediators of beta-cell intracellular signalling (AKT, CREB and PKA) was evaluated by immunoblotting. Irisin increased GSIS approximately 2-fold (p<0.05) in both ND and T2DM islets. Notably, in T2DM islets, characterized by reduced GSIS, this was restored to the levels of ND islets. In addition, irisin increased insulin content (1.6-fold in ND, 1.9-fold in T2DM, p<0.05) and tendentially decreased apoptosis levels only in T2DM islets. Moreover, irisin was able to activate AKT and CREB under conditions of high glucose in the ND islets, without changing cytoplasmic calcium levels. In contrast, in the T2DM islets, irisin did not induce AKT or CREB activation. In conclusion, the myokine irisin is able to increase the secretory function and survival of human pancreatic islets and to improve the functional defects typical of the T2DM islets. The mechanisms underlying the secretagogue effects of irisin in the T2DM islets do not apparently involve the known signaling machinery of this novel hormone. Disclosure N. Marrano: None. A. Natalicchio: Consultant; Self; AstraZeneca, Novo Nordisk Inc., Sanofi-Aventis. G. Biondi: None. A. Cignarelli: None. L. Vincenti: None. L.G. Lupo: None. S. Perrini: None. L. Laviola: Advisory Panel; Self; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis. Speaker’s Bureau; Self; Abbott, Medtronic. F. Giorgino: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., LifeScan, Inc., MedImmune, Merck Sharp & Dohme Corp., Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Development Centre Europe Ltd. Funding European Union (AIM1810057)

Development of erianin-loaded dendritic mesoporous silica nanospheres with pro-apoptotic effects and enhanced topical delivery

BackgroundPsoriasis is a malignant skin disease characterized as keratinocyte hyperproliferation and aberrant differentiation. Our previous work reported that a bibenzyl compound, erianin, has a potent inhibitory effect on keratinocyte proliferation. To improve its poor water-solubility, increase anti- proliferation activity, and enhance the skin delivery, erianin loaded dendritic mesoporous silica nanospheres (E/DMSNs) were employed.ResultsIn this work, DMSNs with pore size of 3.5 nm (DMSN1) and 4.6 nm (DMSN2) were fabricated and E/DMSNs showed pore-size-dependent, significantly stronger anti-proliferative and pro-apoptotic effect than free erianin on human immortalized keratinocyte (HaCaT) cells, resulting from higher cellular uptake efficiency. In addition, compared to free erianin, treatment with E/DMSNs was more effective in reducing mitochondrial membrane potential and increasing cytoplasmic calcium levels, which were accompanied by regulation of mitochondria and endoplasmic reticulum stress (ERS) pathway. Porcine skin was utilized in the ex vivo accumulation and permeation studies, and the results indicated higher drug retention and less drug penetration in the skin when administered as the E/DMSNs-loaded hydrogel compared to the erianin-loaded hydrogel.ConlusionsThis work not only illustrated the further mechanisms of erianin in anti-proliferation of HaCaT cells but also offer a strategy to enhance the efficiency of erianin and the capacity of skin delivery through the DMSNs drug delivery systems.

Toward Decoding Bioelectric Events in Xenopus Embryogenesis: New Methodology for Tracking Interplay Between Calcium and Resting Potentials In Vivo

Although chemical signaling during embryogenesis is readily addressed by a plethora of available techniques, the developmental functions of ionic signaling are still poorly understood. It is increasingly realized that bioelectric events in nonneural cells are critical for pattern regulation, but their study has been hampered by difficulties in monitoring and manipulating them in vivo. Recent developments in visualizing electrical signaling dynamics in the field of neuroscience have facilitated functional experiments that reveal instructive developmental bioelectric signals. However, there is a pressing need for additional tools to explore time-dependent ionic signaling to understand complex endogenous dynamics. Here, we present methodological advances, including 4D imaging and data analysis, for improved tracking of calcium flux in the Xenopus laevis embryo, lowering the barrier for in vivo physiology work in this important model system. Using these techniques, we investigated the relationship between bioelectric ion channel activity and calcium, finding that cell hyperpolarization and depolarization both induce persistent static elevation of cytoplasmic calcium levels that fade over developmental time. These calcium changes correlate with increased cell mobility in early embryos and abnormal craniofacial morphology in later embryos. We thus highlight membrane potential modulation as a tractable tool for modulation of signaling cascades that rely on calcium as a transduction mechanism. The methods we describe facilitate the study of important novel aspects of developmental physiology, are extendable to numerous classes of existing and forthcoming fluorescent physiological reporters, and establish highly accessible, inexpensive protocols for their investigation.

Alpha-asarone improves cognitive function of aged rats by alleviating neuronal excitotoxicity via GABAA receptors

Alzheimer's disease (AD), the most common form of dementia, still lacks effective treatment at present. Alpha-asarone (ASA) is the major compound isolated from the Chinese medicinal herb Acorus gramineus. It has been reported to enhance cognitive function in rodent models, yet its mechanism was not fully understood. In this work, we demonstrated that ASA improved the spatial memory, reduced the neuronal injury, and decreased the level of Aβ1-42 in the hippocampus of aged rats. The results also showed that ASA had the neuroprotective effects against glutamate toxicity and decreased cytoplasmic calcium level in primary hippocampal neurons. By comparing the multiple properties of ASA and propofol (PPF) via computer modelling, we speculated that ASA may bind to the PPF binding site of type A gamma (γ)-aminobutyric acid receptors (GABAARs). This was further supported by the whole-cell patch-clamp recording. Our results suggested that ASA, as a GABAAR positive allosteric modulator (PAM), can improve cognitive function of aged rats by alleviating the neuronal overexcitation. Furthermore, the binding mode of ASA on GABAAR may lay a foundation for structure-based drug design in AD therapy.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

Investigation of the Protective Effects of Amlodipine on the Structure and Function of Testicular Tissue following Experimental Unilateral Cryptorchidism in Rats

Objective- Cryptorchidism, common birth defect of the male genital tract, is one of the causes of fertility problems. The elevation of tissue temperature following of cryptorchidism could induce oxidative stress which influences the cellular and tissue degeneration. Amlodipine is a third-generation of calcium channel blockers which has antioxidant activity. The aim of this study was to evaluate the protective effects of amlodipine on testicular tissue alterations in an animal model of cryptorchidism. Design- Experimental study Animals- Thirty adult male Sprague-Dawley rats weighing 200-220g Procedure- Experimental cryptorchidism was induced in adult rats. Amlodipine (10 mg/kg b.w.) was administrated orally for two and four consecutive weeks. The experimental groups consisted of non-treated cryptorchidism (n=10) and treated cryptorchidism (n=10) groups. Testicular tissue samples were collected on days 14 and 28 following of cryptorchidism form non-treated and treated groups. Histopathological and morphometrical studies with the evaluation of microscopic indices of spermatogenesis were prepared on tissue samples. Results- Tubular atrophy with germinal epithelium disarrangement was observed in cryptorchidism groups. These changes were reduced dose-dependently in treated animals. The mean of Sertoli cells was reduced significantly (p=0.025) in four weeks non-treated and the mean of germ cells lineage was reduced significantly (p<0.0001) in four weeks non-treated and two weeks treated cryptorchidism groups compared to the control group. Similarly, all microscopic indices of spermatogenesis were reduced following the induction of cryptorchidism. These alterations were reduced time-dependently in amlodipine treated groups. Conclusion and clinical relevance- The results of this study revealed that the administration of amlodipine as an antioxidant agent, time-dependently could be effective on the reduction of cellular and tissue damages of testicular tissue induced by cryptorchidism. It seems some parts of these protective effects may be done through its activity as calcium blocker which declines apoptotic processes by reduction of cytoplasmic calcium levels.

Effect of PEGylated Magnetic PLGA-PEI Nanoparticles on Primary Hippocampal Neurons: Reduced Nanoneurotoxicity and Enhanced Transfection Efficiency with Magnetofection

Despite broad application of nanotechnology in neuroscience, the nanoneurotoxicity of magnetic nanoparticles in primary hippocampal neurons remains poorly characterized. In particular, understanding how magnetic nanoparticles perturb neuronal calcium homeostasis is critical when considering magnetic nanoparticles as a nonviral vector for effective gene therapy in neuronal diseases. Here, we address the pressing need to systematically investigate the neurotoxicity of magnetic nanoparticles with different surface charges in primary hippocampal neurons. We found that unlike negative and neutral nanoparticles, positively charged magnetic nanoparticles (magnetic poly(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanoparticles, MNP-PLGA-PEI NPs) rapidly elevated cytoplasmic calcium levels in primary hippocampal neurons, mainly via extracellular calcium influx regulated by voltage-gated calcium channels. We went on to show that this perturbation of intracellular calcium homeostasis elicited serious cytotoxicity in primary hippocampal neurons. However, our next experiment demonstrated that PEGylation on the surface of MNP-PLGA-PEI NPs shielded the surface charge, thereby preventing the perturbation of intracellular calcium homeostasis. That is, PEGylated MNP-PLGA-PEI NPs reduced nanoneurotoxicity. Importantly, biocompatible PEGylated MNP-PLGA-PEI NPs under an external magnetic field enhanced transfection efficiency (>7%) of plasmid DNA encoding GFP in primary hippocampal neurons compared to NPs without external magnetic field mediation. Moreover, under an external magnetic field, this system achieved gene transfection in the hippocampus of the C57 mouse. Overall, this study is the first to successfully employ biocompatible PEGylated MNP-PLGA-PEI NPs for transfection using a magnetofection strategy in primary hippocampal neurons, thereby providing a nanoplatform as a new perspective for treating neuronal diseases or modulating neuron activities.