Changes In Intracellular Calcium Levels Research Articles

Calcium signaling in astrocytes and gliotransmitter release.

Glia are as numerous in the brain as neurons and widely known to serve supportive roles such as structural scaffolding, extracellular ionic and neurotransmitter homeostasis, and metabolic support. However, over the past two decades, several lines of evidence indicate that astrocytes, which are a type of glia, play active roles in neural information processing. Astrocytes, although not electrically active, can exhibit a form of excitability by dynamic changes in intracellular calcium levels. They sense synaptic activity and release neuroactive substances, named gliotransmitters, that modulate neuronal activity and synaptic transmission in several brain areas, thus impacting animal behavior. This "dialogue" between astrocytes and neurons is embodied in the concept of the tripartite synapse that includes astrocytes as integral elements of synaptic function. Here, we review the recent work and discuss how astrocytes via calcium-mediated excitability modulate synaptic information processing at various spatial and time scales.

Activation of the human TRPA1 channel by different alkylating sulfur and nitrogen mustards and structurally related chemotherapeutic drugs

An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.

The glycocalyx and calcium dynamics in endothelial cells.

The endothelial glycocalyx is a dynamic surface layer composed of proteoglycans, glycoproteins, and glycosaminoglycans with a key role in maintaining endothelial cell homeostasis. Its functions include the regulation of endothelial barrier permeability and stability, the transduction of mechanical forces from the vascular lumen to the vessel walls, serving as a binding site to multiple growth factors and vasoactive agents, and mediating the binding of platelets and the migration of leukocytes during an inflammatory response. Many of these processes are associated with changes in intracellular calcium levels that may occur through mechanisms that alter calcium entry in the endothelium or the release of calcium from the endoplasmic reticulum. Whether the endothelial glycocalyx can regulate calcium dynamics in endothelial cells is unresolved. Interestingly, during cardiovascular disease progression, changes in calcium dynamics are observed in association with the degradation of the glycocalyx and with changes in barrier permeability and vascular reactivity. Herein, we aim to provide a summarized overview of what is known regarding the role of the glycocalyx as a regulator of endothelial barrier and vascular reactivity during homeostatic and pathological conditions and to provide a perspective on how such processes may relate to calcium dynamics in endothelial cells, exploring a possible connection between components of the glycocalyx and calcium-sensitive pathways in the endothelium.

OR2AT4 and OR1A2 counterregulate molecular pathophysiological processes of steroid-resistant inflammatory lung diseases in human alveolar macrophages

BackgroundTherapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets.MethodsHuman primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry.ResultsWe demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines.ConclusionThese are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.

Fear conditioning in invertebrates.

Learning to identify and predict threats is a basic skill that allows animals to avoid harm. Studies in invertebrates like Aplysia californica, Drosophila melanogaster, and Caenorhabditis elegans have revealed that the basic mechanisms of learning and memory are conserved. We will summarize these studies and highlight the common pathways and mechanisms in invertebrate fear-associated behavioral changes. Fear conditioning studies utilizing electric shock in Aplysia and Drosophila have demonstrated that serotonin or dopamine are typically involved in relaying aversive stimuli, leading to changes in intracellular calcium levels and increased presynaptic neurotransmitter release and short-term changes in behavior. Long-term changes in behavior typically require multiple, spaced trials, and involve changes in gene expression. C. elegans studies have demonstrated these basic aversive learning principles as well; however, fear conditioning has yet to be explicitly demonstrated in this model due to stimulus choice. Because predator-prey relationships can be used to study learned fear in a naturalistic context, this review also summarizes what is known about predator-induced behaviors in these three organisms, and their potential applications for future investigations into fear conditioning.

Transcriptional Genetically Encoded Calcium Indicators in Drosophila.

Knowing which neurons are active during behavior is a crucial step toward understanding how nervous systems work. Neuronal activation is generally accompanied by an increase in intracellular calcium levels. Therefore, intracellular calcium levels are widely used as a proxy for neuronal activity. Many types of synthetic components and bioluminescent or fluorescent proteins that report transient and long-term changes in intracellular calcium levels have been developed over the past 60 years. Calcium indicators that enable imaging of the dynamic activity of a large ensemble of neurons in behaving animals have revolutionized the field of neuroscience. Among these, transcription-based genetically encoded calcium indicators (transcriptional GECIs) have proven easy to use and do not depend on sophisticated imaging systems, offering unique advantages over other types of calcium indicators. Here, we describe the two currently available fly transcriptional GECIs-calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC)-and review studies that have used them. In the accompanying protocol, we present step-by-step details for generating CaLexA- and TRIC-ready flies and for imaging CaLexA and TRIC signals in dissected brains after experimental manipulations of intact free-moving flies.

FC026: Chronic High Phosphate Level Impairs Cardiac Health

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) suffer from disturbed mineral metabolism in which the chronic phosphate load leads to the elevation of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). Persistent hyperphosphatemia predisposes to cardiovascular events and increased morbidity and mortality rates. It is discussed that even in the general population, consumption of highly processed food increases phosphate level, which in turn may increase cardiovascular risk. Here, we aim to elucidate the pathological mechanisms of how chronic high phosphate intake mediates cardiac remodeling and dysfunction, respectively. METHOD Male C57BL/6 wild type mice received a high phosphate diet containing 2% inorganic phosphate (HPD) or a 0.8% normal phosphate diet (NPD) for 6 months. Echocardiographic analysis and ex-vivo measurements in cardiomyocytes using IonOptix system were assessed to evaluated cardiac function and contractility. Bulk RNA sequencing of whole heart tissue followed by pathway analysis was done to identify affected relevant signaling pathways, which were verified by in vitro studies using neonatal rat ventricular myocytes (NRVM). RESULTS Chronic phosphate overload resulted in increased level of serum and urinary phosphate and high plasma PTH and FGF23 concentrations. HPD caused impaired heart function as shown by reduced ejection fraction, increased end-systolic volumes and enlarged systolic left ventricular internal diameter accompanied by impaired left ventricular relaxation as seen in Doppler echocardiography. Histological analysis showed increased cardiomyocyte area and length in HPD-fed mice compared with NPD demonstrating cellular hypertrophy. RNA sequencing revealed differentially expressed genes of calcium signaling pathways in the HPD group. Besides, immunoblot analysis of whole heart tissue showed altered Phospholamban activity after 6 months of HPD compared with NPD, indicating altered calcium pump in cardiomyocytes. Preliminary analysis of sarcomere movements and changes of intracellular calcium levels in single cardiomyocytes isolated from NPD and HPD mice after 6 months suggested impaired contractility. Detailed results will be presented here. In vitro stimulation of isolated NRVM with high phosphate induced a hypertrophic growth response, verifying a direct effect of high phosphate on the heart. CONCLUSION HPD in mice causes a dilated cardiac phenotype including cellular hypertrophy, altered contractility and differentially expressed genes involved in calcium signaling pathways, indicating that a chronic high phosphate load is harmful for the heart also in the general population.

Noncanonical necrosis in 2 different cell types in a Caenorhabditis elegans NAD+ salvage pathway mutant.

Necrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1 NAD+ salvage deficient Caenorhabditis elegans model excess nicotinamide induces excitotoxic death in uterine-vulval uv1 cells and OLQ mechanosensory neurons. We sought to characterize necrosis in our pnc-1 model in the context of well-characterized necrosis, apoptosis, and autophagy pathways in C. elegans. We confirmed that calpain and aspartic proteases were required for uv1 necrosis, but changes in intracellular calcium levels and autophagy were not, suggesting that uv1 necrosis occurs by a pathway that diverges from mec-4d-induced touch cell necrosis downstream of effector aspartic proteases. OLQ necrosis does not require changes in intracellular calcium, the function of calpain or aspartic proteases, or autophagy. Instead, OLQ survival requires the function of calreticulin and calnexin, pro-apoptotic ced-4 (Apaf1), and genes involved in both autophagy and axon guidance. In addition, the partially OLQ-dependent gentle nose touch response decreased significantly in pnc-1 animals on poor quality food, further suggesting that uv1 and OLQ necrosis differ downstream of their common trigger. Together these results show that, although phenotypically very similar, uv1, OLQ, and touch cell necrosis are very different at the molecular level.

Withdrawn: Safranal induces autophagy by AMPK activation and protects neurons against amyloid beta in Alzheimer’s disease

This article has been withdrawn by the Editor
 Purpose: To investigate autophagic induction by safranal and neuroprotection against amyloid beta in Alzheimer’s disease. 
 Methods: Primary neurons and SH-SY5Y cells were used in this study. Assessment of cell proliferation and neuroprotection by safranal against amyloid beta was done by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. AMPK activation and mTOR inhibition were determined by western blot. Changes in intracellular calcium level, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were assessed by flow cytometry. 
 Results: Safranal protected neurons against amyloid beta toxicity. Furthermore, safranal activated AMPK pathway by activation of calcium/calmodulin-dependent protein kinase (CaMKKβ) to induce autophagy in both cell lines. The toxicity induced by amyloid beta in primary neurons and SH-SY5Y cells were attenuated by safranal. Moreover, amyloid beta-induced calcium levels were significantly decreased by safranal while ROS and MMP loss produced by amyloid beta was attenuated by safranal. 
 Conclusion: These findings suggest that safranal protects neurons against amyloid beta by inducing autophagy via AMPK pathway. Therefore, safranal is a probable therapeutic target for Alzheimer’s disease.

Decreased cardiac pacemaking and attenuated β-adrenergic response in TRIC-A knockout mice.

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ± 15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ± 5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a β-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to β-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.

Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux

BackgroundThe distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106–126)-induced neurotoxicity.MethodsWestern blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope.ResultsWe examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide.ConclusionOur data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases.3p5PCBHnn1RWkUoaQCoeATVideo

An evaluation of the activity of biologically synthesized silver nanoparticles against bacteria, fungi and mammalian cell lines

Biogenic silver nanoparticles are used for a number of applications due to their size, surface characteristics and strong antimicrobial properties. The present study aimed to investigate the synthesis of silver nanoparticles from the indigenous bacterial strain Bacillus sp. MB353 (PRJNA357966). Detailed characterization of silver nanoparticles was performed by UV–vis Spectrophotometer, FTIR, SEM and XRD. Biogenic silver nanoparticles were crystalline with average size 49−53 nm. These silver nanoparticles demonstrated good antibacterial activity against Gram-positive and Gram-negative bacteria (E. coli, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecium, Enterococcus faecalis and Streptomyces laurentii). The nanoparticles also showed excellent antifungal activity against Aspergillus niger, Aspergillus fumigatus, Fusarium soleni. The silver nanoparticles showed negligible antioxidant activity, but ROS generation pointed to a possible mode of antimicrobial activity. Incubation of silver nanoparticles with mammalian cell lines (Rhabdomyosarcomas and fibroblast) showed cell death and inhibition of proliferation. Cytotoxicity was most likely a result of ROS generation and changes in intracellular calcium levels. These findings suggested that biogenic silver nanoparticles could be used as alternative agents for biomedical purposes such as antibacterial and antifungal agents. However, given the effects on normal mammalian cells, it is probable that they could be used as anticancer agents if applied in targeted therapies.

Cadmium-Related Effects on Cellular Immunity Comprises Altered Metabolism in Earthworm Coelomocytes.

The heavy metal cadmium (Cd) is known to modulate the immune system, challenging soil-dwelling organisms where environmental Cd pollution is high. Since earthworms lack adaptive immunity, we determined Cd-related effects on coelomocytes, the cellular part of innate immunity, which is also the site of detoxification processes. A proteomics approach revealed a set of immunity-related proteins as well as gene products involved in energy metabolism changing in earthworms in response to Cd exposure. Based on these results, we conducted extracellular flux measurements of oxygen and acidification to reveal the effect of Cd on coelomocyte metabolism. We observed a significantly changing oxygen consumption rate, extracellular acidification, as well as metabolic potential, which can be defined as the response to an induced energy demand. Acute changes in intracellular calcium levels were also observed, indicating impaired coelomocyte activation. Lysosomes, the cell protein recycling center, and mitochondrial parameters did not change. Taken together, we were able to characterize coelomocyte metabolism to reveal a potential link to an impaired immune system upon Cd exposure.

P3561Detection and functional characterization of angiotensin receptor type 1 autoantibodies: establishment and clinical translation

Abstract Circulating AT1R autoantibodies (AT1R-aabs) directed against the ECL2 of the AT1R with agonist-like activity are supposed to play a pathophysiological role in diseases associated with vascular and renal damage, such as preeclampsia and severehypertension (HT), but they are also thought to be involved in heart failure and primary hyperaldosteronism (PHA). Methods High-throughput screening assays aiming at a reliable detection of AT1R-aabs in sera from patients with HT and PHA were established. The agonist-like activity of AT1R-aabs was assessed by changes in intracellular calcium-levels using Fura2-QBT dye; the AlphaLISA Assay was used to assess induction of ERK1/2-phosphorylation in stably transfected AT1R-HEK-cells or in adrenocortical NCI-H295R cells. Results IgG isolated from sera of n=60 patients with PHA and n=164 with HT were screened for their capacity to increase [Ca2+]i or to activate ERK1/2. Sixteen out of 60 PHA-patients increased [Ca2+]i compared to none of the HT-patients, whereas in both disease-entities we detected AT1R-aabs inducing ERK1/2-activation with a similar prevalence (PHA: 41%, HT: 42%), indicating the existence of differentially acting AT1R-aabs. PHA-patients positive for ERK1/2-activating AT1R-aabs have significantly lower serum potassium- (3,8±0,1 vs. 4,1±0,1 mmol/l, p<0,05) and renin-levels (2,7±0,5 vs. 4,5±0,7 ng/l, p<0,05) together with an increased aldosterone concentration (341±37 vs. 236±20 ng/l, p<0,01) concordant with the disease phenotype. Similarly, higher BP values are observed in AT1R-aab positive HT-patients (syst/diast: 148/85 vs. 167/93 mmHg, p<0,0001) accompanied byhigher aldosteroneserum-levels (93±7 vs. 74±3 ng/l, p<0,05). In addition, ERK1/2-activation induced by either angiotensin II or by IgG isolated from patients with PHA or HT could be differentially blocked by the use of various signaling inhibitors. In order to elucidate if stimulating AT1R-aabs could be involved in an over-secretion of aldosterone due to sustained receptor-activation, we investigatedtheir effects on NCI-H295R-cells. At the transcriptional level, AT1R-aabs were able to induce a time-dependent upregulation of the key steroidogenic enzymes involved in aldosterone biosynthesis CYP21A1-, HSD3B2-, CYP11B1-, and in particular CYP11B2-mRNA (2fold over basal), with the maximum level achieved after 8 to 12 hours. Concordant withan agonist-stimulated internalization of AT1R,AT1R-mRNA was downregulated by AT1R-aabs (up to 25% of basal) providing direct evidence of a chronic receptor-stimulation by AT1R-aabs. Conclusion Functional assays based on AT1R-activation (Ca2+ measurements & ERK1/2-phosphorylation) are able to detect AT1R-aabs in 41% or 42% of patients with HT or PHA, respectively. Moreover, our data provide evidence that AT1R-aabs stabilize a specific AT1R-conformation distinct from that induced by angiotensin II thereby triggering a different intracellular signaling pattern resulting in chronic aldosterone production. Acknowledgement/Funding BMBF grant

Mouse transient receptor potential channel type 6 selectively regulates agonist-induced platelet function

While changes in intracellular calcium levels is a central step in platelet activation and thrombus formation, the contribution and mechanism of receptor-operated calcium entry (ROCE) via transient receptor potential channels (TRPCs) in platelets remains poorly defined. In previous studies, we have shown that TRPC6 regulates hemostasis and thrombosis, in mice. In the present studies, we employed a knockout mouse model system to characterize the role of TRPC6 in ROCE and platelet activation. It was observed that the TRPC6 deletion (Trpc6−/−) platelets displayed impaired elevation of intracellular calcium, i.e., defective ROCE. Moreover, these platelets also exhibited defects in a host of functional responses, namely aggregation, granule secretion, and integrin αIIbβ3. Interestingly, the aforementioned defects were specific to the thromboxane receptor (TPR), as no impaired responses were observed in response to ADP or the thrombin receptor-activating peptide 4 (TRAP4). The defect in ROCE in the Trpc6−/− was also observed with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Finally, our studies also revealed that TRPC6 regulates clot retraction. Taken together, our findings demonstrate that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Thus, TRPC6 may serve as a novel target for the therapeutic management of thrombotic diseases.

The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4-/-Rdh8-/-), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.

Calcium signalling and related ion channels in neutrophil recruitment and function.

The recruitment of neutrophils to sites of inflammation, their battle against invading microorganisms through phagocytosis and the release of antimicrobial agents is a highly coordinated and tightly regulated process that involves the interplay of many different receptors, ion channels and signalling pathways. Changes in intracellular calcium levels, caused by cytosolic Ca2+ store depletion and the influx of extracellular Ca2+ via ion channels, play a critical role in synchronizing neutrophil activation and function. In this review, we provide an overview of how Ca2+ signalling is initiated in neutrophils and how changes in intracellular Ca2+ levels modulate neutrophil function.

Morphological and molecular effects of phenolic extract from coconut kernel on human prostate cancer cell growth in vitro

Coconut is an indispensable ingredient in the diet and traditional medicine of individuals belonging to the Indian subcontinent. Coconut is of high nutritional value owing to the presence of all essential dietary components, viz, saturated fatty acids, arginine rich proteins, fibre and minor components like vitamin E, phytosterols, polyphenols and flavonoids. The polyphenolic content present in coconut kernel is of particular interest due to their numerous reported beneficial effects such as reduction of oxidative stress, combating cancer and in modulating anti-inflammatory pathways. Therefore, in the present study the cytotoxic effect of the polyphenol rich fraction from coconut kernel (CKf) was evaluated in human prostate cancer (DU-145) cells. Individual components present in CKf was determined by LC-MS analysis. It showed that CKf contained several bioactive molecules which have potential anticancer activity viz, coumaric acid, myristin, chlorogenic acid and triterpenoid methyl esters. The cytotoxic effect of CKf at various concentrations (2.5–20 μg/ml) on DU-145 was assessed using MTT assay, AO/EB staining, mitochondrial superoxide/ROS production and changes in intracellular calcium levels, 24 hrs post treatment. Changes in the cell morphology and nucleus were observed using Scanning Electron Microscopy and Confocal microscopy. ROS and mitochondrial superoxide levels was evaluated using DCHF-DA and MitoSOX staining respectively. The impact of ROS on changes in cellular calcium levels was also studied using Fura-2-AM. LDH leakage from C K f treated and control cells were observed colorimetrically. Further, PCR analysis was done to detect changes in mitochondria associated apoptotic gene expression. It was also observed that C K f treatment increased the expression of pro-apoptotic genes - Bax, Bid, Bak and p53 in a dose-dependent manner. Based on the above results, it can be concluded that C K f may be used as a part of a dietary regime for controlling the progression of prostate cancer.

Mechanism of adrenomedullin 2/intermedin mediated vasorelaxation in rat main pulmonary artery

Adrenomedullin 2/intermedin (AM2/IMD) is a member of calcitonin related gene peptide family and an important nitric oxide mediated vasorelaxant in various vascular beds. However, the mechanism of post receptor-interaction is not clear and may differ depending on tissue type and species. In this study, we aimed to investigate the exact mechanism and the role of BKCa and calcium channels on the vasorelaxant effect of AM2/IMD in rat PA. Changes in the AM2/IMD-mediated vasorelaxation were evaluated in the presence of various inhibitors. CGRP(8-37) (10−6 M), L-NAME (10−4 M), ODQ (10−5 M), SQ22536 (10−4 M), H89 (10−6 M), TEA (10−2 M), iberiotoxin (3 × 10−7 M), and verapamil (10−5 M), all partly or completely inhibited the vasorelaxation. The relaxation was also abolished by removal of the endothelium, or in KCl precontracted PAs. AM2/IMD did not elicit vasorelaxation in the Ca2+-free conditions. However, the vasorelaxation was not inhibited with AM(22-52) (10−6 M), 4-AP (3 × 10−3 M), glibenclamide (10−5 M), apamin (3 × 10−7 M), TRAM-34 (10−5 M), and La+3 (10−4 M). AM2/IMD −induced changes in intracellular calcium levels and isometric force were monitored simultaneously in fura-2-loaded, endothelium-intact PAs. The AM2/IMD-induced increase in intracellular Ca2+ concentration was inhibited in the presence of iberiotoxin and verapamil, whereas no change was observed with La3+ incubation. Our data suggest that the cAMP/PKA pathway is one of the important pathways AM2/IMD-induced vasorelaxation. AM2/IMD acts through activation of endothelial BKCa and subsequently causes hyperpolarization of the endothelial cell membrane. The hyperpolarization induces Ca2+ influx, which leads to NO production and subsequent vasorelaxation.

Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex

BackgroundExposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function.MethodsFcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes.ResultsFcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG.ConclusionsOur data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.