Intracellular Ca Concentration Research Articles

Substitution of bridge carbons for sulphur in calix[4]arene-bis-α-hydroxymethylphosphonic acid transformed mobile carrier into ionic channel accompanied with evoked muscle contraction and impaired neurotransmission powered by membrane action of resulting thiocalix[4]arene-bis-α-hydroxymethylphosphonic acid

The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode. Comparative analysis with the membrane action by calix[4]arene-bis-α-hydroxymethylphosphonic acid (C-99) has shown that the substitution of bridge carbons for sulphur and addition of another methyl group to two alkyl tales in the lower rim of former dipropoxycalix[4]arene C-99 transformed mobile carrier that C-99 created in lipid bilayer (Shatursky et al., 2014) into a transmembrane pore as exposure of the bilayer membrane to sulphur-containing derivative dibutoxythiocalix[4]arene C-1193 resulted in microscopic transmembrane current patterns indicative of a channel-like mode of facilitated diffusion. Within all calix[4]arenes tested a net steady-state voltage-dependent transmembrane current was readily achieved only after addition of calix[4]-arene C-1193. In comparison with the membrane action of C-99 the current induced by calix[4]-arene C-1193 exhibited a much weakened anion selectivity passing slightly more current at positive potentials applied from the side of bilayer membrane to which the calix[4]-arene was added. Testing C-1193 for the membrane action against smooth muscle cells of rat uterus or swine myometrium and synaptosomes of rat brain nerve terminals revealed an increase in intracellular concentration of Ca2+ with reduction of the effective hydrodynamic diameter of the smooth muscle cells and enhanced basal extracellular level of neurotransmitters (glutamate and γ-aminobutyric acid) after C-1193-induced depolarization of the nerve terminals.

Corydalis tomentella Franch. Exerts anti-inflammatory and analgesic effects by regulating the calcium signaling pathway

Ethnopharmacological relevanceCorydalis tomentella Franch. is a perennial cespitose plant commonly used to treat stomachaches as a folk medicine. The C. tomentella total alkaloids have good protective effects against acute liver injury and potential anti-hepatoma and anti-Alzheimer's disease activities. Aim of the studyTo establish an effective purification process for total alkaloids from C. tomentella and investigate the mechanism of their anti-inflammatory effects. Materials and methodsCorydalis tomentella were purified using macroporous resin. Then the crude and purified C. tomentella extracts (cCTE and pCTE) were qualitatively analyzed using UPLC-Triple-TOF-MS/MS. The cCTE and pCTE were used to investigate and compare their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW264.7 cells. Doses at 100, 200 and 400 mg/kg/d of pCTE were used to study their anti-inflammatory and analgesic activities in mice with xylene-induced ear swelling and acetic acid-induced writhing tests. Content of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were determined both in RAW264.7 cells and mice. Network pharmacology was used to predict the anti-inflammatory mechanism of C. tomentella, and the key enzymes were validated using qPCR and Western Blot analysis. Concentration of intracellular Ca2+ was detected using flow cytometric analysis. ResultsThe C. tomentella total alkaloid purity increased from 6.29% to 47.34% under optimal purification conditions. A total of 54 alkaloids were identified from CTE. Both cCTE and pCTE could suppress the LPS-induced production of NO, IL-6, IL-1β, and TNF-α in RAW264.7 cells. The pCTE exhibited a more potent anti-inflammatory effect; it also inhibited pain induced by xylene and acetic acid in mice. The calcium signaling pathway is associated with the anti-inflammatory and analgesic activities of C. tomentella. The mRNA expression of nitric oxide synthase (NOS) 2, NOS3 and calmodulin1 (CALM1) was regulated by C. tomentella through the reduction of inflammation-induced Ca2+ influx, and it also exhibited a more pronounced effect than the positive control (L-NG-nitro arginine methyl ester). ConclusionsPurified C. tomentella extract shows anti-inflammatory effect both in vitro and in vivo. It exerts anti-inflammatory and analgesic effects through the calcium signaling pathway by down-regulating NOS2 and CALM1 expression and up-regulating NOS3 expression in LPS-induced RAW264.7 cells, and decreasing intracellular Ca2+ concentration.

3-Iodothyronamine, a trace amine-associated receptor agonist, regulates intracellular Ca2+ increases via CaMK II through Epac2 in rat cerebral arterioles.

Trace amines (TAs) in the nervous system bind to TA-associated receptors (TAARs) and are involved in the regulation of monoaminergic functions. Among TAAR subtypes, TAAR1 has been implicated in the development of neurological disorders, such as schizophrenia. The present study investigated the effects of the TAAR1 agonist, 3-iodothyronamine (T1AM) on cerebral arterioles using fluctuations in the intracellular concentration of Ca2+ ([Ca2+]i) as an index of contractile responses. In cerebral arterioles, most of the TAAR agonists did not increase [Ca2+]i, while only T1AM elevated [Ca2+]i in vascular smooth muscle cells. This increase involved extracellular Ca2+ influx through T-type Ca2+ channels and inositol trisphosphate- and ryanodine-receptor-mediated Ca2+ release from intracellular stores. The inhibition of the cAMP sensor, exchange protein directly activated by cAMP (Epac) 2, and calmodulin kinase (CaMK) II strongly inhibited Ca2+ elevations. The present study revealed that T1AM acted not only on the TAAR1 receptor as previously suggested, but also on other G-protein coupled receptors and/or signal transduction systems to increase intracellular Ca2+ in cerebral arteriole smooth muscle cells. These results suggest that when using T1AM in clinical practice, attention should be paid to the early rise in blood pressure.

Synergistic effect of schizandrin A and DNase I knockdown on high glucose induced beta cell apoptosis by decreasing intracellular calcium concentration.

To explore the synergistic effect of deoxyribonuclease I (DNase I) knockdown combined with Schizandrin A (Sch A) in protecting islet beta-cells (β-cells) from apoptosis under high-glucose (HG) conditions. The concentration of Sch A was detected by Cell Counting Kit-8 (CCK-8). High glucose-cultured rat insulinoma beta cell line (RIN-M5F) cells were treated with Sch A and transfected with DNase I small interfering RNA (siRNA). Cell apoptosis rate and apoptosis-related protein level were examined by flow cytometry and Western blot method respectively. In addition, Na-K-adenosine triphosphatease (Na-K-ATPase) and Ca-Mg-ATPase activity, cell membrane potential, and intracellular Ca concentration was also examined respectively. Our study revealed that HG stimulation can cause a significant increase in DNase I level and cell apoptosis rate. However, Sch A combined with DNase I knockdown can significantly decrease the cell apoptosis rate and apoptosis-related protein levels such as BAX ( 0.05) and Caspase-3 ( 0.01). In addition, we also found that the combination of Sch A and DNase I knockdown can dramatically increase cell membrane potential level, Na-K-ATPase, and Ca-Mg-ATPase activity. Meanwhile, intracellular Ca concentration was also found to be significantly decreased by the synergistic effect of Sch A and DNase I knockdown. Overall, our study reveals a synergistic effect of Sch A and DNase I knockdown in protecting β-cells from HG-induced apoptosis.

DIFFERENT EXPERIMENTAL MODELS FOR HEPATOTOXICITY; A REVIEW A REVIEW

The Hepatic is midst the best industrious and pennon organs in the feasible body. Hepatotoxicity is a symbol intermediary of morbidity and lenity, and its extent is leave off increment appointment by steady old-fashioned in the industrialized nations. Hepatotoxicity is characterized by atomic pyknosis and eosinophilic cytoplasm, followed by copious rash hepatic poison, pudginess changes, lipid peroxidation leads to hepatic centrilobular necrosis. Paracetamol, reluctant tubercular drugs, demon rum, and azathioprine are meditate on to be the tricky venture factors implicated in the progression of hepatotoxicity. Unusual signaling mechanisms, such as activation of transmissible represent encode alike Kupffer cells, simple hew to pieces (NK) cells, and NKT, incendiary mediators, intracellular Ca2 concentration and reactive oxygen species are involved in the pathogenesis of hepatotoxicity. At realistic, helter-skelter is petite aglow panacea is at hand to squeamish patients give hepatotoxicity becoming to deficiency of colleague of signaling culprits involved in the pathogenesis of hepatotoxicity. Gross models are zoological seasoned to reform esteem the plague pathogenesis and develop drugs for hepatotoxicity. In the physical study, we take on submit discrete extremist models for hepatotoxicity, which may frankly vistas for developing new drugs to treat hepatotoxicity.

To study the Hepatoprotective Activity of the Fruit of Exogenous Plant of Xanthophyceae family Against Carbon Tetrachloride Induced Hepatotoxicity in Albino Rats.

The Hepatic is midst the best industrious and pennon organs in the feasible body. Hepatotoxicity is a symbol intermediary of morbidity and lenity, and its extent is leave off increment appointment by steady old-fashioned in the industrialized nations. Hepatotoxicity is characterized by atomic pyknosis and eosinophilic cytoplasm, followed by copious rash hepatic poison, pudginess changes, lipid peroxidation leads to hepatic centrilobular necrosis. Paracetamol, reluctant tubercular drugs, demon rum, and azathioprine are meditate on to be the tricky venture factors implicated in the progression of hepatotoxicity. Unusual signaling mechanisms, such as activation of transmissible represent encode alike Kupffer cells, simple hew to pieces cells, incendiary mediators, intracellular Ca2 concentration and reactive oxygen species are involved in the pathogenesis of hepatotoxicity. At realistic, helter-skelter is petite aglow panacea is at hand to squeamish patients give hepatotoxicity becoming to deficiency of colleague of signaling culprits involved in the pathogenesis of hepatotoxicity. Gross models are zoological seasoned to reform esteem the plague pathogenesis and develop drugs for hepatotoxicity. In the physical study, we take on submit discrete extremist models for hepatotoxicity, which may frankly vistas for developing new drugs to treat hepatotoxicity.

Calpain Regulates Reactive Oxygen Species Production during Capacitation through the Activation of NOX2 and NOX4.

Capacitation is a series of physiological, biochemical, and metabolic changes experienced by mammalian spermatozoa. These changes enable them to fertilize eggs. The capacitation prepares the spermatozoa to undergo the acrosomal reaction and hyperactivated motility. Several mechanisms that regulate capacitation are known, although they have not been fully disclosed; among them, reactive oxygen species (ROS) play an essential role in the normal development of capacitation. NADPH oxidases (NOXs) are a family of enzymes responsible for ROS production. Although their presence in mammalian sperm is known, little is known about their participation in sperm physiology. This work aimed to identify the NOXs related to the production of ROS in guinea pig and mouse spermatozoa and define their participation in capacitation, acrosomal reaction, and motility. Additionally, a mechanism for NOXs' activation during capacitation was established. The results show that guinea pig and mouse spermatozoa express NOX2 and NOX4, which initiate ROS production during capacitation. NOXs inhibition by VAS2870 led to an early increase in the capacitation and intracellular concentration of Ca2+ in such a way that the spermatozoa also presented an early acrosome reaction. In addition, the inhibition of NOX2 and NOX4 reduced progressive motility and hyperactive motility. NOX2 and NOX4 were found to interact with each other prior to capacitation. This interaction was interrupted during capacitation and correlated with the increase in ROS. Interestingly, the association between NOX2-NOX4 and their activation depends on calpain activation, since the inhibition of this Ca2+-dependent protease prevents NOX2-NOX4 from dissociating and ROS production. The results indicate that NOX2 and NOX4 could be the most important ROS producers during guinea pig and mouse sperm capacitation and that their activation depends on calpain.

Role of the NMDAR-GluN2B subunits in the function of supramolecular NMDAR-BK channel complexes.

Large conductance calcium- and voltage-activated potassium channels (BK) are widely expressed across many tissues, contributing to many physiological functions. Membrane depolarization and relatively high (micromolar) intracellular concentrations of Ca2+ are required for their activation. Such concentrations are reached in the vicinity of Ca2+-permeant ion channels, to which BK proteins are closely located in various tissues, including the brain. N-methyl-D-aspartate receptors (NMDAR) are sodium (Na+)- and Ca2+-conducting glutamate-activated ion channels that are functionally coupled to BK channels in the olfactory bulb and the dentate gyrus, contributing to decreased neuronal intrinsic excitability. Recently, our group demonstrated that postsynaptic NMDAR-BK complexes at basal dendrites of somatosensory cortex layer 5 pyramidal neurons regulate synaptic transmission and long-term plasticity. However, the specific contribution of different NMDAR subunits (GluN1 and GluN2A/GluN2B) to the NMDAR-BK interaction remains elusive. Defects in this association may be of pathological relevance in the context of EIEE27 syndrome (epileptic encephalopathy, early infantile, 27), which has been related to mutations in the GluN2B-encoding gene (GRIN2B). In this work I aimed to understand the role of GluN2B subunits in the function of NMDAR-BK complexes and, more specifically, if GluN2B-related human mutations modify NMDAR-BK coupling. Function of NMDAR-BK complexes containing different EIEE27-related GluN2B mutations was examined using electrophysiology in heterologous expression systems. Proximity between channel proteins within complexes was assessed using in situ proximity ligation assay (PLA). Membrane expression efficiency was studied employing TIRF microscopy (Total Internal Reflection Fluorescence). Our results reveal some disease-related GluN2B mutations reduce the NMDAR-BK interaction, either by altered interactions with BK channels and/or by functional uncoupling between the channels within the NMDAR-BK complexes.

Astrocytes Reduce Store-Operated Ca2+ Entry in Microglia under the Conditions of an Inflammatory Stimulus and Muscarinic Receptor Blockade.

Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca2+ entry (SOCE) in neurons, astrocytes, and microglia following cholinergic dysfunction in combination with (or without) an inflammatory stimulus and to investigate the effects of linalyl acetate (LA) on this process. We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca2+ was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na+-K+ ATPase inhibitor ouabain and the Na+/Ca2+ exchanger (NCX) inhibitor Ni2+. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na+-K+ ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.

Effects of the gaseous signalling molecule nitroxyl (HNO) on myenteric neurons governing intestinal motility.

The main function of myenteric neurons is the control of gut motility. As we recently showed that nitroxyl (HNO) induces intestinal smooth muscle relaxation, it was of interest to evaluate the effects of this signalling molecule on myenteric neurons in order to distinguish its properties in regard to myocytes. Myenteric neurons isolated from the ileum of 4-10days old rats were used. HNO-induced changes in intracellular concentration of Ca2+ or membrane potential and ion currents were measured using the Ca2+-sensitive fluorescent dye fura-2 AM or by electrophysiological whole-cell recordings, respectively. Changes in intracellular thiol groups pool were evaluated using thiol tracker violet. Angeli's salt was used as HNO donor. The HNO donor Angeli's salt induced a significant increase in the cytosolic Ca2+ concentration at the concentration 50µM and a membrane hyperpolarization from a resting membrane potential of-56.1±8.0mV to-63.1±8.7mV (n=7). Although potassium channels primarily drive membrane potential changes in these cells, outwardly rectifying potassium currents were not significantly affected by 50µM Angeli's salt. Fast inward sodium currents were slightly but not significantly reduced by HNO. In more sensitive cells, HNO tended to reduce the pool of thiol groups. As in the case of smooth muscle cells, HNO causes hyperpolarization of myenteric neurons, an effect also associated with an increase in intracellular Ca2+ concentration. Pathways other than activation of potassium currents appear to drive the hyperpolarization evoked by HNO.

Inhibition of plasma membrane Сa(2+),Mg(2+)-АТРase by сalixarene sulfonylamidines. Structure-activity relationship

Previously we have already shown that tetrasulfonylamidinecalixarene C-90 inhibited plasma membrane Са2+,Mg2+-АТРаse of smooth muscle cells selectively to other ATPases of plasma membrane. To inhance the inhibitory effect of calixarenes several alkoxycalixarene sulfonylamidines structurally similar to calixa­rene C-90 were synthesized and their effects on the mentioned enzyme activity, the level of cytoplasmic Ca2+ concentration and hydrodynamic diameter of isolated smooth muscle cells were checked. It was shown that sulfonylamidino groups are crucial for Са2+,Mg2+-АТРаse inhibition, the efficiency of inhibition depends on their quantity and spatial orientation at the upper rim of calixarene macrocycle. Introduction of phenyl or tert-butyl groups into the upper rim and of long alkyl chains into the lower rim led to only slightl increase of inhibition efficiency. The inhibitory effect of studied calixarenes on Са2+,Mg2+-АТРаse correlated with effects on cytosolic Ca2+ concentration and hydrodynamic diameter of smooth muscle cells. The obtained results are important for creation of more effective and selective inhibitors of plasma membrane Са2+,Mg2+-АТРаse as regulators of smooth muscle contractility. Keywords: calixa­rene sulfonylamidines, intracellular Ca(2+) concentration, plasma membrane Са(2+);Mg(2+)-АТРаse, smooth muscle

N-Formyl-L-aspartate mediates chemotaxis in sperm via the beta-2-adrenergic receptor.

Chemotaxis is a highly conserved physiological event required for directed sperm movement during fertilization. Recently, studies from our laboratory have identified N-formyl-L-aspartate (NFA) as a sperm chemoattractant. NFA is a known agonist for the beta-2-adrenergic receptor (β-2-AR) that regulates cAMP production and Ca2+ mobilization in somatic cells. As these downstream signaling molecules are also reported to be involved in sperm chemotaxis, in the present study we investigated the putative mechanism/s by which NFA may mediate chemotaxis. Toward this, the expression and localization of β-2-AR in sperm were studied by Western blot and indirect immunofluorescence, respectively. The responses of sperm to various concentration gradients of NFA and ICI-118,551, a β-2-AR specific antagonist, were evaluated using the microfluidics device-based chemotaxis assay. The intracellular concentration of Ca2+, on exposure to NFA, was analyzed using FURA-2 AM-based fluorimetric assay. Furthermore, the effect of NFA on sperm capacitation and acrosome reaction was evaluated using Western blot and immunofluorescence. NFA exhibited a bell-shaped dose-response curve typical of chemotaxis, with maximum response observed at 0.01M NFA, beyond which it was inhibitory; β-2-AR localization was seen on the sperm head and the mid-piece region of the flagella. Inhibition of sperm chemotaxis by ICI-118,551 confirms that sperm respond chemotactically to NFA via β-2-AR. Interestingly, at the concentration used for chemotaxis, NFA induced an increase in the intracellular Ca2+ but decreased cAMP in capacitating sperm. However, NFA per se did not induce capacitation as seen from the lack of effect on tyrosine phosphorylation and membrane potential of uncapacitated sperm. Acrosome evaluation of NFA-treated sperm using PSA-FITC staining showed no effect on the acrosome structure. Our data thus provide evidence indicating that NFA induces sperm chemotaxis and the chemotactic response of sperm to NFA from the ovulatory phase of oviductal fluid is mediated through the β-2-AR on sperm possibly via non-canonical signaling.

Resolvin D2 uses multiple Ca2+ -dependent signaling pathways to stimulate mucin secretion in rat and human conjunctival goblet cells.

The mucin layer of the tear film is produced by goblet cells in the conjunctiva to protect the ocular surface and maintain homeostasis. The pro-resolving lipid mediator resolvin D2 (RvD2) biosynthesized from an omega 3 fatty acid actively terminates inflammation and regulates mucin secretion from conjunctival goblet cells. Our objective was to determine which Ca2+ -dependent signaling pathways RvD2 uses to stimulate conjunctival goblet cell function (CGC). We hypothesize that RvD2 activates multiple intracellular Ca2+ signaling pathways to stimulate CGC secretion. Rat and human CGCs were cultured from conjunctival explants. The amount of RvD2 receptor GPR18/DRV2 message and protein were determined. The intracellular concentration of Ca2+ ([Ca2+ ]i ) was measured in CGCs using a fluorescent Ca2+ dye and mucin secretion was determined by measuring protein secretion enzymatically with a lectin. Goblet cells were incubated with signaling pathway inhibitors before stimulation with RvD2 and [Ca2+ ]i or secretion was measured. In rat and human CGCs RvD2 receptor and in rat CGCs IP3 (a molecule that releases Ca2+ from intracellular organelles) receptors 1-3 were detected. In both species of CGC RvD2 increased [Ca2+ ]i similarly to RvD1. In rat CGCs, the increase in [Ca2+ ]i and secretion stimulated by RvD2 was significantly blocked by inhibitors to phospholipase (PL-) C and IP3 -receptor, but not protein kinase C. Increase in [Ca2+ ]i was blocked by the PLD inhibitor, but not the PLA2 inhibitor. Secretion was blocked by PLA2 inhibitor, but not the PLD inhibitor. An inhibitor of the epidermal growth factor receptor blocked the increase in [Ca2+ ]i by RvD2 in both species of CGCs. In CGCs RvD2 activates multiple intracellular signaling pathways that are Ca2+ -dependent, along with one Ca2+ -independent and one cAMP/protein kinase A-dependent pathway. Activation of these pathways stimulate mucin secretion from rat and human CGCs into the tear film contributing to ocular surface homeostasis and health.

Study of the Ca2+-dependent gene expression of EuPrt, an extracellular metalloprotease produced by the psychro-tolerant bacterium Exiguobacterium undae Su-1.

The effect of a Ca2+ ion on the gene expression of an on-demand type of metalloprotease from psychrotrophic Exiguobacterium undae Su-1 (EuPrt) was studied. We first established a modified m m9 medium for strain Su-1 to examine its effect in more detail. Then, when the strain was cultured in m m9 medium and 1.0 m m CaCl2 was added, we detected the mature EuPrt and its precursor proteins via Western blotting analysis and found the relative protease activity and its transcription increased by 50-fold and 7-fold, respectively, at the peak. Furthermore, the intracellular concentration of Ca2+ ions was analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) with other metal ions along the growth of strain Su-1. The intracellular concentration of Ca2+ ion was found to increase as much as 3-fold in response to the addition of an extracellular Ca2+ ions, indicating that euPrt gene expression is regulated by sensing its intracellular concentration.

Sacubitril-valsartan improves longitudinal strain and ejection fraction in preclinical models treated with anthracyclines through NLRP3, MyD88 pathways resulting in a reduction of myocardial IL-1β, IL-6, TNF-α and growth factors.

12063 Background: Doxorubicin-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sacubitril-valsartan (LCZ 696) is a combination drug, made up of neprilysin inhibitor sacubitril and angiotensin II receptor blocker valsartan, used for the treatment of heart failure in patients with a reduced ejection fraction. We hypothesized that LCZ 696, administered during doxorubicin, could improve cardiac function and cardiac inflammation in preclinical models. Methods: Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination with LCZ-696 (100 mM) for 72 h. After the incubation period, we performed the following tests: cell viability, apoptosis and necrosis; expression of malondialdehyde and 4-hydroxynonenal and concentration of intracellular Ca2+. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; NF-κB). C57Bl/6 mice were untreated (Sham, n = 6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n = 6), LCZ-696 at 60 mg/kg (LCZ, n = 6) or doxorubicin combined to LCZ-696 (DOXO-LCZ, n = 6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, NF-kB and 13 chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified. Results: LCZ-696 exerts cardioprotective effects, enhancing cell viability of 48-54.6% compared to only doxorubicin-treated cells (p < 0,001 for all); LCZ 696 decreased NLRP3, MyD88 and NF-kB expression in cardiac cells. In preclinical study, LCZ 696 improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88 and NF-kB in cardiac tissues was seen in DOXO-LCZ group compared to DOXO mice (p < 0.001). Cardiac expression of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with LCZ-696 indicating anti-inflammatory properties. Conclusions: LCZ-696 exerts direct beneficial effects in cardiomyocytes exposed to doxorubicin. In preclinical models, LCZ-696 reduced inflammation and cytokine expression involved in doxorubicin-mediated cardiotoxicity.

C49 SACUBITRIL–VALSARTAN IMPROVES LONGITUDINAL STRAIN AND EJECTION FRACTION IN MICE TREATED WITH DOXORUBICIN THROUGH NLRP3, MYD88 PATHWAYS RESULTING IN A REDUCTION OF MYOCARDIAL IL–1Β, IL–6, TNF–Α, G–CSF AND GM–CSF LEVELS

Abstract Introduction Doxorubicin–mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sacubitril–valsartan (LCZ 696) is a combination drug, made up of neprilysin inhibitor sacubitril and angiotensin II receptor blocker valsartan, used for the treatment of heart failure in patients with a reduced ejection fraction. Purpose We hypothesized that LCZ 696, administered during doxorubicin, could improve cardiac function and cardiac inflammation in preclinical models Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination with LCZ–696 (100 mM) for 72 h. After the incubation period, we performed the following tests: cell viability, apoptosis and necrosis; expression of malondialdehyde and 4–hydroxynonenal and concentration of intracellular Ca2+. Moreover, pro–inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; NF–κB). C57Bl/6 mice were untreated (Sham, n = 6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n = 6), LCZ–696 at 60 mg/kg (LCZ, n = 6) or doxorubicin combined to LCZ–696 (DOXO–LCZ, n = 6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, NF–kB and 13 chemokines (IL–1α, IL–1β, IL–2, IL–4, IL–6, IL–10, IL–12, IL17–α, IL–18, IFN–γ, TNF–α, G–CSF, and GM–CSF) were quantified. Results LCZ–696 exerts cardioprotective effects, enhancing cell viability of 48–54.6% compared to only doxorubicin–treated cells (p < 0,001 for all); LCZ 696 decreased NLRP3, MyD88 and NF–kB expression in cardiac cells. In preclinical study, LCZ 696 improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88 and NF–kB in cardiac tissues was seen in DOXO–LCZ group compared to DOXO mice (p < 0.001). Cardiac expression of IL–1β, IL–6, TNF–α, G–CSF and GM–CSF were significantly reduced after treatment with LCZ–696 indicating anti–inflammatory properties. Conclusion LCZ–696 exerts direct beneficial effects in cardiomyocytes exposed to doxorubicin. In preclinical models, LCZ–696 reduced inflammation and cytokine expression involved in doxorubicin–mediated cardiotoxicity.

Protective effect of Celastrus paniculatus on cognitive function in glutamate-induced brain-injured mice by reducing the intracellular influx of Ca.

Present investigation evaluates the protective effect of Celastrus paniculatus (CP) on the cognitive function in neuronal injured mice. Neuronal injury was induced by oral administration of monosodium glutamate (MSG) at a dose of 1.66 g/kg/day for 30 days. Mice in the CP-treated group receives CP 30 mg/kg ip and CP + GGA-treated group received CP 30 mg/kg ip and glutamic acid (GGA, 1.5mg/kg, ip) 30 min prior to the administration of MSG for 30 days. Assessment of cognitive function was done using Morris water maze. Level of inflammatory cytokines and production of reactive oxygen species (ROS) was estimated in the brain tissue of brain-injured mice. Moreover, intracellular concentration of Ca+ ion was estimated in the brain tissue and expression of Bcl-2, Bax, and caspase-3 protein was estimated in the brain tissue by western blot assay. Cognitive function was attenuated in CP-treated glutamate-injured mice. Data of the study suggest that treatment with CP reduces the level of inflammatory cytokines and production of ROS in the brain tissue compared to negative control group. There was reduction in the concentration of Ca+ ion in the neuronal cells in CP-treated group than negative control group of mice. Treatment with CP ameliorates the expression of Bax, Bcl-2, and caspase-3 in the brain tissue of glutamate-induced brain-injured mice. In conclusion, data of the study suggest that treatment with CP attenuates the cognitive function and neuronal apoptosis in glutamate-induced neuronal injury by reducing the concentration of intracellular Ca+ ion.

Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5'AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701-1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKKα/β in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST-CaMKKα/β, which could bind to TIM-127-sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose-dependent manner. In contrast to GST-CaMKKα, GST-CaMKKβ was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKKβ without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKKα prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKKα mutant (residues 126-434) lacking the regulatory domain (residues 438-463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca2+/CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.

Nanoparticle pre- or co-exposure affects bacterial ingestion by the protozoan Tetrahymena thermophila

Although nanoparticles’ (NPs) toxicity has been intensively studied, their effects on bacterial ingestion by protozoans (as an important component of the microbial loop) is unknown. This study investigated the effects of NPs of different chemical composition [hematite (HemNPs), anatase (AnaNPs), and silica (SiNPs) NPs] and size [SiNPs with particle size of 20 (Si-20), 100 (Si-100), and 500 (Si-500) nm] on the ingestion of Escherichia coli by the protozoan Tetrahymena thermophila. Potential differences between pre- vs. co-exposure were also assessed. Pre-exposure to HemNPs had no effects on bacterial ingestion but the other NPs caused a significant inhibition, due to their inhibition of ATP synthesis and the down-regulation of phagocytosis-related genes (ACT1 and CTHB). Contrastively, co-exposure to HemNPs and Si-20 didn’t affect bacterial ingestion while co-exposure to AnaNPs (Si-100 and Si-500) induced (inhibited) ingestion. The stimulatory effect of AnaNPs was due to their induction of an increase in the intracellular Ca concentration of T. thermophila whereas the inhibitory effects of Si-100 and Si-500 were attributable to ATP synthesis reduction, enhanced bacterial cell aggregation, and competition between the bacterial cells and the NPs. These findings provide insights into the mechanisms underlying the environmental risks of NPs.

Exercise improves the body function and protects the neuronal injury in Parkinson's disease rats by activating calpain 1 and kallikrein 6.

Parkinson's disease (PD) is a neurodegenerative disease, which alters body and cognitive functions. The present study evaluates the effect of exercise on body function and neuronal injury against a 6-hydroxydopamine hydrobromide (6-OHDA) induced PD rat model and postulates a possible molecular mechanism of its action. Parkinson's disease was induced by administration of (20 µg/5 µl at the rate of 1 µl/min) 6-OHDA and exercise training was given to mice by motorized rodent treadmill for a period of 14 days after the confirmation of PD. Behavioural changes were observed by apomorphine-induced rotation and motor function was assessed using the rotarod apparatus. The effect of exercise was observed on the levelof neurochemicals and the expression of calpain-1 (CAPN1) and kallikrein 6 (KLK6) was estimated in brain tissue of PD rats using western blot assay. A more significant improvement in the motor and cognitive function was observed in the PD + exercise group than in the PD group of rats. Exercise attenuates the altered level of g-aminobutyric acid (GABA), dopamine (DA) and glutamate in brain tissue of PD rats. Intracellular concentration of Ca+ ion was reduced significantly in brain tissue of the PD + exercise group compared to PD rats. Moreover, exercise activates the expression of KLK6 and CAPN1 protein in brain tissue of PD rats. In conclusion, data of the study reveal that exercise protects neuronal injury by reducing intracellular concentration Ca+ ion and activates KLK6 and CAPN1 in brain tissue of PD rats and thereby improves motor and cognitive functions.