Channel Pathway Research Articles

Influence of the location of discrete macro-encapsulated thermal energy storage on the performance of a double pass solar plate collector system

In this work, an outdoor experimental analysis is conducted to determine the impact on the useful heat gain when discrete cylindrical energy storage units are directly integrated into the solar collector. The collector has a double-pass airflow channel pathway, and the storage is intended to serve only for a short-term. The location of storage inside the collector is always a major concern. This study seeks to determine whether the thermodynamic performance of the system is effective by the location of cylindrical energy storage (paraffin wax) capsules on the upper or the lower airflow channel pathway. The obtained results suggest that due to asymmetric channel depth, the thermodynamic performance of the collector was not greatly influenced by the placement of capsules, unlike with symmetric channel depths. The amount of useful heat gain when storage was placed in the upper (Case A) and lower (Case B) airflow pathways was 0.35 kW and 0.4 kW. For Case A and Case B, the average collector thermal efficiency was 62.9% and 73.7%, and the exergy efficiency was 44.3% and 47.5%. The energy payback time for the collector based on energy calculations is nine months, and that on exergy analysis is 34 months and 20 days.

The conduction pathway of potassium channels is water free under physiological conditions.

Ion conduction through potassium channels is a fundamental process of life. On the basis of crystallographic data, it was originally proposed that potassium ions and water molecules are transported through the selectivity filter in an alternating arrangement, suggesting a "water-mediated" knock-on mechanism. Later on, this view was challenged by results from molecular dynamics simulations that revealed a "direct" knock-on mechanism where ions are in direct contact. Using solid-state nuclear magnetic resonance techniques tailored to characterize the interaction between water molecules and the ion channel, we show here that the selectivity filter of a potassium channel is free of water under physiological conditions. Our results are fully consistent with the direct knock-on mechanism of ion conduction but contradict the previously proposed water-mediated knock-on mechanism.

Involvement of the L-arginine/Nitric Oxide/Cyclic GMP/KATP Channel Pathway and PPARγ Receptors in the Peripheral Antinociceptive Effect of Carbamazepine

Carbamazepine has been shown to exert analgesic effects in clinical and experimental pain situation. This study was conducted to evaluate its potential peripheral antinociceptive effects and the possible involvement of L-arginine/NO/cGMP/KATP channel pathway and PPARγ receptors in an animal model of pain. The antinociceptive effect induced by intraplantar administration of carbamazepine (100-1 000 μg/paw) was assessed using the formalin test in rats. To evaluate the involvement of L-arginine/NO/cGMP/KATP channel pathway in the antinociceptive action of carbamazepine, rats were pre-treated intraplantarlly with L-arginine (a nitric oxide precursor, 100 and 200 μg/paw), L-NAME (NOS inhibitor, 50 and 100 μg/paw), methylene blue (guanylyl cyclase inhibitor, 100 and 200 μg/paw), glibenclamide (KATP channel blocker, 100 and 200 μg/paw), and diazoxide (400 μg/paw). Moreover, to investigate the possible involvement of PPARγ receptors, pioglitazone (10 μg/paw; a PPARγ agonist) alone or in combination with GW9662 (3 μg/paw; a PPARγ antagonist) were pre-treated with carbamazepine. The local ipsilateral, but not contralateral, administration of carbamazepine into the hind paw produced dose-related analgesia during both early and late phases of formalin test. Moreover, pre-treatment with L-NAME, methylene blue, and glibenclamide dose-dependently prevented carbamazepine (300 μg/paw)-induced antinociception in both phases of the test. In addition, administration of L-arginine and diazoxide before the sub-effective dose of carbamazepine (100 μg/paw) produced an antinociceptive effect. Also, antinociception induced by carbamazepine plus pioglitazone (10 μg/paw) was blocked by GW-9662 in both phases of the test. In conclusion, carbamazepine induced a peripheral antinociceptive effect through PPARγ receptors and L-arginine/NO/cGMP/KATP channel pathway, with potential for a new topical analgesic drug.

Abstract 3069: Efficacy of RP4010, a calcium release-activated calcium (CRAC) channel inhibitor, in preclinical models of gastrointestinal cancers

Abstract Background: Calcium Ca(2+) signals and the underlying Ca(2+) channels and transporters are known to influence development, growth, and metastasis of many cancers. Calcium release-activated calcium (CRAC) channel proteins, STIM and Orai, have been shown to play a role in cancer progression and metastasis. Here we investigate the impact of RP4010, a CRAC channel inhibitor, in GI [pancreatic ductal adenocarcinoma (PDAC), gastric cancer (GC) and pancreatic neuroendocrine tumors (PNETs)] cellular and patient derived tumor models. Methods: GI cancer cell lines [3 PDAC and one normal human pancreatic ductal adenocarcinoma (HPDE); 3 GC cells and 2 PNET cell lines] were exposed to RP4010 in the presence or absence of standard of care drugs such as gemcitabine, nab-paclitaxel, or everolimus. Cell growth inhibition was evaluated using MTT. Apoptosis was determined by Annexin V FITC and 7AAD assays. Alterations in markers associated with the CRAC channel pathway were evaluated using RT-PCR and Western Blotting. In vivo efficacy was evaluated in gastric cancer sub-cutaneous xenograft as well as a hyaluronan positive PDAC patient derived xenograft in the presence or absence of chemotherapeutic agents. Results: CRAC channel inhibition by RP4010 resulted in cancer cell selective growth inhibition (IC50s in the low micro M range). Growth inhibition was concurrent with apoptosis at similar doses. RP4010 synergized with gemcitabine and nab-paclitaxel in PDAC; nab-paclitaxel and docetaxel in GC (CI<1), and significantly enhanced the activity of everolimus in PNETs. RT-PCR and Western Blotting revealed that RP4010 caused a reduction in NFATC1, NFAT2, Akt, mTOR and related markers associated with CRAC channel pathway signaling. The combination treatment led to superior suppression of several CRAC channel regulated genes. RP4010 (given at 50 mg/kg daily for 4 weeks) as a single agent reduced tumor weight/volume in a hyaluronan positive (stroma +ve) PDAC PDx model. Anti-tumor activity of RP4010 was enhanced in the presence of gemcitabine-nab-paclitaxel (RP4010 50 mg/kg orally+50 mg/kg i.v once a week for 3 weeks and 30 mg/kg nab-paclitaxel once a week for 3 weeks). Residual tumor tissue analysis to capture CRAC channel pathway inhibition in vivo is currently ongoing. Conclusion: Our studies indicate that RP4010, a novel CRAC channel inhibitor could be a viable therapeutic option in GI cancers that need further clinical evaluation. A multi-center Phase I/IB dose-escalation trial evaluating the safety and efficacy of RP4010, a CRAC channel inhibitor in patients with relapsed or refractory Lymphomas is currently ongoing in USA (ClinicalTrials.gov Identifier: NCT03119467). Citation Format: Irfana Muqbil, Rachel Sexton, Gabriel Mpilla, Anteneh Tesfaye, Steve Kim, Philip A. Philip, Amro Aboukameel, Srikant Viswanadah, Kumar V. Penmesta, Ramzi M. Mohammad, Asfar S. Azmi. Efficacy of RP4010, a calcium release-activated calcium (CRAC) channel inhibitor, in preclinical models of gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3069.

Submarine channels ‘swept’ downstream after bend cutoff in salt basins

AbstractChannel‐bend expansion and downstream translation, as well as vertical movements by aggradation and incision, set the stratigraphic architecture of channelized depositional systems. Early work on submarine‐channel evolution has suggested that downstream translation is rare. It is proposed here that downstream translation of bends might be common in deep‐water salt‐tectonic provinces, where complex topography can localize channel pathways that promote meander cutoffs and the generation of high‐curvature bends. Three‐dimensional seismic‐reflection data from a region with salt‐influenced topography in the Campos Basin, offshore Brazil, is used to characterize the structural geometry of a salt diapir and stratigraphic architecture of an adjacent submarine‐channel system that extends for 18 km. The combined structural and stratigraphic evolution is interpreted, including meander‐cutoff development near the salt diapir followed by ~10 km of downstream translation of a channel bend. The stratigraphic evolution is tested with a simple numerical model of channel meandering. This integrated subsurface characterization and stratigraphic modelling study sheds light on the processes and controls of submarine‐channel downstream translation, which might be common in rapidly deforming settings, such as salt basins, that promote localized subsidence, meander cutoffs, and rapidly translating, high‐curvature bends.

Essential oil from Lippia microphylla Cham. modulates nitric oxide pathway and calcium influx to exert a tocolytic effect in rat uterus

The essential oil of Lippia microphylla (LM-OE) presents several pharmacological activities. This work evaluates the tocolytic effect of LM-OE on rats. LM-OE inhibited phasic contractions and relaxed tonic contractions on rat uterus. Considering that nitric oxide (NO) pathway regulates uterine contraction, LM-OE potency was attenuated in the presence of NO synthase (NOS) inhibitor and this reduction was reversed in the presence of a NOS substrate. Similarly, the relaxant potency of LM-OE was reduced in the presence of soluble guanylyl cyclase (sGC) and protein kinase G (PKG) inhibitors. LM-OE also demonstrates a positive modulation of large and small conductance calcium-activated, voltage-gated and adenosine triphosphate-sensitive potassium channels and inhibited curves to CaCl2 as well as relaxed the uterus pre-contracted by S-(-)-Bay K8644, suggesting voltage-gated calcium channels type-1 (CaV1) blockade. Thus, the tocolytic effect of LM-OE on rat involves positive modulation of NO/NOS/sGC/PKG/K+-channels pathway and Ca2+ influx blockade through CaV1.

GABAA receptor, KATP channel and L-type Ca2+ channel is associated with facilitation effect of H2S on the baroreceptor reflex in spontaneous hypertensive rats.

We aimed to investigate whether the facilitating effect of H2S on the baroreceptor reflex is associated with the GABAA receptor, KATP channel and L-type Ca2+ channel pathway. Spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were used to investigate the facilitating effect of H2S on the baroreceptor reflex by perfusing the isolated carotid sinus. The mechanism by which H2S facilitated the baroreceptor reflex was determined by using Bay K8644 (an agonist of calcium channels), glibenclamide (Gli, a KATP channel blocker), and picrotoxin (PIC, a blocker of γ-aminobutyric acid [GABA]A receptor). As compared with WKY rats, SHRs showed impaired baroreceptor reflex sensitivity, as demonstrated by a right and upward shift of the functional curve for the intrasinus pressure-arterial blood pressure relation. H2S perfusion (25, 50, or 100 μmol/L) dose-dependently ameliorated the impaired sensitivity of the baroreceptor reflex. Bay K8644 (500 nmol/L), Gli (20 μmol/L) and PIC (50 μmol/L) all prevented H2S ameliorating the impaired baroreceptor reflex. H2S facilitating the baroreceptor reflex might be associated with activating the GABAA receptor, opening the KATP channel, and closing the L-type Ca2+ channel. These areas should provide new targets for preventing and treating hypertension.

(Invited) Intermediate Transport on Flexible Cascade Surfaces with Electrostatic Channeling

Biocatalytic cascades can potentially utilize the full bond energy stored in complex molecules using sequential active sites. This results in an enhanced electrocatalytic efficiency, enabling biofuel cells of high energy density and biosensors of high sensitivity. Mass transport of cascade intermediates is enhanced by electrostatic channeling, wherein where charged intermediate was transported along an oppositely charged pathway [1]. Natural cascade has be studied for decades and in 2017 we reported the first cascade with artificially introduced electrostatic channeling [2]. That finding highlighted a surface-channeled diffusing mechanism on a 1D bridge. However, intermediate hopping between an artificial bridge and an enzyme binding site is a flexible 2D surface with multiple pathways and increased potential for leakage [3]. This process is of great significance to design and optimization of electrostatic pathways. Here, we further map the channeling pathway between the bridge and intermediate active site for two different enzymes of varying overall electrostatic charge. Enhanced molecular dynamics sampling methods are used to elucidate the intermediate’s ergodicity in this process; the multiple pathways detected by MD are analyzed via a novel Markov State Model approach. Finally, the kinetic Monte Carlo method is used to quantify the overall kinetics, from which the leaking contribution from each channeling area can be evaluated. I. Wheeldon, S. D. Minteer, S. Banta, S. C. Barton, P. Atanassov, and M. Sigman, Nat. Chem., 8, 299–309 (2016). doi:10.1038/nchem.2459Y. Liu, D. P. Hickey, J.-Y. Guo, E. Earl, S. Abdellaoui, R. D. Milton, M. S. Sigman, S. D. Minteer, and S. Calabrese Barton, ACS Catal., 7, 2486–2493 (2017). doi:10.1021/acscatal.6b03440Y. Liu, I. Matanovic, D. P. Hickey, S. D. Minteer, P. Atanassov, and S. C. Barton, ACS Catal., 8, 7719–7726 (2018). doi:10.1021/acscatal.8b01041 Figure 1

Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments.

Cytochrome c oxidase (C cO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven C cO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other C cOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase.

Estrogen regulation of cardiac cAMP-L-type Ca2+ channel pathway modulates sex differences in basal contraction and responses to \u03b22AR-mediated stress in left ventricular apical myocytes

Backgrounds/AimMale and female hearts have many structural and functional differences. Here, we investigated the role of estrogen (E2) in the mechanisms of sex differences in contraction through the cAMP-L-type Ca2+channel pathway in adult mice left ventricular (LV) apical myocytes at basal and stress state.MethodsIsolated LV apical myocytes from male, female (Sham) and ovariectomised mice (OVX) were used to investigate contractility, Ca2+ transients and L-type Ca2+ channel (LTCC) function. The levels of β2AR, intracellular cAMP, phosphodiesterase (PDE 3 and PDE 4), RyR2, PLB, SLN, and SERCA2a were compared among the experimental groups.ResultsWe found that (1) intracellular cAMP, ICaL density, contraction and Ca2+ transient amplitudes were larger in Sham and OVX + E2 myocytes compared to male and OVX. (2) The mRNA expression of PDE 3 and 4 were lower in Sham and OVX + E2 groups compared with male and OVX groups. Treatment of myocytes with IBMX (100 μM) increased contraction and Ca2+ transient amplitude in both sexes and canceled differences between them. (3) β2AR-mediated stress decreased cAMP concentration and peak contraction and Ca2+ transient amplitude only in male and OVX groups but not in Sham or OVX + E2 groups suggesting a cardioprotective role of E2 in female mice. (4) Pretreatment of OVX myocytes with GPR30 antagonist G15 (100 nM) abolished the effects of E2, but ERα and ERβ antagonist ICI 182,780 (1 μM) did not. Moreover, activation of GPR30 with G1 (100 nM) replicated the effects of E2 on cAMP, contraction and Ca2+ transient amplitudes suggesting that the acute effects of E2 were mediated by GPR30 via non-genomic signaling. (5) mRNA expression of RyR2 was higher in myocytes from Sham than those of male while PLB and SLN were higher in male than Sham but no sex differences were observed in the mRNA of SERCA2a.ConclusionCollectively, these results demonstrate that E2 modulates the expression of genes related to the cAMP-LTCC pathway and contributes to sex differences in cardiac contraction and responses to stress. We also show that estrogen confers cardioprotection against cardiac stress by non-genomic acute signaling via GPR30.

Simvastatin exerts antidepressant-like activity in mouse forced swimming test: Role of NO-cGMP-KATP channels pathway and PPAR-gamma receptors

Simvastatin, one of the lipophilic statins, has been shown to be effective in reducing depression in rodents. The present study aimed to investigate the potential antidepressant-like activity of simvastatin and the possible involvement of NO-cGMP-KATP channels pathway and PPARγ using forced swimming test (FST) in mice. In addition, the interaction between simvastatin and fluoxetine as a reference drug was examined. After assessment of locomotor behavior in the open-field test (OFT), FST was applied for evaluation of depressive behavior in mice. Simvastatin at doses (20, 30, and 40 mg/kg, i.p.) was administrated 30 min before the OFT or FST. To evaluate the involvement of NO-cGMP-KATP channels pathway, mice were pre-treated intraperitoneally with l-arginine (a nitric oxide precursor, 750 mg/kg), L-NAME (a NOS inhibitor, 10 mg/kg), methylene blue (guanylyl cyclase inhibitor, 20 mg/kg), sildenafil (a PDE-5 inhibitor, 5 mg/kg), glibenclamide (ATP-sensitive K+ channel blocker, 1 mg/kg), and diazoxide (K+ channels opener, 10 mg/kg). Moreover, to clarify the probable involvement of PPARγ receptors, pioglitazone, a PPARγ agonist (5 mg/kg, i.p.), and GW9662, a PPARγ antagonist (2 mg/kg, i.p.), were pre-treated with simvastatin. Immobility time was significantly decreased after simvastatin injection. Administration of L-NAME, methylene blue, glibenclamide and pioglitazone in combination with the sub-effective dose of simvastatin (20 mg/kg, i.p.) reduced the immobility time in the FST compared to drugs alone, while co-administration of effective doses of simvastatin (30 mg/kg, i.p.) with l-arginine, sildenafil, diazoxide, and GW9662 prevented the antidepressant-like effect of simvastatin. In addition, simvastatin (20 mg/kg) potentiated the antidepressant-like effect of fluoxetine through the NO pathway. None of the drugs produced any significant alterations in locomotor activity using OFT. These results demonstrated that NO-cGMP-KATP channels pathway and PPARγ receptors may be involved in the antidepressant-like effect of simvastatin.

Crystal structure of human mitochondrial trifunctional protein, a fatty acid β-oxidation metabolon

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α2β2 The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two β-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the β2 dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3'-AMP-PPi moiety is guided by the positively charged residues located along the "ceiling" of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.

Calcitonin gene-related peptide inhibits anoxia/reoxygenation-induced increases in the enzymatic activity of caspase-3 and caspase-9 through the protein kinase C-mitochondrial ATP sensitive potassium channel pathway

Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on the apoptosis of rat cardiomyocytes induced by anoxia/reoxygenation (A/R) and its related signaling pathways. Methods A total of 60 male healthy SD rats aging 1 d to 3 d were selected to establish a primary model of cardiomyocytes in vitro. The animals were randomly divided ten groups before A/R experiments (n=6): ① a control group, ② an A/R group, ③ a CGRP+A/R group, ④ a CGRP8-37+CGRP+A/R group, ⑤ an 5-HD+CGRP+A/R group, ⑥ a DZ+A/R group, ⑦ an H-89+CGRP+A/R group, ⑧ a chelerythine+CGRP+A/R group, ⑨ a glibenclamide+CGRP+A/R group and ⑩ an HMR-1098+CGRP+A/R group. The activities of caspase-3 and caspase-9 were measured by spectrophotometry. Results Compared with the control group, the A/R group showed significantly increased activities of caspase-3 and caspase-9 (P 0.05). Compared with the CGRP+A/R group, the chelerythine+CGRP+A/R group produced significantly increased activities of caspase-3 and caspase-9 (P 0.05). Conclusions CGRP relieves cardiomyocyte A/R injury and plays an important role in cardiomyocyte apoptosis by activating protein kinase C-mitochondrial ATP sensitive potassium channel and decreasing the expression of caspase-3 and caspase-9. Key words: Calcitonin gene-related peptide; Anoxia-reoxygenation injury; ATP sensitive potassium channel; Caspases; Cardiomyocytes

Effect of Oxidative Stress on the Estrogen-NOS-NO-KCa Channel Pathway in Uteroplacental Dysfunction: Its Implication in Pregnancy Complications.

During pregnancy, the adaptive changes in uterine circulation and the formation of the placenta are essential for the growth of the fetus and the well-being of the mother. The steroid hormone estrogen plays a pivotal role in this adaptive process. An insufficient blood supply to the placenta due to uteroplacental dysfunction has been associated with pregnancy complications including preeclampsia and intrauterine fetal growth restriction (IUGR). Oxidative stress is caused by an imbalance between free radical formation and antioxidant defense. Pregnancy itself presents a mild oxidative stress, which is exaggerated in pregnancy complications. Increasing evidence indicates that oxidative stress plays an important role in the maladaptation of uteroplacental circulation partly by impairing estrogen signaling pathways. This review is aimed at providing both an overview of our current understanding of regulation of the estrogen-NOS-NO-KCa pathway by reactive oxygen species (ROS) in uteroplacental tissues and a link between oxidative stress and uteroplacental dysfunction in pregnancy complications. A better understanding of the mechanisms will facilitate the development of novel and effective therapeutic interventions.

Erythromycin relaxes BALB/c mouse airway smooth muscle

AimsBitter taste receptor (TAS2R) agonists have bronchodilatory potentials. Erythromycin is a ligand of TAS2R10, but its relaxant profile is unknown. This study was performed to understand the relaxant effects of erythromycin and its potential mechanism. Main methodsAirway resistance was tested by the whole body plethysmography in the ovalbumin-aluminum hydroxide induced asthma model mice. Tracheal ring segment myography was used to investigate the isometric tension of the smooth muscle. The cyclic adenosine monophosphate (cAMP) concentration was measured by enzyme immunoassay kit. Changes in the calcium influx in airway smooth muscle cells (ASMCs) were surveyed using a real-time confocal microscopy. Key findingsErythromycin significantly relieved airway hyperreactivity in asthma model mice. Erythromycin relaxed mouse tracheal segments precontracted with carbachol, KCl, 5-hydroxytryptamine and U46619, and further dilated the tracheal rings relaxed by isoprenaline or atropine. Epithelium removal, indomethacin or NS-398 partially reduced the relaxation. U73122, 2-APB, iberiotoxin or ouabain did not change the concentration-relaxation curves of erythromycin on tracheal segments. Erythromycin didn't elevate cAMP level. CaCl2-induced contraction in the K+-rich solution was impaired by erythromycin in the Ca2+-free solution. The intercellular Ca2+ level in the ASMCs was decreased by erythromycin, which was partly inhibited by Bay K8644 but not gallein. SignificanceErythromycin had marked bronchodilatory effect. The relaxation might be related to the L-type voltage-dependent calcium channel, but not the gustducin-associated βγ/phospholipase-Cβ/inositol 1,4,5-tri-phosphate receptor/large conductance Ca2+-activated K+ channel pathway or a cAMP-dependent way.

Ion Permeation through ORAI Proteins

Under depleted calcium levels in the endoplasmic reticulum, calcium release-activated calcium (CRAC) channels generate a sustained calcium influx crucial for several cellular responses such as gene expression, cell migration, and growth. Abnormal CRAC channel activity has been linked to various diseases, including severe combined immunodeficiency and autoimmunity disorders, allergy, and inflammatory bowel disease. CRAC channels are composed of two proteins: Orai proteins, that form the pore subunit, and Stim proteins, that activate the pore. Recent crystal structures of Orai proteins have shed light onto the nature of the permeation pathway of CRAC channels but details of the ion permeation mechanism are yet to be revealed. In this study, we use Umbrella Sampling and the Weighted Histogram Analysis Method to investigate the permeation of calcium, potassium and sodium ions through Orai pores. We extend the calculations to both Orai hexamer and tetramer assemblies, since previous studies have implicated different multimeric states of Orai as functional CRAC channels. In addition, we investigate the effect of rotation of the pore-forming transmembrane helices (suggested to be responsible for gating) on the free energy cost of ion permeation. Our simulations indicate that both hexamer and tetramer assemblies form conducting ion pores in which the free energy barrier is mainly set by the hydrophobic core of the pore. In all cases, helix rotation lowers the energetic cost of translocating an ion.

Evaluation of anti-nociceptive and anti-inflammatory activities of the methanol extract of Holigarna caustica (Dennst.) Oken leaves

Ethnopharmacological relevanceHoligarna caustica (Dennst.) is commonly used in traditional medicine to treat a variety of painful conditions such as eye irritation, inflammation, arthritis, skin diseases, cuts and wounds. Aim of the studyThe present study was undertaken to investigate the anti-nociceptive and anti-inflammatory activities of the methanol extract of H. caustica leaves and to elucidate its possible mechanism(s) of action. Materials and methodsFresh leaves of H. caustica were collected, dried, and extracted with methanol (MEHC). MEHC was subjected to activity testing, using chemical-induced (acetic acid and formalin test) and heat-induced (hot plate and tail immersion test) pain models. To determine the possible mechanism behind the anti-nociceptive activity of MEHC, the opioid antagonist naltrexone was used to evaluate the involvement of opioid receptors in the case of formalin, hot plate and tail immersion tests, while the involvement of the cGMP and ATP-sensitive K+ channel pathways were assessed using methylene blue and glibenclamide respectively, in the acetic acid-induced writhing test. In parallel, the carrageenan-induced paw oedema model was used to determine the anti-inflammatory potential of the extract. Exploratory and motor behaviours were evaluated by the open-field test. Various bioactive compounds potentially responsible for the anti-nociceptive and anti-inflammatory activities were ascertained using GC-MS analysis. ResultsMEHC showed strong, significant and dose-dependent anti-nociceptive activity in all chemical-induced and heat-induced pain models at all experimental doses. The association of opioid receptors with the observed anti-nociceptive effects was confirmed by using naltrexone. The cGMP and ATP-sensitive K+ channel pathway was also shown to be involved in the anti-nociceptive activity of MEHC. In addition, MEHC exhibited a dose-dependent inhibition of inflammatory oedema induced by carrageenan. MEHC was not connected with changes in either the locomotor activity or motor responses of mice. In a GC-MS analysis, 40 compounds were identified, among which twelve are documented bioactive compounds with potent analgesic and anti-inflammatory properties. ConclusionsOur current study revealed that MEHC possesses strong central and peripheral anti-nociceptive as well as anti-inflammatory activity. It may also be concluded that both opioid receptors as well as the cGMP and ATP-sensitive K+ channel pathway are involved in the anti-nociceptive mechanism of MEHC. This study rationalizes the ethnomedicinal use of H. caustica leaves in various painful conditions.

Mechanistic Pathways of ATP Sensitive Potassium Channels Referring to Cardio-Protective Effects and Cellular Functions.

A study of potassium channels correlates the fundamentals of mechanistic pathways and various physiological functions. The knowledge of these pathways provides the background, how to determine unit cell functions and to affect cardio protection. ATP sensitive potassium channels adjust excitability of membrane and functions as per metabolic status of cell. A lot of energy consumption primarily occurred in skeletal muscles which also express high number of potassium channels. The increase in calcium release and high heat production is occurred due to loss of potassium channels. Such type of mediations determines metabolic changes and energy required in the dissipation. IPC reduces infarct size in anesthetized mice. In ischemic-reperfusion, pressure in left ventricle was watched while contractile power recovery did not happen. It was seen that elements of intact potassium channel are fundamental for Ischemic preconditioning (IPC). If more prominent is enactment of potassium channels and their cardiologic effects create high heart rate. All the more as of late, it has been suggested that mitochondrial ATP sensitive potassium channels are critical as closing stage effectors which trigger IPC as opposed to sarcolemmal potassium channels. Nevertheless, the importance of the potassium channels reconsidered in cardio-protection in present findings. These discoveries recommend that potassium channels in the adjusting ischemic-reperfusion damage in mice. The heart rate of the mouse occurred during ischemia; enhance watchful extrapolation applied to larger warm blooded animals.

Detecting the structural assembly pathway of human antimicrobial peptide pores at single-channel level.

The pore-forming structures of an anionic human antimicrobial peptide dermcidin (DCD) in a membrane environment has not been demonstrated previously. Using single-channel electrical recordings, we characterized the structural and functional properties of the DCD peptide channel in lipid membranes. We show that a 48-residue, 8 nm long anionic DCD-1L peptide is folded in the right conformation in sodium dodecyl sulfate (SDS) that spontaneously inserts into lipid bilayers to form well-defined channels. However, the DCD-1L peptides are not properly folded in n-dodecyl-β-d-maltoside (DDM), resulting in unstable channels suggesting the significance of specific detergent in stable channel formation. Furthermore, a 25-residue cationic DCD SSL-25 peptide formed channels both in SDS and DDM micelles as the length of the peptide matches with the thickness of the membrane. Finally, we quantified the permeation of small molecules through the DCD channels in liposome assays. Accordingly, we propose a molecular model demonstrating the structural self-assembly of the DCD channels in the membrane. We suggest that an understanding of the mechanism of action of DCD peptides at single-channel resolution will lead to developing peptide-based therapeutics.

Antinociceptive and anti-inflammatory properties of Tetracera alnifolia Willd. (Dilleniaceae) hydroethanolic leaf extract.

Background Tetracera alnifolia Willd. (Dilleniaceae) is used in traditional African Medicine for the treatment of headache, abdominal pain, and rheumatism. Hence, this study sought to investigate the antinociceptive and anti-inflammatory effects of the hydroethanolic leaf extract of T. alnifolia (HeTA) in rodents. Methods Antinociceptive activity was evaluated using the acetic acid-induced writhing, formalin-/capsaicin-induced paw licking and hot plate tests in mice. The contribution of opioidergic, l-arginine-nitric oxide, and ATP-sensitive potassium channel pathways in HeTA-induced antinociception was also evaluated. The anti-inflammatory effect was assessed using the carrageenan-induced paw edema, xylene ear edema, cotton pellet granuloma, and complete Freund's adjuvant (CFA)-induced arthritis in rats. Results HeTA (100, 200, and 400 mg/kg, p.o.) produced significant (p<0.05) decrease in mean number of acetic acid-induced writhing, time spent licking paw in formalin, and capsaicin tests as well as time course increase in nociceptive reaction latency in hot plate test. HeTA-induced antinociception was prevented by pretreatment of mice with naloxone (non-selective opioid receptor antagonist), l-arginine (nitric oxide precursor), or glibenclamide (ATP-sensitive potassium channel blocker). HeTA (100 mg/kg, p.o.) produced a significant anti-inflammatory effect against carrageenan-induced rat paw edema (1-5 h), xylene-induced ear edema, cotton pellet-induced granuloma formation, and CFA-induced arthritis in rats. The effects of HeTA in various models were similar to the effect of the standard reference drugs. Conclusions Findings from this study showed that HeTA possesses antinociceptive effect possibly mediated through peripheral opioid receptors with activation of l-arginine-nitric oxide and ATP-sensitive potassium channel pathway as well as anti-inflammatory activity.