A central role for cAMP/EPAC/RAP/PI3K/AKT/CREB signaling in LH-induced follicular Pgr expression at medaka ovulation†.

Nuclear progestin receptor (PGR) is a ligand-activated transcription factor that has been identified as a pivotal mediator of many processes associated with ovarian and uterine function, and aberrant control of PGR activity causes infertility and disease including cancer. The essential role of PGR in vertebrate ovulation is well recognized, but the mechanisms by which PGR is rapidly and transiently induced in preovulatory follicles after the ovulatory LH surge are not known in lower vertebrates. To address this issue, we utilized the small freshwater teleost medaka Oryzias latipes, which serves as a good model system for studying vertebrate ovulation. In the in vitro ovulation system using preovulatory follicles dissected from the fish ovaries, we found that inhibitors of EPAC (brefeldin A), RAP (GGTI298), PI3K (Wortmannin), AKT (AKT inhibitor IV), and CREB (KG-501) inhibited LH-induced follicle ovulation, while the PKA inhibitor H-89 had no effect on follicle ovulation. The inhibitors capable of inhibiting follicle ovulation also inhibited follicular expression of Pgr and matrix metalloproteinase-15 (Mmp15), the latter of which was previously shown to not only be a downstream effector of Pgr but also a proteolytic enzyme indispensable for follicle rupture in medaka ovulation. Further detailed analysis revealed for the first time that the cAMP/EPAC/RAP/PI3K/AKT/CREB signaling pathway mediates the LH signal to induce Pgr expression in preovulatory follicles. Our data also showed that phosphorylated Creb1 is a transcription factor essential for pgr expression and that Creb1 phosphorylated by Akt1, rather than PKA, may be preferably used to induce pgr expression.

Effects of hypoxic acclimation, muscle strain, and contraction frequency on nitric oxide-mediated myocardial performance in steelhead trout (Oncorhynchus mykiss).

Whether hypoxic acclimation influences nitric oxide (NO)-mediated control of fish cardiac function is not known. Thus, we measured the function/performance of myocardial strips from normoxic- and hypoxic-acclimated (40% air saturation; ∼8 kPa O2) trout at several frequencies (20-80 contractions·min-1) and two muscle strain amplitudes (8% and 14%) when exposed to increasing concentrations of the NO donor sodium nitroprusside (SNP) (10-9 to 10-4 M). Further, we examined the influence of 1) nitric oxide synthase (NOS) produced NO [by blocking NOS with 10-4 M NG-monomethyl-l-arginine (l-NMMA)] and 2) soluble guanylyl cyclase mediated, NOS-independent, NO effects (i.e., after blockade with 10-4 M ODQ), on myocardial contractility. Hypoxic acclimation increased twitch duration by 8%-10% and decreased mass-specific net power by ∼35%. However, hypoxic acclimation only had minor impacts on the effects of SNP and the two blockers on myocardial function. The most surprising finding of the current study was the degree to which contraction frequency and strain amplitude influenced NO-mediated effects on myocardial power. For example, at 8% strain, 10-4 SNP resulted in a decrease in net power of ∼30% at 20 min-1 but an increase of ∼20% at 80 min-1, and this effect was magnified at 14% strain. This research suggests that hypoxic acclimation has only minor effects on NO-mediated myocardial contractility in salmonids, is the first to report the high frequency- and strain-dependent nature of NO effects on myocardial contractility in fishes, and supports previous work showing that NO effects on the heart (myocardium) are finely tuned spatiotemporally.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na+/H+ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

SDHI Fungicide Toxicity and Associated Adverse Outcome Pathways: What Can Zebrafish Tell Us?

Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agriculture to combat molds and fungi, two major threats to both food supply and public health. However, the essential requirement for the succinate dehydrogenase (SDH) complex-the molecular target of SDHIs-in energy metabolism for almost all extant eukaryotes and the lack of species specificity of these fungicides raise concerns about their toxicity toward off-target organisms and, more generally, toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish (Brachydanio rerio) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil, isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicate that these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimes at developmentally relevant concentrations. Adverse effects include developmental toxicity, cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes in metabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggesting that glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most of these SDHIs. However, other adverse outcome pathways, possibly involving unsuspected molecular targets, are also suggested. Lastly, we note that because of their recent arrival on the market, the number of studies addressing the toxicity of these compounds is still scant, emphasizing the need to further investigate the toxicity of all SDHIs currently used and to identify their adverse effects and associated modes of action, both alone and in combination with other pesticides.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Identification of Phenolic Compounds by LC-MS/MS and Evaluation of Bioactive Properties of Two Edible Halophytes: Limonium effusum and L. sinuatum.

This work aimed to evaluate the phenolic content and in vitro antioxidant, antimicrobial and enzyme inhibitory activities of the methanol extracts and their fractions of two edible halophytic Limonium species, L. effusum (LE) and L. sinuatum (LS). The total phenolic content resulted about two-fold higher in the ethyl acetate fraction of LE (522.82 ± 5.67 mg GAE/g extract) than in that of LS (274.87 ± 1.87 mg GAE/g extract). LC-MS/MS analysis indicated that tannic acid was the most abundant phenolic acid in both species (71,439.56 ± 3643.3 µg/g extract in LE and 105,453.5 ± 5328.1 µg/g extract in LS), whereas hyperoside was the most abundant flavonoid (14,006.90 ± 686.1 µg/g extract in LE and 1708.51 ± 83.6 µg/g extract in LS). The antioxidant capacity was evaluated by DPPH and TAC assays, and the stronger antioxidant activity in ethyl acetate fractions was highlighted. Both species were more active against Gram-positive bacteria than Gram negatives and showed considerable growth inhibitions against tested fungi. Interestingly, selective acetylcholinesterase (AChE) activity was observed with LE and LS. Particularly, the water fraction of LS strongly inhibited AChE (IC50 = 0.199 ± 0.009 µg/mL). The ethyl acetate fractions of LE and LS, as well as the n-hexane fraction of LE, exhibited significant antityrosinase activity (IC50 = 245.56 ± 3.6, 295.18 ± 10.57 and 148.27 ± 3.33 µg/mL, respectively). The ethyl acetate fraction and methanol extract of LS also significantly inhibited pancreatic lipase (IC50 = 83.76 ± 4.19 and 162.2 ± 7.29 µg/mL, respectively). Taken together, these findings warrant further investigations to assess the potential of LE and LS as a bioactive source that can be exploited in pharmaceutical, cosmetics and food industries.

The reduction of lipid-sourced energy production caused by ATGL inhibition cannot be compensated by activation of HSL, autophagy, and utilization of other nutrients in fish.

The adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL)-mediated lipolysis play important roles in lipid catabolism. ATGL is considered the central rate-limiting enzyme in the mobilization of fatty acids in mammals. Currently, severe fat accumulation has been commonly detected in farmed fish globally. However, the ATGL-mediated lipolysis and the potential synergy among ATGL, HSL, and autophagy, which is another way for lipid breakdown, have not been intensively understood in fish. In the present study, we added Atglistatin as an ATGL-specific inhibitor into the zebrafish diet and fed to the fish for 5 weeks. The results showed that the Atglistatin-treated fish exhibited severe fat deposition, reduced oxygen consumption, and fatty acid ß-oxidation, accompanied with increased oxidative stress and inflammation. Furthermore, the Atglistatin-treated fish elevated total and phosphorylation protein expressions of HSL. However, the free fatty acids and lipase activities in organs were still systemically reduced in the Atglistatin-treated fish, and the autophagy marker LC3 was also decreased in the liver. On the other hand, glycogenolysis was stimulated but blood glucose was higher in the Atglistatin-treated fish. The transcriptomic analysis also provided the hint that the protein turnover efficiency in Atglistatin-treated fish was likely to be accelerated, but the protein content in whole fish was not affected. Taken together, ATGL plays crucial roles in energy homeostasis such that its inhibition causes loss of lipid-sourced energy production, which cannot be compensated by activation of HSL, autophagy, and utilization of other nutrients.

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish.

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver-Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

Cbx2, a PcG Family Gene, Plays a Regulatory Role in Medaka Gonadal Development.

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish.

Acute effects of sex steroids on visual processing in male goldfish.

Elevations of sex steroids induced by social cues can rapidly modulate social behavior, but we know little about where they act within the nervous system to produce such effects. In male goldfish, testosterone (T) rapidly increases approach responses to the visual cues of females through its conversion to estradiol. Because aromatase is expressed in the retina, we tested if T can acutely influence retina responses to visual stimuli, and investigated the receptor mechanisms that may mediate such effects. Specifically, we measured FOS protein immunoreactivity to determine if T affects cellular responses to visual stimuli that include females, and used electrophysiology to investigate whether T can generally affect light sensitivity. We found that T acutely increased FOS responses to the simultaneous onset of light and the presence of female visual stimuli, both of which would normally be associated with early morning spawning, and increased electrophysiological responses to low intensity light pulses. Both effects were blocked by an estrogen receptor beta (ERß) antagonist, indicating that T is likely being converted to estradiol (E2) and acting through an ERß mediated mechanism to acutely modulate visual processing. Changes in sensory processing could subsequently influence approach behavior to increase reproductive success in competitive mating environments.

The inhibition of GSK-3ß promotes the production of reactive oxygen species via ß-catenin/C/EBPα signaling in the spleen of zebrafish (Danio rerio).

In this study, the mechanism that the inhibition of glycogen synthase kinase-3ß (GSK-3ß) promotes the production of reactive oxygen species (ROS) via ß-catenin/CCAAT/enhancer binding protein α (C/EBPα) signaling was investigated in the spleen of zebrafish (Danio rerio). The results demonstrated that the inhibition of GSK-3ß induced the mRNA expression of ß-catenin and C/EBPα by lithium (Li) treatments or GSK-3ß RNA interference. The levels of hydrogen peroxide (H2O2), superoxide anion (O2.-), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT) and glutathione peroxidase (GSH-PX) were decreased in the spleen and ZF4 cells of zebrafish by Li+ treatments. In addition, GSK-3ß RNA interference increased ROS levels and decreased the activities of CAT and GSH-PX in the spleen. The fluorescence intensity of ROS was increased but the mitochondrial membrane potential (MMP) was decreased by Li+ treatments in ZF4 cells labeled with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and Rhodamine-123, respectively. The results of present study indicated that the inhibition of GSK-3ß promoted the ROS production via ß-catenin/C/EBPα signaling in the spleen of zebrafish, and the balance between ROS and antioxidants could be destroyed by the GSK-3ß/ß-catenin/C/EBPα signaling. The results may be a valuable contribution to understanding the modulatory mechanism of GSK-3ß/ß-catenin/C/EBPα signaling on the antioxidant system in fish species.

Effects of aryl methanesulfonate derivatives on acetylcholinesterase and butyrylcholinesterase.

There is a dire need for new treatments for Alzheimer's disease (AD). Principal drugs have reached maturity, and the number of people affected by AD is growing at a rapid rate. After years of research and many clinical trials, only symptomatic treatments are available. An effective disease-modifying drug for AD needs to be discovered. The research presented in this paper aims to facilitate in the discovery of new potential targets that could help in the ongoing AD research. Aryl methanesulfonate derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. IC50 values between 0.660 and 3.397 µM against AChE and 0.885 and 2.596 µM against BuChE were obtained.

Inhibition effects of pesticides on glutathione-S-transferase enzyme activity of Van Lake fish liver.

Glutathione-S-transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4-dichlorophenoxyacetic acid dimethylamine salt (2,4-D DMA), haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg-1 specific activity and 10.76% yield using GSH-agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. Ki constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4-D DMA, haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin, respectively. λ-Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ-cyhalothrin were competitive, while the other pesticides were noncompetitive.

Peroxiredoxin 1 (Prx1) is a dual-function enzyme by possessing Cys-independent catalase-like activity.

Peroxiredoxin (Prx) was previously known as a Cys-dependent thioredoxin. However, we unexpectedly observed that Prx1 from the green spotted puffer fish Tetraodon nigroviridis (TnPrx1) was able to reduce H2O2 in a manner independent of Cys peroxidation and reductants. This study aimed to validate a novel function for Prx1, delineate the biochemical features and explore its antioxidant role in cells. We have confirmed that Prx1 from the puffer fish and humans truly possesses a catalase (CAT)-like activity that is independent of Cys residues and reductants, but dependent on iron. We have identified that the GVL motif was essential to the CAT-like activity of Prx1, but not to the Cys-dependent thioredoxin peroxidase (POX) activity, and generated mutants lacking POX and/or CAT-like activities for individual functional validation. We discovered that the TnPrx1 POX and CAT-like activities possessed different kinetic features in the reduction of H2O2 The overexpression of wild-type TnPrx1 and mutants differentially regulated the intracellular levels of reactive oxygen species (ROS) and the phosphorylation of p38 in HEK-293T cells treated with H2O2 Prx1 is a dual-function enzyme by acting as POX and CAT with varied affinities towards ROS. This study extends our knowledge on Prx1 and provides new opportunities to further study the biological roles of this family of antioxidants.

Involvement of xanthine oxidase inhibition with the antioxidant property of nanoencapsulated Melaleuca alternifolia essential oil in fish experimentally infected with Pseudomonas aeruginosa.

Nanoencapsulated Melaleuca alternifolia essential oil (tea tree oil, TTO) is a natural alternative treatment, with 100% therapeutic efficacy in fish experimentally infected with Pseudomonas aeruginosa, and has also potent protective effects linked with antioxidant properties. However, the pathways responsible for the antioxidant capacity remain unknown. Thus, this study evaluated whether the inhibition of seric xanthine oxidase (XO) activity can be considered a pathway involved in the antioxidant capacity of nanoencapsulated TTO in fish experimentally infected with P. aeruginosa. Seric samples from fish infected with P. aeruginosa showed increased XO activity, as well as increased uric acid and reactive oxygen species (ROS) levels. In contrast, the prophylactic treatment with nanoencapsulated TTO prevented these infection-induced alterations. Based on the evidence obtained, the upregulation of seric XO activity induced pro-oxidative effects in the serum of fish experimentally infected with P. aeruginosa, due to excessive formation of uric acid, which stimulates the release of ROS. This treatment was able to prevent the upregulated seric XO activity and, consequently, the excessive formation of uric acid and ROS. In summary, inhibition of seric XO activity can be considered a pathway involved in the antioxidant capacity of nanoencapsulated TTO in fish experimentally infected with P. aeruginosa.

Inhibitory properties of some heavy metals on carbonic anhydrase I and II isozymes activities purified from Van Lake fish (Chalcalburnus Tarichi) gill.

In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring "CO2-hydratase activity". Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu2+, Ag+, Cd2+, Ni2+ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu2+, Ag+, Cd2+, and Ni2+ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC50 values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC50 values were determined as 3.39, 6.38, 13.52, and 206 µM for CA I isozyme and 6.16, 20.29, 46, and 223 µM for CA II isozyme respectively.

Conjugates of Tacrine and Its Cyclic Homologues with p-Toluenesulfonamide as Novel Acetylcholinesterase and Butyrylcholinesterase Inhibitors.

Using the acylation reaction with tosyl chloride of N-aminopropyl analogues of tacrine and its cyclic homologues with different size of the aliphatic cycle (5-8), we synthesized a number of new derivatives of p-toluenesulfonamide. It is shown that the synthesized hybrid compounds of tacrine and p-toluenesulfonamide are effective inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with the preferential inhibition of BChE. They also displace propidium from the peripheral anionic site of the electric eel AChE (Electrophorus electricus). The characteristics of the efficiency and selectivity of cholinesterase inhibition by the test compounds were confirmed by the results of molecular docking.

Determination of affinity and efficacy of acetylcholinesterase inhibitors using isothermal titration calorimetry.

BACKGROUND: Acetylcholinesterase (AChE), an enzyme rapidly terminating nerve signals at synapses of cholinergic neurons is an important drug target in treatment of Alzheimer's disease and related memory loss conditions. Here we present comprehensive use of isothermal titration calorimetry (ITC) for investigation of AChE kinetics and AChE-inhibitor interactions. METHODS: Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus was assayed for interactions with five well known AChE inhibitors, galanthamine, tacrine, donepezil, edrophonium and ambenonium. In ITC experiments the inhibitors were injected to the enzyme solution solely (for thermodynamic characterization of binding) or in presence of the substrate, acetylcholine (for determination of inhibitors potency). RESULTS: Detailed description of various experimental protocols is presented, allowing evaluation of inhibitors potency (in terms of IC50 and Ki) and thermodynamic parameters of the binding. The potency of tested inhibitors was in nano to micromolar range which corresponded to activities determined in conventional method. Binding of all inhibitors showed to be enthalpy driven and obtained Ka values demonstrated good correlation with the data from standard Ellman's assay. CONCLUSIONS: Obtained results confirmed the usability of the ITC technique for comprehensive characterization of AChE-inhibitor interactions and AChE kinetics. The method reduced the complexity of reaction mixture and interference problems with the advantage of using natural substrates. GENERAL SIGNIFICANCE: The work reports complete thermodynamic characteristics of the AChE - inhibitor complexes. Due to the universal character of ITC measurements, described protocols can be easily adapted to other enzymatic systems.

Cortisol Induces Reactive Oxygen Species Through a Membrane Glucocorticoid Receptor in Rainbow Trout Myotubes.

Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc.

Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus).

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca2+-dependent K+ current (IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca2+, and was reduced by 100 µM Cd2+ A voltage-dependent inward current in Ba2+ solution, with peak at 0 mV, confirmed the presence of Ca2+ channels. A voltage-dependent K+ current (IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K+ current (IKB) with open rectification. Mean resting membrane potential of -45.2 ± 11.6 mV did not change upon administration of hypoxia (Po2 = 11 mmHg), nor were any of the K+ currents sensitive to changes in Po2 during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca2+ concentration and initiated synaptic vesicle activity. 100 µM Cd2+ and 50 µM nifedipine eliminated uptake of FM 1-43. We conclude that Ca2+ influx via L-type Ca2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia.NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca2+ channels and three types of K+ channels, one of which was dependent upon intracellular Ca2+ Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca2+ and synaptic vesicle activity.