Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL₁₇₇ and PRL₁₈₈; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6 h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na⁺/Cl^? cotransporter and a Na⁺/H⁺ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na⁺/K⁺/2Cl^? cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6 h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6 h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.     

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K⁺) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K⁺ supplementation (1.31 ± 0.04 mmol/L and 2.06 ± 0.04 mmol/L K⁺; mean ± SEM), one was maintained with crystal salt and no K⁺ supplementation (0.33 ± 0.05 mmol/L K⁺), the fourth system was maintained using a standard marine mix salt (2.96 ± 0.04 mmol/L K⁺), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na⁺/K⁺-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na⁺/K⁺/2Cl^? cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K⁺ supplementation resulted in 100% mortality within 24 h. Mortality and dry mass were significantly influenced by K⁺ concentration (P ≤ 0.05). No differences were observed among treatment groups for NKA activity. At 1 dph NKA mRNA expression was higher in the 0.3 mmol [K⁺] group than in other treatment groups and at 7 dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.     

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl^? through the transporters including the Na⁺, K⁺-ATPase (NKA), Na⁺, K⁺, 2Cl^? cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl^? secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (> 1 day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24 h, the Cl^? secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.     

Effect of fasting on body composition and responses to stress in sunshine bass

The integrated responses of the hormonal regulation of growth and stress in sunshine bass (Morone chrysops X Morone saxatilis) as regulated by feed deprivation were investigated. Groups of fish were fed 1.5% of the body weight per day or offered no feed for 4 weeks. Another group of fish was not fed for 3 weeks and feed was offered during the fourth week. Fish in each group were sampled immediately before or after a 15-min low water confinement stressor after each week of the experiment. Liver mass and liver glycogen content were decreased after one week of fasting and remained low until the end of the study. However, both recovered after a week of refeeding. Intraperitoneal fat was significantly lower after two weeks of fasting and did not recover after a week of refeeding. None of these components were affected by confinement stress. Plasma glucose in unstressed fish was generally unaffected by fasting or refeeding; however, plasma glucose increased after confinement stress in fed but not in fasted fish. The cortisol stress response was unaltered by fasting and remained robust. Plasma IGF-I generally decreased in fasted fish but was not significantly lower than fed fish until the fourth week. A week of refeeding did not restore plasma IGF-I concentrations. Plasma IGF-I concentrations were higher in confinement stressed fed fish after two and four weeks but were unchanged in the fourth week. There was no change in the plasma IGF-I concentrations in fasted or refed fish due to the stress. Liver weight and liver glycogen were essentially depleted after 2 weeks of fasting. The reduction of liver glycogen greatly reduced the glucose response to stress; however, the cortisol stress response was maintained for at least four weeks of fasting. Intraperitoneal fat was decreased very little after 4 weeks of fasting. Plasma IGF-I concentrations were reduced only after 3 weeks of fasting.     

Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation

The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na⁺/K⁺-ATPase (NKA) and vacuolar-type H⁺-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na⁺ concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model.     

Expression of sodium/hydrogen exchanger 3 and cation-chloride cotransporters in the kidney of Japanese eel acclimated to a wide range of salinities

Reabsorption of monovalent ions in the kidney is essential for adaptation to freshwater and seawater in teleosts. To assess a possible role of Na⁺/H⁺ exchanger 3 (NHE3) in renal osmoregulation, we first identified a partial sequence of cDNA encoding NHE3 from the Japanese eel kidney. For comparison, we also identified cDNAs encoding kidney specific Na⁺–K⁺–2Cl^? cotransporter 2 (NKCC2α) and Na⁺–Cl^? cotransporter (NCCα). In eels acclimated to a wide range of salinities from deionized freshwater to full-strength seawater, the expression of NHE3 in the kidney was the highest in eel acclimated to full-strength seawater. Meanwhile, the NCCα expression exhibited a tendency to increase as the environmental salinity decreased, whereas the NKCC2α expression was not significantly different among the experimental groups. Immunohistochemical studies showed that NHE3 was localized to the apical membrane of epithelial cells composing the second segments of the proximal renal tubule in seawater-acclimated eel. Meanwhile, the apical membranes of epithelial cells in the distal renal tubule and collecting duct showed more intense immunoreactions of NKCC2α and NCCα, respectively, in freshwater eel than in seawater eel. These findings suggest that renal monovalent-ion reabsorption is mainly mediated by NKCC2α and NCCα in freshwater eel and by NHE3 in seawater eel.     

Short-term mercury exposure on Na+/K+-ATPase activity and ionoregulation in gill and brain of an Indian major carp,Cirrhinus mrigala

Recently mercury pollution has been increased considerably in aquatic resources throughout the world and it is a growing global concern. In this study, the 96 h LC50 value of waterborne mercuric chloride for Cirrhinus mrigala was found to be 0.34 mg/L (with 95% confidence limits). Fingerlings of C. mrigala were exposed to 0.068 and 0.034 mg/L of mercuric chloride for 96 h to assess the Na⁺/K⁺-ATPase activity and ionoregulation (Na⁺, K⁺ and Cl^?) in gill and brain. Results showed that Na⁺/K⁺-ATPase activity and ionic levels (Na⁺, K⁺ and Cl^?) in gill and brain of fish exposed to different concentrations of mercuric chloride were found to be significantly (p < 0.05) decreased throughout the study period. Mercury inactivates many enzymes by attaching to sulfur atoms in which the enzyme Na⁺/K⁺-ATPase is highly sensitive to mercury. The inhibition of gill and brain Na⁺/K⁺-ATPase activity might have resulted from the physicochemical alteration of the membrane due to mercury toxicity. Moreover, inhibition of Na⁺/K⁺-ATPase may affect the ion transport and osmoregulatory function by blocking the transport of substances across the membrane by active transport. The present study indicates that the alterations in these parameters can be used in environmental biomonitoring of mercury contamination in aquatic ecosystem.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [³H]-ouabain binding (to analyze K⁺ site of Na⁺, K⁺-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [³H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2 mg/kg) and clozapine (3, 10 and 30 mg/kg). Animals were sacrificed 18 h later, cerebral cortices harvested, membrane fractions prepared and high affinity [³H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10 mg/kg clozapine. Rats were administered with neurotensin (3, 10 y 30 μg, i.c.v.) and 60 min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [³H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30 μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [³H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2 mg/kg, i.p.) or clozapine (10 mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na⁺, K⁺-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na⁺, K⁺-ATPase at central synapses.     

Ion and water balance in Gryllus crickets during the first twelve hours of cold exposure

Insects lose ion and water balance during chilling, but the mechanisms underlying this phenomenon are based on patterns of ion and water balance observed in the later stages of cold exposure (12 or more hours). Here we quantified the distribution of ions and water in the hemolymph, muscle, and gut in adult Gryllus field crickets during the first 12 h of cold exposure to test mechanistic hypotheses about why homeostasis is lost in the cold, and how chill-tolerant insects might maintain homeostasis to lower temperatures. Unlike in later chill coma, hemolymph [Na⁺] and Na⁺ content in the first few hours of chilling actually increased. Patterns of Na⁺ balance suggest that Na⁺ migrates from the tissues to the gut lumen via the hemolymph. Imbalance of [K⁺] progressed gradually over 12 h and could not explain chill coma onset (a finding consistent with recent studies), nor did it predict survival or injury following 48 h of chilling. Gryllus veletis avoided shifts in muscle and hemolymph ion content better than Gryllus pennsylvanicus (which is less chill-tolerant), however neither species defended water, [Na⁺], or [K⁺] balance during the first 12 h of chilling. Gryllus veletis better maintained balance of Na⁺ content and may therefore have greater tissue resistance to ion leak during cold exposure, which could partially explain faster chill coma recovery for that species.     

A novel three-TMH Na+/H+ antiporter and the functional role of its oligomerization

Na⁺/H⁺ antiporters are a category of ubiquitous transmembrane proteins with various important physiological roles in almost all living organisms ranging from bacteria to humans. However, the knowledge of novel Na⁺/H⁺ antiporters remains to be broadened, and the functional roles of oligomerization in these antiporters have not yet been thoroughly understood. Here, we reported functional analysis of an unknown transmembrane protein composed of 103 amino acid residues. This protein was found to function as a Na⁺(Li⁺, K⁺)/H⁺ antiporter. To the best of our knowledge, this antiporter is the minimal one of known Na⁺/H⁺ antiporters and thus designated as NhaM to represent the minimal Na⁺/H⁺ antiporter. NhaM and its homologs have not yet been classified into any protein family. Based on phylogenetic analysis and protein alignment, we propose NhaM and its homologs to constitute a novel transporter family designated as NhaM family. More importantly, we found that NhaM is assembled with parallel protomers into a homo-oligomer and oligomerization is vital for the function of this antiporter. This implies that NhaM may adopt and require an oligomer structure for its normal function to create a similar X-shaped structure to that of the NhaA fold. Taken together, current findings not only present the proposal of a novel transporter family but also positively contribute to the functional roles of oligomerization in Na⁺/H⁺ antiporters.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia

We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl^? secretion in human submucosal serous Calu-3 cells under a Na⁺/K⁺/2Cl^? cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl^? by diminishing the activity of NKCC1 without blockade of apical Cl^? channel (TEI-589a > TEI-602a > TEI-589b), while any other tested compounds including ambroxol had no effects on Cl^? secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl^? secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl^? secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.     

Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.     

Synthesis and ionophoric activities of functionalized bis(choloyl) conjugates with a rigid core

Three bis(choloyl) conjugates bearing a rigid p-phenylenediamine/p-bis(aminomethyl)benzene linker and amino/acetamido groups were synthesized, and fully characterized on the basis of ¹H NMR, ESI-MS and HRMS. Their ionophoric activities were investigated by means of pH discharge assay. The results indicate that these conjugates exhibit potent ionophoric activities across egg-yolk l-α-phosphatidylcholine (EYPC)-based liposomal membranes, via a cation/proton antiport mechanism. They show moderate ion selectivity among alkali metal ions. Of the three conjugates, the ones having amino groups transport alkali metal ions in the order of Na⁺ > Li⁺ > K⁺ ≈ Rb⁺ ≈ Cs⁺, whereas the one having acetamido groups functions in the order of Li⁺ > Na⁺ > K⁺ ≈ Rb⁺ ≈ Cs⁺.     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

IGF-I mediated inhibition of leptin receptor expression in porcine hepatocytes

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from swine and seeded into T-25 flasks. Cultures were established in medium containing fetal bovine serum for one day and switched to serum-free medium (William's E medium and 1 ng/mL insulin) for the remainder of the 3 d culture period. For the final 24 h, medium was supplemented with porcine growth hormone (GH, 100 or 500 ng/mL), insulin-like growth factor 1 (IGF-1, 50 to 250 ng/mL) or triiodothyronine (T3, 100 ng/mL). RNA was extracted and relative quantitative RT-PCR was performed with primers for long form leptin receptor. Receptor expression was calculated relative to 18S rRNA. Insulin had no effect (P > 0.05), while T3 increased leptin receptor mRNA abundance (P < 0.05). Treatment with GH or IGF-I reduced leptin receptor expression (P < 0.05). Phosphorylation of ERK1/2 in response to acute leptin treatment was inhibited by previous exposure to GH or IGF-I. Hepatocytes secreted IGF-I under basal conditions and this was enhanced by GH addition. These data suggest porcine hepatocytes may be less sensitive to leptin stimulation due to the actions of endogenous IGF-I on leptin receptor expression.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

Morphofunctional modifications in gill mitochondria-rich cells of Mozambique tilapia transferred from freshwater to 70% seawater, detected by dual observations of whole-mount immunocytochemistry and scanning electron microscopy

Acute responses of gill mitochondria-rich (MR) cells to direct transfer from freshwater to 70% seawater were examined in a euryhaline teleost Mozambique tilapia (Oreochromis mossambicus). Scanning electron microscopic (SEM) observations revealed that apical openings of MR cells were morphologically classified into an apical pit, a convex apical surface, a concave apical surface, and a transitory apical surface. Meanwhile, in whole-mount immunocytochemistry with anti-Na⁺/K⁺-ATPase (NKA), T4 antibody (detecting apical Na⁺/Cl^? cotransporter (NCC) and basolateral Na⁺/K⁺/2Cl^? cotransporter (NKCC)), and anti-Na⁺/H⁺ exchanger-3 (NHE3), NKA-immunoreactive MR cells were functionally classified into immature cells without both NKCC/NCC and NHE3 (type I), ion-absorptive cells with apical NCC (type II), those with apical NHE3 (type III), and ion-secretory cells with basolateral NKCC (type IV). Dual observations of whole-mount immunocytochemistry and SEM clearly showed morphofunctional alterations in MR cells. After transfer to 70% seawater, type-II MR cells with a convex surface or pit closed their apical openings to suspend ion absorption. Type-III MR cells with a concave surface or pit were transformed into type-IV MR cells with an enlarged pit, via a transitory surface. Our findings indicate functional plasticity of type-III/IV MR cells to switch ion-transport functions, whereas type-II MR cells are considered to be specific for freshwater adaptation.