Ontogeny and osmoregulatory function of the urinary system in the Persian sturgeon,Acipenser persicus (Borodin, 1897)

The structure of the kidney and the localization of Na⁺, K⁺-ATPase (NKA) immunopositive cells were examined throughout the postembryonic development of the Persian sturgeon, Acipenser persicus, from newly hatched prelarvae (10 mm) to 20 days post hatch (20 DPH) larvae (31 mm). Investigations were conducted through histology and immunohistochemistry by using the light and immunofluorescence microscopy. The pronephros was observed in newly hatched prelarvae. The cells lining the distal pronephric tubules and their collecting ducts showed laterally expressed NKA immunofluorescence that later extended throughout the whole cytoplasm. Mesonephrogenous placodes and pre-glomeruli were distinguished at 2 DPH along the collecting ducts posteriorly. Their tubules were formed and present in kidney mesenchyma, differentiated into neck, proximal, distal and collecting segments at 7 DPH when NKA immunopositive cells were observed. Their distal and collecting tubules showed an increasing immunofluorescence throughout their cytoplasm while the glomeruli remained unstained. From D 9 to D 17, the epithelial layer of pronephric collecting duct changed along the mesonephros to form ureters. Ureters, possessing isolated strong NKA immunopositive cells, appeared as two sac-like structures hanging under the trunk kidney. Since NKA immunopositive cells were not observed on the tegument or along the digestive tract of newly hatched prelarva, and also the gills are not formed yet, the pronephros is the only osmoregulatory organ until 4 DPH. At the larval stage, the pronephros and mesonephros are functional osmoregulatory organs and actively reabsorb necessary ions from the filtrate.     

Application of microsatellite markers for genetic conservation and management of Persian sturgeon (Acipenser persicus,Borodin, 1897) in the Caspian Sea

A study was conducted to ascertain the genetic structure and the level of heterozygosity of Acipenser persicus in the Caspian Sea. A total of 167 fish were randomly collected from Turkmenistan, Russia and two regions of Iran. The number of alleles of eleven microsatellite markers ranged from 3 to 21 and the mean observed values of heterozygosity were 0.56 ± 0.20, 0.64 ± 0.14, 0.67 ± 0.16, and 0.64 ± 0.11. The observed heterozygosity was lower than the expected levels. The observed low genetic differentiation indicates that all populations are closely related. Hence, inbreeding is a potential problem, which should be taken into consideration in future breeding programs to avoid a further decline in genetic diversity.     

Growth performance and body composition of sub-yearling Persian sturgeon, (Acipenser persicus, Borodin, 1897), fed different dietary protein and lipid levels

In order to evaluate the protein and energy requirement of Persian sturgeon (Acipenser persicus) sub‐yearlings, eight experimental diets containing two protein levels (40% and 45%) and four lipid levels (10%, 15%, 20% and 25%) were tested. Sturgeons (W₀ = 136.8 g) were fed the experimental diets to satiation four times daily for 150 days, resulting in a final mean weight of 375.8 g. Growth was significantly affected by lipid content of the diets. At 40% protein level, weight gain and specific growth rate (% per day) were significantly improved (P < 0.05) by increasing the dietary lipid (energy) content. Protein efficiency ratio (PER) was significantly affected by different dietary treatments for each dietary protein level tested, reaching a mean value of 3.58 in fish fed high lipid diets and a PER of 2.77 in low lipid diets. Results obtained in the present study suggest that the optimum dietary protein content for Persian sturgeon is 40%, with an estimated optimum protein‐to‐energy ratio of 18–20 mg kJ⁻¹.     

Effect of starvation and re‐feeding on growth performance and content of plasma lipids,glucose and insulin in cultured juvenile Persian sturgeon (Acipenser persicus Borodin, 1897)

The effect of starvation and subsequent re‐feeding to satiation on compensatory growth performance, insulin and blood serum values were investigated in juvenile Persian sturgeon (Acipencer persicus) with an average weight 108.04 ± 0.28 g (mean ± SEM) and in the same rearing condition over an 8‐week period. Sturgeons were allocated to one of five feeding treatments: controls (C, continuous feeding), W1 (1 week starvation), W2 (2 weeks starvation), W3 (3 weeks starvation) and W4 (4 weeks starvation), followed by a single 4 weeks of re‐feeding to satiation. Changes in growth performance and blood serum indices were examined at the end of weeks 4 and 8. Body weight, specific growth rate (SGR), condition factor (CF) and weight gain were determined to have significantly decreased during starvation. Fish starved for 1 week reached the same weight as the control fish after re‐feeding for 4 weeks, indicating that complete compensatory growth occurred. Although the specific growth rate in W2, W3 and W4 fish was greater than that in the control fish after re‐feeding, W2, W3 and W4 fish did not reach the same body weight as control fish at the end of re‐feeding period, and showed partial compensation only. Blood plasma, glucose and insulin concentrations did not change significantly during starvation and re‐feeding (P > 0.05). This suggests that sturgeon are able to maintain glycaemia during starvation, probably due to their non‐carbohydrate dietary source. Plasma total lipid and triglyceride levels increased in starvation treatments, whereas the increases were significant only in W3 treatment (P < 0.05). After a 4‐week re‐feeding period, their levels decreased in comparison to the starvation periods. Increases in plasma total lipid and triglyceride levels appear to be due to their roles as preferred nutrients for mobilization in Persian sturgeon and the magnitude and duration of compensatory growth depended on the length of food deprivation.     

Anesthesia of wild female Persian sturgeon,Acipenser persicus (Borodin, 1897) breeders during controlled propagation: effects on hematological parameters,stress response and reproductive performance

This study examined the effects of anesthesia on the hematological and biochemical parameters as well as the reproductive performance of wild female Persian sturgeon, Acipenser persicus, during controlled spawning. Fourteen mature females were divided into two groups: ‘anesthetized’ and ‘non‐anesthetized’. All activities including transportation, catheterizing and handling were performed with both groups: (i) under anesthesia (150 ppm clove oil), and (ii) without anesthesia. After 10 days storage and handling, blood samples were taken from all fish after anesthesia. No significant differences were found in the reproductive performance of either group. However, differences were found in the hematological parameters, with values being significantly higher in the non‐anesthetized group, including neutrophils (34.36 ± 6.33% vs 23.63 ± 5.22%), monocytes (2.84 ± 1.70% vs 1.27 ± 0.64%), mean corpuscular volume (321.3 ± 39.40 pg vs 228.0 ± 24.46 pg) and mean corpuscular hemoglobin (106.9 ± 15.70 fl vs 76.50 ± 7.50 fl). Significantly lower values were found in the non‐anesthetized group for lymphocytes (60.68 ± 7.25% vs 73.54 ± 4.80%), Hb (4.62 ± 0.74 mg dl^?1 vs 6.28 ± 1.21 mg dl^?1), Hct (13.86 ± 1.76% vs 18.84 ± 3.85%), red blood cell (0.43 ± 0.05 cell mm^?3 vs 0.85 ± 0.13 × 10⁶ cell mm^?3) and white blood cell (22 403 ± 2240 cell mm^?3 vs 35 318 ± 3084 cell mm^?3). The non‐anesthetized fish had significantly higher cortisol levels compared to the anesthetized group (62.33 ± 8.85 ng ml^?1 vs 46.12 ± 8.07 ng ml^?1). There was no difference in plasma glucose levels between groups. It is concluded that the use of clove oil as an anesthetic is suitable for handling of wild female Persian sturgeon in controlled propagation programmes. However, further research is needed to determine a standardized protocol for the safe application of anesthesia for use in sturgeons in general.     

Monthly fluctuations during the breeding season of sperm density,volume, motility,and composition of seminal and coelomic fluid in broodfish of Persian sturgeon,Acipenser persicus Borodin, 1897

Obtaining knowledge on the gamete quality of certain species is essential for appropriate management and conservation of wild stocks of the species concerned. In the present study, eighteen breeders (nine males and nine females) of Acipenser Persicus Borodin, 1897 were selected for recording breeding season changes in gamete quality in terms of the parameters: spermatozoa motility indices, sperm volume and density, ionic composition and osmolality of seminal and coelomic fluids. Stripping was performed at the beginning of March and during April and May. Results indicated that sperm volume (ml per male) and density (×10⁹ spz ml^?1) decreased towards the end of the spawning season. There was no significant change in osmolality (mOsmol kg^?1) or in the concentrations (as mm ) of sodium, potassium, magnesium or calcium ions in either the seminal or the coelomic fluids during the breeding season. A significant difference in chloride ion concentration in the coelomic fluid was noted at different times during the spawning season. The percentage of motile spermatozoa at 45 s post‐activation was not significantly different for samples taken at different dates, but the maximum duration of spermatozoa movement, at 15 s post‐activation, was observed in March. This value decreased significantly towards the end of the reproductive season. In conclusion, changes observed in A. persicus sperm parameters during the breeding season suggest an association between such changes and the sperm aging processes.     

Effects of different concentrations of 2‐phenoxyethanol on primary and secondary stress responses in Persian sturgeon,Acipenser persicus

The anaethetic effects of 2‐phenoxyethanol (2‐PE) on possible primary (cortisol level) and secondary (hematological indices and glucose level) stress responses were studied in Persian sturgeon (Acipenser persicus). Fish were first exposed to 0.1, 0.3, 0.5, 0.7, 0.9 and 1.1 ml L^?1 2‐PE, and the time to induction (deep anaethesia) and recovery were measured. At a concentration of 0.1 ml L^?1, 2‐PE failed to induce deep anaethesia in fish, whereas at concentrations of 0.7, 0.9 and 1.1 ml L^?1 all fish were anaethetized within 3 min of exposure. For assessing possible stress effects caused by effective concentrations of 2‐PE, the hematological indices, serum cortisol and glucose were determined in the deeply anaethetized fish as stress indicators. The 2‐PE exposure resulted in significant increases in red blood cell (RBC) values at 0.3 and 0.5 ml L^?1; parallel increases in hemoglobin values were also observed at these concentrations (P < 0.01). Moreover, a lower concentration of 2‐PE (0.3 ml L^?1) caused a significant increase in hematocrit values (P < 0.05). Among the hematological indices, mean corpuscular volume (MCV) values decreased at 0.5 ml L^?1 when compared with the control and other groups (P < 0.05). Serum cortisol level was elevated at 0.3, 0.5 and 0.7 concentrations of 2‐PE (P < 0.01). The glucose level followed a trend similar to that observed for cortisol. The outcome of these experiments shows that 2‐PE at a concentration of 0.9 ml L^?1 is a suitable anaesthetic for Persian sturgeon. This study demonstrates that rapid induction of deep anaethesia with a relatively high concentration of 2‐PE (0.9 and 1.1 ml L^?1) was associated with the lowest effects on signs of physiological stress in Persian sturgeon.     

Phosphoregulation of K+‐Cl− cotransporter 4 during changes in intracellular Cl− and cell volume

It has long been stated that the K⁺‐Cl^? cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N‐ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C_VOL) and intracellular [Cl^?] ([Cl^?]_i) on the activity and phosphorylation levels (P_LEV) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)‐sensitive effectors. We found that (1) low [Cl^?]_i or low C_VOL leads to decreased activity but increased P_LEV, (2) high C_VOL leads to increased activity but no decrease in P_LEV and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P_LEV. Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl^?]_i or C_VOL modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A‐sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787–796, 2009. © 2009 Wiley‐Liss, Inc.     

Different neuronal Na+/K+‐ATPase isoforms are involved in diverse signaling pathways

Inhibition of rat neuronal Na⁺/K⁺‐ATPase α3 isoform at low (100 nM) ouabain concentration led to activation of MAP kinase cascade via PKC and PIP₃ kinase. In contrast to ouabain‐sensitive α3 isoform of Na⁺/K⁺‐ATPase, an ouabain‐resistant α1 isoform (inhibition with 1 mM of ouabain) of Na⁺/K⁺‐ATPase regulates MAP kinase via Src kinase dependent reactions. Using of Annexin V‐FITC apoptotic test to determine the cells with early apoptotic features allows to conclude that α3 isoform stimulates and α1 suppresses apoptotic process in cerebellum neurons. These data are the first demonstration showing participation of ouabain‐resistant (α1) and ouabain‐sensitive (α3) Na⁺/K⁺‐ATPase isoforms in diverse signaling pathways in neuronal cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of a VxP Targeting Signal in the Flagellar Na+/K+‐ATPase

Na⁺/K⁺‐ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes‐specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin‐B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co‐immunoprecipitates with ankyrin‐B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP‐NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes‐specific NKA isoform.      

Phosphoregulation of K+‐Cl− cotransporters during cell swelling: Novel insights

The K⁺‐Cl^? cotransporters (KCCs) belong to the cation‐Cl^? cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K⁺ and Cl^? ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl^?] (Cl_i), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K⁺‐Cl^? cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.     

Involvement of the K+‐Cl− co‐transporter KCC2 in the sensitization to morphine‐induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti‐anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K⁺‐Cl^? co‐transporter 2 (KCC2) in the sensitization to morphine‐induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ‐aminobutyric acid A‐type receptor (GABA_AR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine‐induced hyperlocomotion, which is accompanied by the up‐regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down‐regulation of protein phosphatase‐1 (PP‐1) as well as the up‐regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP‐1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre‐treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine‐induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ‐PP‐1‐KCC2 pathway by chronic treatment with zolpidem.     

The reverse operation of Na+/Cl−‐coupled neurotransmitter transporters – why amphetamines take two to tango

Sodium‐chloride coupled neurotransmitter transporters achieve reuptake of their physiological substrate by exploiting the pre‐existing sodium‐gradient across the cellular membrane. This terminates the action of previously released substrate in the synaptic cleft. However, a change of the transmembrane ionic gradients or specific binding of some psychostimulant drugs to these proteins, like amphetamine and its derivatives, induce reverse operation of neurotransmitter:sodium symporters. This effect eventually leads to an increase in the synaptic concentration of non‐exocytotically released neurotransmitters [and – in the case of the norepinephrine transporters, underlies the well‐known indirect sympathomimetic activity]. While this action has long been appreciated, the underlying mechanistic details have been surprisingly difficult to understand. Some aspects can be resolved by incorporating insights into the oligomeric nature of transporters, into the nature of the accompanying ion fluxes, and changes in protein kinase activities.     

Comparative genetics of Na+/K+‐ATPase in monarch butterfly populations with varying host plant toxicity

Herbivores have evolved numerous behavioural and physiological adaptations to host plants; however, molecular adaptations are still poorly understood. One well‐studied case comprises the specialist insects that feed on cardenolide‐containing plants. Here, convergent molecular evolution in the Na⁺/K⁺‐ATPase results in a reduced sensitivity to cardenolides across four insect orders. Because different plant species and genotypes differ in toxicity, Na⁺/K⁺‐ATPase may be under differential selection from geographically varying host plants. We examined the α subunit of Na⁺/K⁺‐ATPase in monarch butterflies (Danaus plexippus) from six worldwide populations to test whether differences in their host plant chemistry result in local adaptation at the molecular level. Although our study revealed multiple synonymous changes, we did not find these to be population‐specific, nor did we identify nonsynonymous changes. Additionally, we compared the amino acid sequence of this subunit across 19 species. We identified two novel changes at sites 836 (K836N) and 840 (E840R) in the αM7‐αM8 regions in the genus Danaus. Although previous studies focused on the first two trans‐membrane domains, C‐terminal domains may also interact with cardenolides. These results reveal a lack of molecular evolution of Na⁺/K⁺‐ATPase at the population level, and call for additional attention regarding the C‐terminal regions of this important enzyme.     

Bumetanide-sensitive Na+ /K+ /Cl− transporter is stimulated by phorbol ester and different mitogens in quiescent human skin fibroblasts

In this study we investigated the correlation between the mitogenic effect and stimulation of Rb + (K + ) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide-sensitive and ouabain-sensitive, are stimulated 2-3-fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G₀/G₁ human skin fibroblasts. Three groups of mitogens were compared: (i) the phorbol ester 2-O-tetradecanoyl-phorbol-13-acetate (TPA); (ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C—fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and α-thrombin; and (iii) growth factors that do not activate kinase C—insulin-like growth factor-1 (IGF-1), and transforming like growth-factor-α (TGF-α). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose-dependent fashion. The results indicate that both the bumetanide-sensitive and the ouabain-sensitive Rb+ fluxes are stimulated by protein kinase C-dependent and by the protein kinase C-independent pathways of the mitogenic signal.     

No signs of Na+/K+‐ATPase adaptations to an invasive exotic toxic prey in native squamate predators

Invasions by exotic toxic prey, like the release of the South American cane toad (Bufo (Rhinella) marinus) to the toad‐free Australian continent in 1935, have been shown to result in massive declines in native predator numbers. Due to minor nucleotide mutations of the Na⁺/K⁺‐ATPase gene most Australian squamate predators are highly susceptible to cane toad toxin. However, in spite of this, predators like yellow‐spotted goannas (Varanus panoptes) and red‐bellied black snakes (Pseudechis porhyriacus) still persist in parts of Queensland where they, in some areas, have co‐existed with cane toads for more than 70 years. Here, we show that the amino acids of the Na⁺/K⁺‐ATPase enzyme in the two species do not provide toad toxin resistance, and hence the two Queensland predators are still highly susceptible to cane toad toxin. Both yellow‐spotted goannas and lace monitors (Varanus varius) have, however, been recorded avoiding feeding on cane toads in areas where they co‐exist with this toxic amphibian. Moreover, both varanids have also been shown to learn to avoid feeding on toads when first subjected to conditioned taste aversion. Such behavioural shifts may therefore explain why yellow‐spotted goannas and red‐bellied black snakes still exist in cane toad infested areas of Queensland. The process appears, however, to be unable to rapidly restore varanid populations to pre‐toad population numbers as even after 10 years of co‐existence with cane toads in the Northern Territory, we see no signs of an increase in yellow‐spotted goanna numbers.