Sodium dependent ion transporters in trout gills

Biochemical procedures developed to isolate plasma membranes from the branchial epithelium of rainbow trout (Oncorhynchus mykiss) yield membrane fractions that are specifically enriched in the plasma membrane marker enzyme Na⁺/K⁺-ATPase. As the bulk of the branchial Na⁺/K⁺-ATPase is assumed to be confined to the mitochondria-rich chloride cells, such membrane preparations must contain the essence of the enzymatic machinery of the chloride cells. Basal Na⁺ activity in branchial (chloride) cells is around 10 millimolar and, accordingly, we find a K_m for Na⁺ of the Na⁺/K⁺-ATPase of 13 millimolar, indicating that the enzyme may be regulated by changes in cytosolic sodium. The Na⁺-gradient across the serosal plasma membrane created by this pump provides energy for 3Na⁺/Ca²⁺-exchange and bumetanide-sensitive Na⁺/K⁺/2Cl^--cotransport. Here we further postulate the presence of a Na⁺/Cl^--cotransporter, indicated by thiazide-sensitive, bumetanide-insensitive transport of Na⁺ and Cl^-; this cotransporter activity awaits the characterization of its kinetics. The Na⁺/Ca²⁺-exchanger has kinetic characteristics compatible with a regulatory role of cytosolic Na⁺ in the activity of this carrier. Both Na⁺/Ca²⁺-exchange and Ca²⁺-ATPase activity may contribute to transport of Ca²⁺, the former having lower affinity for calcium but a higher capacity than the latter carrier. The Na⁺/K⁺/2Cl^--cotransporter has kinetics that favor a regulatory role for plasma K⁺ in the activity of this carrier. Seawater adaptation leads to increased activity of cotransporter molecules in the plasma membrane fractions (the activity increases relative to that of the Na⁺/K⁺-ATPase) and this may reflect a function in Cl^--extrusion performed by the chloride cells in a seawater environment. A function for the cotransporter in the gills of freshwater fish may be the regulation of cell volume.     

Characterization of transport Na+-ATPases in gills of freshwater tilapia

Branchial plasma membranes from the freshwater cichlid teleostOreochromis mossambicus (tilapia) contain two Na⁺-dependent ATPases: Na⁺/K⁺ ATPase, and an amiloride-sensitive ATPase which is postulated to operate as a Na⁺/H⁺ (–NH₄ ⁺) ATPase. It is suggested that both enzyme activities are located in the basolateral membrane system of the chloride cells. K⁺ has opposing effects on the two enzymes: it stimulates Na⁺/K⁺ ATPase and inhibits Na⁺/H⁺ (–NH₄ ⁺) ATPase activity. Na⁺/H⁺ ATPase appears more sensitive to NH₄ ⁺ at low concentrations than Na⁺/K⁺ ATPase and the stimulatory effect by NH₄ ⁺ ions on the first enzyme could be important in facilitating NH₄ ⁺ excretion by tilapia gills under physiological conditions.In vitro maximum stimulation by NH₄ ⁺ is similar for the two enzymes (200%). In contrast to Na⁺/K⁺ ATPase, Na⁺/H⁺ ATPase activity is inhibited by supra-physiological (>20 mM) concentrations of NH₄ ⁺.     

Enhanced hypoosmoregulatory response to growth hormone after cortisol treatment in immature rainbow trout,Salmo gairdneri

The growth-independent effect of ovine growth hormone (oGH) and oGH + cortisol treatment on seawater (SW) adaptation in immature rainbow trout, Salmo gairdneri was investigated. Fish were injected every second day with saline, 2.0 μg oGH/g or 2.0 μg oGH + 8.0 μg cortisol/g for a maximum of 8 injections in freshwater (FW). Subgroups were transferred to 28‰ SW after 4 or 8 injections, and changes in plasma Na⁺ and Cl⁻, muscle water content and gill Na⁺/K⁺-ATPase activity were measured. In both of the hormone-treated groups retained in FW, gill Na⁺/K⁺-ATPase activity and interlamellar chloride cell density increased. The effects were most pronounced in the oGH + cortisol group after 2 weeks of treatment. After transfer to SW most of the control fish died due to the osmotic stress, whereas in the hormone-treated groups, mortality was low and there was a positive correlation between pretransfer gill Na⁺/K⁺-ATPase and the ability to maintain ionic-osmotic homeostasis after SW transfer. After two weeks of oGH + cortisol treatment, gill Na⁺/K⁺-ATPase activity was maximal. In contrast, after SW transfer, Na⁺/K⁺-ATPase activity increased further in the oGH-treated group. This group regulated ionic-osmotic parameters less effectively than the oGH + cortisol-treated group. The data indicate that GH and cortisol are important hormones in the regulation of hypoosmoregulatory mechanisms in S. gairdneri.     

Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K⁺ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na⁺, K⁺-ATPase was attained at 10 mol l^–1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K⁺ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K⁺ influx increased, but was inhibited to about 15% by 30 mol l^–1 sulfide. In RBCs of crucian carp neither Na⁺, K⁺-ATPase nor ouabain-resistant K⁺ influx were affected by sulfide concentrations up to 850 mol l^–1. Differences in sulfide-sensitivity of K⁺ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na⁺, K⁺-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K⁺ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species.     

Lipid composition and microsomal ATPase activities in gills and kidneys of warm- and cold-acclimated sea bass (Dicentrarchus labrax L.)

The response to cold of gill and kidney membrane lipid composition and microsomal (Na⁺+K⁺)-ATPase, Na⁺-ATPase and Mg²⁺-ATPase activities in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was carried out according to the seasonal cycle from August to March. No cold-promoted increase in fatty acid unsaturation was shown in gill and kidney polar lipids and in total lipids of mitochondria and microsomes. In both tissues the (Na⁺+K⁺)-ATPase exhibited positive compensation for cold acclimation whereas the Na⁺-ATPase displayed negative compensation. The Mg²⁺-ATPase showed no compensation in the gills and positive compensation in the kidneys. During cold acclimation the break in the Arrhenius plot of the (Na⁺+K⁺)-ATPase decreased, whereas breaks of both the Na⁺-ATPase and the Mg²⁺-ATPase activities remained unchanged. The results indicate that the sea bass does not adopt membrane unsaturation as a cold-facing strategy. The cold-promoted enhancement of (Na⁺+K⁺)-ATPase activity in osmoregulatory tissues may be advantageous to maintain efficient osmoregulation under thermodynamically unfavourable conditions.     

盐度驯化对史氏鲟鳃Na+/K+ ATP酶活力、血清渗透压及离子浓度的影响

对史氏鲟在盐度驯化过程中鳃Na⁺/K⁺ATP酶活力、血清渗透压及血清离子（Na⁺、K⁺、Cl^-）浓度进行了检测和分析，探讨了史氏鲟驯化过程中血清渗透压调节机制。研究表明：史氏鲟在不同盐度（10、20、25）下经过驯化，鳃Na⁺/K⁺ATP酶活力显著高于对照组鳃Na⁺/K⁺ATP酶活力(P<0.05），其活力是对照组的2～2.5倍。驯化过程中，3种不同盐度阶段下鳃Na⁺/K⁺ATP酶活力首先表现为下降，随着驯化时间的延长，活力逐渐增加，最后下降并趋于平稳。血清渗透压也随盐度的增加而上升，盐度10时最高，达到(328.77±26.78) mmol·kg^-1，此后逐渐下降并稳定在290 mmol·kg^-1左右，略高于淡水中血清渗透压。不同盐度下，血清渗透压和鳃Na⁺/K⁺ATP酶活力的变化趋势相同。3种不同盐度下史氏鲟血清K⁺浓度平均值保持在3.00～3.30 mmol·L^-1之间，与对照组相比无显著差异（P>0.05）。3种盐度下血清Na⁺和Cl^-浓度变化趋势基本一致，随着盐度的增高而增高，盐度20时达到最高。盐度20以下血清Na⁺和Cl^-含量没有显著差异（P>0.05）。史氏鲟血清渗透压调节可以分为3个阶段：一是应激反应阶段，主要表现为鳃Na⁺/K⁺ATP酶活力受到抑制，陡然下降；二是主动调节阶段，鳃Na⁺/K⁺ATP酶被重新激活，且活力逐渐上升；三是适应阶段，鳃Na⁺/K⁺ ATP酶趋于平稳。     

Effects of Na/K and Mg/Ca ratios in saline groundwaters on Na-K-ATPase activity, survival and growth of Marsupenaeus japonicus postlarvae

The effects of the Na⁺/K⁺ and Mg²⁺/Ca²⁺ ratios in saline groundwaters on Na⁺-K⁺-ATPase activity, survival and growth of Marsupenaeus japonicus postlarvae were investigated. The results indicate that the Na⁺-K⁺-ATPase activity, survival rate and weight gain of postlarvae were significantly affected by the Na⁺/K⁺ and Mg²⁺/Ca²⁺ ratios (P < 0.05). The Na⁺-K⁺-ATPase activity of postlarvae, in every treatment, changed corresponding to Na⁺/K⁺ and Mg²⁺/Ca²⁺ ratios, and came to a stable level after 24 h. There was a negative relation between Na⁺-K⁺-ATPase activity and Na⁺/K⁺ ratio, while there was a positive relation between Na⁺-K⁺-ATPase activity and Mg²⁺/Ca²⁺ ratio. Compared with seawater (the Na⁺/K⁺ and Mg²⁺/Ca²⁺ ratios are 27.8 and 4.64 respectively), the Na⁺-K⁺-ATPase activity of the Na⁺/K⁺ ratio 30 treatment showed no significant difference, while the Mg²⁺/Ca²⁺ ratio 4.5 treatment showed distinct difference. The survival rates and weight gain of postlarvae increased markedly when the suitable amount of K⁺ and Ca²⁺ was added to test water, and arrived at their maximum in the Na⁺/K⁺ ratio 20-30 or Mg²⁺/Ca²⁺ ratio 4.5 treatment, having no significant difference compared with normal seawater. Therefore, considering the Na⁺/K⁺, Mg²⁺/Ca²⁺ ratios and the absolute concentration of Mg²⁺, Ca²⁺ in the experimental saline groundwaters applied to Marsupenaeus japonicus farming, it should be modulated to around 30, 4.5 and 1312 mg/l, 291 mg/l, respectively.     

黄斑蓝子鱼LC-PUFA 合成代谢与渗透压调节的关系研究

为探讨黄斑蓝子鱼(Siganus canaliculatus)长链多不饱和脂肪酸(long-chain polyunsaturated fatty acids,LC-PUFA)合成代谢与渗透压调节的关系,本研究以鱼油(FO)和混合植物油(苏子油与双低菜籽油,VO)为脂肪源配制两种等氮等脂饲料,投喂饲养在3种盐度(10、20和32)下的黄斑蓝子鱼幼鱼8周后,分析了各处理组幼鱼的生长性能和鳃的磷脂脂肪酸组成、Na+/K+-ATPase(NKA)活力及其基因表达。结果显示,相同盐度下,VO组和FO组鱼的生长性能差异不显著(P0.05);FO组鱼鳃磷脂中的n-3 LC-PUFA含量显著高于VO组(P0.05),但VO组鱼的n-6 LC-PUFA水平显著高于FO组(P0.05);VO组鱼鳃的NKA酶活力及其m RNA表达量都显著高于FO组(P0.05)。不同盐度下,无论VO组还是FO组的鱼,盐度10组鱼的生长性能显著低于盐度20和32组(P0.05),而其鳃的LC-PUFA含量、NKA酶活力及其m RNA表达量都显著高于盐度20和32组(P0.05),各指标在后两个盐度组之间差异不显著(P0.05)。由此可见,盐度10对黄斑蓝子鱼具有一定的胁迫性,导致其生长性能较差。摄食鱼油脂肪源饲料,可以提高鱼鳃磷脂的n-3 LC-PUFA水平;而摄食植物脂肪源饲料时,鱼体可能通过自身合成的n-6LC-PUFA调控鳃的NKA基因表达及其酶活力以调节渗透压。     

中华绒螯蟹中肠和后肠内AchE和NOS神经元的观察以及 Ach、NO含量和Na+,K+-ATP活性的分析

对中华绒螯蟹中肠和后肠肠壁分别进行分层铺片,应用乙酰胆碱酯酶(AchE)和NADPH-黄递酶组织化学染色方法分别观察中肠和后肠中AchE和一氧化氮合酶(NOS)阳性神经元的分布和形态,并对其相应递质乙酰胆碱(Ach)和一氧化氮(NO)的含量和Na⁺,K⁺-ATP酶活性进行测定。结果显示:①对所获得铺片进行形态学观察发现,中肠肌层较后肠薄,肌纤维较细,肌纤维间隔明显;肠道黏膜下层细胞分布密集,后肠黏膜下层细胞分布较中肠更为密集。②AchE和NOS阳性神经元广泛分布于中肠和后肠的黏膜下层,而肌层和基膜未见分布,两种神经元在后肠黏膜下层的分布均较中肠密集。AchE阳性产物为棕色沉淀,阳性神经元大小为3~10 μm,中肠中AchE形态多样,多为圆形、卵圆形或梭形;后肠胞体阳性神经元呈圆形或者椭圆形,无明显胞突。NOS阳性产物为蓝色沉淀,阳性胞体的大小不等,呈不同形态,少量细胞有胞突伸向邻近细胞,中肠阳性神经元多呈条状或点状分散分布,而后肠阳性神经元常呈块状分布。③中肠Ach和NO含量分别为(1.28±0.41)和(1.84±0.25)μg/mg prot,显著低于后肠Ach(1.62±0.27) μg/mg prot和NO(2.10±0.25) μg/mg prot,而Na⁺,K⁺-ATP活性在中肠为(1.12±0.17)μmol Pi/(mg prot·h),显著高于后肠的(0.62±0.18) μmol Pi/(mg prot·h)。研究表明,中华绒螯蟹肠道黏膜下层是AchE和NOS阳性神经元分布的部位,两种神经元递质含量和Na⁺,K⁺-ATP酶活性在中肠和后肠间存在显著差异。     

Mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone release from dispersed goldfish pituitary cells

The mechanisms of pituitary adenylate cyclase activating polypeptide (PACAP) action on goldfish growth hormone (GH) release were investigated by examining GH release responses from dispersed goldfish pituitary cells to a synthetic mammalian (m)PACAP₃₈ peptide. It was established that GH release stimulated by 2-h exposure to mPACAP₃₈ was concentration-dependent, attenuated by the PACAP receptor antagonist mPACAP_6–38, and subject to neuroendocrine modulation by somatostatin. Maximal mPACAP₃₈-stimulated GH release was not additive to the responses elicited by either the adenylate cyclase activator forskolin or the cyclic (c)AMP analog 8-bromo-cAMP. The GH responses to mPACAP₃₈, forskolin and 8-bromo-cAMP, either alone or in combination, were abolished by H89, a protein kinase A (PKA) inhibitor. SQ22536, an adenylate cyclase inhibitor, attenuated forskolin- and mPACAP₃₈-stimulated GH release. In contrast, mPACAP₃₈-stimulated GH release were additive to the responses to two protein kinase C (PKC) activators and unaffected by two PKC inhibitors. These results suggest that the stimulatory action of PACAP on GH secretion is mediated through a cAMP- / PKA-dependent mechanism, whereas the involvement of PKC appears unlikely. The ability of mPACAP₃₈ to further enhance maximal GnRH (PKC)-dependent GH release, but not dopamine D1 agonist (PKA)-dependent GH secretion, is consistent with this hypothesis. A possible involvement of Ca²⁺ in PACAP action is also suggested. Two inhibitors of voltage-sensitive Ca²⁺ channel reduced the GH responses to mPACAP₃₈ in static incubation; conversely, mPACAP₃₈ increased intracellular [Ca²⁺] in identified, single goldfish somatotropes.     

Biochemical and molecular differences in diploid and triploid ocean-type chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) smolts

There is increasing evidence for complex dosage effects on gene expression, enzyme activity and phenotype resulting from induced ploidy change. In this study, ocean-type chinook salmon were bred using a 2 × 2 factorial mating design to create four families and test whether triploidization resulted in changes in growth performance and smolting. Eggs were pressure shocked after fertilization to create triploid fish from a subset of each family. In June, fish were sampled for size, plasma insulin-like growth factor 1 (IGF-1), gill Na⁺–K⁺-ATPase activity, and expression of two Na⁺–K⁺-ATPase α subunits in the gill. Diploids were significantly heavier than triploids, and there were significant differences due to family. Despite a significant positive correlation between plasma IGF-1 and fish size, plasma IGF-1 did not differ between diploid and triploid smolts. Diploids also had significantly greater gill Na⁺–K⁺-ATPase enzyme activities than triploids and there was a strong family effect. Gill Na⁺–K⁺-ATPase α1b isoform expression differed significantly by family, but not ploidy, and generally families with lower Na⁺–K⁺-ATPase enzyme activity had higher α1b isoform gene expression. Na⁺–K⁺-ATPase α1a isoform expression did not differ among any of the groups. Although diploids were larger and had higher specific activities of Na⁺–K⁺-ATPase in the gills, there was no difference in gene expression or circulating hormone levels. The strong family effect, however, suggests that strain selection may be useful in improving performance of triploids for aquaculture.     

Osmoregulation in the mudskipper,Boleophthalmus boddaerti I. Responses of branchial cation activated and anion stimulated adenosine triphosphatases to changes in salinity

The mudskipperB. boddaerti, was able to survive in waters of intermediate salinities (4–27). Fish submerged in dechlorinated tap water suffered 60% mortality by the fifth day while 60% of those in 100% sea-water (sw) died after the third day of exposure. After being submerged in 50% or 80% sw for 7 days, the plasma osmolality, plasma Na⁺ and Cl^– concentrations and the branchial Na⁺ and K⁺ activated adenosine triphosphatase (Na⁺,K⁺-ATPase) activity were significantly higher than those of fish submerged in 10% sw for the same period. However, the activities of the branchial HCO₃ ^– and Cl^– stimulated adenosine triphosphatase (HCO₃ ^–,Cl^–-ATPase) and carbonic anhydrase of the latter fish were significantly greater than those of the former. Such correlation suggests that Na⁺,K⁺-ATPase is important for hyperosmotic adaptation in this fish while HCO₃ ^–-Cl^–-ATPase and carbonic anhydrase may be involved in hypoosmotic survival.     

Osmoregulation in the mudskipper,Boleophthalmus boddaerti II. transepithelial potential and hormonal control

Boleophthalmus boddaerti submerged in 10%, 50% and 80% seawater (sw) for 7 days, had whole body transepithelial potentials (TEP) of 3.3, 18.3 and 22.9 mV, respectively. Hypophysectomy significantly decreased the TEP ofB. boddaerti and reversed the polarity of the TEP of the fish exposed to 10% sw.Hypophysectomy also significantly decreased the branchial Na⁺-K⁺ activated adenosine triphosphatase (Na⁺,K⁺-ATPase) activity but increased the activity of branchial HCO₃ ^–-Cl^– stimulated adenosine triphosphatase (HCO₃ ^–,Cl^–-ATPase) inB. boddaerti exposed to 10% sw. However, survival in 10% sw was not significantly impaired by hypophysectomy and no significant change in plasma osmolality and plasma Na⁺ and Cl^– concentrations was observed.Various doses of ovine-prolactin or salmon-prolactin were unable to restore the TEP of hypophysectomizedB. boddaerti in 10% sw to that of the sham-operated fish. However, cortisol increased TEP to a positive value in hypophysectomizedB. boddaerti, though it was still lower than the sham-operated control. Cortisol treatment also affected the plasma osmolality, plasma Na⁺ and Cl^– contents and branchial Na⁺,K⁺-ATPase and HCO₃ ^–,Cl^–-ATPase activities. Overall, the hormonal control of osmoregulation inB. boddaerti appeared to differ from that of other teleosts.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.