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1.
It has been established that secretion of gonadotropin (GtH) and growth hormone (GH) release in goldfish are both stimulated by GtH-releasing hormone (GnRH); in addition GtH secretion is inhibited by dopamine D2 mechanisms. In the present study, depletion of protein kinase C (PKC) in goldfish pituitary cells reduced the GtH and GH responses to GnRH and an activator of PKC in static culture. In perifusion studies, GtH released in response to sGnRH analog was greatly attenuated in PKC-depleted cells, however, hormone responses to forskolin were enhanced. Stimulation of dopamine D2 receptors reduced the GtH, but not the GH, responses elicited by PKC activators. These results indicate that PKC participates in the GtH and GH responses to natural neuroendocrine regulators in the goldfish.
Résumé Il a été établi que chez le poisson rouge, les sécrétions de gonadotropine (GtH) et d'hormone de croissance (GH) sont toutes les deux stimulées par la gonadolibérine (GnRH); de plus, la sécrétion de GtH est inhibée par des mécanismes dopaminergiques de type D2. Dans le présent travail, la déplétion de la teneur en protéine kinase C (PKC) dans des cellules hypophysaires de poisson rouge réduit les résponses en GtH et GH au GnRH et à un activateur de la PKC de cellules maintenues en incubation statique. Dans des cellules maintenues en périfusion et soumises à une déplétion en PKC, la GtH libérée en réponse à un analogue du sGnRH est fortement diminuée, cependent les réponses hormonales à la forskoline sont augmentées. La stimulation des récepteurs dopaminergiques D2 réduit, dans le cas d'action d'activateur de la PKC, la réponse en GtH mais pas en GH. Ces résultats indiquent que la PKC est impliquée dans les mécanismes de régulation de GtH et GH par des facteurs neuroendocriniens naturels.
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2.
The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala6, Pro9-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca2+ ionophore (A23187; 1–100 μM) or ionomycin (0.02–10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1–10 μM) indicating that Ca2+ influx via voltage-sensitive Ca2+ channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16–160 μM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25–125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33–330 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro2,6, Trp3]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca2+ influx through voltage-sensitive Ca2+ channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.  相似文献   

3.
The tilapia prolactin (PRL) cell responds rapidly (10–20 min) to small physiological changes in medium osmotic pressure (OP), releasing increasing quantities of hormone as medium OP is reduced. This release is rapidly (≤ 10 min) inhibited by somatostatin (SRIF). There is now extensive evidence that tilapia PRL cell function is regulated through the second messengers Ca++ and cAMP. Our studies have shown that PRL release is augmented by treatments that lead to increased levels of intracellular Ca++ or cAMP. On the other hand, PRL release is blocked when tissues are incubated in Ca++-depleted medium or upon the addition of Co++, an inhibitor of Ca++-mediated processes. The use of45Ca++ to characterize the movement of Ca++ into PRL cells has provided evidence that an increase in the influx of extracellular Ca++ may participate in PRL release upon exposure to hyposmotic medium. Our studies have also shown that SRIF suppresses the increase in45Ca++ accumulation that is brought about when OP is reduced. We have also examined the effects of OP and SRIF on cAMP levels. The reduction of medium OP did not alter cAMP metabolism during 20 min of incubation. By contrast, cAMP accumulation in the presence of IBMX was enhanced at 1 hr of incubation in reduced OP. Thus, an increase in cAMP turnover may play a role in maintaining PRL release under sustained stimulation. SRIF reduced the accumulation of cAMP during 10 min of incubation with IBMX and also reduced the forskolin-stimulated increase in cAMP. Thus, SRIF may suppress adenylate cyclase activity. Finally, our studies have revealed that the forskolin-stimulated increase in cAMP levels is not dependent upon medium Ca++. The presence of Ca++ in the medium is required, however, for PRL release even when the cAMP messenger system has been activated. Moreover, cAMP accumulation was augmented when intracellular Ca++ was increased. This raises the possibility that reduced OP may stimulate an increase in cAMP turnover indirectly through its action(s) on cytosolic Ca++.  相似文献   

4.
Earlier studies have established that polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid and docosahexaenoic acid inhibit steroid production in the goldfish testis. As PUFA inhibit testicular steroidogenesis in the rat through activation of protein kinase C (PKC), the present studies were undertaken to characterize the properties of PKC in the goldfish testis and to test the effects of selected PUFA on PKC activity. PKC activity was quantified in goldfish testis homogenate following partial purification by DEAE-cellulose chromatography by determining the transfer of radiolabelled phosphate from [γ - 32P]ATP to histone III-S. Testicular PKC activity was defined by the amount of protein phosphorylation in the presence of phosphatidylserine, phasphatidylcholine, Ca2+ ions and diolein (a 1,2-diacylglycerol analog) above that obtained in response to Ca2+ ions alone. Western blot analysis of a crude testis homogenate using an antibody specific to the α and β isoforms of mammalian PKC led to the identification a single band of protein (80 kD) that co-migrated with PKC from rabbit brain cytosol. Addition of arachidonic, eicosapentaenoic or docosahexaenoic acids failed to activate PKC. However, PKC activity stimulated by phospholipid, Ca2+ ions and diolein was inhibited in a dose related fashion by all of these fatty acids. These studies suggest that the inhibitory effects of EPA and DHA on testicular steroidogenesis are not mediated by activation of PKC. The lack of effect of PUFA on PKC activity in the goldfish testis suggests that either the distribution of PKC isoforms differs between the testis of mammals and fish or that PKC is not activated by PUFA in the goldfish.  相似文献   

5.
Changes in Ca2+ content and flux, and the development of skin chloride cells in embryos and larvae of tilapia, Oreochromis mossambicus, were studied. Tilapia embryos hatched within 96h at an ambient temperature of 26–28°C. Total body Ca2+ content was maintained at a constant level, about 4–8 nmol per individual, during embryonic development. However, a rapid increase in body Ca2+ level was observed after hatching, 12.8 to 575.3 nmol per individual from day 1 to day 10 after hatching. A significant influx and efflux of Ca2+ occurred during development, with the average influx rate for Ca2+ increasing from 5.9 pmol mg−1 h−1 at 48h postfertilization to 47.8 pmol mg−1 h−1 at 1 day posthatching. The skin was proposed as the main site for Ca2+ influx before the development of gills, and the increased Ca2+ influx may be ascribed to gradual differentiation of skin surface and chloride cells during embryonic development. Ca2+ efflux was 16–56 pmol mg−1 h−1 in 1-day-old larvae. The resulting net influx of Ca2+, 10–12 pmol mg−1 h−1, accounted for the increased Ca2+ content after hatching. When comparing the measured and estimated ratios of efflux and influx, active transport was suggested to be involved in the uptake of Ca2+. Chloride cells, which may be responsible for the active uptake of Ca2+, started to differentiate in the skin of embryos 48h after fertilization, and the density of chloride cells increased following the development. A possibility of active transport for Ca2+ in early developmental stages of tilapia is suggested.  相似文献   

6.
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 Km 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.  相似文献   

7.
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 Km 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.  相似文献   

8.
Effects of environmental calcium concentrations on the survival, growth, body calcium content and calcium uptake kinetics in developing tilapia (Oreochromis mossambicus) larvae were studied. Fertilized eggs were incubated in high- and low-calcium artificial freshwater (0.88–0.96 mmol l–1 vs. 0.02–0.03 mmol l–1 CaCl2 or CaSO4) until 3 days after hatching. Tilapia larvae showed similar hatching rates and wet weights in either high- or low-calcium medium, indicating neither the development nor the growth in tilapia larvae was affected by the environmental calcium levels. The body calcium content in low-calcium groups was about 90–95% that of high-calcium groups, No matter what calcium source was used (CaCl2 or CaSO4), acclimation to low calcium medium caused a stimulation of calcium uptake (measured in 0.2 mmol l–1 calcium),i.e., 1.2–1.3 fold higher than that of high calcium groups. This enhanced calcium uptake capacity was characterized by a 50% decrease in Km and a 25% increase in Jmax. Effect of different calcium salts on calcium influx was significant only in low calcium level,i.e., calcium influx in low-CaCl2 group higher than that in low-CaSO4 group. These results suggest that tilapia larvae are able to modulate their calcium uptake mechanism to maintain normal body calcium content and growth in environments with different levels of calcium.  相似文献   

9.
虾夷扇贝闭壳肌和外套膜肌原纤维蛋白的特性分析   总被引:4,自引:2,他引:2  
吴忠  刘俊荣  田元勇 《水产学报》2015,39(11):1640-1649
为探索采捕后活品虾夷扇贝品质变化与其肌肉蛋白质生理特性变化间的关联,本研究以虾夷扇贝2个可食部肌肉为研究对象,以肌原纤维蛋白ATPase活性为指标(Ca2+-ATPase,Mg2+-ATPase),对扇贝肌原纤维蛋白(Mf)的稳定性进行了系统探索。首先,分别提取闭壳肌肌原纤维(A-Mf)和外套膜肌原纤维(M-Mf);然后,考察了不同因素(离子强度I、pH、温度)对Mf的ATPase活性的影响规律;对A-Mf及M-Mf的稳定性进行了探索;进一步比较了闭壳肌和外套膜肌原纤维蛋白ATPase的失活特性。研究结果表明:(1)虾夷扇贝闭壳肌与外套膜的Mf的理化性质相似,A-Mf与M-Mf的pI均在5.0附近,粘度分析发现A-Mf热稳定性高于M-Mf。(2)ATPase活性变化规律的结果发现,与脊椎动物中的鱼类一样,作为无脊椎动物的扇贝,与Mg2+-ATPase相比,Ca2+-ATPase更能准确地反映Mf的稳定性。(3)闭壳肌和外套膜二者的Mf的Ca2+-ATPase呈现出共同特性,在pH为中性时活性最高;A-Mf与M-Mf的差异性则表现为前者的Ca2+-ATPase在较低离子强度(I=0.2)下活性最高,后者则在较高离子强度(I=0.5)下活性最高;离子强度对A-Mf的热稳定性影响不明显,而M-Mf的热稳定性明显受到离子强度的影响,其在较低离子强度下表现出更好的稳定性。(4)Ca2+-ATPase失活速率的研究发现,无论是闭壳肌还是外套膜,其稳定性与离子强度I和温度均呈现显著正相关(R2=0.8181、0.8436和R2=0.9887、0.9557);二者在pH 7.0左右的稳定性最好,偏离中性会促使Ca2+-ATPase失活,与碱性条件相比,酸性对蛋白质稳定性的破坏更加明显。  相似文献   

10.
pH值和Ca2+浓度对日本沼虾生长和能量收支的影响   总被引:19,自引:1,他引:18  
董双林  堵甫山  赖伟 《水产学报》1994,18(2):118-123
本文报道了不同pH值(6.5、7.5和8.5)和不同Ca^2+浓度(38.8、61.1和78.8ppm)对日本沼是生长和能量收支的影响,实验结果表明,pH值对该虾的生长有一定的影响,Ca^2+和pH值在影响其生长的过程中可能有一定的交互作用,pH值和Ca^2+对该虾生长的影响主要是通过影响其能量摄入量实现的,本实验条件下,该虾摄入的能量平均有15.8%用于生长,1.8%作为粪便排出体外,其余用于呼  相似文献   

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