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Water salinity has effects on growth and metamorphosis of anuran species, including Hoplobatrachus rugulosus. Previously, we reported that cultured H. rugulosus tadpoles at low salinities (2–4‰) were of bigger size and grew faster than those in fresh water (FW). However, at a higher salinity level of 6‰, their sizes were reduced and the metamorphosis was delayed. It was therefore hypothesized that high salinity-induced osmotic stress affected secretion of prolactin (PRL), which acts as osmoregulatory hormone and a regulator of metamorphosis in amphibians. In this study, transferring tadpoles into 4‰ and 6‰ brackish water increased the PRL levels by ~1.2- and ~twofold, respectively, as compared to FW group. These osmotic challenges also increased the total body fluid osmolality and levels of Na+, Cl− and Ca2+. The contents of triiodothyronine (T3) were significantly reduced in 4‰ and 6‰ groups, but not 2‰ group. Three sizes (49, 29 and 23 kDa) of H. rugulosus PRL receptors (PRLRs) were detected, and their protein expression was found in the skin, gill, tail fin, brain, intestine, heart, liver and kidney. The expression of PRLR-49k protein was significantly higher in both skin and gills in 2–4‰ groups, whereas that of PRLR-29k and PRLR-23k were higher in gills and lower in skin of the 6‰ group than FW group. In conclusion, salinity challenge, particularly 4‰ and 6‰, increased the levels of PRL, while decreasing the T3 levels, which could explain why salinity markedly modulated growth, metamorphosis and survival of tadpoles.

NaCl-rich rock salt dissolved in natural water source leads to salinity fluctuation that profoundly affects freshwater ecosystem and aquatic fauna. The snakehead (Channa striata) can live in saline water, but the osmoregulatory mechanisms underlying this ability remain unclear. Herein, we found that exposure to salinities ≥ 10‰ NaCl markedly elevated plasma cortisol and glucose levels, and caused muscle dehydration. In a study of time-dependent response after being transferred from fresh water (0‰ NaCl, FW) to salt-dissolved brackish water (10‰ NaCl, SW), FW-SW, cortisol increased rapidly along with elevations of plasma glucose and lactate. Interestingly, plasma cortisol returned to baseline after prolonged exposure, followed by a second peak that probably enhanced the branchial Na(+)/K(+)-ATPase activity. Under SW-FW condition, Na(+)/K(+)-ATPase activity was not altered as compared to SW-adapted fish. In conclusion, salinity change, especially FW-SW, induced a stress response and hence cortisol release in C. striata, which might increase plasma glucose and lactate to energize the branchial Na(+)/K(+)-ATPase.

Water salinity affects survival, growth and metamorphosis of anuran tadpoles. Hoplobatrachus rugulosus is considered not only as a freshwater amphibian but is also found in brackish wetlands. However, whether salinity change interferes with hatching, survival, body mass and development of H. rugulosus tadpoles is unknown. We found that salinity levels of 5‰. After a 3-week experiment, body weights of tadpoles exposed to 2‰ and 4‰ salinities were higher but that of the 6‰ group was lower compared with the 0‰ group. More than 90% of tadpoles exposed to 2‰ and 4‰ salinity showed complete metamorphosis. Salinity levels

Besides being a Ca2+-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl−, HCO3−, and Na+, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO3−secretion. CFTR served as the principal route for PTH-stimulated apical HCO3−efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCO3−was probably supplied by basolateral uptake via the electrogenic Na+-HCO3−cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H+might be extruded by both apical and basolateral Na+/H+exchangers. Furthermore, the PTH-stimulated HCO3−secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14–22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca2+chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO3−secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 μg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca2+-ATPase (PMCA) inhibitor (trifluoperazine) as well as small interfering RNA targeting voltage-dependent L-type calcium channel Cav1.3, but not TRPV6 or calbindin-D9k. As demonstrated by45Ca uptake study, PI3K and PKC, but not ROCK, were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKCζknockdown or exposure to inhibitors of PKCζ, but not of PKCα, PKCβ, PKCε, PKCμ, or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY-294002) and ROCK (Y-27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Cav1.3 and PMCA, presumably through PI3K and PKCζpathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 μg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

Besides being a Ca²-regulating hormone, parathyroid hormone (PTH) has also been shown to regulate epithelial transport of certain ions, such as Cl, HCO₃, and Na, particularly in the kidney. Although the intestinal epithelium also expressed PTH receptors, little was known regarding its mechanism in the regulation of intestinal ion transport. We investigated the ion regulatory role of PTH in intestinal epithelium-like Caco-2 monolayer by Ussing chamber technique and alternating current impedance spectroscopy. It was found that Caco-2 cells rapidly responded to PTH within 1 min by increasing apical HCO₃- secretion. CFTR served as the principal route for PTH-stimulated apical HCOV efflux, which was abolished by various CFTR inhibitors, namely, NPPB, glycine hydrazide-101 (GlyH-101), and CFTRinh-172, as well as by small interfering RNA against CFTR. Concurrently, the plasma membrane resistance was decreased with no changes in the plasma membrane capacitance or paracellular permeability. HCOV was probably supplied by basolateral uptake via the electrogenic Na⁺-HCO₃⁻ cotransporter and by methazolamide-sensitive carbonic anhydrase, while the resulting intracellular H⁺ might be extruded by both apical and basolateral Na/H exchangers. Furthermore, the PTH-stimulated HCO₃-secretion was markedly reduced by protein kinase A (PKA) inhibitor (PKI 14-22 amide) and phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY-294002), but not by intracellular Ca²⁺ chelator (BAPTA-AM) or protein kinase C inhibitor (GF-109203X). In conclusion, the present study provided evidence that PTH directly and rapidly stimulated apical HCO₃- secretion through CFTR in PKA- and PI3K-dependent manner, which was a novel noncalciotropic, ion regulatory action of PTH in the intestinal epithelium.

The milk-producing hormone prolactin (PRL) increases the transcellular intestinal calcium absorption by enhancing apical calcium uptake through voltage-dependent L-type calcium channel (Ca(v)) 1.3. However, the redundancy of apical calcium channels raised the possibility that Ca(v)1.3 may operate with other channels, especially transient receptor potential vanilloid family calcium channels (TRPV) 5 or 6, in an interdependent manner. Herein, TRPV5 knockdown (KD), TRPV5/TRPV6, TRPV5/Ca(v)1.3, and TRPV6/Ca(v)1.3 double KD, and TRPV5/TRPV6/Ca(v)1.3 triple KD Caco-2 monolayers were generated by transfecting cells with small interfering RNAs (siRNA). siRNAs downregulated only the target mRNAs, and did not induce compensatory upregulation of the remaining channels. After exposure to 600 ng/mL PRL, the transcellular calcium transport was increased by ~2-fold in scrambled siRNA-treated, TRPV5 KD and TRPV5/TRPV6 KD monolayers, but not in TRPV5/Ca(v)1.3, TRPV6/Ca(v)1.3 and TRPV5/TRPV6/Ca(v)1.3 KD monolayers. The results suggested that Ca(v)1.3 was the sole apical channel responsible for the PRL-stimulated transcellular calcium transport in intestine-like Caco-2 monolayer.

During pregnancy and lactation, the enhanced intestinal Ca2+absorption serves to provide Ca2+for fetal development and lactogenesis; however, the responsible hormone and its mechanisms remain elusive. We elucidated herein that prolactin (PRL) markedly stimulated the transcellular and paracellular Ca2+transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a two-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic levels by upregulating the expression of genes related to transcellular transport, e.g., TRPV5/6 and calbindin-D9k, and the paracellular transport, e.g., claudin-3, thereby raising Ca2+absorption rate to a new “baseline” (Step 1). During suckling, PRL surge further increased Ca2+absorption to a higher level (Step 2) in a nongenomic manner to match Ca2+loss in milk. PRL-enhanced apical Ca2+uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca2+movement. Such nongenomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil-forming kinase pathways. In conclusion, two-step stimulation of intestinal Ca2+absorption resulted from long-term PRL exposure, which upregulated Ca2+transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge, which further increased Ca2+transport to the maximal capacity. The present findings also suggested that Ca2+supplementation at 15–30 min prior to breastfeeding may best benefit the lactating mother, since more Ca2+could be absorbed as a result of the suckling-induced PRL surge.

Prolactin (PRL) is reported to stimulate calcium absorption in the rat’s small intestine. However, little is known regarding its effects on the cecum, a part of the large intestine with the highest rate of intestinal calcium transport. We demonstrated herein by quantitative real-time polymerase chain reaction and Western blot analysis that the cecum could be a target organ of PRL since cecal epithelial cells strongly expressed PRL receptors. In Ussing chamber experiments, PRL enhanced the transcellular cecal calcium absorption in a biphasic dose–response manner. PRL also increased the paracellular calcium permeability and passive calcium transport in the cecum, which could be explained by the PRL-induced decrease in transepithelial resistance and increase in cation selectivity of the cecal epithelium. PRL actions in the cecum were abolished by inhibitors of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK), but not inhibitors of gene transcription and protein biosynthesis. In conclusion, PRL directly enhanced the transcellular and paracellular calcium transport in the rat cecum through the nongenomic signaling pathways involving PI3K, PKC, and ROCK.

Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestine-like Caco-2 monolayer. However, its mechanism was not completely understood. Here, we investigated nongenomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in the Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes and paracellular calcium permeability within 60 min after exposure but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1-β-d-ribobenzimidazole), confirming that PRL actions were nongenomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF-109203X and Y-27632, respectively) abolished the stimulatory effect of PRL on transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor, diminished the PRL effect on paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and the permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated transepithelial calcium transport through PRLR-L and increased paracellular permeability to cations in the Caco-2 monolayer. These nongenomic actions of PRL were mediated by the PKC and ROCK signaling pathways.

BackgroundZebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development.ResultsUsing transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number.ConclusionOur results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network.