The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.

The biochemical and biological properties of a novel neuroendocrine-associated phosphatase (NEAP) were characterized. NEAP had a sequence characteristic of a dual-specificity phosphatase (DSP), and was preferentially expressed in neuroendocrine cells/tissues as well as in skeletal muscle and heart. Expression of NEAP was up-regulated in nerve growth factor (NGF)-treated, differentiated PC12 cells. NEAP was cytosolic and did not apparently have effects against extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase activated by various stimuli. Although NEAP and MAPK phosphatase (MPK)-1 showed similar phosphatase activity towards p-nitro phenylphosphate (pNPP), in contrast to MKP-1, NEAP did not dephosphorylate JNK and p38-MAPK in vitro. Overexpression of NEAP, but not the C152S mutant, in PC12 cells suppressed NGF-induced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and Akt activation. Overexpression of NEAP also suppressed neurite outgrowth induced by NGF and sensitized PC12 cells to cisplatin-induced apoptosis. Suppression of NEAP by RNA interference enhanced NGF-induced neurite outgrowth and Akt activation. Our results indicated that, unlike other DSPs, down-regulation of conventional MAPKs was not the major function of NEAP. Furthermore, NEAP might be involved in neuronal differentiation via regulation of the PI3K/Akt signaling. Keywords: dual-specificity phosphatase, mitogen-activated protein kinase, nerve growth factor, neurite outgrowth, neuroendocrine-associated phosphatase. Abbreviations used: AIF, apoptosis-inducing factor; ATF, activating transcription factor; CIP, calf intestinal phosphatase; DAPI, 4¢,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DSP, dual-specificity phosphatase; DTT, dithiothreitol; EGF, epidermal growth factor; EST, expression sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; IP, immunoprecipitation; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; NEAP, neuroendocrine-associated phosphatase; NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis; pAKT, phospho-Akt; PARP, poly (ADP-ribose)polymerase; PBS, phosphate-buffered saline; (p)ERK, (phospho-)extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; (p)JNK, (phospho-)c-Jun N-terminal kinase; PMA, phorbol 12 myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; (p)mTOR, (phospho)mammalian target of rapamycin; pNPP, p-nitro phenylphosphate; pp38, phospho-p38 MAPK; PPase, phosphatase; pTyr, phosphotyrosine; SCLC, small cell lung cancer; SDS, sodium dodecyl sulfate; TMDP, testis and skeletal muscle-specific dual-specificity phosphatase; TNF-a, tumour necrosis factor-a; UV-C, ultraviolet C; VHR, vaccinia virus H1 phosphatase gene-related phosphatase; WB, western blot.

Changes in expression of Na, K-ATPase (NKA) and morphometry of mitochondrion-rich (MR) cells in gills of tilapia were investigated on a 96-hr time course following transfer from seawater (SW) to fresh water (FW). A transient decline in plasma osmolality and Na þ , Cl À concentrations occurred from 3 hrs onward. Gills responded to FW transfer by decreasing NKA activity as early as 3 hrs from transfer. This response was followed by a significant decrease in the NKA isoform a1-mRNA abundance, which was detected by real-time PCR at 6 hrs post transfer. Next, a decrease of a1-protein amounts were observed from 6 hrs until 24 hrs post transfer. Additionally, during the time course of FW transfer, modifications in number and size of subtypes of gill MR cells were observed although no significant difference was found in densities of all subtypes of MR cells. These modifications were found as early as 3 hrs, evident at 6 hrs (exhibition of 3 subtypes of MR cells), and mostly completed by 24 hrs post transfer. Such rapid responses (in 3 hrs) as concurrent changes in branchial NKA expression and modifications of MR cell subtypes are thought to improve the osmoregulatory capacity of tilapia in acclimation from hypertonic SW to hypotonic FW.

The objective of the present study was to test the hypothesis that fish gills can express more than one isoform of the Na+-K+-ATPase a subunit responsible for ion regulation in seawater and freshwater environments. Using rapid amplification of complementary DNA ends (RACE), we cloned and sequenced full-length cDNAs encoding Na+-K+-ATPase alpha 1 and alpha 3 subunits of tilapia (Oreochromis mossambicus). Clone TG33 is 3390 bp in length and encodes a polypeptide of 1023 amino acids, while clone TH3 is 3581 bp in length and encodes a protein of 1010 amino acids. Clones TG33 and TH3 showed 91% and 88% identities at the amino acid level with previously described animal Na+-K+-ATPase alpha 1 and alpha 3 subunits, respectively. Northern blot and reverse transcriptase polymerase chain reaction analyses indicated that the alpha 1 subunit is expressed predominantly in kidney and intestine, while the alpha 3 subunit is expressed mainly in brain and heart. However, lower levels of expression of both genes were detected in other tissues such as gill, spleen, and testis. The amounts of both alpha 1 and alpha 3 subunit messenger RNA in gill tissue increased with the level of environmental salinity. This provides direct evidence of enhanced transcription of N+-K+-ATPase alpha 1 and alpha 3 subunit genes upon salinity challenge.