ABSTRACT
The nonapeptide arginine vasotocin (AVT) regulates osmotic balance in teleost fishes, but its mechanisms of action are not fully understood. Recently, it was discovered that nonapeptide receptors in teleost fishes are differentiated into two V1a-type, several V2-type, and two isotocin (IT) receptors, but it remains unclear which receptors mediate AVT's effects on gill osmoregulation. Here, we examined the role of nonapeptide receptors in the gill of the euryhaline Amargosa pupfish (Cyprinodon nevadensis amargosae) during osmotic acclimation. Transcripts for the teleost V1a-type receptor v1a2 were upregulated over fourfold in gill 24 h after transferring pupfish from 7.5 ppt to seawater (35 ppt) or hypersaline (55 ppt) conditions and downregulated after transfer to freshwater (0.3 ppt). Gill transcripts for the nonapeptide degradation enzyme leucyl-cystinyl aminopeptidase (LNPEP) also increased in fish acclimating to 35 ppt. To test whether the effects of AVT on the gill might be mediated by a V1a-type receptor, we administered AVT or a V1-type receptor antagonist (Manning compound) intraperitoneally to pupfish before transfer to 0.4 ppt or 35 ppt. Pupfish transferred to 35 ppt exhibited elevated gill mRNA abundance for cystic fibrosis transmembrane conductance regulator (cftr), but that upregulation diminished under V1-receptor inhibition. AVT inhibited the increase in gill Na+/Cl- cotransporter 2 (ncc2) transcript abundance that occurs following transfer to hypoosmotic environments, whereas V1-type receptor antagonism increased ncc2 mRNAs even without a change in salinity. These findings indicate that AVT acts via a V1-type receptor to regulate gill Cl- transport by inhibiting Cl- uptake and facilitating Cl- secretion during seawater acclimation.
Subject(s)
Fish Proteins/metabolism , Gills/metabolism , Killifishes/metabolism , Osmoregulation , Receptors, Vasopressin/metabolism , Salinity , Salt Tolerance , Vasotocin/metabolism , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystinyl Aminopeptidase/genetics , Cystinyl Aminopeptidase/metabolism , Female , Fish Proteins/genetics , Killifishes/genetics , Male , Oxytocin/analogs & derivatives , Oxytocin/metabolism , Receptors, Vasopressin/genetics , Seawater , Signal Transduction , Solute Carrier Family 12, Member 1/genetics , Solute Carrier Family 12, Member 1/metabolism , Up-RegulationABSTRACT
Pupfishes (genus Cyprinodon) evolved some of the broadest salinity tolerances of teleost fishes, with some taxa surviving in conditions from freshwater to nearly 160 ppt. In this study, we examined transcriptional dynamics of ion transporters and aquaporins in the gill of the desert Amargosa pupfish (Cyprinodon nevadensis amargosae) during rapid salinity change. Pupfish acclimated to 7.5 ppt were exposed to freshwater (0.3 ppt), seawater (35 ppt), or hypersaline (55 ppt) conditions over 4 h and sampled at these salinities over 14 d. Plasma osmolality and Cl- concentration became elevated 8 h after the start of exposure to 35 or 55 ppt but returned to baseline levels after 14 d. Osmolality recovery was paralleled by increased gill Na+/K+-ATPase activity and higher relative levels of messenger RNAs (mRNAs) encoding cystic fibrosis transmembrane conductance regulator (cftr) and Na+/K+/2Cl- cotransporter-1 (nkcc1). Transcripts encoding one Na+-HCO3- cotransporter-1 isoform (nbce1.1) also increased in the gills at higher salinities, while a second isoform (nbce1.2) increased expression in freshwater. Pupfish in freshwater also had lower osmolality and elevated gill mRNAs for Na+/H+ exchanger isoform-2a (nhe2a) and V-type H+-ATPase within 8 h, followed by increases in Na+/H+ exchanger-3 (nhe3), carbonic anhydrase 2 (ca2), and aquaporin-3 (aqp3) within 1 d. Gill mRNAs for Na+/Cl- cotransporter-2 (ncc2) also were elevated 14 d after exposure to 0.3 ppt. These results offer insights into how coordinated transcriptional responses for ion transporters in the gill facilitate reestablishment of osmotic homeostasis after changes in environmental salinity and provide evidence that the teleost gill expresses two Na+-HCO3- cotransporter-1 isoforms with different roles in freshwater and seawater acclimation.
Subject(s)
Acclimatization/genetics , Aquaporins/genetics , Fish Proteins/genetics , Gene Expression , Ion Pumps/genetics , Killifishes/physiology , Salinity , Animals , Aquaporins/metabolism , Female , Fish Proteins/metabolism , Fresh Water , Gills , Ion Pumps/metabolism , Killifishes/genetics , Male , SeawaterABSTRACT
Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3ß or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m6A). Here, we describe an intersection between these components, the regulation of m6A by GSK-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3ß-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS identified the sites on FTO that are phosphorylated in a GSK-3-dependent fashion. GSK-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.
