LY. Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae. In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H ϩ , Na ϩ , and NH 4 ϩ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na ϩ uptake and NH3/NH 4 ϩ excretion were detected at most mitochondrion-rich cells (MRCs). H ϩ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 M) blocked 35% of the NH3/ NH 4 ϩ secretion and 54% of the Na ϩ uptake, suggesting that the Na ϩ /H ϩ exchanger (NHE) is involved in Na ϩ and NH3/NH 4 ϩ transport. Low-Na ϩ water (Ͻ0.001 mM) or high-NH 4 ϩ (5 mM) acclimation simultaneously increased Na ϩ uptake and NH3/NH 4 ϩ excretion but decreased or even reversed the H ϩ gradient at the skin and MRCs. The correlation between NH 4 ϩ production and H ϩ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH 4 ϩ significantly blocked NH 3/NH4 ϩ excretion and Na ϩ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH 4 ϩ excretion and Na ϩ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na ϩ /H ϩ exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na ϩ acclimation. This study suggests a novel Na ϩ /NH 4 ϩ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H ϩ gradients across apical membranes of MRCs.

Shih TH, Horng JL, Liu ST, Hwang PP, Lin LY. Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water.

Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na(+),K(+)-ATPase-rich (NaR), Na(+)-Cl(-) cotransporter-expressing (NCC), H(+)-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca(2+) uptake, Cl(-) uptake, Na(+) uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca(2+) or low Cl(-) freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca(2+) channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na(+), a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

Liu ST, Tsung L, Horng JL, Lin LY. Proton-facilitated ammonia excretion by ionocytes of medaka (Oryzias latipes) acclimated to seawater. Am J Physiol Regul Integr Comp Physiol 305: R242-R251, 2013. First published May 15, 2013 doi:10.1152/ajpregu.00047.2013.-The proton-facilitated ammonia excretion is critical for a fish's ability to excrete ammonia in freshwater. However, it remains unclear whether that mechanism is also critical for ammonia excretion in seawater (SW). Using a scanning ion-selective electrode technique (SIET) to measure H ϩ gradients, an acidic boundary layer was detected at the yolk-sac surface of SW-acclimated medaka (Oryzias latipes) larvae. The H ϩ gradient detected at the surface of ionocytes was higher than that of keratinocytes in the yolk sac. Treatment with Tricine buffer or EIPA (a NHE inhibitor) reduced the H ϩ gradient and ammonia excretion of larvae. In situ hybridization and immunochemistry showed that slc9a2 (NHE2) and slc9a3 (NHE3) were expressed in the same SW-type ionocytes. A real-time PCR analysis showed that transfer to SW downregulated branchial mRNA expressions of slc9a3 and Rhesus glycoproteins (rhcg1, rhcg2, and rhbg) but upregulated that of slc9a2. However, slc9a3, rhcg1, rhcg2, and rhbg expressions were induced by high ammonia in SW. This study suggests that SW-type ionocytes play a role in acid and ammonia excretion and that the Na ϩ /H ϩ exchanger and Rh glycoproteins are involved in the proton-facilitated ammonia excretion mechanism. mitochondrion-rich cell; gill; fish; embryos; skin AMMONIA IS A NITROGENOUS PRODUCT of amino acid metabolism. Ammonia excretion in fish is largely accomplished by their gill epithelium, which has a large surface area and a small diffusion distance. Ammonia exists as two distinct chemical species, dissolved ammonia gas (NH 3 ) and ammonium ions (NH 4 ϩ ). The conventional term, "ammonia," used in this article refers to both NH 3 and NH 4 ϩ . In freshwater (FW) fish, it is generally accepted that ammonia excretion occurs by diffusion of nonionic NH 3 down a favorable gradient across the gill epithelium (7,11,35). The excreted NH 3 can be trapped via acid secretion into the unstirred layer of water on gill surfaces. This "acidtrapping" or "proton-facilitated" mechanism maintains a favorable NH 3 gradient across the gill epithelium (35). Although phospholipid membranes are permeable to nonionic NH 3 , the existence of NH 3 channels in cell membranes provides an efficient and regulated pathway. In recent years, Rhesus glycoproteins (Rh proteins) in cell membranes of the gill and skin epithelium were found to facilitate ammonia excretion in fishes (11,12,39). The human Rh antigen on blood cells has long been associated with blood typing; however, the role of Rh proteins in ammonia transport of erythrocytes and nonerythrocytes was discovered in the past decade. The human erythroid RhAG and its nonerythroid homologues, RhBG and RhCG, were demonstrated to function as ammonia transporters or channels (31, 32). In teleosts, Nakada et al. (24) (3...

Ionocytes in the skin and gills of seawater (SW) teleosts are responsible for both salt and acid secretion. However, the mechanism through which ionocytes secrete acid is still unclear. Here, we hypothesized that apical Na+/H+ exchangers (NHE2/3), carbonic anhydrase (CA2-like), and basolateral HCO3−/Cl− exchanger (AE1) are involved in acid secretion. In addition, the hypothesized involvement of basolateral AE1 suggested that acid secretion may be linked to Cl− secretion by ionocytes. The scanning ion-selective electrode technique (SIET) was used to measure H+ and Cl− secretion by ionocytes in the skin of medaka larvae acclimated to SW. Treatment with inhibitors of NHE, CA, and AE suppressed both H+ and Cl− secretion by ionocytes. Short-term exposure to hypercapnic SW stimulated both H+ and Cl− secretion. mRNA of CA2-like and AE1 were localized to ionocytes in the skin. Branchial mRNA levels of NKCC1a, CA2-like, and AE1a increased together with the salinity to which fish were acclimated. In addition, both AE1a and AE1b mRNA increased in fish acclimated to acidified (pH 7) SW; NKCC1a mRNA increased in fish acclimated to pH 9 SW. This study reveals the mechanism of H+ secretion by ionocytes, and refines our understanding of the well-established mechanism of Cl− secretion by ionocytes of SW fish.