We have identi¢ed cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the ¢rst time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C-terminus, which had an intramolecular ring formed by an S^S bond and a possibly amidated C-terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon^intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pu¡er¢sh AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body £uid regulation.

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

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in the distal nephrons of euryhaline and seawater pufferfishes. Am J Physiol Regul Integr Comp Physiol 300: R284 -R297, 2011. First published November 17, 2010 doi:10.1152/ajpregu.00725.2009.-The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na ϩ -K ϩ -Cl Ϫ cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na ϩ -Cl Ϫ cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7ϫ decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater. distal tubule; collecting duct; NKCC2; NCC; fish kidney; dilute urine FRESHWATER FISHES LIVE IN hypo-osmotic environments, resulting in significant influx of water into the body, mainly across the gills. To maintain body fluid homeostasis, freshwater fishes import ions from surrounding water through the gills (13,22) or from the diet, and excrete excess water in hypotonic urine (ϳ10 mM Na ϩ and Cl Ϫ ) through the kidneys (30). To increase urine volume (i.e., water excretion), freshwater fishes secrete NaCl in the proximal tubule and draw interstitial water into the tubular lumen (9); this proximally introduced NaCl should be reabsorbed in the distal nephron. Reabsorption of Na ϩ and Cl Ϫ ions from urine under water-impermeable conditions is, therefore, essential for net water excretion and the survival of fishes in freshwater. Na ϩ and Cl Ϫ reabsorption is also important for seawater fishes. Unlike freshwater fishes, however, seawater fishes experience passive water efflux. To balance water loss, seawater fishes absorb water and ions through the intestine by drinking large amounts of seawater (17). Surplus ions are excreted mainly through the gills (13, 22), although divalent ions (Mg 2ϩ and SO 4 2Ϫ ) are excreted through isotonic urine (4, 30). To produce urine with high concentrations of Mg 2ϩ (ϳ140 mM) ...