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Kato A, Chang MH, Kurita Y, Nakada T, Ogoshi M, Nakazato T, Doi H, Hirose S, Romero MF. Identification of renal transporters involved in sulfate excretion in marine teleost fish. Am J Physiol Regul Integr Comp Physiol 297: R1647-R1659, 2009. First published October 7, 2009; doi: 10.1152/ajpregu.00228.2009.--Sulfate (S[O.sup.2-.sub.4]) is the second most abundant anion in seawater (SW), and excretion of excess S[O.sup.2.sub.4] from ingested SW is essential for marine fish to survive. Marine teleosts excrete S[O.sup.2-.sub.4] via the urine produced in the kidney. The S[O.sup.2-.sub.4] transporter that secretes and concentrates S[O.sup.2- .sub.4] 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 Sic 13 and Slc26 families from mefugu (Takifugu obscurus). We compared Sic 13 and [CI.sup.-]S[O.sup.2-.sub.4] 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.sup.-]/S[O.sup.2- .sub.4], [Cl.sup.-]/[oxalate.sup.2-], and [Cl.sup.-]/nHCO.sup.-.sub.3] exchanges and electroneutral [Cl.sup.-]/[formate.sup.-] exchange. Two-electrode voltage-clamp experiments demonstrated that the S[O.sup.2-.sub.4]-elicited currents of mfSlc26a6A is quite large (~35 [micro]A at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and [HCO.sup.-.sub.3] are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest S[O.sup.2-.sub.4] 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 S[O.sup.2-.sub.4] transporter that mediates S[O.sup.2-.sub.4]-secretion in the kidney of marine teleosts. mefugu; proximal tubule; Slcl3; Slc26; sulfate homeostasis; oxalate doi: 10.1152/ajpregu.00228.2009.

The teleost adrenomedullin (AM) family consists of three groups, AM1/AM4, AM2/AM3, and AM5. In the present study, we examined the effects of homologous AM1, AM2, and AM5 on drinking and renal function after peripheral or central administration in conscious freshwater eels. AM2 and AM5, but not AM1, exhibited dose-dependent (0.01-1 nmol/kg) dipsogenic and antidiuretic effects after intra-arterial bolus injection. The antidiuretic effect was significantly correlated with the degree of associated hypotension. To avoid the potential indirect osmoregulatory effects of AM-induced hypotension, infusion of AMs was also performed at nondepressor doses. Drinking was enhanced dose-dependently at 0.1-3 pmol x [kg.sup.-1] x [min.sup.-1] of AM2 and AM5, matching the potency and efficacy of angiotensin II (ANG II), the most potent dipsogenic hormone known thus far. AM2 and AM5 infusion also induced mild antidiuresis, while AM I caused antinatriuresis. Additionally, AMs were injected into the third and fourth ventricles of conscious eels to assess their site of dipsogenic action. However, none of the AMs at 0.05-0.5 nmol induced drinking, while ANG II was highly dipsogenic. AM2 and ANG II injected into the third ventricle increased arterial pressure while AM5 decreased it in a dose-dependent manner, and both AM2 and AM5 decreased blood pressure when injected into the fourth ventricle. These data suggest that circulating AM2 and AM5 act on a target site in the brain that lacks the blood-brain barrier. Collectively, the present study showed that AM2 and AM5 are potent osmoregulatory hormones in the eel, and their actions imply involvement in seawater adaptation of this euryhaline species. drinking; antidiuresis; angiotensin; intracerebroventricular administration; teleost fish

Adrenomedullin (AM), known as a multifunctional hormone in mammals, forms a unique family of five paralogous peptides in teleost fish. To examine their cardiovascular effects using homologous AMs in eels, we isolated cDNAs encoding four eel AMs, and named AM1 (ortholog of mammalian AM), AM2, AM3 (paralog of AM2 generated only in teleost lineage), and AM5 according to the known teleost AM sequences. Unlike pufferfish, not only AM1 but AM2/3 and AM5 were expressed ubiquitously in various eel tissues. Synthetic mature AM1, AM2, and AM5 exhibited vasodepressor effects after intra-arterial injections, and the effects were more potent at dorsal aorta than at ventral aorta. This indicates that AMs preferentially act on peripheral resistance vessels rather than on branchial arterioles. The potency was in the order of AM2 = AM5 >> AM 1 in both freshwater (FW) and seawater (SW) eels, which is different from the result of mammals in which AM1 is as potent as, or more potent than, AM2 when injected peripherally. The minimum effective dose of AM2 and AM5 in eels was 1/10 that of AM1 in mammals. The hypotension reached 50% at 1.0 nmol/kg of AM2 and AM5, which is much greater than atrial natriuretic peptide (20%), another potent vasodepressor hormone. Even with such hypotension, AMs did not change heart rate in eels. In addition, AM1 increased blood pressure at ventral aorta and dorsal aorta immediately after an initial hypotension at 5.0 nmol/kg, but not with AM2 and AM5. These data strongly suggest that specific receptors for AM2 and AM5 exist in eels, which differ from the AM1 receptors identified in mammals. calcitonin gene-related peptide; teleost; ventral aorta; dorsal aorta; vasodepressor action