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The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AMl (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AMl), CLR2RAMP2 (AMl), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells). calcitonin receptor-like receptor; pillar cell; proximal tubule; receptor activity-modifying proteins

Abstract: Endothelin, a vasoconstrictor peptide, plays important roles not only in the mammalian circulatory system but also in non-mammalian systems, such as the gill lamellar vascular network with complex structural characteristics. Here, we show that (i) the contraction of pillar cells that delimit the lamellar vasculature is controlled by endothelin through the type A endothelin receptor (ETA) linked to the intracellular calcium signaling system and (ii) ETA receptor is also highly expressed on fugu erythrocytes, a hitherto unexpected finding. Database mining revealed the presence of five endothelin receptor (ETR) sequences in the fugu genome. By Northern blotting, cDNA cloning, and fura-2 monitoring, the branchial ETR subtype was shown to be ETA able to induce a Ca2+ transit. Immunohistochemistry revealed its pillar cell and erythrocyte localization. These results suggest an endothelin/ETA-mediated coordinated regulation of the pillar cell shape and erythrocyte membrane flexibility. [Copyright &y& Elsevier]

A new type of urea transporter was identified by a database search and shown to be highly expressed in the renal proximal tubule cells of teleosts; proximal tubule-type urea transporters have not been describe previously. We first identified urea trans- porter-like sequences in the fugu genome and in an EST database of rainbow trout. Based on these pieces of sequence information, we obtained a full-length cDNA for the eel ortholog, consisting of 378 amino acid residues, and named it eUT-C. Although its sequence similarity to the known urea transporters is low (∼35%), its heterologous expression in Xenopus laevis oocytes indicated that it is a facilitative urea transporter sensitive to phloretin. Its activity is not dependent on Na+. Northern blot analysis showed that expression Of eUT-C is highly restricted to the kidney, with weak expression in the stomach. In both tissues, eUT-C mRNA was strongly induced when eels were transferred from freshwater to seawater. Immunohistochem- istry and in situ hybridization histochemistry revealed proximal tubule cell localization of eUT-C. Taking into account that 1) urea is mainly secreted from the gill where another type of urea transporter (eUT) has been identified and 2) fish excrete a very small volume of urine in seawater, we propose that eUT-C cloned here is a key component working in combination with the gill transporter to achieve an efficient urea excretory system in fish, namely, eUT-C reabsorbs urea from glomerular filtrate and sends it to the gill, through the circulation, for excretion. [ABSTRACT FROM AUTHOR]