Multiple urea transporter proteins in the kidney of holocephalan elephant fish (Callorhinchus milii)

Reabsorption of filtered urea by the kidney is essential for retaining high levels of urea in marine cartilaginous fish. Our previous studies on the shark facilitative urea transporter (UT) suggest that additional UT(s) comprising the urea reabsorption system could exist in the cartilaginous fish kidney. Here, we isolated three cDNAs encoding UTs from the kidney of elephant fish, Callorhinchus milii, and termed them efUT-1, efUT-2 and efUT-3. efUT-1 is orthologous to known elasmobranch UTs, while efUT-2 and efUT-3 are novel UTs in cartilaginous fish. Two variants were found for efUT-1 and efUT-2, in which the NH₂-terminal intracellular domain was distinct between the variants. Differences in potential phosphorylation sites were found in the variant-specific NH2-terminal domains. When expressed in Xenopus oocytes, all five UT transcripts including the efUT-1 and efUT-2 variants induced more than a 10-fold increase in [¹⁴C] urea uptake. Phloretin inhibited dose-dependently the increase of urea uptake, suggesting that the identified UTs are facilitative UTs. Molecular phylogenetic analysis revealed that efUT-1 and efUT-2 had diverged in the cartilaginous fish lineage, while efUT-3 is distinct from efUT-1 and efUT-2. The present finding of multiple UTs in elephant fish provides a key to understanding the molecular mechanisms of urea reabsorption system in the cartilaginous fish kidney.

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule

In marine cartilaginous fish, reabsorption of filtered urea by the kidney is essential for retaining a large amount of urea in their body. However, the mechanism for urea reabsorption is poorly understood due to the complexity of the kidney. To address this problem, we focused on elephant fish (Callorhinchus milii) for which a genome database is available, and conducted molecular mapping of membrane transporters along the different segments of the nephron. Basically, the nephron architecture of elephant fish was similar to that described for elasmobranch nephrons, but some unique features were observed. The late distal tubule (LDT), which corresponded to the fourth loop of the nephron, ran straight near the renal corpuscle, while it was convoluted around the tip of the loop. The ascending and descending limbs of the straight portion were closely apposed to each other and were arranged in a countercurrent fashion. The convoluted portion of LDT was tightly packed and enveloped by the larger convolution of the second loop that originated from the same renal corpuscle. In situ hybridization analysis demonstrated that co-localization of Na(+),K(+),2Cl(-) cotransporter 2 and Na(+)/K(+)-ATPase α1 subunit was observed in the early distal tubule and the posterior part of LDT, indicating the existence of two separate diluting segments. The diluting segments most likely facilitate NaCl absorption and thereby water reabsorption to elevate urea concentration in the filtrate, and subsequently contribute to efficient urea reabsorption in the final segment of the nephron, the collecting tubule, where urea transporter-1 was intensely localized.

Osmoregulation in elephant fish Callorhinchus Milii (Holocephali), with special reference to the rectal gland

Osmoregulatory mechanisms in holocephalan fishes are poorly understood except that these fish are known to conduct urea-based osmoregulation as in elasmobranchs. We, therefore, examined changes in plasma parameters of elephant fish Callorhinchus milii, after gradual transfer to concentrated (120%) or diluted (80%) seawater (SW). In control fish, plasma Na and urea concentrations were about 300 mmol l^–1 and 450 mmol l^–1, respectively. These values were equivalent to those of sharks and rays, but the plasma urea concentration of elephant fish was considerably higher than that reported for chimaeras, another holocephalan. After transfer to 120% SW, plasma osmolality, urea and ion concentrations were increased, whereas transfer to 80% SW resulted in a fall in these parameters. The rises in ion concentrations were notable after transfer to 120% SW, whereas urea concentration decreased predominantly following transfer to 80% SW. In elephant fish, we could not find a discrete rectal gland. Instead, approximately 10 tubular structures were located in the wall of post-valvular intestine. Each tubular structure was composed of a putative salt-secreting component consisting of a single-layered columnar epithelium, which was stained with an anti-Na⁺,K⁺-ATPase serum. Furthermore, Na⁺,K⁺-ATPase activity in the tubular structures was significantly increased after acute transfer of fish to concentrated SW (115%). These results suggest that the tubular structures are a rectal gland equivalent, functioning as a salt-secreting organ. Since the rectal gland of elephant fish is well developed compared to that of Southern chimaera, the salt-secreting ability may be higher in elephant fish than chimaeras, which may account for the lower plasma NaCl concentration in elephant fish compared to other chimaeras. Since elephant fish have also attracted attention from a viewpoint of genome science, the availability of fish for physiological studies will make this species an excellent model in holocephalan fish group.

Mechanisms of vasodilation in the dorsal aorta of the elephant fish, Callorhinchus milii (Chimaeriformes: Holocephali)

This study investigated vasodilator mechanisms in the dorsal aorta of the elephant fish, Callorhinchus milii, using anatomical and physiological approaches. Nitric oxide synthase could only be located in the perivascular nerve fibres and not the endothelium of the dorsal aorta, using NADPH histochemistry and immunohistochemistry. In vitro organ bath experiments demonstrated that a NO/soluble guanylyl cyclase (GC) system appeared to be absent in the vascular smooth muscle, since the NO donors SNP (10⁻⁴ mol l⁻¹) and SIN^-1 (10⁻⁵ mol l⁻¹) were without effect. Nicotine (3 × 10⁻⁴ mol l⁻¹) mediated a vasodilation that was not affected by ODQ (10⁻⁵ mol l⁻¹), _l-NNA (10⁻⁴ mol l⁻¹), indomethacin (10⁻⁵ mol l⁻¹), or removal of the endothelium. In contrast, the voltage-gated sodium channel inhibitor, tetrodotoxin (10⁻⁵ mol l⁻¹), significantly decreased the dilation induced by nicotine, suggesting that it contained a neural component. Pre-incubation of the dorsal aorta with the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP_8–37 (10⁻⁶ mol l^−  1) also caused a significant decrease in the nicotine-induced dilation. We propose that nicotine is mediating a neurally-derived vasodilation in the dorsal aorta that is independent of NO, prostaglandins and the endothelium, and partly mediated by CGRP.

Osmoregulation in elephant fish, CALLORHYNCHUS MILLII (HOLOCEPHALI)

Osmoregulatory mechanisms in holocephalan fishes are unknown except that they conduct urea-based osmoregulation as in elasmobranchs. We, therefore, examined changes in plasma parameters of elephant fish, Callorhynchus millii, after gradual transfer to concentrated (120%) or diluted (80%) seawater (SW). In control fish, plasma Na and urea concentrations were about 300mM and 450mM, respectively. These values were equivalent to those of sharks and rays, but the plasma urea concentration of elephant fish was considerably higher than that reported for chimaeras, another holocephalan. After transfer to 120% SW, the plasma Na concentration markedly increased, while a conspicuous decrease in plasmaurea concentration was observed following transfer to 80% SW. In elephant fish, we could not find a discrete rectal gland. Instead, approximately 10 tubular structures were located in the wall of post-valvular intestine. Each tubular structure was composed of a putative salt-secreting component consisting of a single-layered columnar epithelium, which was stained with anti-Na+,K±ATPase serum. It is most likely that the tubular structures in the posterior intestine represent a primitive form of the rectal gland in elephant fish. In addition, we have identified two C-type natriuretic peptides (CNPs) from the heart and brain of elephant fish, which may contribute to the control of NaCl excretion from the rectal gland of elephant fish as it does in elasmobranchs.

Urea-based osmoregulation in the developing embryo of oviparous cartilaginous fish (Callorhinchus milii) : contribution of the extraembryonic yolk sac during the early developmental period

Marine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in the extra-embryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in the YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish.

The Myosin Heavy Chain specific A4.1025 antibody discriminates different cardiac segments in ancient groups of gnathostomes: Morphological and evolutionary implications

The pan-Myosin Heavy Chain (pan-MyHC) marker MF20 have been reported to show similar, homogeneous signal in the myocardial segments of the heart of teleosts and tetrapods. However, in an ongoing study of the myocardial structure of the dogfish (Scyliorhinus canicula; Chondrichthyes), we observed differential immunostaining of the cardiac segments using another pan-MyHC, the A4.1025 antibody. In order to investigate the relevance of this finding for better understanding of the morphology and evolution of the vertebrate heart, we performed immunohistochemistry, slot blot and western blot in several species of chondrichthyans, actinopterygians and mammals using the above mentioned antibodies. In the dogfish heart, A4.1025 and MF20 specifically recognized MyHC isoforms, although with different degree of affinity. MF20 reactivity was homogeneous and high in all the myocardial segments. However, A4.1025 reactivity was heterogeneous. It was high in the sinus venosus (external layer), atrium and atrioventricular region, low in the ventricle and conus arteriosus, and null in the internal layer of the sinus venosus. A heterogeneous pattern of A4.1025 immunoreactivity was also detected in two other elasmobranchs, a holocephalan, a polypteryform and an acipenseriform. In all of these species, MF20 immunoreactivity was homogeneous. In addition, both markers showed a homogeneous immunoreactivity pattern in teleosts and mammals. Our results indicate that in the hearts of ancient gnathostomes, in all of which a conspicuous conus arteriosus exists, one or more MyHC isoforms with low affinity for A4.1025 show segment-specific distributions. Thus, A4.1025 appears to be an appropriated marker to identify the cardiac segments and their boundaries. We propose that the segmentspecific distribution of MyHC isoforms may generate a particular type of myocardial contractility associated with the presence of a conus arteriosus.

Sulfate transporters involved in sulfate secretion in the kidney are localized in the renal proximal tubule II of the elephant fish (Callorhinchus milii)

Most vertebrates, including cartilaginous fishes, maintain their plasma SO4 (2-) concentration ([SO4 (2-)]) within a narrow range of 0.2-1 mM. As seawater has a [SO4 (2-)] about 40 times higher than that of the plasma, SO4 (2-) excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO4 (2-) via the kidney. However, little is known about the underlying mechanisms for SO4 (2-) transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO4 (2-) transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO4 (2-) transporter, while cmSlc26a6 is an apically located, electrogenic Cl(-)/SO4 (2-) exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO4 (2-) was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO4 (2-) by the kidney of elephant fish. Possible mechanisms for SO4 (2-) secretion in the PII segment are discussed.