Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii)

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Abstract

Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed.

Introduction

Cartilaginous fish occupy an important position in vertebrate evolution as they are the oldest extant group of jawed vertebrates (the superclass Gnathostomata). The class Chondrichthyes comprise two subclasses, Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays), which are thought to have divided in the Silurian about 420 Ma ago (Inoue et al., 2010). Marine elasmobranchs overcome the hyperosmotic stress of seawater by retaining urea to maintain their plasma isoosmotic or slightly hyperosmotic to the surrounding seawater (Hazon et al., 1997); they are thus recognized as ureosmotic organisms. To maintain high internal urea levels, de novo production of urea as well as reduced urea loss from the kidney and gills is required (Griffith, 1991; reviewed by Walsh and Mommsen, 2001). In contrast to elasmobranchs, little is known about the osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation as in elasmobranchs (Fänge and Fugelli, 1962, Robertson, 1976, Hyodo et al., 2007).

The liver is considered to be the primary organ for production of urea in elasmobranchs as it expresses the full complement of ornithine-urea cycle (OUC) enzymes, such as the rate-limiting carbamoyl phosphate synthetase III (CPSIII), ornithine transcarbamylase (OTC) and arginase (ARG). In addition, a reduction of environmental salinity concomitantly decreased plasma urea levels and lowered hepatic urea synthesis, further supporting the importance of the liver in maintaining a high plasma urea level (Anderson et al., 2005). Recently, an extra-hepatic contribution to urea production has been reported in several elasmobranch species. In particular, skeletal muscle has been shown to contribute significantly to urea production; in Squalus acanthias, as CPSIII activity in muscle was 1.6 to 4.7-fold greater than that in the liver (Kajimura et al., 2006).

A growing body of evidence shows that ARG and the accessory enzyme glutamine synthetase (GS) are encoded by multiple genes in vertebrates (Jenkinson et al., 1996, Murray et al., 2003). In teleost fish, expression of these multiple genes varies among tissues (Walsh et al., 2003, Joerink et al., 2006) and during development (Essex-Fraser et al., 2005), suggesting functional diversity of these multiple genes. However, in holocephalan fishes, there is little information on the molecular basis of urea production and the OUC enzymes.

Recently, the holocephalan elephant fish (or elephant shark, Callorhinchus milii), has attracted attention as a model for genome studies of cartilaginous fish (Venkatesh et al., 2005). The haploid cellular DNA content of the elephant fish was found to be much smaller than other cartilaginous fish genomes (Hinegardner, 1976). Furthermore, the aggregation of large numbers of elephant fish in the shallow waters of southern Australia, Tasmania and New Zealand during the egg-laying season enables the performance of physiological studies on holocephalan fish (Hyodo et al., 2007). We have previously examined plasma parameters of the elephant fish after transfer to different salinity environments. Plasma osmolality, Na+, Cl and urea concentrations were equivalent to those typically reported in elasmobranchs. Elephant fish have the osmoregulatory ability to adjust their plasma ions, urea and osmolality in altered environmental salinity, which indicates that they are a useful model for the study of the biology of holocephalan fishes (Hyodo et al., 2007, Kakumura et al., 2009).

In the present study, we cloned cDNAs encoding C. milii (cm) CPSIII, cmOTC and multiple cmGSs and cmARGs. Expression of the cloned mRNAs, including alternatively spliced variants of the cmGS1 mRNA, was examined in various tissues from fish acclimated in different salinity environments, and fish of peri-hatching stages. Our results revealed the contribution of hepatic and extrahepatic tissues to nitrogen metabolism in the elephant fish, and that the multiple GS mRNA products have distinct function in separate tissues.

Section snippets

Fish

Elephant fish, C. milii Bory de Saint-Vincent, 1823 of both sexes (total length: 75.7 ± 3.2 cm and body mass: 1.9 ± 0.2 kg) were collected in Western Port Bay, Victoria, Australia, using recreational fishing equipment, and were transported to Primary Industries Research Victoria, Queenscliff, using a 1000 L fish transporter. Elephant fish embryos in their egg case were gathered in Western Port Bay by professional divers. The adult fish were kept in a 10,000 L round tank with running seawater (SW) under

Identification of urea cycle enzyme cDNAs

We obtained partial OUC enzyme cDNAs by cDNA cloning using specific primers designed on DNA fragments obtained from the elephant fish genome database (Table 1). Since we obtained two distinct cDNAs for cmGS and cmARG, the whole sequences of GS and ARG cDNAs were determined. The nucleotide and amino acid sequences of elephant fish OUC cDNAs were shown to have considerable homology to other vertebrate OUC enzymes, and were designated as cmcpsIII (GenBank accession no. AB603761), cmotc (AB622984),

Discussion

This is the first report of the sequence of OUC enzyme mRNAs from a holocephalan fish, including the rate-limiting enzyme, CPSIII, and the accessory enzyme, GS. We found that the elephant fish has two separate gs (cmgs1 and cmgs2) and arg (cmarg1 and cmarg2) genes. Furthermore, alternatively spliced variants were found for cmGS1 mRNA. Expression of multiple gs and arg transcripts varied among tissues, suggesting that nitrogen metabolism is intricately regulated in the elephant fish.

Acknowledgment

We sincerely thank Ms. Elizabeth McGrath and Mr. Rod Watson of VMSC, and Ms. Camila Martin of Monash University, Australia, for their kind support. This study was supported by a Grant-in-Aid for Scientific Research (C), and by the Japan-Australia Research Cooperative Program from the Japan Society for the Promotion of Science to S.H.

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