Systemic Distribution of Salusin Expression in the Rat

1255 Hypertens Res Vol.30 (2007) No.12 p.1255-1262 Original Article Systemic Distribution of Salusin Expression in the Rat Noriko SUZUKI1), Masayoshi SHICHIRI1), Takumi AKASHI2), Kengo SATO3), Maya SAKURADA1), Yuki HIRONO1), Takanobu YOSHIMOTO1), Takatoshi KOYAMA3), and Yukio HIRATA1) Salusin-α and salusin-β are multifunctional bioactive peptides with hypotensive and bradycardic effects. They were originally identified from full-length human cDNAs by bioinformatics analyses. Salusin peptides are expressed in human tissues at the mRNA level, but no information is available about their systemic distributions in any species. We examined the distributions of preprosalusin mRNA and the salusin peptides in a variety of normal rat organs. Whereas preprosalusin mRNA was expressed ubiquitously, immunoreactive salusin-β was detected most strongly in the hypothalamus and posterior pituitary, and less abundantly in the anterior pituitary and gastrointestinal, immune, and hematopoietic systems. Salusin-β–positive cells appeared to be of either hematopoietic or endocrine origin, and many hematopoietic cells were also stained with anti-CD68, which specifically recognizes macrophages. Salusin-α–like immunoreactivity was not detected in any of the rat tissues. These results indicate that rat salusin is immunologically similar to human salusin-β and widely expressed, especially in the immune, gastrointestinal, and central nervous systems and mainly in endocrine- and hematopoietic-derived cells. (Hypertens Res 2007; 30: 1255–1262) Key Words: salusin, immunohistochemistry, real-time quantitative polymerase chain reaction, expression, macrophage Introduction Salusins are bioactive peptides that were originally identified using in silico analyses of a full-length human cDNA library (1). Salusin-β causes rapid and temporary decreases in blood pressure and heart rate in rats, while the hemodynamic effects of salusin-α are far less potent (1). Salusin-β’s effects are mediated mainly by parasympathetic stimulation rather than direct suppression of cardiac contractility (2). Human salusins have been shown to promote cardiomyocyte hypertrophy (3) as well as the growth of vascular smooth muscle cells and fibroblasts (1), and to protect against apoptotic death of car- diomyocytes (4). However, the exact mechanisms underlying these actions by exogenous human salusin peptides are largely unknown, and salusin receptors have not yet been identified (5). Both salusins are considered to be concomitantly biosynthesized as a result of alternative splicing from the torsion dystonia–related gene, TOR2a (DYT1), with subsequent frameshift reading and processing at dibasic amino acids (1). We previously reported the distribution of preprosalusin mRNA in human tissues as well as the concomitant presence of immunoreactive salusin-α and salusin-β in human kidney tissues (1). In nonhuman species, salusin-β– like immunoreactivity (LI) was found to coexist in vasopressin-expressing neurons of the rat posterior pituitary and From the 1)Department of Clinical and Molecular Endocrinology, 2)Department of Pathology, and 3)Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan. This work was supported in part by Grants-in-Aid for Scientific Research A (M.S., Y.H.), Scientific Research on Priority Areas “Applied Genomics” (M.S.) and for Exploratory Research (M.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Address for Reprints: Masayoshi Shichiri, M.D., Ph.D., Tokyo Medical and Dental University, 1–5–45 Yushima, Bunkyo-ku, Tokyo 113–8519, Japan. E-mail: Received June 12, 2007; Accepted in revised form July 25, 2007. 1256 Hypertens Res Vol. 30, No. 12 (2007) Fig. 1. Structures and distribution of rat salusin. A: Schematic representation of TOR2a and preprosalusin mRNAs. Numbers indicate the exon numbers, and red arrows indicate the RT-PCR primer positions. Indicated between exons 3 and 5 is the position of the TaqMan probe, labeled with the fluorescent dye 6-carboxy-fluoroscein (FAM) at its 5′ end and quencher dye 6-carboxy-tetramethylrhodamine (TAMRA) at its 3′ end for use in real-time quantitative RT-PCR. B: Comparison of the putative rat salusin with the human salusin-β and salusin-α amino acid sequences. Identical residues are shown in red. The antigenic peptide sequence used to raise the polyclonal antibody is boxed. C: Conventional RT-PCR analysis for detection of preprosalusin expression in rat tissue samples. The arrows indicate the positions of bands corresponding to the sizes of TOR2A (250 bp) and preprosalusin (122 bp) after agarose gel electrophoresis. D: Distribution of preprosalusin transcripts in various rat tissues as measured by real-time quantitative PCR using a TaqMan probe. The quantified transcripts are expressed as copy numbers relative to the corresponding copy numbers in an epididymis extract. Each column with a bar represents the mean ± SEM obtained from 8 independent rats. Suzuki et al: Systemic Distribution of Salusin Expression hypothalamus, suggesting its possible neural secretion into the systemic circulation via axon terminals (6). However, the systemic distributions of salusin peptides remain unclarified in any species, and preprosalusin gene expression in nonhuman species has not yet been investigated. Thus, it remains unknown whether or not the TOR2a gene undergoes alternative splicing, resulting in salusin peptide biosynthesis, in nonhuman species. The present study was designed to determine the presence and distribution of rat preprosalusin transcripts and immunoreactive rat salusin in a variety of rat tissues. The predicted N-terminal amino acid residues were highly homologous to human salusin-β, thereby allowing efficient detection of immunoreactive rat salusin in rat tissue specimens. Methods Animals Adult male Sprague-Dawley rats, weighing 250–300 g (Charles River Japan, Shiga, Japan), were used in all experiments. All procedures were performed in accordance with the Tokyo Medical and Dental University Guidelines for the Care and Use of Experimental Animals. Conventional Reverse Transcription–Polymerase Chain Reaction for Detecting TOR2a/Preprosalusin Transcripts After homogenization of the tissues, total RNA was extracted using QIAzol (Qiagen, Valencia, USA), which includes a DNase incubation step. RNA was quantified by spectrophotometry (Biochrom Ltd., Cambridge, UK), and diluted to 50 μg/mL for use in reverse transcription (RT)–polymerase chain reaction (PCR) assays. First-strand cDNA was synthesized using a Quantitect® Reverse Transcription kit (Qiagen) as described (7). Specific primers used for amplification and located in exon 3 (5′-ATCGCAAAGCCATTTTCATC-3′) and exon 5 (5′-ACACAGTGGCGCACATGAT-3′), and that detect both unspliced TOR2a (250 bp) and spliced preprosalusin (122 bp) (Fig. 1A), were synthesized by FASMAC Co. Ltd. (Atsugi, Japan). The amplified products were subjected to agarose gel el (...truncated)