Short sequence-paper Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells 1 Kazuya Sobue a;b; *, Naoki Yamamoto b;c , Kazuhiro Yoneda b;c , Kaori Fujita b , Yutaka Miura b , Kiyofumi Asai b , Takako Tsuda a , Hirotada Katsuya a , Taiji Kato b a Department of Anesthesiology and Resuscitology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan b Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan c Department of Pharmaceutics, Faculty of Pharmaceutical Science, Nagoya City University, Mizuho-ku, Nagoya 467-8601, Japan Received 21 June 1999; received in revised form 16 September 1999; accepted 29 September 1999 Abstract Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5P-genomic sequence analysis showed that the 5P-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Aquaporin ; Gene cloning ; Mercurial-insensitive ; Water channel ; Bovine ; Endothelial cell Aquaporin-4 (AQP4), a member of the water-se- lective membrane channel family, is expressed in a wide variety of animal tissues including brain, spinal cord, retina, lung, kidney, skeletal muscle, trachea, stomach, and colon [1^3]. AQP4 as a member of the AQP family is characterized by its high water permeability and mercurial-insensitive water channel (MIWC) [4]. In the central nervous system (CNS) it is expressed predominantly in ependymal cells [5] and astrocytes [6]. Accordingly, it is speculated that AQP4 mediates transmembrane water movement to- gether with other members of the AQP family at the blood-brain barrier (B-BB) and brain-cerebrospinal £uid interface. Considering that the B-BB structure is composed of endothelial cells and astrocytes, there should be a certain level of AQP4 expression in the endothelial cells directly facing the intravascular space. However, AQP4 expression has not been de- tected in endothelial cells. Although rat [7], human [8], and mouse [9] AQP4 cDNAs have been cloned, it has not been cloned from bovine whose brain endo- thelial cells are most commonly used for fundamen- tal studies of the B-BB in vitro [10,11]. Therefore in 0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4781(99)00194-3 Abbreviations : AQP, aquaporin ; B-BB, blood-brain barrier ; MIWC, mercurial-insensitive water channel * Corresponding author, at address b. Fax: +81 (52) 8423316; E-mail : kazsobue@med.nagoya-cu.ac.jp 1 DDBJ, EMBL, and GenBank accession Nos. AB015947 (bAQP4-A) and AB028642 (bAQP4-B). Biochimica et Biophysica Acta 1489 (1999) 393^398 www.elsevier.com/locate/bba