Cloning and characterization of muscarinic receptor genes from the nile tilapia (Oreochromis niloticus)
2009
Seo, J.S. (National Fisheries Research and Development Institute, Busan, Republic of Korea) | Kim, M.S. (Pukyong National University, Busan, Republic of Korea) | Park, E.M. (Pukyong National University, Busan, Republic of Korea) | Ahn, S.J. (Pukyong National University, Busan, Republic of Korea) | Kim, N.Y. (Pukyong National University, Busan, Republic of Korea) | Jung, S.H. (National Fisheries Research and Development Institute, Busan, Republic of Korea) | Kim, J.W. (National Fisheries Research and Development Institute, Busan, Republic of Korea) | Lee, H.H. (Pukyong National University, Busan, Republic of Korea) | Chung, J.K. (Pukyong National University, Busan, Republic of Korea), E-mail: [email protected]
To investigate the regulatory mechanism underlying the contractile response in the intestinal smooth muscle of the nile tilapia (Orechromis niloticus), we used pharmacologic and molecular approaches to identify the muscarinic subreceptors and the intracellular signaling pathways involved in this motility. Myography assays revealed that an M1- and M3-subtype selective antagonist, but not a M2-subtype selective antagonist, inhibited carbachol HCl (CCH)-induced intestinal smooth muscle contraction. In addition, a phospholipase C inhibitor, but not an adenylate cyclase inhibitor, blocked the contractile response to CCH. We also cloned five muscarinic genes (OnM2A, OnM2B, OnM3, OnM5A, and OnM5B) from the nile tilapia. In the phylogenetic analysis and sequence comparison to compare our putative gene products (OnMs) with the sequences obtained from the near complete teleost genomes, we unexpectedly found that the teleost fish have respectively two paralogous genes corresponding to each muscarinic subreceptor, and other teleost fish, except zebrafish, do not possess muscarinic subreceptor M1. In addition, the expression pattern of the nile tilapia muscarinic subreceptor transcripts during CCH-induced intestinal smooth muscle contraction in the proximal intestinal tissue was analyzed by real-time PCR surveys and it was demonstrated that CCH increased the OnMs mRNA expression rapidly and transiently.
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