Distribution of alpha-1 adrenoceptor subtypes in RNA and protein in rabbit eyes

We investigated subtypes of alpha-1 adrenoceptor (AR) in rabbit ocular tissues using reverse transcription-polymerase chain reaction (RT – PCR), in situ hybridization (ISH) and binding studies. Competitive RT – PCR assays specific for the subtypes of alpha-1 AR revealed that the mRNA expression of alpha-1a AR was dominant, and that of each alpha-1b and alpha-1d was less than 10% and 0.5% of total alpha-1 ARs mRNA, respectively, in the iris, ciliary body, choroid and retina. In alpha-1a AR splice isoform-specific RT – PCR assays, we found a distinct proportion of each isoform mRNA in the iris, ciliary body and choroid. The results of the ISH assays for alpha-1a AR subtype showed that hybridization signals were clearly observed in the iris dilator muscle and in the epithelium of the ciliary processes. In binding studies, alpha-1A AR was a dominant subtype in the iris, choroid and retina in contrast to the ciliary body that had more alpha-1B than alpha-1A AR subtype at protein level. Keywords: Alpha-1 adrenoceptor, eye, iris, dilator muscle, ciliary body Introduction Alpha-1 ARs constitute a heterogeneous family of receptors and play critical roles in the regulation of the sympathetic system. Molecular cloning techniques revealed the existence of three alpha-1 AR subtypes (alpha-1a, alpha-1b and alpha-1d AR) in many species (Cotecchia et al., 1988; Schwinn et al., 1990; Lomansney et al., 1991; Perez et al., 1991) and pharmacological studies suggested that these recombinant subtypes correspond to native alpha-1A, alpha-1B, and alpha-1D AR subtypes, respectively (Bylund et al., 1994; Hieble et al., 1995). Furthermore, splice isoforms of the alpha-1a AR subtype were found in humans; alpha-1a HSA.1-, 2-, 3- and 4-AR (Hirasawa et al., 1995; Chang et al., 1998) and in rabbits; alpha-1a OCU.1-, 2- and 3-AR (Suzuki et al., 2000). Alpha-1 ARs play important functions in the eyes. The contraction of the iris dilator muscle in rabbit eyes is mediated by alpha-1 ARs (Konno & Takayanagi, 1986; Nakamura et al., 1999). In vivo studies have revealed that alpha-1 ARs are involved in the regulation of intraocular pressure (IOP) in rabbits and monkeys (Kiuchi et al., 1992; Nishimura et al., 1993; Wang et al., 1997; Zhan et al., 1998; Okada & Gregory, 2001). Moreover, alpha-1 ARs have been found to regulate cellular functions in cultured endothelial cells from various ocular regions in several species (Walkenbach et al., 1992; Moroi-Fetters et al., 1995; Ryan et al., 1998). However, which subtype(s) of alpha-1 AR is involved in the ocular functions remains to be clarified. The purpose of this study was to identify and characterize alpha-1 AR subtypes in rabbit ocular tissues by RT – PCR, ISH and binding studies.