IMMORTALIZATION OF CAT IRIS SPHINCTER SMOOTH-MUSCLE CELLS BY SV40 VIRUS - GROWTH, MORPHOLOGICAL, BIOCHEMICAL AND PHARMACOLOGICAL CHARACTERISTICS

The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating oc ular receptors and their signal transduction pathways. Cultured cat ir is sphincter muscle cells were immortalized by viral transformation wi th SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transforme d cell clone, SV-CISM-2, was further characterized biochemically and p harmacologically. The normal muscle cells showed characteristics of sm ooth muscle cells, as judged by their growth and the presence of smoot h muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained t heir proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirme d by their expression of the large T-antigen, the incorporation of vir al DNA into cellular DNA, growth in agarose and in low-serum medium, a nd complete loss of contact inhibition. The immortalized cells express ed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-ad renergic, peptidergic (substance P and endothelin), Platelet-activatin g factor, and prostaglandin (PG). Muscarinic stimulation of these cell s resulted in: (a) a dose-dependent increase in the release of arachid onic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphoryl ation (118%), an indicator of smooth muscle contractility. The stimula tory effects of carbachol on these responses were completely blocked b y atropine, a muscarinic receptor antagonist. This study constitutes t he first successful immortalization of iris sphincter smooth muscle ce lls. The SV-CISM-2 cells can serve as an important model system for in vestigations on the biochemical and pharmacological properties of rece ptors and their signal transduction pathways in smooth muscle. The adv antage of these cells over normal iris sphincter cells is that they ca n be propagated over many generations without alterations in their mor phological, biochemical and phgsiological characteristics. (C) 1995 Ac ademic Press Limited