Generation and initial characterization of conditionally immortalized chromaffin cells

Adrenal chromaffin cells have been successfully used to attenuate chronic p ain when transplanted near the spinal cord, but primary cells are neither h omogeneous nor practical for routine use in human therapy. Conditional immo rtalization with the temperature-sensitive allele of the large T antigen (t sTag) and creation of stable chromaffin cell lines would advance our unders tanding of both the use and limits of cell lines that contain this immortal ization gene for such therapies. Cultures of embryonic day 17 rat adrenal a nd neonatal bovine adrenal cells were immortalized with the temperature-sen sitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyros ine hydroxylase (TH), the first enzyme in the synthetic pathway for catecho lamines, dopa-beta-hydroxylase (D beta H), and phenylethanolamine-N-methylt ransferase (PNMT). At permissive temperature (33 degrees C), these chromaff in cells are proliferative, have a typical rounded chromaffinlike morpholog y, and contain detectable TH-, D beta H-, and PNMT-lr. At nonpermissive tem perature (39 degrees C), these cells stop proliferating, decrease Tag expre ssion, and change the expression of TH-, D beta H-, and PNMT-ir in vitro, s uggesting increased differentiation at nonpermissive temperature. The chrom affin cell lines also express immunoreactivity for the opioid met-enkephali n (ENK) at permissive and nonpermissive temperatures. The expression of TH- ir in the bovine chromaffin cells is upregulated by the addition of dexamet hasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of D beta H- ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not det ectable by high-performance liquid chromatography, suggesting incomplete di fferentiation under current conditions, or influence by continued low level s of Tag expression. Both cell lines have been carried over many passages i n vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaf fin cells that can maintain the phenotype of primary chromaffin cells in vi tro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management . (C) 2000 Wiley-Liss, Inc.