Endcapped antisense oligonucleotides decrease chromaffin cell scinderin expression and stimulation-evoked F-actin disassembly and exocytosis.

Abstract

We have recently designed a 20-mer scinderin phosphorothioate endcapped substituted antisense oligodeoxynucleotide (ODN) sequence, which encompasses the scinderin initiation (ATG) site, and evaluated its impact on scinderin expression and amine output from cultured chromaffin cells. Chromaffin cells cultured for 48 hours in serum-free medium were treated with 2$\mu$M 20-mer scinderin phosphorothioate endcapped antisense ODN or a 20-mer mismatch phosphorothioate endcapped ODN sequence for 2-4 days. Cellular viability was not compromised when chromaffin cells were plated in serum-free medium or in serum-free medium containing antisense. Uptake of the antisense was evident when 5$\sp\prime$-fluorescein labelled 20-mer scinderin phosphorothioate endcapped antisense ODN entered chromaffin cells within 1 hour of treatment and remained highly concentrated in the nucleus for 24 hours post-treatment suggesting that the antisense was targeting gene expression. Moreover, a 60% decrease in scinderin mRNA levels was accompanied by a decrease of 50% in scinderin levels when chromaffin cells were treated with the 20-mer scinderin phosphorathioate endcapped antisense ODN compared to those chromaffin cells treated with the 20-mer mismatch phosphorothioate endcapped ODN sequence. Furthermore, stimulation of chromaffin cells with a depolarizing concentration of K$\sp+$ showed that both F-actin disassembly and evoked release of amines in 20-mer phosphorothioate endcapped scinderin antisense treated chromaffin cells was 50% lower than those chromaffin cells untreated or treated with the 20-mer phosphorothioate endcapped mismatch sequence. (Abstract shortened by UMI.)