REVERSIBLE PERMEABILIZATION - A NOVEL TECHNIQUE FOR THE INTRACELLULARINTRODUCTION OF ANTISENSE OLIGODEOXYNUCLEOTIDES INTO INTACT SMOOTH-MUSCLE

Antisense oligodeoxynucleotides (ODNs) have been used to modify gene e xpression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN-mediated changes in prote in expression of cultured cells, use of these compounds to achieve ant isense regulation of specific proteins in intact tissue has been limit ed. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of fo rce-generating capabilities and normal ultrastructure and (2) to devel op a method for efficient introduction of antisense ODNs into intact t issue. Sheets of ODN-containing, reversibly permeabilized rat outer lo ngitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membra ne depolarization or carbachol stimulation; the G protein-coupled calc ium sensitization pathway was also intact after 7 days. Reversible per meabilization, a method previously used to load smooth and cardiac mus cle with aequorin and heparin, was effective for loading >95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5'-A AGGGCCATTTTGTT-FITC-3'). Confocal microscopy of reversibly permeabiliz ed smooth muscle loaded with fluorescent antisense ODNs revealed inten se nuclear fluorescence and less intense, homogeneous, cytoplasmic flu orescence. Internally radiolabeled ODNs (homologous to the above seque nce) showed complete degradation between 4 and 16 hours after introduc tion into the cells. In summary, we have demonstrated methods for long -term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential u tility for investigating many questions in smooth muscle biology. At p resent, however, a major limitation of this approach is the short half -life of phosphorothioated ODNs.