Cell layer-specific expression of cyclic nucleotide phosphodiesterases in rat arteries: Molecular cloning using isolated cell layers from paraformaldehyde-fixed tissues

Abstract

We describe a method for the isolation of small quantities of large poly (A)⁺ mRNA from blood vessels of the rat as well as from distinct cell layers of the rat aorta. The poly (A)⁺ mRNA isolated by this method is suitable for use in reverse transcription polymerase chain reaction (RT-PCR) amplification of low abundance messages. In this method, anesthetized rats are perfused with ice-cold phosphate-buffered paraformaldehyde to allow for the in situ fixation of many of the main arteries of the rat. Following the in situ fixation of the rat vasculature, selected blood vessels can be removed, cleaned, and poly (A)⁺ mRNA purified. In addition, the distinct cell layers of the paraformaldehyde-fixed aorta can be mechanically separated and poly (A)⁺ mRNA purified selectively from each. The application of this method to the study of enzymes involved in cyclic nucleotide-mediated cell-signaling is illustrated by the cloning of two cyclic AMP phosphodiesterases from rat arteries, and from the selective amplification of message for these enzymes from different cell layers isolated from the rat aorta. This method should be applicable to determine if selected mRNAs are present in selected blood vessels of the rat, or within distinct cell layers of particular large blood vessels.