CELL LAYER-SPECIFIC EXPRESSION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES IN RAT ARTERIES - MOLECULAR-CLONING USING ISOLATED CELL-LAYERS FROM PARAFORMALDEHYDE-FIXED TISSUES

We describe a method for the isolation of small quantities of large po ly (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 me thod is suitable for use in reverse transcription polymerase chain rea ction (RT-PCR) amplification of low abundance messages. In this method , anesthetized rats are perfused with ice-cold phosphate-buffered para formaldehyde to allow for the in situ fixation of many of the main art eries of the rat. Following the in situ fixation of the rat vasculatur e, selected blood vessels can be removed, cleaned, and poly (A)(+) mRN A purified. In addition, the distinct cell layers of the paraformaldeh yde-fixed aorta can be mechanically separated and poly (A)(+) mRNA pur ified selectively from each. The application of this method to the stu dy 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 thes e enzymes from different cell layers isolated from the rat aorta. This method should be applicable to determine if selected mRNAs are presen t in selected blood vessels of the rat, or within distinct cell layers of particular large blood vessels.