RETROVIRUS-MEDIATED TRANSFER OF AN ANGIOTENSIN TYPE-I RECEPTOR (AT(1)-R) ANTISENSE SEQUENCE DECREASES AT(1)-RS AND ANGIOTENSIN-II ACTION IN ASTROGLIAL AND NEURONAL CELLS IN PRIMARY CULTURES FROM THE BRAIN

The AT(1)-R has been implicated in many cellular and physiological actions of angiotensin II (AII) in the brain. A retrovirus vector (LNSV) containing an AT(1B)-R antisense sequence (AT(1B)-AS) (termed LNSV-AT(1B)-AS) was constructed and used to determine the feasibility of using viral-mediated gene transfer to control AT(1)-Rs and AII actions in astroglial and neuronal cells in primary cultures from rat brain. Briefly, a 1.26-kb antisense sequence corresponding to nt -132 to +1128 of AT(1)-R cDNA was cloned into the LNSV vector, the vector was transfected into PA317 cells, and transfected cells were selected in G418. Incubation of brain cells with culture medium containing LNSV-AT(1B)-AS viral particles showed that AT(1B)-AS was integrated into the genome and transcribed in brain cells. This was associated with a significant decrease in AT(1)-Rs and in the AII-stimulated increase of c-fos mRNA, a measure of AT(1)-R function. These observations show that the AT(1B)-AS gene can be transferred into astroglial cells in culture by LNSV and that such a transfer inhibits AT(1)-Rs and the AII stimulation of cellular activities. In addition, the usefulness of this approach to study AII-dependent pathophysiology in primary neuronal cultures from brain, in particular, is established.