Retrovirus-mediated transfer of an angiotensin type I receptor (AT1-R) antisense sequence decreases AT1-Rs and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain.
The AT1-R has been implicated in many cellular and physiological actions of angiotensin II (AII) in the brain. A retrovirus vector (LNSV) containing an AT1B-R antisense sequence (AT1B-AS) (termed LNSV-AT1B-AS) was constructed and used to determine the feasibility of using viral-mediated gene transfer to control AT1-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 AT1-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-AT1B-AS viral particles showed that AT1B-AS was integrated into the genome and transcribed in brain cells. This was associated with a significant decrease in AT1-Rs and in the AII-stimulated increase of c-fos mRNA, a measure of AT1-R function. These observations show that the AT1B-AS gene can be transferred into astroglial cells in culture by LNSV and that such a transfer inhibits AT1-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.
Lu
D
D
Department of Physiology, University of Florida, College of Medicine, Gainesville 32610.
Yu
K
K
Raizada
M K
MK
D000803:Angiotensin I / Q000378:metabolism*; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D051381:Rats; D011921:Rats, Inbred WKY; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D000803:Angiotensin I