Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP.
Studies in vitro have underestimated the importance of cGMP-dependent protein kinase (PKG) in the modulation of vascular smooth muscle cell (SMC) proliferation and apoptosis in vivo. This is attributable, in part, to a rapid decline in PKG levels as vascular SMC are passaged in culture. We used a recombinant adenovirus encoding PKG (Ad.PKG) to augment kinase activity in cultured rat pulmonary artery SMC (RPaSMC). Incubation of Ad. PKG-infected RPaSMC (multiplicity of infection = 200) with 8-Br-cGMP decreased serum-stimulated DNA synthesis by 85% and cell proliferation at day 5 by 74%. The effect of 8-Br-cGMP on DNA synthesis in Ad.PKG-infected RPaSMC was blocked by KT5823 (PKG inhibitor), but not by KT5720 (cAMP-dependent protein kinase inhibitor). A nitric oxide (NO) donor compound, S-nitrosoglutathione, at concentrations as low as 100 nM, inhibited DNA synthesis in Ad. PKG-infected RPaSMC, but not in uninfected cells or in cells infected with a control adenovirus. In addition, 8-Br-cGMP and S-nitrosoglutathione induced apoptosis in serum-deprived RPaSMC infected with Ad.PKG, but not in uninfected cells or in cells infected with a control adenovirus. These results demonstrate that modulation of PKG levels in vascular SMC can alter the sensitivity of these cells to NO and cGMP. Moreover, these observations suggest an important role for PKG in the regulation of vascular SMC proliferation and apoptosis by NO and cGMP.
Chiche
J D
JD
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. chiche@etherdome
Schlutsmeyer
S M
SM
Bloch
D B
DB
de la Monte
S M
SM
Roberts
J D
JD
Filippov
G
G
Janssens
S P
SP
Rosenzweig
A
A
Bloch
K D
KD
D000256:Adenoviridae; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000201:enzymology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D026422:S-Nitrosoglutathione; D014162:Transfection
D018696:Neuroprotective Agents; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011994:Recombinant Proteins; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D017869:Cyclic GMP-Dependent Protein Kinases; D005978:Glutathione; D006152:Cyclic GMP