Protein kinase G regulates potassium chloride cotransporter-4 [corrected] expression in primary cultures of rat vascular smooth muscle cells.
K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated by the cGMP signaling pathway. Expression of KCC mRNAs (KCC1-KCC4) in rat vascular smooth muscle cells (VSMCs) is unknown. We have reported the presence of KCC1 and KCC3 mRNAs in primary cultures of VSMCs by specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 mRNA was undetectable. Semiquantitative reverse transcription-polymerase chain reaction revealed a 2:1 KCC1/KCC3 mRNA ratio in VSMCs. Depletion of protein kinase G (PKG)-1 from VSMCs did not change KCC3 mRNA expression. Analogous results were obtained with PKG-1-catalytic domain- and vector only-transfected VSMCs lacking endogenous PKG, suggesting no involvement of PKG-1 in the maintenance of basal KCC3 mRNA expression. However, 8-bromo-cGMP, a PKG stimulator, acutely increased KCC3 mRNA expression in a concentration- and time-dependent fashion; this effect was blocked by the PKG inhibitor KT5823 but not by actinomycin D. These findings show that VSMCs express mainly two mRNA isoforms, KCC1 and KCC3, and suggest that PKG participates post-transcriptionally in the acute KCC3 mRNA regulation. The role of KCC3 on cell volume and electrolyte homeostasis in response to PKG modulators remains to be determined.
Di Fulvio
M
M
Department of Pharmacology, Wright State University, School of Medicine, Dayton, Ohio 45435-0002, USA.
Lincoln
T M
TM
Lauf
P K
PK
Adragna
N C
NC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001921:Brain / Q000378:metabolism; D002227:Carbazoles; D002352:Carrier Proteins / Q000235:genetics*; D020134:Catalytic Domain; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D007211:Indoles; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000096922:K Cl- Cotransporters
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000470:Alkaloids; D002227:Carbazoles; D002352:Carrier Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007211:Indoles; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D027981:Symporters; D000096922:K Cl- Cotransporters; C492281:Slc12a6 protein, rat; C492283:Slc12a7 protein, rat; C073601:KT 5823