KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.
K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.
Adragna
N C
NC
Department of Pharmacology and Toxicology, Wright State University, School of Medicine, Dayton, OH, 45435-0002, USA. norma.adragna@wright.edu
Zhang
J
J
Di Fulvio
M
M
Lincoln
T M
TM
Lauf
P K
PK
D000085:Acetates / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism* / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D007192:Indenes / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
D000085:Acetates; D017869:Cyclic GMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide; D005665:Furosemide; D007192:Indenes; D027981:Symporters; D000096922:K Cl- Cotransporters; C095651:((2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy)acetic acid