Submitted on May 21, 2008
Revised on September 30, 2008
Accepted on October 23, 2008
Prolonged Treatment of Porcine Pulmonary Artery with Nitric Oxide Decreases cGMP Sensitivity and cGMP-dependent Protein Kinase Specific Activity
William John Perkins1*, David O. Warner2, and Keith A. Jones3
1 Mayo Clinic College of Medicine
2 Mayo Clinic & Foundation
3 University of Alabama
* To whom correspondence should be addressed. E-mail: perkinsw{at}mayo.edu.
A cultured porcine pulmonary artery model (PA) was used to examine the effects of prolonged NO treatment on the response to acutely applied NO, cGMP analog or atrial natriuretic peptide (ANP). 24h treatment with the NO-donor DETA-NO resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, Sp-8-Br-PET-cGMPS, and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10-6 M ANP. 24h in culture and treatment with DETA-NO decreased total cGKI mRNA levels compared with that in freshly prepared PA, 1.05±0.12, 0.42±0.08 and 0.11±0.01 attomoles/µg, respectively. Total cGKI protein levels were decreased to a lesser extent by 24h in culture and further decreased by 24h DETA-NO treatment compared with that in freshly prepared PA, 361±33, 272±20 and 238±25 ng/mg total protein, respectively. Maximal cGMP-stimulated phosphotransferase activity was reduced in 24h cultured and DETA-NO treated PA, 986±84, 815±81 and 549±78 pmol Pi•min-1•mg-1 soluble protein, but the [cGMP] resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24h, compared with freshly prepared and 24h cultured PA, 1.95±0.22, 2.64±0.25 and 2.85±0.28 pmol Pi•min-1•ng cGKI-1, respectively. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.
