Nitric oxide (NO) is widely used by surgeons, anesthesiologists and critical care physicians managing critically-ill patients (e.g., inhaled NO for treatment of pulmonary hypertension and nitrovasodilators for decreasing afterload and for improving myocardial perfusion). Tolerance to these NO-based therapies occurs clinically and the extent to which this NO-induced NO hyporesponsiveness is due to decreased activity in the vascular smooth muscle NO-cGMP-cGK signaling system is unknown. The objective of this proposal is to determine the mechanisms by which chronic exposure to NO affects this signaling system in vascular smooth muscle. Two broad categories of mechanism for the development of chronic NO-induced NO hyporesponsiveness involving the vascular smooth muscle NO- cGMP-cGK signaling system are possible: 1) changes in [cGMP]i regulation and 2) reductions in the sensitivity to cGMP (cGMP sensitivity). To explore these mechanisms, we will utilize a cultured pulmonary artery preparation (PA) which permits experiments in fully functional, differentiated vascular smooth muscle cells. This cultured vessel preparation makes it possible to directly relate chronic NO-induced changes in the vascular smooth muscle NO-cGMP-cGK signaling system to chronic NO-induced decreases in NO responsiveness. Chronic NO exposure will be achieved using both NO-donors, and endogenously-derived NO following nitric oxide synthase gene transfer. Greater insight into the factors responsible for chronic NO-induced NO hyporesponsiveness will aid in the development of future NO-based therapeutic strategies. This is of particular relevance in the pulmonary circulation, in which inhaled NO is administered as a therapy for pulmonary hypertension, yet a large fraction of these patients are unresponsive to the NO. Methods used in the proposed studies include isometric force measurements, RT-PCR to isolate mRNA for all soluble guanylyl cyclase subunits (sGC) and cGMP-dependent protein kinase (cGK) isoforms, quantitative RT-PCR and immunoblotting to measure expression of each, and sGC and cGK enzyme activity measurement. We will use a combination of pharmacological probes and novel antisense oligomers to determine the mechanism by which chronic NO treatment decreases the expression and activity of sGC subunits and cGK isoforms.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL069968-01
Application #
6461608
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Harabin, Andrea L
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$289,000
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Perkins, William J; Warner, David O; Jones, Keith A (2009) Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity. Am J Physiol Lung Cell Mol Physiol 296:L121-9
Perkins, William J; Kost, Susan; Danielson, Mark (2009) Prolonged NO treatment decreases alpha-adrenoreceptor agonist responsiveness in porcine pulmonary artery due to persistent soluble guanylyl cyclase activation. Am J Physiol Lung Cell Mol Physiol 296:L666-73
Perkins, William J; Taniguchi, Miwa; Warner, David O et al. (2007) Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide. Am J Physiol Lung Cell Mol Physiol 293:L84-95
Kwak, Young L; Jones, Keith A; Warner, David O et al. (2006) Prolonged relaxation consistent with persistent soluble guanylyl cyclase activation in canine pulmonary artery following brief treatment with nitric oxide donors. Life Sci 79:2001-9
Kwak, Young L; Jones, Keith A; Warner, David O et al. (2006) NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation. Am J Physiol Lung Cell Mol Physiol 290:L200-8
Perkins, William J (2006) Regulation of soluble guanylyl cyclase: looking beyond NO. Am J Physiol Lung Cell Mol Physiol 291:L334-6
Taniguchi, Miwa; Kwak, Young Lan; Jones, Keith A et al. (2006) Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness. Am J Physiol Lung Cell Mol Physiol 290:L1018-27
Sakihara, Chie; Perkins, William J; Warner, David O et al. (2004) Anesthetics inhibit acetylcholine-promoted guanine nucleotide exchange of heterotrimeric G proteins of airway smooth muscle. Anesthesiology 101:120-6