Chronic nitrates blunt the effects of not only nitric oxide but also natriuretic peptides in cardiac myocytes

Abstract

Exposure to nitrates causes tachyphylaxis to nitric oxide (NO), which reduces the effects of the second messenger cyclic guanosine-3',-5'-monophosphate (cyclic GMP). We tested the hypothesis that prolonged exposure to NO would also blunt the effects of natriuretic peptides. Cardiac myocytes were isolated from control (N=7) and chronic nitroglycerin (patched, N=7) rabbits. Patched animals received a transdermal nitroglycerin patch (0.3mg/h for 5 days). Myocyte function was determined at baseline, after C-type natriuretic peptide (CNP, 10(-8) and 10(-7)M) or brain natriuretic peptide (BNP, 10(-8) and 10(-7)M) or S-nitroso-N-acetyl-penicilliamine (SNAP, a NO donor, 10(-6) and 10(-5)M) followed by KT5823 (a cyclic GMP protein kinase inhibitor, 10(-6)M). Soluble and particulate guanylyl cyclase activities were measured in vitro and phosphoprotein analysis was performed. In control animals, CNP 10(-8)M (5.14+/-0.5%) and 10(-7)M (4.4+/-0.7%) significantly reduced percentage shortening from baseline (6.1+/-1.6%). KT5823 restored percentage shortening to 4.9+/-0.8%. Similar data were obtained with BNP and SNAP. In patched animals, CNP, BNP, SNAP had no significant effects on percentage shortening. The data on maximal rate of shortening and relaxation were consistent with these results. Guanylyl cyclase activities were not different in the control and patched animals. The myocytes from control and patched animals had similar protein phosphorylation patterns. Our data suggested that in addition to NO, the responses to both natriuretic peptides were downregulated after chronic exposure to nitroglycerin, but these effects were not due to changes in either guanylyl cyclase or cyclic GMP protein kinase, suggesting an altered downstream pathway.

Figure 1

Effects of CNP (Top), BNP (Middle) and SNAP (Bottom) on percentage shortening in freshly isolated ventricular myocytes from control and patched rabbits. Data are shown as mean ± S.E.M. (N=7). *P<0.05 vs. baseline, +P<0.05 vs. control rabbits.

Figure 2

Particulate and soluble guanylyl cyclase activity in ventricular myocytes from control and patched rabbits at baseline and after stimulation with 0.1% Triton X-100 (particulate) and 0.1 mM SNAP (soluble) (N=5).

Figure 3

Effects of cyclic GMP-dependent protein phosphorylation on ventricular myocytes from control (lanes 1–3) and patched (lanes 4–6) groups of rabbits. An equal amount of protein (5 μg) was loaded on each lane. Lanes 1 and 4 show the basal activities for protein phosphorylation of the two groups. Lane 2 and 5 show protein phosphorylation after addition (+) of 8-Br-cGMP. Lane 3 and 6 show phosphorylation in the presence of both 8-Br-cGMP and the cyclic GMP protein kinase inhibitor KT5823. Molecular weights standards are shown on the left. Note the increased phosphorylation with 8-Br-cGMP and the effect of KT5823 in reducing the degree of phosphorylation. This result indicated that the myocytes of the control and patched animals had a similar pattern of cyclic GMP-dependent protein phosphorylation