Natriuretic peptides are potent stimulants of cGMP production by particulate guanylate cyclase (pGC). Atrial natriuretic peptide (ANP) is of cardiac origin, while C-type natriuretic peptide (CNP) is a recently described natriuretic peptide which is synthesized in vascular endothelium. These peptides activate pGC by distinct NPR-A and NPR-B receptors, which have high affinity for ANP and CNP, respectively. A third receptor, NPR-C, binds ANP and CNP with equal affinity, but does not activate pGC. We recently reported that ANP and CNP are dilators of the pulmonary vasculature of the developing lamb, although with distinct sites of action. Further, both ANP and CNP are more potent dilators of PA from juvenile compared to fetal lambs. In the current study, we examined the mechanisms of action of ANP and CNP, as well as their receptor content and localization in lungs isolated from late gestation fetal lambs (138 days gestation, n=5) and 6 week old juvenile lambs (n=5). Fifth generation PA and PV were studied using conventional tissue both techniques. Rings were preconstricted with norepinephrine following pretreatment with indomethacin and LNA to block cGMP production by NO-soluble guanylate cyclase. CNP completely relaxed PV from both fetal and juvenile lambs at a concentration of 3×10-8 M. These relaxations were significantly blocked by the cGMP kinase inhibitor Rp-8Br-PET-cGMPS (3×10-5M, which inhibits relaxations to exogenous cGMP), and by the pGC inhibitor HS-142-1. CNP relaxed PA significantly less than PV (62 ± 8% at 10-7 M, p<0.05 vs PA). Relaxations to CNP in PA from juvenile lambs were not inhibited by Rp-8Br-PET-cGMPS or HS-142-1. In contrast, ANP relaxed PA to a greater degree than PV. In both vessel types, ANP relaxations were significantly inhibited by Rp-8Br-PET-cGMPS and HS-142-1. Using immunohistochemistry, we examined the localization for NPR-A (anti-rat NPR-A R1214) and NPR-B (anti-rat NPR-B Z657, both from D Garbers and K Hamra). In both fetal and juvenile lung sections, NPR-A localized predominantly to arterial walls, and NPR-B localized to both venous and arterial walls. In further studies, RT-PCR of 1 µg total cellular RNA was performed to assess mRNA expression for NPR-A, NPR-B and NPR-C receptors in lung homogenates. NPR-A and NPR-B expression was similar in lungs from fetal and juvenile lambs. However, NPR-C was significantly increased greater in juvenile vs fetal lungs (Arbitrary units, normalized to β-actin mRNA: 0.408 ± 0.03 vs 0.274 ± 0.02, p<0.05). We conclude that ANP exerts its vascular effects on both PA and PV by activation of NPR-A and pGC. CNP is a potent pulmonary venodilator by a mechanism dependent on activation of NPR-B and pGC. CNP dilates PA, but by a mechanism that is independent of NPR-B and pGC. We speculate that the increase in NPR-C expression during development may provide an alternate pGC-independent mechanism for the effects of CNP in PA.
Funded by NIH 54705 and AHA 9740024N
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Lakshminrusimha, S., Nielsen, L., D'Angelis, C. et al. Mechanisms of Action of Natriuretic Peptides in the Developing Ovine Lung. Pediatr Res 45, 68 (1999) doi:10.1203/00006450-199904020-00407