C-type natriuretic peptide system in fetal ovine pulmonary vasculature.

C-type natriuretic peptide (CNP) is a recently described endothelium-derived relaxing factor. CNP relaxes vascular smooth muscle and inhibits smooth muscle proliferation by binding to natriuretic peptide receptor (NPR) type B (NPR-B) and producing cGMP. Lung parenchyma and fifth-generation pulmonary arteries (PA) and veins (PV) were isolated from late-gestation fetal lambs. All three types of NPR mRNA were detected in PA and PV by RT-PCR. CNP and NPR-B immunostaining was positive in pulmonary vascular endothelium and medial smooth muscle. CNP concentration-response curves of PA and PV were compared with those of atrial natriuretic peptide (ANP) by use of standard tissue bath techniques. CNP relaxed PV significantly better than PA. ANP relaxed PA and PV equally, but ANP relaxed PA significantly better than CNP. Pretreating PA and PV with natriuretic peptide receptor blocker (HS-142-1) or cGMP-dependent protein kinase inhibitor Rp-beta-phenyl-1- N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate significantly inhibited the CNP relaxation response, indicating that the response was mediated through the NPR-cGMP pathway. We conclude that CNP is important in mediating pulmonary venous tone in the fetus.

Pulmonary hypertension alters soluble guanylate cyclase activity and expression in pulmonary arteries isolated from fetal lambs.

The nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway plays an important role in the pulmonary vascular transition at birth. We studied pulmonary arteries and veins isolated from normal late-gestation fetal lambs and from fetal lambs with persistent pulmonary hypertension (PPHN) following prenatal ligation of the ductus arteriosus. We additionally used double immunolabeling and immunoblot analysis to determine relative vascular contents of endothelial nitric oxide synthase (NOS-III) and soluble guanylate cyclase (sGC). Cyclic GMP content and sGC activity were significantly lower in arteries from hypertensive lambs than controls. A rank order for contents of both soluble guanylate cyclase and NOS-III was observed by both immunolabeling and immunoblotting: Control vein = Hypertensive vein > Control artery > Hypertensive artery. Our data demonstrate that the relative expression of sGC correlates well with the relative expression of NOS-III, and indicate the potential importance of soluble guanylate cyclase in the regulation of the perinatal pulmonary circulation. These data may help us understand vascular mechanisms producing PPHN, as well as patterns of response to exogenous NO.

Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension.

Congenital heart disease associated with increased pulmonary blood flow produces pulmonary hypertension. To characterize vascular alterations in the nitric oxide (NO)-cGMP cascade induced by increased pulmonary blood flow and pulmonary hypertension, 10 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). When the lambs were 4-6 wk of age, we assessed responses of pulmonary arteries (PAs) and pulmonary veins (PVs) isolated from lungs of control and shunted lambs. PVs from control and shunted lambs relaxed similarly to exogenous NO (S-nitrosyl-acetyl-penicillamine), to NO produced endogenously (zaprinast and A-23187), and to cGMP (atrial natriuretic peptide). In contrast, relaxations to A-23187 and zaprinast were blunted in PAs isolated from shunted lambs relative to controls. Inhibitors of NO synthase (NOS) and soluble guanylate cyclase constricted control but not shunt PAs, indicating reduced basal NOS activity in shunt PAs. Pretreatment of shunt PAs with the substrates L-arginine and sepiapterin, a precursor for tetrahydrobiopterin synthesis, did not augment A-23187 relaxations. However, pretreatment with superoxide dismutase and catalase significantly enhanced A-23187 relaxations in shunt PAs. We conclude that increased pulmonary blood flow induces an impairment of endothelium-dependent relaxation that is selective to PAs. The impaired relaxation may be mediated in part by excess superoxide production.

cGMP inhibition of endothelin-stimulated inositol phosphate production in the fetal lamb pulmonary artery.

Endothelin-1 (ET-1) stimulates inositol phosphate production in vascular smooth muscle. In the present study, interactions between cyclic GMP (cGMP), cyclic AMP (cAMP) and ET-1 in fetal lamb pulmonary arteries were investigated using phosphoinositide hydrolysis studies and tissue bath techniques. ET-1 was found to be a potent vasoconstrictor of these vessels, with an EC50 of 15.8 nM. ET-1 stimulated total inositol phosphate (IP) production; basal IP production was 68 cpm/mg vs. 247 cm/mg with 1 microM ET-1. 8-bromo-cGMP (2 mM) significantly increased the threshold of ET-1 concentration for pulmonary artery contraction, but had no effect on IP production. Zaprinast (a selective type V phosphodiesterase inhibitor, 60 microM) did not affect ET-1-induced contractility or IP production. IBMX (0.5 mM), a non-specific phosphodiesterase inhibitor, inhibited the potent and maximal effects of ET-1 in arterial contraction and decreased ET-1-stimulated IP production by 49%, while forskolin had a lesser effect in the tissue bath and no effect on IP production. Thus, 8-bromo-cGMP and IBMX alter the contractile effects of ET-1 in the fetal pulmonary artery and IBMX also inhibits inositol phosphate production. The cross-talk mechanisms of these agents require further investigation.

Endothelium-dependent relaxations to adenosine in juvenile rabbit pulmonary arteries and veins.

We studied the actions of adenosine and its analogues 5'-(N-ethylcarboxamido)-adenosine (NECA) and N6-cyclohexyladenosine (CHA) in pulmonary vessels isolated from juvenile rabbits. Pulmonary arteries relaxed in a concentration-dependent fashion to all three compounds. Pretreatment with the methylxanthine 8-p-sulfophenyltheophylline shifted the concentration-response curves to adenosine and NECA rightward, indicating that the vasodilator effects were mediated by the adenosine receptor. The order of potency of adenosine compounds was NECA > adenosine > CHA, indicating that the A2-receptor mediates relaxations to adenosine in rabbit pulmonary arteries. Endothelium rubbing attenuated relaxations to adenosine at concentrations of < or = 3 x 10(-7) M and to all NECA concentrations. Inhibition of nitric oxide synthase with NG-nitro-L-arginine (L-NNA) similarly attenuated relaxations at concentrations of < or = 3 x 10(-7) M for adenosine and < or = 3 x 10(-8) M for NECA. With the use of the same methods, a substantial endothelial contribution was additionally observed in pulmonary veins to the vasodilator effects of NECA. We conclude that adenosine, and the more specific A2-receptor agonist NECA, dilate pulmonary arteries and veins isolated from young rabbits via a mechanism that is partially dependent on endothelium-derived nitric oxide.

Developmental differences in endothelium-dependent responses in isolated ovine pulmonary arteries and veins.

Despite evidence for an important role for endothelium-derived relaxing factor (EDRF) in transitional circulation, previous in vitro studies of newborn pulmonary arteries have demonstrated diminished EDRF activity when compared with arteries from older animals. We studied pulmonary arteries and veins isolated from early newborn and juvenile sheep using standard tissue bath techniques. Incubation of vessels with the nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NNA) constricted veins but not arteries from both age groups. Further studies using preconstricted vessels revealed that arteries relaxed to acetylcholine (ACh), with significantly greater responses observed in juvenile arteries. Veins from both age groups contracted to ACh. Pretreatment with prostaglandin inhibitors (indomethacin and SQ 29,548) diminished ACh relaxations in pulmonary arteries from both age groups, greatly enhanced relaxations to ACh in newborn pulmonary veins, and depressed contractions in juvenile pulmonary veins. Removal of endothelium mechanically or functionally with prostaglandin inhibitors and L-NNA eliminated relaxations to ACh in pulmonary arteries from both age groups and resulted in contractions in veins. We conclude that isolated pulmonary veins from newborn sheep exhibit both baseline and stimulated release of EDRF, and we speculate that these venous responses may be important in the transitional pulmonary circulation.

Characterization and mechanisms of H2O2-induced contractions of pulmonary arteries.

We studied H2O2-induced contractions of isolated rabbit intrapulmonary arteries mounted in standard tissue baths. All vessels were pretreated with a thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29,548, to block immediate transient contractions to H2O2 and to isolate slowly developing sustained contractions. When exposed to H2O2 (0.1, 0.2, 0.3, 0.6, and 1.0 mM) for 30 min, vessels contracted in (0.1, 0.2, 0.3, 0.6, and 1.0 mM) for 30 min, vessels contracted in a concentration-dependent fashion between 0.1 and 0.3 mM H2O2; contractions at 0.6 and 1.0 mM H2O2 were not significantly different from those at 0.3 mM H2O2. During recovery (90 min) from H2O2 exposures, baseline tension was significantly greater, but active tension (10 microM phenylephrine) was significantly less for vessels previously exposed to 0.6 and 1.0 mM H2O2. Contractions to 0.3 mM H2O2 were not blunted by the following interventions: 1) endothelium rubbing, 2) incubation in Ca(2+)-free 100 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) Krebs-Ringer solution, 3) incubation in the Ca(2+)-free solution and depletion of ryanodine (20 microM)-sensitive Ca(2+) stores, or 4) pretreatment with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-3-methyl-piperazine (20 microM). However, contractions were depressed by approximately 50% when vessels were pretreated with the phospholipase C/serine esterase inhibitor 2-nitro-4-carboxy-phenyl-N,N-diphenylcarbamate (50 microM). These results suggest that slow-developing contractions to H2O2 are concentration dependent and may result, in part, from activation of a serine esterase(s) and/or phospholipase C.