Hypoxic pulmonary vasoconstriction is impaired in rats with nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Pulmonary hypertension and persistent fetal circulation contribute to the high mortality rate associated with congenital diaphragmatic hernia (CDH). Morphological alterations of the pulmonary vasculature in infants with CDH are thought to contribute to exaggerated vasoconstrictor responses to normal vasoconstrictor stimuli. In the pulmonary circulation, hypoxia is a potent vasoconstrictor. Under pathological conditions, hypoxia-induced vasoconstriction may contribute to the development of pulmonary hypertension. METHODS: The authors have used the nitrofen-induced model of congenital diaphragmatic hernia in rats to investigate the magnitude of the hypoxic vasoconstrictor response. Congenital diaphragmatic hernias were induced in fetal rats by feeding nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) to pregnant Sprague-Dawley rats at midgestation. Hypoxia-induced vasoconstriction was measured in isolated, perfused third-generation pulmonary arterioles from normal rats and from rats with nitrofen-induced CDH. RESULTS: The hypoxic vasoconstrictor response was significantly blunted in the pulmonary arterioles of fetal rats with nitrofen-induced (2% +/- 1% vasoconstriction), as compared with the responses observed in normal fetal rats (15% +/- 3% vasoconstriction, P = .004). CONCLUSION: Blunting of the hypoxic pulmonary vasoconstrictor response may contribute to ventilation-perfusion mismatching in infants with CDH.

Pulmonary arterioles from rats with congenital diaphragmatic hernias are hypoplastic but not hyperresponsive.

BACKGROUND: Infants born with congenital diaphragmatic hernias (CDH) frequently die as a result of pulmonary hypertension and persistent fetal circulation. The pulmonary vessels of infants with CDH have decreased total cross-sectional area, increased muscle content, and muscularization of intra-acinar arterioles that are normally not muscularized. These structural alterations are believed to result in exaggerated responses to normal vasoconstrictor stimuli. METHODS: The authors used the nitrofen-induced CDH model in rats to determine whether the vasoconstrictor responses of pulmonary arterioles are exaggerated in this animal model of CDH. The authors compared the responses of isolated third-generation pulmonary arterioles from normal rats and from rats with nitrofen-induced CDH to K+-induced depolarization, phenylephrine, angiotensin II, serotonin, and the thromboxane A2 agonist, U46619. RESULTS: It was found that the intraluminal diameter of third-generation pulmonary arterioles from CDH rats was significantly less than in controls (129 +/- 5 micron v 152 +/- 9 micron, respectively). In addition, the ratio of wall thickness to vessel internal diameter was increased in the third-generation pulmonary arterioles of rats with nitrofen-induced CDH (0.62 +/- 0.4 v 0.50 +/- 0.5 for controls). Responses to K+-induced depolarization, phenylephrine, angiotensin II, serotonin, and U46619, however, were not different for pulmonary arterioles from control and CDH rats. CONCLUSION: These data suggest that the structural alterations of the pulmonary vasculature observed in infants with CDH may not cause exaggerated vasoconstrictor responses to normal vasoconstrictor stimuli.

Endothelin-1 pulmonary vasoconstriction in rats with diaphragmatic hernia.

BACKGROUND: Pulmonary hypertension is an important cause of mortality in infants with congenital diaphragmatic hernia (CDH). Endothelin-1 has been implicated as a mediator of pulmonary hypertension. ET-A receptors are increased in the nitrofen model of CDH in rats. We hypothesized that vasoconstrictor responses to endothelin-1 are increased in pulmonary arterioles of rats with nitrofen-induced CDH. MATERIALS AND METHODS: CDH was induced in fetal rats by feeding nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) to pregnant rats at midgestation. Third-generation pulmonary arterioles were isolated on the final day of gestation. Arterioles were cannulated and perfused at constant pressure with a physiologic salt solution. Diameters of arterioles from control animals (n = 8), CDH animals (n = 5), and animals exposed to nitrofen but without CDH (n = 4) were measured. Responses to endothelin-1 concentrations of 10(-12) to 10(-8) M were compared by Student's t test. RESULTS: CDH arterioles constricted more than controls in response to endothelin-1 at concentrations of 10(-11) M (29 +/- 11% vs 5 +/- 3%, P = 0.02) and 10(-10) M (40 +/- 14% vs 9 +/- 6%, P = 0.04). The log concentration of endothelin-1 that induced half-maximal response (ED50) was lower for CDH arterioles than for control arterioles (-10.3 +/- 0.6 vs -9.1 +/- 0.2, P = 0.03). Responses of arterioles from animals exposed to nitrofen but without CDH were not different from controls (P > or = 0.05). CONCLUSIONS: Exaggerated vasoconstrictor responses to endothelin-1 may contribute to pulmonary hypertension in CDH.

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation.

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

Interaction of thyroid hormone and retinoic acid receptors on the regulation of the rat growth hormone gene promoter.

Retinoic acid transcriptionally regulate growth hormone (GH) gene expression through sequences located in the 5'-flanking region of the gene. A partial induction by retinoic acid was obtained with the -181 bp of the rat GH promoter, and sequences up to -209 were required for a full response. These sequences contain the previously identified thyroid hormone responsive element. The retinoid X receptor RXR increased transactivation by T3 and RA. The retinoid was relatively more effective in stimulating the native GH promoter than an heterologous promoter which contains the response element, thus showing the importance of the promoter context on transactivation by the nuclear receptors.