Maria R Pierce

Limb Reduction Defects after Prenatal Inhibition of Nitric Oxide Synthase in Rats

ABSTRACT: To determine the influence of nitric oxide (NO) on vascular tone during fetal development, timed pregnant rats received the NO synthase inhibitor NG-nitro-L-arginine methyl ester for consecutive 4, 7, or 14 d before parturition (postorganogenesis). Offspring demonstrated limb reduction defects (incidence, 53%) involving either or both hindlimbs, whereas forelimbs were uniformly spared. Defects were dose-dependent but independent of the duration of administration occurring with equal frequency in 4-, 7-, and 14-d treatment groups. Histologic analysis revealed features characteristic of vascular disruption with hemorrhagic necrosis and loss of structure. The defects were prevented by concurrent maternal administration of L-arginine or the NO donors S-nitroso-N-acetyl-penicillamine and sodium nitroprusside. Defects were not seen after prenatal treatment with aminoguanidine. To study basal and agonist-mediated NO release, newborn femoral and brachial arteries were cannulated with a glass micropipette under constant pressure, and changes in intraluminal diameter (micrometers) were measured in response to acetylcholine and the NO synthase inhibitor Nω-nitro-L-arginine. Newborn femoral and brachial vessels demonstrated a dramatic (59%) decrease in resting diameter compared with adult vessels (16%). These findings suggest that basal NO release is upregulated during fetal development concurrent with the processes that increase maternal NO release. The data also suggest that up-regulation of NO release occurs throughout the fetal systemic circulation and is not restricted to hindlimbs. This is the first study to demonstrate inhibition of NO release in the pathogenesis of limb reduction defects.

Fatal Carnitine Palmitoyltransferase II Deficiency in a Newborn: New Phenotpic Features

We describe the term male infant of asymptomatic, healthy nonconsanguineous parents presenting on the first day of life with nonketotic hypoglycemia, seizures, hepatomegaly, cardio-megaly with biventricular hypertrophy, and ventricular arrhythmias. Cranial ultrasound revealed cystic dysplasia with several foci of hyperechogenicity within the right basal ganglia. Free carnitine was markedly decreased in the urine and plasma with a pronounced elevation of plasma long-chain acylcarnitines. Fibroblast carnitine palmitoyltransferase II activity was reduced to 26% and 38% in the father and mother, respectively. The infant expired on day 5 of life from malignant ventricular tachy-arrhythmias. Diffuse lipid accumulation was evident at autopsy, including in the liver, heart, kidney, adrenal cortex, skeletal muscle, and lungs. This new case of infantile CPT-1I deficiency illustrates the severity of the early onset form of CPT-II deficiency.

Report of a new patient with transposition of the great arteries with deletion of 22q11.2

We report on a new patient with d-transposition of the great arteries who was found to have deletion of 22q11.2. He had minor facial anomalies, normal T- and B-cell subsets, and transient hypocalcemia. Similar to rare previous reports, our patients extracardiac manifestations were relatively mild.

Nitric oxide synthase inhibition decreases tolerance to hyperoxia in newborn rats

We evaluated the effects of sustained perinatal inhibition of NO synthase (NOS) on hyperoxia induced lung injury in newborn rats. NG-nitro-Larginine-methyl-ester (L-NAME) or untreated water was administered to pregnant rats for the final 7 days of gestation and during lactation; followed by postnatal exposure to hyperoxia (>95% O2) or room air. The survival rate of L-NAME treated pups when placed in > 95% O2 at birth was significantly lower than controls from day 4 (L-NAME, 87%; control pups, 100%, p < 0.05) to 14 (L-NAME, 0%; control pups, 53%, p < 0.05). Foetal pulmonary artery vasoconstriction was induced by L-NAME with a decrease in internal diameter from 0.88 ± 0.03 mm to 0.64 ± 0.01 mm in control vs. L-NAME groups (p < 0.05), respectively. We conclude that perinatal NOS inhibition results in pulmonary artery vasoconstriction and a decreased tolerance to hyperoxia induced lung injury in newborn rats.

ADRENOMEDULLIN MEDIATED NITRIC OXIDE RELEASE IN THE REGULATION PLACENTAL ARTERIAL VASCULAR TONE. • 451

The role of agonist and constitutive nitric oxide (NO) release in the regulation of human placental vascular tone are not known. ADM is a recently discovered hypotensive peptide isolated from pheochromocytoma cells that shares structural homology to Calcitonin Gene Related Peptide, (CGRP). Adrenomedullin has been shown to produce vascular relaxation through NO and cyclic AMP mediated intracellular signal pathways. We investigated in vitro responses to both peptides in cannulated chorionic plate arteries (CPA) from term uncomplicated pregnancies. Vessels were cannulated on micropipettes under constant intraluminal pressure and changes in diameter (<400 microns) were measured through a videodimension analyzer. Percent relaxation in response to 0.1uM synthetic human ADM and CGRP were observed following submaximal preconstriction with U46619. Vessels were rechallenged with either peptide following equilibration with 0.1mM of the NO synthase inhibitor L-NNA. An additional group was studied following removal of the endothelium (EC-) by perfusing the lumen with lcc air at atmospheric pressure. Results shown below are Mean ± SEM (n=6) percent increase lumen diameter (*, p<0.05 vs. control). Membrane receptors and intracellular signal pathways to both peptides are present in term CPA. The in vitro response to ADM is mediated by endothelial NO release. Selective inhibition of the response to ADM but not CGRP by L-NNA strongly suggests two distinct receptor populations. Constitutive NO release may play an important role in the regulation of placental vascular tone in vivo. Table

IS PEROXYNITRITE-INDUCED APOPTOSIS INVOLVED IN INTRAUTERINE GROWTH RETARDATION? † 1429

Placental vasoconstriction has been proposed as a primary mechanism for fetal growth retardation. To challenge this hypothesis, we developed a model of fetal growth retardation characterized by increased release of vasodilators: maternal exposure to low dose endotoxin (LPS). This study addressed the hypothesis that LPS administration compromised fetal growth by inducing apoptosis in the utero-placental unit. METHODS: Timed-pregnant dams were randomly assigned to a control or endotoxin treated groups (30μg/kg i.p. daily). At term, fetal and placental weights, immunohistochemical evidence for nitrotyrosine (a marker for peroxynitrite formation) and apoptosis (TUNEL) in the utero-placental unit were determined. Positive TUNEL immunoreactivity results from the incorporation of digoxygenin-labeled UTP to 3′ ends of DNA by the enzyme terminal deoxynucleotide transferase. Multiple 3′ ends is a hallmark of apoptosis. NO release was also determined in explants of uterus and placenta by electrochemistry. RESULTS: Endotoxin administration resulted in growth retardation of both the fetus and placenta. NO release from explants was increased in placenta (6-fold) and uterus (21-fold) in response to LPS. Nitrotyrosine and DNA fragmentation(TUNEL) were absent in control animals but marked in placenta and uterus following endotoxin administration. CONCLUSIONS: Maternal exposure to LPS results in fetal growth retardation. Fetal growth retardation may occur independent of placental vascular insufficiency but in response to DNA damage induced by free radicals and oxidants (possibly peroxynitrite) leading to apoptosis and a net reductin in fetal and placental growth.

INDUCIBLE NITRIC OXIDE SYNTHASE REGULATES FETAL GREAT VESSEL CALIBER. 420

Background Nitric oxide plays a role in the regulation of fetal great vessels and ductus arteriosus caliber near the end of gestation but the enzyme source is unknown. Aims: To determine if inducible nitric oxide synthase (iNOS) is present in the cardiovascular system of fetal rats, and to compare the effects ofL-NG-(1-lminoethyl)lysine (L-NIL) a selective inhibitor of iNOS, with lipopolysacharide (LPS), sodium nitroprusside (SNP) and untreated controls near the end of gestation.Methods and Results iNOS inthe rat fetus was demonstrated in heart/great vessels and placenta using RT-PCR. Rats in the last week of pregnancy were treated for 5 d with L-NIL (1, 10, and 100 μg/ml in the drinking water). LPS 30 μg/kg od ip 5 d. SNP in mini-osmotic pumps to deliver 10 μg/kg/min. Control group was undisturbed. On day 21 of gestation, dams were anesthetized, fetuses delivered by cesarean section and rapidly frozen in isopentane chilled in liquid nitrogen. Cryostat sections(10μm) were used to reconstruct a computer generated 3D image of the great vessels and ductus arteriosus, calibers were measured with electronic calipers in mm. Table shows Mean±SEM Significant dose-dependent constriction of the great vessels and ductus arteriosus was observed with L-NIL while both LPS and SNP significantly dilated the vessels.Conclusions Nitric oxide, generated by inducible nitric oxide synthase plays a significant role in the control of major vessels and ductus arteriosus caliber in the fetal rat. In this regard nitrergic regulation of vascular tone in the fetus differs from the adult.