A receptor for vascular endothelial growth factor that stimulates endothelial apoptosis.

Vascular endothelial growth factor (VEGF) is a dimeric angiogenic factor that is overexpressed by many tumors and stimulates tumor angiogenesis. VEGF initiates signaling by dimerizing the receptors VEGFR-1 and VEGFR-2. The Fas receptor stimulates apoptosis, and artificial dimerization of the Fas cytoplasmic domain has been shown to induce apoptosis. We constructed a chimeric receptor (VEGFR2Fas) combining the extracellular and transmembrane domains of VEGFR-2 with the cytoplasmic domain of Fas receptor. When VEGFR2Fas was stably expressed in endothelial cells in vitro, treatment with VEGF rapidly induced cell death with features characteristic of Fas-mediated apoptosis. These findings demonstrate that VEGFR2Fas functions as a VEGF-triggered death receptor and raise the possibility that introduction of VEGFR2Fas into tumor endothelium or tumor cells in vivo may convert tumor-derived VEGF from an angiogenic factor into an antiangiogenesis agent.

Altered regulation of the ET-1 cascade in lambs with increased pulmonary blood flow and pulmonary hypertension.

Plasma concentrations of endothelin-1 (ET-1) are increased in children with congenital heart disease associated with increased pulmonary blood flow. However, the role of ET-1 in the pathophysiology of pulmonary hypertension remains unclear. Preproendothelin-1 gene expression is increased in adults with advanced pulmonary hypertension. To characterize potential early molecular alterations in the ET-1 cascade induced by increased pulmonary blood flow and pulmonary hypertension, fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). RNase protection assays and Western blot analysis were performed on lung tissue prepared from 4-wk-old shunt lambs and age-matched controls. Endothelin-converting enzyme-1 [the enzyme responsible for the production of active ET-1 from big ET-1, mRNA (411%, p<0.05)] and protein (170%, p<0.05) were increased in lung tissue prepared from shunt lambs, compared with age-matched controls. Endothelin type A receptor (the receptor that mediates vasoconstriction), mRNA (246%, p<0.05), and protein (176%, p<0.05) also were increased in lung tissue prepared from shunt lambs compared with age-matched controls. Conversely, endothelin type B receptor (the receptor that mediates vasodilation), mRNA (46%, p<0.05), and protein (65%, p<0.05) were decreased in shunt lambs. Both the mRNA and protein levels for preproendothelin-were unchanged. Thus we conclude that increased pulmonary blood flow and pulmonary hypertension induce early alterations in the ET-1 cascade that result in increased ET-1 production, increased ET-1-mediated vasoconstriction, and decreased vasodilation. These early alterations in gene expression may contribute to the development of pulmonary hypertension and its associated enhanced pulmonary vascular reactivity.

Altered endothelial function in lambs with pulmonary hypertension and acute lung injury.

Acute lung injury produces pulmonary hypertension, altered vascular reactivity, and endothelial injury. To determine whether acute lung injury impairs the endothelium-dependent regulation of pulmonary vascular tone, 16 lambs were studied during U46619-induced pulmonary hypertension without acute lung injury, or air embolization-induced pulmonary hypertension with acute lung injury. The hemodynamic responses to endothelium-dependent (acetylcholine, ATP, ET-1, and 4 Ala ET-1 [an ETb receptor agonist]) and endothelium-independent (nitroprusside and isoproterenol) vasodilators were compared. During U46619-induced pulmonary hypertension, all vasodilators decreased pulmonary arterial pressure and vascular resistance (P < 0.05). During air embolization-induced pulmonary hypertension, the pulmonary vasodilating effects of acetylcholine, ATP, and 4 Ala ET-1 were attenuated (P < 0.05); the pulmonary vasodilating effects of nitroprusside and isoproterenol were unchanged; and the pulmonary vasodilating effects of ET-1 were reversed, producing pulmonary vasoconstriction (P < 0.05). During air embolization, the pulmonary vasoconstricting effects of ET-1 were blocked by BQ 123, an ETa receptor antagonist. The systemic effects of the vasoactive drugs were similar during both conditions. We conclude that pulmonary hypertension with acute lung injury induced by air embolization results in endothelial dysfunction; there is selective impairment of endothelium-dependent pulmonary vasodilation and an altered response to ET-1 from pulmonary vasodilation to vasoconstriction. This altered response to ET-1 is associated with decreased ETb receptor-mediated vasodilation and increased ETa receptor-mediated vasoconstriction. Endothelial injury and dysfunction account, in part, for the altered regulation of pulmonary vascular tone during pulmonary hypertension with acute lung injury.

Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs.

Ligation of the ductus arteriosus in utero produces fetal and neonatal pulmonary hypertension and alterations in the hemodynamic responses to nitric oxide and endothelin-1 in fetal and newborn lambs. To determine whether fetal pulmonary hypertension alters the expression of the genes of the nitric oxide and endothelin-1 pathways, seven fetal lambs (123-126-d gestation) underwent ligation of the ductus arteriosus. Near-term (138-139-d gestation), total lung RNA, and protein were prepared from control and ductal ligation fetal lambs for RNase protection assays and Western blotting. Ligation of the ductus arteriosus was associated with decreased expression of endothelial nitric oxide synthase mRNA and protein, and the alpha1 and the beta1 subunits of soluble guanylate cyclase protein; and with increased expression of phosphodiesterase V mRNA. Ligation of the ductus arteriosus was also associated with increased expression of preproendothelin-1 mRNA and with decreased expression of endothelin B receptor (ET(B)) mRNA. These results suggest that there is coordinated regulation of genes of the nitric oxide pathway, which would decrease nitric oxide and cGMP concentration, thereby decreasing pulmonary vasodilator activity. There is also coordinated regulation of genes of the endothelin-1 pathway, which would increase endothelin-1 concentration and limit ET(B) receptor activation, thereby increasing pulmonary vasoconstrictor activity. These alterations in gene expression would increase fetal pulmonary vascular resistance, contributing to the development of pulmonary hypertension after birth.

Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension.

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart defects and increased pulmonary blood flow. Nitric oxide (NO) is an important mediator of pulmonary vascular reactivity. The objective of this study was to characterize potential early alterations in expression, localization, and activity of endothelial NO synthase (eNOS) induced by increased pulmonary blood flow and pulmonary hypertension. Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have established a unique animal model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Ten fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). RNase protection assays and Western blotting were performed on lung tissue prepared from 4-wk-old shunt lambs and age-matched controls. eNOS mRNA (2.4:1, P < 0.05) and protein (2. 08:1, P < 0.05) were increased in lungs of shunt lambs. In situ hybridization and immunohistochemistry revealed that the increase was confined to the endothelium of pulmonary arteries. eNOS protein (1.55:1, P < 0.05) and tissue cGMP concentrations (2.1:1, P < 0.05) were also increased in isolated fifth-generation pulmonary arteries of shunt lambs. In addition, total lung eNOS activity was increased (2.9:1, P < 0.05). Thus we report a previously undescribed, early upregulation of eNOS gene expression and activity in lambs with increased pulmonary blood flow and pulmonary hypertension.

Ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs.

At birth, ventilation and oxygenation immediately decrease pulmonary vascular resistance (PVR) and increase pulmonary blood flow (PBF); more gradual changes occur over the next several hours. Nitric oxide, produced by endothelial nitric oxide synthase (eNOS), mediates these gradual changes. To determine how ventilation and oxygenation affect eNOS gene expression, 12 fetal lambs were ventilated for 8 h without changing fetal descending aortic blood gases or pH (rhythmic distension) or with 100% oxygen (O2 ventilation). Vascular pressures and PBF were measured. Total RNA, protein, and tissue sections were prepared from lung tissue for RNase protection assays, Western blotting, and in situ hybridization. O2 ventilation increased PBF and decreased PVR more than rhythmic distension (P < 0.05). Rhythmic distension increased eNOS mRNA expression; O2 ventilation increased eNOS mRNA expression more and increased eNOS protein expression (P < 0.05). To define the mechanisms responsible for these changes, ovine fetal pulmonary arterial endothelial cells were exposed to 1, 21, or 95% O2 or to shear stress. 95% O2 increased eNOS mRNA and protein expression (P < 0.05). Shear stress increased eNOS mRNA and protein expression (P < 0.05). Increased oxygenation but more importantly increased PBF with increased shear stress induce eNOS gene expression and contribute to pulmonary vasodilation after birth.

Frequency of family history of acute myocardial infarction in patients with acute myocardial infarction. Argentine FRICAS (Factores de Riesgo Coronario en America del Sur) Investigators.

The relation between family history of acute myocardial infarction (AMI) and the risk of AMI was analyzed using data of a case-control study conducted in Argentina between 1992 and 1994. Case patients were 1,060 subjects with AMI admitted to 35 coronary care units, and controls were 1,071 subjects admitted to the same network of hospitals where cases had been identified, for a wide spectrum of acute conditions unrelated to known or likely risk factors for AMI: 31% of cases versus 15% of controls reported > or = 1 first-degree relative with history of AMI. Compared with subjects without family history of AMI, the odds ratio (OR) of AMI, after allowance for age, sex, cholesterolemia, smoking, diabetes, hypertension, body mass index, education, social class, and physical exercise, was 2.18 (95% confidence interval [CI] 1.74 to 2.74) for those with family history of AMI. The OR was 2.04 (95% CI 1.60 to 2.60) for subjects with 1 relative, and 3.18 (95% C 1.86 to 5.44) for those reporting > or = 2 relatives with AMI. In women the OR for any family history of AMI was 2.83, and in men 2.01. The association was of similar magnitude if the mother (OR 1.98), the father (OR 2.13), or a sibling (OR 2.48) had had an AMI. The association with family history was stronger at a younger age because the OR for subjects reporting > or = 2 more relatives with a history of AMI was 4.42 for subjects aged < 55 years, and 3.00 for those aged > or = 55 years. The association between AMI and family history of AMI was consistent across separate strata of education, social class, smoking, and serum cholesterol, but was less strong in subjects with history of diabetes and hypertension. When the interaction of known risk factors with family history of AMI was analyzed, hypercholesterolemia, hypertension, and smoking had approximately multiplicative effects on the relative risk. The OR was 4.50 for subjects with family history and cholesterol > or = 240 ml/dl, 4.52 for those with hypertension, and 5.77 for current smokers with family history of AMI. Thus, this study confirms that a family history of AMI is a strong and independent risk factor for AMI. In this population from Argentina, family history accounted for 14% of all cases of AMI in men and 26% in women.

Heart rate is a marker of amiodarone mortality reduction in severe heart failure. The GESICA-GEMA Investigators. Grupo de Estudio de la Sobrevida en la Insuficiencia Cardiaca en Argentina-Grupo de Estudios Multicéntricos en Argentina.

OBJECTIVES: The impact of amiodarone on mortality in patients with severe congestive heart failure (CHF) (New York Heart Association functional classes II [advanced], III and IV; left ventricular ejection fraction < 35%) In the Grupo de Estudio de la Sobrevida en la Insuficiencia Cardiaca en Argentina (GESICA) trial was analyzed in relation to initial mean baseline heart rate (BHR) and its change after 6 months of follow-up. BACKGROUND: Trials of amiodarone therapy in CHF have produced discordant results, suggesting that the effect is not uniform in all patient subgroups with regard to survival. METHODS: The present analysis was carried out in 516 patients randomized to receive amiodarone, 300 mg/day (n = 260), or nonantiarrhythmic therapy (n = 256, control group) and followed up for 2 years. Survival was evaluated for patients with a BHR > or = 90 beats/min (control: n = 132; amiodarone: n = 122) and < 90 beats/min (control: n = 124; amiodarone: n = 138). Survival was also analyzed according to heart rate reduction at 6 months for 367 patients. RESULTS: For patients with a BHR > or = 90 beats/min, amiodarone therapy reduced mortality to 38.4% compared with 62.4% in control patients (relative risk [RR] 0.55, 95% confidence interval [CI] 0.35 to 0.95, p < 0.002). Both sudden death (RR 0.46, 95% CI 0.24 to 0.90, p < 0.02) and progressive heart failure death (RR 0.60, 95% CI 0.30 to 1.03, p < 0.06) were reduced, and functional capacity was improved. In patients with a BHR < 90 beats/min, amiodarone did not alter survival. Among 367 patients who completed 6 months of follow-up, amiodarone reduced 2-year mortality only in those with a BHR > or = 90 beats/min, which was reduced at 6 months. CONCLUSIONS: Elevated rest heart rates in severe CHF identify a subgroup of patients who benefit from treatment with amiodarone. Amiodarone-induced heart rate slowing may be an important benefit for patients.

Nonsustained ventricular tachycardia in severe heart failure. Independent marker of increased mortality due to sudden death. GESICA-GEMA Investigators.

BACKGROUND: The goal of the study was to determine the prognostic value of nonsustained ventricular tachycardia (NSVT) in total mortality in severe congestive heart failure (CHF) and in death modes. NSVT is associated with an increased mortality in CHF. However, the predictive value of NSVT as a marker for sudden death or death due to progressive heart failure has not been determined. METHODS AND RESULTS: Five hundred sixteen patients from the GESICA trial (33.4% with NSVT) were initially studied with the results of 24-hour Holter and 2 years of follow-up. Within 2 years, 87 of 173 patients (50.3%) with NSVT and 106 of 343 patients (30.9%) without NSVT died. Relative risk (RR) was 1.69 (95% confidence interval [CI], 1.27 to 2.24; P < .0002), and Cox proportional hazard analysis was 1.62 (95% CI, 1.22 to 2.16; P < .001). Sudden death increased from 8.7% (30 of 343) to 23.7% (41 of 173) in patients with NSVT (RR, 2.77; 95% CI, 1.78 to 4.44; P < .001). Progressive heart failure death was also increased from 17.5% (60 of 343) to 20.8% (36 of 173) (P = .22). Quantitative analysis of 24-hour Holter (first 295 patients) demonstrated that couplets had a similar RR to that of NSVT for both total mortality (RR, 1.81; 95% CI, 1.22 to 2.66; P < .002) and sudden death (RR, 3.37; 95% CI, 1.57 to 7.25; P < .0005). Couplets and/or NSVT (ventricular repetitive beats) were even more predictive for sudden death (RR, 10.1; 95% CI, 1.91 to 52.7; P < .01). CONCLUSIONS: In patients with CHF, NSVT is an independent marker for increased overall mortality rate and sudden death. The absence of NSVT and ventricular repetitive beats in a 24-hour Holter indicates a low probability of sudden death.

Expression of neuronal nitric oxide synthase corresponds to regions of selective vulnerability to hypoxia-ischaemia in the developing rat brain.

Nitric oxide (NO) has been implicated in the pathogenesis of brain injury from hypoxia-ischaemia. In the brain, the enzyme responsible for NO synthesis is neuronal nitric oxide synthase (nNOS). Using in situ hybridization, immunohistochemistry and NADPH diaphorase histochemistry, we examined the spatial and temporal expression of nNOS during development of the rat brain to determine whether the expression of nNOS delineates the areas of the brain that are selectively vulnerable to hypoxic-ischaemia injury. The expression of nNOS was localized to discrete areas of the brain. nNOS could be detected in the developing forebrain in the 10-day-old embryo (E10). From E14 to E18, the highest level of expression was in the cortical plate, where the majority of neurons were positive. However, this expression diminished with time; in the adult there were only a few nNOS-positive neurones in the deep layers of the cortex. Expression of nNOS was not detected prenatally in the basal ganglia. There was transient high-level expression during the first postnatal week. Thereafter, the basal ganglia exhibited the adult pattern of expression. Expression of nNOS could be detected in the hippocampus at E16. This expression remained constant with regional localization in layers CA1 and CA3 in the adult. Similarly, nNOS expression in the developing cerebellum was observed only after birth. From the first day after birth (P1) to P6, expression was limited to the molecular cell layer. As the cerebellum matured, nNOS expression could be detected in the inner granular layer. By P21, the adult distribution of nNOS expression was observed. All regions expressing nNOS mRNA also demonstrated nNOS protein expression and NADPH diaphorase catalytic activity. Our results demonstrate that nNOS expression in the developing brain correlates with regions of selective vulnerability to hypoxic-ischaemic injury, and, therefore, supports a role for NO in hypoxic-ischaemic injury in the developing brain.

Endothelinb receptor agonists produce pulmonary vasodilation in intact newborn lambs with pulmonary hypertension.

The hemodynamic effects of endothelin-1 (ET-1) are mediated by at least two distinct receptors: ETa and ETb receptors. Recently, ETb receptor agonists (4 Ala ET-1 and IRL 1620) were developed. To investigate the role of ETb receptor activation on the pulmonary and systemic circulations, we studied the hemodynamic effects of intrapulmonary arterial injections of these receptor agonists in 10 intact newborn lambs. At rest, 4 Ala ET-1 (290-1,725 ng/kg) changed no hemodynamic variables. IRL 1620 (180-1,095 ng/kg) decreased mean pulmonary arterial pressure (PAP, 16.8% +/- 15.0 and 17.8% +/- 8.5, p < 0.05) and left pulmonary artery blood flow (21.6% +/- 22.1 and 33.4% +/- 27.7, p < 0.05) at the two highest doses only. During U46619-induced pulmonary hypertension, both 4 Ala ET-1 (3.2% +/- 8.0 to 15.9% +/- 6.4, p < 0.05) and IRL 1620 (8.7% +/- 6.3 to 21.9% +/- 4.1, p < 0.05) produced selective dose-dependent decreases in PAP. The decrease in mean PAP induced by 4 Ala ET-1 and IRL 1620 was attenuated by N omega-nitro-L-arginine [an inhibitor of endothelium-derived nitric oxide (EDNO) synthesis] (16.6% +/- 3.5 vs. 5.9% +/- 2.3 and 16.2% +/- 3.4 vs. 6.6% +/- 2.8, p < 0.05) and by glybenclamide (a blocker of ATP-dependent potassium channels) (18.2% +/- 7.9 vs. 7.5% +/- 8.3 and 14.7% +/- 3.6 vs. 6.3% +/- 3.2, p < 0.05). ETb receptor activation produces selective pulmonary vasodilation during pulmonary hypertension in intact newborn lambs. The vasodilating properties are mediated in part by release of ENDO and by potassium channel activation.

Developmental effects of endothelin-1 on the pulmonary circulation in sheep.

Endothelin-1 (ET-1) is a polypeptide that has potent hemodynamic effects on the pulmonary circulation. To determine whether there are changes in these effects with increasing postnatal age, we investigated the effects of ET-1 (250 ng/kg) at rest and during pulmonary hypertension in eight lambs (< 1 wk old) and 11 juvenile sheep (6-12 mo old). At rest, ET-1 did not change pulmonary arterial pressure in lambs, but increased pulmonary arterial pressure by 64.0 +/- 37.5% (p < 0.05) in sheep. During pulmonary hypertension, ET-1 produced greater decreases in pulmonary arterial pressure in lambs than in sheep (26.6 +/- 3.4% versus 18.7 +/- 8.3%, p < 0.05). In juvenile sheep, the increase in resting pulmonary arterial pressure produced by ET-1 was inhibited by meclofenamic acid, an inhibitor of prostaglandin synthesis (40.3 +/- 9.9% versus 2.3 +/- 4.7%, p < 0.05); during pulmonary hypertension, the decrease in pulmonary arterial pressure produced by ET-1 was inhibited by N omega-nitro-L-arginine, an inhibitor of endothelium-derived nitric oxide synthesis (21.4 +/- 10.7% versus 8.0 +/- 3.6%, p < 0.05) and by glybenclamide, an ATP-dependent potassium-channel blocker (18.8 +/- 8.4% versus 4.0 +/- 4.4%, p < 0.05). The hemodynamic effects of ET-1 on the pulmonary circulation are dependent on postnatal age. Pulmonary vasoconstriction is mediated by prostaglandin production, and pulmonary vasodilation is mediated, in part, by release of endothelium-derived nitric oxide and activation of ATP-dependent potassium channels.

Use of ATP-MgCl2 in the evaluation and treatment of children with pulmonary hypertension secondary to congenital heart defects.

BACKGROUND: Pulmonary hypertension results in increased morbidity and mortality in children after surgical repair of congenital heart defects. Various vasodilators have been unsuccessful in providing preferential pulmonary vasodilation in these patients. Identification of a more preferential pulmonary vasodilator would improve the assessment, management, and outcome of these children. To determine whether ATP-MgCl2 is a preferential pulmonary vasodilator in children with pulmonary hypertension secondary to congenital heart defects, ATP-MgCl2 was administered during routine cardiac catheterization, and the effects were compared with tolazoline. In addition, ATP-MgCl2 was infused intravenously during episodes of postoperative pulmonary hypertension. METHODS AND RESULTS: During cardiac catheterization in 28 children, the effect of ATP-MgCl2 on the pulmonary artery pressure (PAP) and pulmonary vascular resistance index (Rp) was compared with tolazoline. ATP-MgCl2 (0.1 mg of ATP per kilogram per minute) decreased mean PAP by 24% (P < .05) and Rp by 47% (P < .05) without changing mean systemic arterial pressure or systemic vascular resistance. These effects were comparable to those of tolazoline (1 mg/kg). ATP-MgCl2 produced no significant side effects; tolazoline caused tachycardia, nausea, and vomiting. After cardiac surgery in 7 patients, ATP-MgCl2 decreased PAP by 14% (P < .05) and systemic arterial pressure by 6% (P < .05) and eliminated pulmonary hypertensive crises in 3 of 3 patients. CONCLUSIONS: ATP-MgCl2 is a safe, effective, and preferential pulmonary vasodilator in children with pulmonary hypertension secondary to congenital heart defects. It is useful for evaluating pulmonary vasoreactivity during cardiac catheterization and for treating pulmonary hypertension after cardiac surgery.

Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs.

Persistent pulmonary hypertension of the newborn (PPHN) is associated with chronic intrauterine events. Acute nitric oxide (NO) inhibition attenuates the normal increase in pulmonary blood flow at birth. We investigated whether chronic NO inhibition in utero causes persistent pulmonary hypertension. 11 fetal lambs received either a continuous infusion of N omega-nitro-L-arginine (an NO synthesis inhibitor) or 0.9% saline. Before infusion, acetylcholine (dependent upon endogenous NO production) and sodium nitroprusside (which releases its own NO) produced potent pulmonary vasodilation. After 10.5 +/- 1.5 d of infusion, acetylcholine did not produce pulmonary vasodilation in N omega-nitric-L-arginine-treated fetal lambs, but did in saline-treated fetal lambs; sodium nitroprusside produced pulmonary vasodilation in both groups. Immediately after birth, at 140 d of gestation, during the 3-h study period, mean pulmonary arterial pressure did not decrease in N omega-nitro-L-arginine-treated lambs; the increase in pulmonary blood flow and decrease in pulmonary vascular resistance were markedly attenuated compared to saline-treated lambs. These hemodynamic derangements were reversed by L-arginine. There were no anatomic abnormalities in the pulmonary circulation. Chronic NO inhibition in utero reproduces many of the physiologic derangements of PPHN. Intrauterine events which result in endothelial dysfunction and inhibition of NO may produce the physiologic derrangements of PPHN.

HA1004, an intracellular calcium antagonist, selectively attenuates pulmonary hypertension in newborn lambs.

HA1004, an isoquinolinesulfonamide and a cyclic nucleotide-dependent protein kinase inhibitor, is an intracellular calcium antagonist that produces vascular smooth muscle (VSM) relaxation in vitro. We studied the hemodynamic effects of intravenous (i.v.) infusions of HA1004 (0.1-2.0 mg/kg) in vivo in 8 newborn lambs, at rest and during pulmonary hypertension induced either by the i.v. infusion of U46619, a thromboxane A2 (TXA2) mimic, or by alveolar hypoxia. For comparison, we also studied the hemodynamic effects of i.v. infusions of nifedipine (15 and 40 micrograms/kg/min), a calcium entry blocker. At rest, HA1004 produced slight but significant changes in pulmonary and systemic arterial pressure (PAP, SAP) and pulmonary and systemic vascular resistances (PVR, SVR) (p < 0.05). During pulmonary hypertension induced by U46619, HA1004 decreased PAP 12-23% and PVR 9-33% (p < 0.05), whereas SAP decreased 7% and SVR decreased 14% at only one dose (p < 0.05). During pulmonary hypertension induced by alveolar hypoxia, HA1004 decreased PAP 6-32% and PVR 11-30% (p < 0.05), whereas SAP decreased 15% only at the highest dose (p < 0.05). Linear regression analysis of the pooled data demonstrated that HA1004 caused selective pulmonary vasodilation during pulmonary hypertension. Nifedipine decreased PAP 6 and 14% and SAP 5 and 17% during pulmonary hypertension. In newborn lambs with pulmonary hypertension, HA1004, an intracellular calcium antagonist, is more selective and potent than nifedipine, a calcium entry blocker, in decreasing PAP and therefore may be useful in treatment of children with pulmonary hypertension.

Diethylamine/nitric oxide (NO) adduct, an NO donor, produces potent pulmonary and systemic vasodilation in intact newborn lambs.

Nitric oxide (NO), a labile humoral factor produced by vascular endothelial cells, is a potent vasodilator and an important mediator of pulmonary vascular tone. Nucleophile/NO adducts are a new class of compounds that spontaneously and predictively release NO. We investigated the hemodynamic effects of intravenous (i.v.) infusions of a recently developed NO-donor drug, the diethylamine-nitric oxide adduct (DEA/NO), in 17 intact newborn lambs. At rest, DEA/NO (1-2 microgram.kg-1.min-1) produced dose-dependent decreases in mean pulmonary (from 10.6 +/- 8.6 to 21.2 +/- 7.9%, p < 0.05) and systemic arterial pressure (from 13.2 +/- 11.7 to 31.0 +/- 15.4%, p < 0.05). Similarly, during pulmonary hypertension induced by infusion of U46619, DEA/NO (0.5-2.0 micrograms.kg-1.min-1) produced dose-dependent decreases in mean pulmonary (from 7.3 +/- 5.6 to 24.1 +/- 13.3%, p < 0.05) and systemic arterial pressure (from 2.2 +/- 3.8 to 20.3 +/- 12.9%, p < 0.05). Cardiac output (CO), heart rate (HR), systemic arterial blood gases, and pH were unchanged; atrial pressures decreased at higher doses. Equimolar infusions of S-nitroso-N-acetyl-penicillamine, nitroglycerin (NTG), and sodium nitroprusside (SNP) produced similar decreases in pulmonary and systemic arterial pressure. The nucleophile/NO adducts are potent vasodilators; their predictable and quantitative release of NO make them potentially useful research tools. In addition, because these compounds may decrease the incidence of tolerance and the risk from toxic metabolites associated with use of other nitrovasodilators, they may be clinically useful.

Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development.

The protease thrombin is a potent agonist for platelet aggregation, mesenchymal cell proliferation, and endothelial production of growth factors and adhesion molecules. Thrombin also modulates neurite outgrowth in neuronal cultures. These apparently disparate responses to thrombin appear to be largely mediated by the recently cloned thrombin receptor. In the adult, thrombin is generated from its zymogen prothrombin at sites of vascular injury when circulating coagulation factors meet extravascular tissue factor. In this context thrombin's varied actions may mediate responses to wounding. Whether thrombin's actions on cells may also play a role in development is unknown. We examined the expression of thrombin receptor, prothrombin, and tissue factor by in situ hybridization in mouse development. Thrombin receptor mRNA was expressed widely in mesenchymal cell populations during early organogenesis (E9.5) and was particularly abundant in developing heart and blood vessels. Robust receptor expression was also noted in the germinal epithelium of the hindbrain. Thrombin receptor expression became more restricted with time and by the fetal growth stage (E16.5) was most readily detected in certain neurons, endocardial and endothelial cells, and within lung and liver. In contrast to the thrombin receptor, prothrombin mRNA was limited to the embryonic liver and was not detected until E12.5, well after the onset of receptor expression. mRNA for tissue factor, one important trigger for thrombin generation in the adult, was detected in embryonic epithelia from E9.5-12.5. In several instances, tissue factor-expressing epithelia were surrounded by thrombin receptor-expressing mesenchyme. These data suggest a possible role for the thrombin receptor in development. The finding of robust thrombin receptor expression before prothrombin mRNA was detected raises the question of whether other proteases or peptide ligands can activate the thrombin receptor.

Endothelin-1 produces pulmonary vasodilation in the intact newborn lamb.

The vascular endothelium mediates, in part, pulmonary vascular tone. Because endothelin-1 (ET-1), a paracrine hormone produced by vascular endothelial cells, has vasoactive properties, we investigated the hemodynamic effects of intrapulmonary injections of ET-1 in eight intact newborn lambs at rest and during pulmonary hypertension. At rest, ET-1 (50-1,000 ng/kg) did not change pulmonary arterial pressure. During pulmonary hypertension induced by the infusion of U46619 (a thromboxane A2 mimic), ET-1 (50-1,000 ng/kg) produced a selective dose-dependent decrease in pulmonary arterial pressure (5.8 +/- 3.9 to 32.9 +/- 6.9%; P < 0.05). Similarly, during pulmonary hypertension induced by alveolar hypoxia, ET-1 (50-500 ng/kg) produced a selective dose-dependent decrease in pulmonary arterial pressure (7.2 +/- 3.6 to 26.1 +/- 3.3%; P < 0.05). The decrease in pulmonary arterial pressure produced by ET-1 (250 ng/kg) was attenuated by N omega-nitro-L-arginine (an inhibitor of endothelium-derived nitric oxide synthesis, 23.7 +/- 3.4 vs. 12.5 +/- 4.7%; P < 0.05) and by glibenclamide (an ATP-gated potassium-channel blocker, 25.2 +/- 5.0 vs. 9.6 +/- 5.3%; P < 0.05) but not by meclofenamic acid (an inhibitor of prostaglandin synthesis). ET-1 is a pulmonary vasodilator during pulmonary hypertension in the intact newborn lamb. The vasodilating properties are mediated, in part, by release of endothelium-derived nitric oxide, and by activation of ATP-gated potassium channels.