Larry G. Thaete

Differential effects of endothelin A and B receptor antagonism on fetal growth in normal and nitric oxide-deficient rats.

Objective: To determine the role of endothelin (ET) in fetal and placental growth in rats with and without long-term nitric oxide synthase (NOS) inhibition. Methods: Pregnant rats were treated with Nω-nitro-L-arginine methyl ester (L-NAME) or saline and with one of three ET receptor antagonists or vehicle. The antagonists included A-182086 (nonselective) as well as A-127722 and FR-139317 (both ETA selective). Treatment was begun on day 14 of gestation. On gestational day 21, a hysterotomy was done. Litter size was recorded, and viability and fetal and placental weights were determined. Results were analyzed by analysis of variance or by a Kruskal-Wallis nonparametric analysis. Results: In the absence of L-NAME, fetal and placental weights were not affected by ETA-selective antagonism but were significantly decreased by nonselective receptor antagonism (P < .001 and P < .05 for fetal and placental weights, respectively). Infusion of L-NAME resulted in fetal and placental growth restriction (P < .001). In the setting of L-NAME infusion, fetal and placental weights were increased by the ETA-selective antagonists (P < .01) but not by the nonselective antagonist, compared with weights from animals treated with L-NAME alone. There were more fetal deaths with L-NAME treatment (P < .05), but their occurrence was not significantly affected by any of the ET receptor antagonists. Conclusions: Endothelin-A antagonism alone did not affect fetal or placental growth, whereas combined ETA plus ETB antagonism produced fetal and placental growth restriction. In the setting of long-term NOS inhibitio, ETA-selective antagonism improved fetal and placental growth, whereas antagonism of both ETA and ETB receptors did not. Endothelin contributes to NOS inhibition-induced growth restriction acting throughthe ETA receptor.

The Fetal Response to Chronic Placental Insufficiency

Fetal growth restriction is most commonly caused by failure of the placenta to meet the increasing demands for oxygen and substrate of the developing fetus, resulting in common fetal compensatory responses. Understanding these responses is helpful in developing a management strategy that will optimize pregnancy outcome.

Endothelin receptor A antagonism prevents hypoxia-induced intrauterine growth restriction in the rat

OBJECTIVE Our purpose was to investigate the hypothesis that endothelin plays a critical role in maternal hypoxia-induced intrauterine growth restriction. STUDY DESIGN Chronic indwelling venous and arterial catheters were placed on day 17 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Twelve rats were infused with saline solution and 12 with 6 mg/kg per day FR139317, an endothelin receptor A-specific antagonist. For gestational days 18 to 21 half the rats in each infusion group were housed in a normoxic environment and the other half in a hypoxic (14% oxygen) environment. On day 21 an arterial blood gas level was determined, the rats were then anesthetized, and a hysterotomy was performed. The weight of each pup and its corresponding placenta was recorded. Statistical significance was determined by analysis of variance. RESULTS Among the rats receiving saline solution infusions, fetal weights were 20% less and placental weights were 11% less for those housed in a hypoxic environment compared with those housed in a normoxic environment (p < 0.003). Among the rats receiving FR139317 infusions, fetal and placental weights were not significantly different for those in a hypoxic environment compared with those in a normoxic environment. The fetal and placental weights for the rats receiving FR139317 infusion in hypoxic or normoxic environments were similar to those receiving saline solution in a normoxic environment. CONCLUSIONS Endothelin plays a critical role in hypoxia-induced intrauterine growth restriction. Infusion of an endothelin antagonist prevents the intrauterine growth restriction caused by chronic hypoxia.

Endothelin and the regulation of uterine and placental perfusion in hypoxia-induced fetal growth restriction.

Objective: Normal placental function is dependent on maintenance of uteroplacental perfusion. Endothelin, a potent vasoconstrictor, is produced in and is active in the uteroplacental vusculature. The purpose of this study was to determine the role of endothelin in the regulation of uteroplacental perfusion under normal conditions, and in hypoxia-induced fetal growth restriction. Methods: Timed-pregnant Sprague-Dawley rats, outfitted with arterial catheters, were maintained in either a normoxic or a normobaric hypoxic (12% oxygen) atmosphere from day 18 to 21 of gestation. During this time, the endothelin receptor A antagonist, A-127722, or its vehicle was administered. Regional blood flow was determined on gestational day 21 using 57Co-labeled microspheres. Data was analyzed by analysis of variance with statistical significance accepted at P < .05. Results: Both placental and uterine placental bed perfusion were significantly decreased by hypoxia and returned to normal values with the endothelin antagonist (P < .01 and P < .05, respectively). Fetal weights were significantly lower in the hypoxic group (P < .001) and restored to control levels by the antagonist. Conclusion: In the rat, endothelin contributes little to the regulation of uteroplacental perfusion under normal conditions. Hypoxia results in a decrease in perfusion of the uteroplacental bed and of the placenta, and perfusion in both of these beds in normalized by endothelin A receptor antagonism. We conclude that endothelin plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction by reducing uteroplacental perfusion.

Expression of Endothelin 1 and Its Receptors in the Hypoxic Pregnant Rat

Abstract Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O2) atmosphere from Gestational Days 18–21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.

Influence of ETB Receptor Antagonism on Pregnancy Outcome in Rats

Objective: To determine the effect of endothelin-B (ETB)-selective receptor antagonism on pregnancy outcome in normal rats. Methods: ETB receptor antagonist (A-192621; 5.0, 10.0, and 15.0 mg/kg per day) or vehicle was infused subcutaneously for 7 days by osmotic pump. Infusion was begun on day 14 of a 22-day gestation. Nonpregnant animals were treated similarly, and blood pressure (BP) responses and plasma antagonist levels were compared to those in pregnant animals. Mean arterial pressure (MAP) was measured on days 1, 4, and 7 of the infusion. Plasma ETB antagonist levels were measured on day 7 of infusion. On gestational day 21, fetal and placental weights and viability were evaluated at hysterotomy. Data were analyzed by analysis of variance and are presented at mean ± standard error of the mean. Results: Fetal and placental weights were significantly lower at doses of 10 and 15 mg/kg per day of the ETB antagonist compared with vehicle-treated controls (P < .001); these effects were less severe at 15 than at 10 mg/kg per day despite a fourfold higher plasma level of antagonist. Mean arterial pressure was significantly higher at 10 and 15 mg/kg per day compared with controls, but only on infusion day 1 (P < .05). In contrast, MAPs for nonpregnant rats were elevated throughout the infusion at all doses of the ETB antagonist (P < .05). Conclusions: ETB receptor antagonism inhibited fetal growth and increased maternal MAP in a dose-dependent manner, although the effect on BP was not sustained in pregnant animals. ETB receptor antagonism is detrimental to pregnancy outcome in the rat.

Pathophysiology of Chronic Nitric Oxide Synthase Inhibition-Induced Fetal Growth Restriction in the Rat

Objective. To evaluate the pathophysiology of chronic nitric oxide synthase (NOS) inhibition-induced fetal growth restriction (FGR) in the rat. Methods. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) with or without endothelin (ET-1) receptor A (ETA) antagonist from day 14 to 21 of gestation. In separate groups, ETA antagonist and/or L-NAME were discontinued on day 18. On day 21 fetal and placental weights, and maternal and fetal plasma nitrate/nitrite (NOx) were determined. Results. L-NAME led to FGR, and decreased maternal and fetal NOx. Maternal NOx was further decreased when ETA antagonist was co-administered with L-NAME. ETA antagonism along with L-NAME did not impact fetal growth. Discontinuation of L‐NAME on day 18 resulted in normal fetal and placental growth at day 21 and an increase of maternal NOx. Simultaneous cessation of both NOS inhibition and ETA antagonism on day 18 produced FGR at day 21, whereas continuation of ETA antagonism after discontinuation of L-NAME resulted in normal fetal growth. Conclusions. NOS inhibition in the pregnant rat leads to decreased maternal and fetal nitric oxide (NO) production and FGR. The effects of NOS inhibition on fetal growth are reversible, and are mediated at least in part by ET-1. With chronic NOS inhibition, ETA antagonism improves but does not normalize fetal growth, and may allow increased access of L-NAME to the fetal compartment. Continued access of L-NAME to the fetal compartment may limit the effect on fetal growth of any therapeutic intervention in this model of FGR.

Endothelin-1-Induced Placental and Fetal Growth Restriction in the Rat

The objective of this study was to evaluate the effect of increased circulating endothelin on fetal and placental growth in the rat. Indwelling arterial and venous catheters were placed on day 14 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Saline, 0.2 nmol/kg/h endothelin, or 0.5 nmol/kg/h endothelin was continuously infused from day 15 through 21 in randomly assigned animals (n = 12 in each group). Maternal arterial blood pressure and serum endothelin levels were evaluated on days 15, 18, and 21 of gestation. On day 21, pregnancy outcome data including fetal and placental weights, litter size, and the occurrence of stillbirths were ascertained, and fetal blood was obtained for serum endothelin levels. All data were compared among the three groups, and statistical significance was determined by analysis of variance. Endothelin infusion resulted in a dose-dependent decrease in fetal and placental weights when compared to saline-treated pregnancies. A significant increase in maternal arterial blood pressure was noted only in the 0.5 nmol/kg/h endothelin group. Fetal and placental growth restriction occurred in the absence of maternal hypertension in the 0.2 nmol/kg/h group. These results demonstrate that endothelin infusion causes restriction of fetal and placental growth, even in the absence of maternal hypertension.

ENDOTHELIN AND BLOOD PRESSURE REGULATION IN THE FEMALE RAT: STUDIES IN NORMAL PREGNANCY AND WITH NITRIC OXIDE SYNTHASE INHIBITION-INDUCED HYPERTENSION

OBJECTIVE To evaluate the role of endothelin (ET) in blood pressure regulation in normal pregnant and nonpregnant rats and with nitric oxide synthase (NOS) inhibition. METHODS Pregnant and nonpregnant Sprague-Dawley rats were treated for 7 days with an ET(A)-selective (A-127722 or FR-139317), ET(B)-selective (A-192621), or ET(A)/ET(B) nonselective (A-182086) endothelin receptor antagonist, and/or with the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME, 2. 5 mg/kg/h). In pregnant rats, the ET antagonists and L-NAME were administered from gestational day 14 through day 21 (term = 22 days). All rats received indwelling arterial catheters for blood pressure measurement. Mean arterial pressures were recorded on infusion days 1, 4, and 7 and these data were compared by analysis of variance among experimental groups with p < 0.05 considered significant. RESULTS The ET(A) receptor antagonism lowered blood pressure in both pregnant and nonpregnant rats (p < 0.05), whereas ET(B) antagonism resulted in hypertension (p < 0.001). ET(B) antagonism-induced hypertension was attenuated by pregnancy (p < 0. 001). Hypertension was induced in all rats treated with L-NAME (p < 0.001). Endothelin receptor antagonism, regardless of specificity, did not ameliorate L-NAME-induced hypertension in pregnant or nonpregnant female rats. The only observed effect of ET(A) antagonism on NOS inhibition-induced hypertension was the prevention of a continued rise at infusion day 7 in nonpregnant rats. CONCLUSIONS Endothelin, acting via both the ET(A) and ET(B) receptors, contributes to blood pressure homeostasis in pregnant and nonpregnant rats. Endothelin receptor antagonism does not ameliorate NOS-inhibition-induced hypertension in pregnant rats.

Tetrahydrobiopterin prevents platelet-activating factor-induced intestinal hypoperfusion and necrosis: Role of neuronal nitric oxide synthase.

Objective:We reported previously that neuronal nitric oxide synthase (nNOS) is the predominant NOS in rat small intestine and is down-regulated by platelet-activating factor (PAF). The severity of the bowel injury induced by PAF is inversely related to its suppressing effect on nNOS. Here, we investigated whether intestinal perfusion is regulated by nNOS and whether tetrahydrobiopterin, a co-factor and stabilizer of nNOS, reverses PAF-induced intestinal hypoperfusion and injury. Setting:Animal laboratory. Design:We first examined nNOS regulation of splanchnic blood flow by measuring the perfusion of the heart, lung, ileum, and kidney in rats after a nNOS inhibitor. We then examined the protective effect of tetrahydrobiopterin on PAF-induced bowel injury, mesenteric hypoperfusion, and systemic inflammation. Subjects:Adult male Sprague-Dawley rats. Intervention:In part 1 of the experiment, rats were given 7-nitroindazole (a specific nNOS inhibitor, 50 mg·kg−1·day−1). In part 2 of the experiment, rats were treated with tetrahydrobiopterin (20 mg/kg) 5 mins before and 30 mins after PAF challenge (2.2 &mgr;g/kg, intravenously) Measurements:Perfusion of the heart, lung, ileum, and kidney was measured at 1 and 4 days after 7-nitroindazole, using fluorescent microspheres. Intestinal injury and inflammation (myeloperoxidase content), blood perfusion, calcium dependent-NOS activity, and systemic inflammation (hypotension and hematocrit increase) were assessed 1 hr after PAF with and without tetrahydrobiopterin treatment. Results:In part 1 of the experiment, 7-nitroindazole induced a long-lasting reduction of blood perfusion and inducible NOS expression selectively in the ileum but not in nonsplanchnic organs such as heart, lungs, and kidneys. In part 2, tetrahydrobiopterin protected against PAF-induced intestinal necrosis, hypoperfusion, neutrophil influx, and NOS suppression. It also reversed hypotension and hemoconcentration. Sepiapterin (2 mg/kg, stable tetrahydrobiopterin precursor) also attenuated PAF-induced intestinal injury. Conclusions:We conclude that nNOS selectively regulates intestinal perfusion. Tetrahydrobiopterin prevents PAF-induced intestinal injury, probably by stabilizing nNOS and maintaining intestinal perfusion.