Loren P. Thompson

Endothelium-derived relaxing factor and indomethacin-sensitive contracting factor alter arterial contractile responses to thromboxane during pregnancy***

OBJECTIVESnPregnancy reduces uterine artery contractile responses to norepinephrine and angiotensin II in many species, including the human and the guinea pig, by release of endothelium-derived relaxing substances. We hypothesized that vascular reactivity to thromboxane during pregnancy would also be reduced by a similar mechanism.nnnSTUDY DESIGNnIsolated ring segments of uterine and carotid arteries from nonpregnant and near-term pregnant guinea pigs were suspended in a myograph for the measurement of isometric tension.nnnRESULTSnUterine but not carotid artery sensitivity to cumulative addition of the thromboxane analog U46619 was decreased during pregnancy. The maximal contractile responses of both vessels were unaltered by pregnancy. N omega-nitro-L-arginine (10(-4) mol/L), an inhibitor of nitric oxide endothelium-derived relaxing factor synthesis, increased the sensitivity of uterine and carotid arteries to U46619 in both pregnant and nonpregnant animals. The maximal contractile response of uterine arteries from pregnant guinea pigs was also increased, but that of nonpregnant ones was not. The maximal U46619 contractile response of the carotid artery was not significantly altered by N omega-nitro-L-arginine. Indomethacin (10(-5) mol/L), a cyclooxygenase inhibitor, reduced both the sensitivity and the maximal response of U46619 in each vessel group. Removal of the endothelium from uterine artery of pregnant animals enhanced both sensitivity and maximal response to U46619. Pretreatment of the denuded segments with indomethacin reduced the sensitivity to U46619. However, indomethacin-treated denuded segments were still more sensitive to U46619 than controls.nnnCONCLUSIONnThe sensitivity of guinea pig uterine artery but not carotid artery to thromboxane is reduced during pregnancy. Although the precise mechanism remains unclear, both endothelium-derived relaxing factor and an indomethacin-sensitive contracting factor are involved. If indomethacin-sensitive contracting factor is released by humans and disease alters that release, it is possible that any enhanced contractile response to thromboxane resulting from the loss of endothelium-derived relaxing agents such as prostacyclin and endothelium-derived relaxing factor would be offset by the loss of indomethacin-sensitive contracting factor.

Chronic hypoxia increases peroxynitrite, MMP9 expression, and collagen accumulation in fetal guinea pig hearts

Introduction:Chronic hypoxia increases the expression of inducible nitric oxide synthase (iNOS) mRNA and protein levels in fetal guinea pig heart ventricles. Excessive generation of nitric oxide (NO) can induce nitrosative stress leading to the formation of peroxynitrite, which can upregulate the expression of matrix metalloproteinases (MMPs). This study tested the hypothesis that maternal hypoxia increases fetal cardiac MMP9 and collagen through peroxynitrite generation in fetal hearts.Results:In heart ventricles, levels of malondialdehyde, 3-nitrotyrosine (3-NT), MMP9, and collagen were increased in hypoxic (HPX) vs. normoxic (NMX) fetal guinea pigs.Discussion:Thus, maternal hypoxia induces oxidative–nitrosative stress and alters protein expression of the extracellular matrix (ECM) through upregulation of the iNOS pathway in fetal heart ventricles. This identifies iNOS-derived NO as an important stimulus for initiating the adverse effects of peroxynitrite in HPX fetal hearts.Methods:Pregnant guinea pigs were exposed to normoxia (room air) or hypoxia (10.5% O2, 14 d) before term (term ≈ 65 d) and administered water, L-N6-(1-iminoethyl)-lysine (LNIL), an iNOS inhibitor, or N-acetylcysteine (NAC), an antioxidant.

Chronic hypoxia increases inducible NOS-derived nitric oxide in fetal guinea pig hearts.

Intrauterine hypoxia impacts fetal growth and organ function. Inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) expression was measured to assess the response of fetal hearts to hypoxic (HPX) stress. Pregnant guinea pigs were housed in a hypoxic chamber (10.5% O2 for 14 d, n = 17) or room air [normoxic (NMX), n = 17]. Hearts of anesthetized near-term fetuses were removed. mRNA [hypoxia-inducible factor, (HIF)-1α, 1β, 2α, 3α, iNOS, and nNOS] and protein levels (HIF-1α, iNOS, and nNOS) of fetal cardiac left ventricles were quantified by real time polymerase chain reaction (PCR) and Western analysis, respectively. Cardiac nitrite/nitrate levels were measured in the presence/absence of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, administered to pregnant sows. Hypoxia significantly increased fetal cardiac HIF-1α and -2α mRNA, HIF-1α protein but not HIF-3α or -1β mRNA levels. Hypoxia increased both iNOS mRNA (by 5×) and protein (by 23%) levels but had no effect on nNOS levels. Nitrite/nitrate levels were increased in HPX hearts by 2.5× and decreased with L-NIL by 67 ± 14%. Thus, up-regulation of iNOS-derived nitric oxide (NO) generation is an important mechanism by which fetal hearts respond to chronic hypoxic stress.

Chronic Hypoxia Decreases Endothelial Nitric Oxide Synthease Protein Expression in Fetal Guinea Pig Hearts

Objectives: The underlying cellular mechanisms mediating hypoxia-induced adaptations in the fetus are poorly understood. We tested the hypothesis that hypoxia up-regulates endothelial nitric oxide synthase (NOS3, type III) protein expression in fetal hearts similar to that observed in adult hearts as a cardioprotective adaptation. This study investigates the effect of chronic hypoxia on NOS3 protein expression in hearts and carotid arteries of fetal guinea pigs exposed to normoxia or intrauterine hypoxia. Methods: Time-mated pregnant guinea pigs (term = 65 days) were housed in either normoxic room air (NMX) or exposed to 12% O2 (hypoxia; HPX) for 14 or 28 days of duration. At near term (∼60 days of gestation), pregnant mothers were anesthetized and fetal guinea pig hearts and carotid arteries were excised from NMX and HPX animals and frozen until ready for study. In addition, hearts were also excised from anesthetized adult nonpregnant female guinea pigs exposed to either NMX or HPX for 14 days. NOS3 protein was extracted from all tissues and quantified using Western blot analysis. Fetal heart samples were also prepared for localization of NOS3 protein using immunohistochemistry. Results: Chronic hypoxia increased both maternal and fetal hematocrit after 28 days of duration. HPX decreased NOS3 protein levels in fetal guinea pig hearts by 29% after 28 days compared to NMX controls. In contrast, HPX increased both NOS3 progein levels in adult hearts by 62% and fetal carotid arteries by fourfold after 14 days of exposure compared to their respective NMX controls. Positive immunostaining of NOS3 protein of fetal hearts was localized in both cardiomyocytes and endothelial cells. Conclusion: Contrary to our hypothesis, the hypoxia-induced decrease in fetal guinea pig heart NOS3 protein contrasts to the protein levels measured in either adult hearts or fetal carotid arteries. These results suggest that the NOS protein expression is altered differently by hypoxia in fetal and adult hearts and in a peripheral fetal artery exposed to the same level of hypoxia. Thus, the functional role of NO in the fetal heart during hypoxia may differ from that of the adult.

Long-term Estradiol Replacement Decreases Contractility of Guinea Pig Coronary Arteries to the Thromboxane Mimetic U46619

BACKGROUNDnEstradiol replacement therapy reduces the incidence of coronary artery disease. Current evidence suggests that estradiol may stimulate the production of endothelium-derived NO and thereby reduce the contractile response of vascular smooth muscle. We investigated the effect of long-term replacement of estradiol on NO release and its effect on coronary artery contractility.nnnMETHODS AND RESULTSnFemale guinea pigs were ovariectomized and allowed to recover for 100 days. Pellets containing 17 beta-estradiol (0.25, 0.5, 1.5, and 7.5 mg released over 21 days) were placed subcutaneously for 19 to 20 days. Animals were then anesthetized, and the coronary arteries were excised and cut into ring segments. Rings were placed in small-vessel myographs for measurement of isometric force. Contractile responses of coronary arteries to cumulative addition of U46619 (10(-10) to 10(-5) mol/L), a thromboxane mimetic, were measured in the presence and absence of nitro-L-arginine (LNA), a selective NO synthase inhibitor, and methylene blue, a guanylate cyclase inhibitor. Low (0.25-mg) but not high (0.5-, 1.5-, or 7.5-mg) doses of estradiol inhibited the maximal contractile responses to U46619 compared with arteries from untreated castrated animals. In addition, both LNA and methylene blue potentiated contractile responses to U46619 of arteries from animals receiving 0.25 and 0.5 mg but not 1.5 and 7.5 mg estradiol. Negative log EC50 values were significantly inhibited at 0.25 and 7.5 mg but unaffected at 0.5 and 1.5 mg estradiol compared with castrated animals.nnnCONCLUSIONSnEstradiol at low doses may protect against vasospasm by stimulating endothelium-derived NO release and inhibiting coronary artery contractility.

Regionalization of Endothelium-Dependent Relaxation in the Thoracic Aorta of Pregnant and Nonpregnant Guinea Pigs

Regional variation in the response of the thoracic aorta to contractile agonists has previously been demonstrated. Since the net contractile response reflects the interaction between smooth muscle activation and the release of endothelial substances, we hypothesize that agonist-stimulated release of endothelium-derived nitric oxide (NO) also varies along the length of the thoracic aorta. The distribution of thoracic aorta estrogen receptors is also regionalized. Since pregnancy augments the release of endothelium-derived NO by acetylcholine (ACh) in some arterial beds, we further hypothesize that pregnancy enhances the stimulated release of NO from the thoracic aorta. Aortae were removed from nonpregnant and near term pregnant guinea pigs and cut into ring segments numbered sequentially proximal to distal. The rings were suspended at their optimal passive tension and submaximally contracted with prostaglandin F2 alpha. Endothelium-derived NO-dependent relaxation to ACh increased moving proximal to distal along the aorta independent of pregnancy and ACh relaxation was unaffected by pretreatment with physostigmine to inhibit cholinesterase. The magnitude of the relaxation to carbachol among the different segments was similar to ACh. Pregnancy decreased the ED50 for ACh of segments from the middle and distal segments of the thoracic aorta. Relaxation to the NO donor sodium nitroprusside and the nonreceptor-mediated endothelium-dependent relaxing agent A23187 was uniform along the length of the aorta and independent of pregnancy. These experiments demonstrate regional variation in the stimulated release of endothelium-derived NO in the guinea pig thoracic aorta which is increased by pregnancy.

Acetylcholine Relaxation of Renal Artery and Nitric Oxide Synthase Activity of Renal Cortex Increase with Fetal and Postnatal Age

Endothelium-derived nitric oxide (NO) regulates hemodynamics in the fetal kidney and modulates renal perfusion during postnatal maturation. We hypothesize that NO release by renal arteries increases with fetal maturation and contributes to the increased renal perfusion before and after birth. We tested the effect of maturation on relaxation to acetylcholine (ACh; 10-9 M to 10-5 M), the prototypic endothelium-dependent relaxing agent, and sodium nitroprusside (10-9 M to 10-5 M), an NO donor, on isolated main renal arteries obtained from anesthetized fetal guinea pigs of varying gestational age (0.5-0.8, 0.8-0.9, and 0.9-0.97 gestation), and neonatal (1-50 d) and reproductively mature adult guinea pigs. The effect of NO synthase inhibition by nitro-L-arginine (LNA; 10-4 M) and cyclooxygenase inhibition by indomethacin (10-5 M) on ACh relaxation was also measured. Ca2+-dependent NO synthase activity was measured in fetal (0.5-0.87 gestation), neonatal (1-10 d), and adult (mature) renal cortex by the conversion of [L-14C]arginine to [L-14C]citrulline and the time course compared with the relaxation responses. Sensitivity and maximal relaxation to ACh increased with fetal age. In neonatal renal arteries, maximal relaxation but not sensitivity to ACh increased relative to the fetal arteries. In adult renal arteries, both sensitivity and maximal relaxation increased compared with fetal arteries. Sensitivity but not maximal responses to sodium nitroprusside increased with age but exhibited a different maturational pattern than ACh relaxation. LNA inhibited ACh relaxation in arteries of all ages. Indomethacin reduced the sensitivity to ACh only in the fetal arteries. Ca2+-dependent NO synthase activity of the renal cortex increased during fetal development reaching levels at near term similar to those found in both the newborn and adult kidneys. These results suggest that endothelium-derived NO release by the renal artery and constitutive NO synthase activity in the renal microvasculature increases with fetal and postnatal maturation. Further, the sensitivity of vascular smooth muscle to NO also increases after birth. Thus, functional adaptations in both the endothelium and the vascular smooth muscle contribute to the maturational changes in mechanisms regulating renal hemodynamics before and after birth.

Chronic Hypoxia Inhibits Contraction of Fetal Arteries by Increased Endothelium-Derived Nitric Oxide and Prostaglandin Synthesis

Objective: Chronic hypoxia causes redistribution of fetal cardiac output by mechanisms poorly understood. We tested the hypothesis that chronic hypoxia alters vascular reactivity of arteries from near-term fetal guinea pigs. Methods: Pregnant guinea pigs (50 days, term = 65 days) were exposed to either normoxia (room air) or hypoxia (12% O2) for 14 days. Carotid artery ring segments from anesthetized fetuses were mounted onto myographs for measurement of force. Contractile responses to cumulative addition of prostaglandin F2α (PGF2α, 10-9 M to 10-5 M), U46619, a thromboxane mimetic (10-12 M to 12-6 M), and KCl (10 to 120 mM) were measured in the presence and absence of INDO (INDO, 10-5 M) alone and INDO plus mintro-L-arginine (LNA, 10-4 M), or INDO plus N6-iminoethyl-L-lysine (LNIL, 5 × 10-5 M, a selective iNOS inhibitor), and measured in endothelium-intact and denuded arteries. Nitric oxide synthase (NOS) activity was measured in isolated arteries by 14C-L-arginine to 14C-L-citrulline conversion. Results: Hypoxia decreased contractile responses to both PGF2α and U46619 under control conditions. Maximal contraction to both agonists was increased in hypoxemic arteries after INDO alone and INDO + LNA compared to normoxic controls. Endothelium-denudation abolished the differences between the groups. KCl contration was unaffected by hypoxia. LNIL potentiated maximal PGF2α contraction but was similar between groups. Hypoxia increased (P < .05) total and Ca2+-dependent NOS activities by 1.7- and 2.1-fold, respectively, but had no effect on Ca2+-independent activity. Conclusion: Chronic hypoxia alters vascular reactivity of fetal carotid arteries by increasing the contribution of both vasodilator prostaglandins and nitric oxide and suggests that changes in local vascular mechanisms may be altered by chronic hypoxia.

Differential effect of intrauterine hypoxia on caspase 3 and DNA fragmentation in fetal guinea pig hearts and brains.

The aim of this study is to quantify the effect of intrauterine hypoxia (HPX) and the role of nitric oxide (NO) on the apoptotic enzyme, caspase 3, and DNA fragmentation in fetal heart and brain. Hypoxia and NO are important regulators of apoptosis, although this has been little studied in the fetal organs. We investigated the effect of intrauterine HPX on apoptosis and the role of NO in both fetal hearts and brains. Pregnant guinea pigs were exposed to room temperature (N = 14) or 10.5% O2 (N = 12) for 14 days prior to term (term = 65 days) and administered water or l-N6-(1-iminoethyl)-lysine (LNIL), an inducible nitric oxide synthase (iNOS) inhibitor, for 10 days. Fetal hearts and brains were excised from anesthetized near-term fetuses for study. Chronic HPX decreased pro- and active caspase 3, caspase 3 activity, and DNA fragmentation levels in fetal hearts compared with normoxic controls. l-N6-(1-iminoethyl)-lysine prevented the HPX-induced decrease in caspase 3 activity but did not alter DNA fragmentation levels. In contrast, chronic HPX increased both apoptotic indices in fetal brains, which were inhibited by LNIL. Thus, the effect of HPX on apoptosis differs between fetal organs, and NO may play an important role in modulating these effects.