Sedentariness and increased visceral adiposity in adult perinatally iron-deficient rats.

BACKGROUND: Perinatal iron deficiency (PID) adversely programs offspring resulting in alterations in adult cardiometabolic function. Increased visceral adiposity is the proposed culprit for these sequelae, and may be potentiated by decreased physical activity. Herein, we determined (i) the effect of PID on visceral adipose tissue (VAT) and locomotor activity, and (ii) whether increased VAT is associated with blood pressure responsiveness to increased dietary sodium. METHODS AND RESULTS: Dams were fed a low iron diet (<10 mg/kg Fe) prior to and throughout gestation. From 12 to 35 weeks of age, locomotor activity (assessed by radiotelemetry) in PID offspring was 25% lower compared with control offspring (P<0.001). At 36 weeks of age, PID offspring had 15% more VAT than controls (P<0.05). Furthermore, the elevation of mean arterial pressure (by radiotelemetry) in response to increased sodium intake was approximately twofold greater in the PID offspring (P<0.05). CONCLUSIONS: PID results in increased visceral adiposity, which was associated with enhanced blood pressure responsiveness to dietary salt, perhaps due to programmed sedentary behavior.

The source of endogenous carbon monoxide formation in human placental chorionic villi.

Carbon monoxide (CO) is proposed to play a role in placental vascular control, as the placenta produces and responds to CO. The mechanism by which CO is formed by the placenta is unclear but could be through heme oxygenase (HO) degradation of heme, lipid peroxidation, or both. Human placental cotyledons were perfused with Kreb s solution to remove blood. Chorionic villi segments were prepared for measurements of CO production in the absence/presence of an exogenous supply of heme substrate (methemalbumin), inhibitors of HO, or inhibitors of lipid peroxidation. HO inhibitors used were chromium mesoporphyrin (CrMP) (0.1 mM, 0.3 mM), and azalanstat (0.1 mM, 0.3 mM). The lipid peroxidation inhibitors used were EDTA (0.1 mM, 0.3 mM) and deferoxamine (0.1 mM). Incubation of villi segments with methemalbumin (0.15 mM, 0.3 mM, 0.45 mM) resulted in a concentration-dependent increase in CO formation above the basal, endogenous rate. CrMP and azalanstat inhibited basal endogenous CO production, whereas EDTA and deferoxamine enhanced CO formation above basal level. These results demonstrate that endogenous CO was formed by human chorionic villi from heme, primarily through the action of HO, and are consistent with the hypothesis that HO plays a role in the regulation of placental vasculature by the formation of heme-derived CO.

Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 microM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased (P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% (P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants (P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content (r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.

Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta.

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.

Comparison of rat and human cytochrome P450 (CYP) sources of N-alkylprotoporphyrin IX. Formation after interaction with porphyrinogenic xenobiotics: studies with cDNA-expressed single CYP enzymes.

1. The porphyrinogenicity of certain xenobiotics is due to mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes, with concurrent formation of N-alkylprotoporphyrins (N-alkylPPs), which disrupt control of haem biosynthesis. An ambiguity arises when extrapolating results obtained with such porphyrinogenic xenobiotics in animals to humans owing to species' differences in CYP enzymes. The objective was to use cDNA-expressed individual rat CYP enzyme preparations in microsomes prepared from baculovirus-infected insect cells to determine which rat CYP enzymes were the source of N-alkylPPs after interaction with three porphyrinogenic xenobiotics and to compare the results with formation of N-alkylPPs in individual human CYP enzyme orthologues. 2. A sensitive fluorometric technique was employed to quantitate N-alkylPP formation after interaction of individual CYP enzymes with a porphyrinogenic xenobiotic. 3. N-alkylPP formation was found following the interaction of three porphyrinogenic xenobiotics with CYP1A2, 2B1, 2C6, 2C11 and 3A2, in amounts ranging from 0.45 to 0.07 nmol N-alkylPP nmol(-1) CYP. The results obtained with rat CYP1A2, 2C6, 2C11 and 3A2 were compared with those previously obtained with the human CYP orthologues 1A2, 2C9 and 3A4. 4. Some results corresponded while others did not, reinforcing a previous recommendation that when dealing with xenobiotics whose porphyrinogenicity depends upon interaction with CYP enzymes resulting in N-alkylPP formation, animal experiments should be augmented with studies using human CYP preparations.

Carbon monoxide decreases perfusion pressure in isolated human placenta.

Carbon monoxide (CO) is one of the metabolites formed via heme oxidation catalysed by the enzyme heme oxygenase (HO). Endogenous formation of CO, mediated by HO, has been noted in both placental and umbilical vessels. In blood vessels from different mammalian sources, it has been proposed that the vasodilator effect of CO is mediated via stimulation of soluble guanylyl cyclase (sGC) and consequent increased cGMP formation. The purpose of the present study was to determine the effect of exogenous CO on placental cotyledon perfusion pressure and to determine the role of sGC in the CO-induced decrease of perfusion pressure using the in vitro human placental perfusion preparation. A thromboxane A2 mimetic (U46619) was added to the foetal perfusion medium to constrict the placental blood vessels. Carbon monoxide was added to the foetal perfusion medium in increasing concentrations to determine its effect on placental perfusion pressure. Carbon monoxide produced a concentration-dependent decrease in placental perfusion pressure. The addition of ODQ, a sGC inhibitor, attenuated the CO-induced decrease in placental perfusion pressure, while addition of YC-1, an activator of sGC, augmented the CO-induced decrease in placental perfusion pressure. The data indicate that CO causes vasorelaxation of placental resistance blood vessels, in large part, via activation of sGC.

An extracellular source of heme can induce a significant heme oxygenase mediated relaxation in the rat aorta.

Carbon monoxide has been under active investigation for a role in controlling vascular tone throughout the last decade because of its ability to induce relaxation in blood vessels. The underlying mechanisms of this response are hypothesized to be mediated by soluble guanylyl cyclase (sGC) and, in some instances, KCa channels. The major source of CO in major blood vessels is the catabolic process of heme degradation, which is catalyzed by heme oxygenase (HO). This heme substrate could be derived from heme sources within vascular smooth muscle cells, such as heme proteins, or by uptake from the extracellular milieu. The current study shows that the isolated rat aorta relaxes upon exposure to pharmacological concentrations of heme in the bathing medium. This response was inhibited by an inhibitor of HO (tin protoporphyrin) and sGC (1-H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one). These observations were interpreted to mean that vascular smooth muscle cells are capable of taking up and utilizing heme for the production of CO.

Endogenous carbon monoxide formation by chorionic villi of term human placenta.

Carbon monoxide (CO) is a novel messenger that is proposed to play a complementary role with nitric oxide in the regulation of placental haemodynamics. In a previous study, CO formation from exogenous haem has been measured in the microsomal fraction of chorionic villi as an index of haem oxygenase activity. The objective of the present study was to determine whether endogenous CO is formed by dissected chorionic villi of term human placenta, to which no exogenous substrate or co-factor had been added. Each sample of freshly isolated chorionic villi (approximately 0.4 g) of term human placenta from caesarean delivery was incubated in a sealed vial containing 1 ml of Krebs' solution (pH 7.4) at 37 degrees C. CO formation was determined by quantitating, using a gas-chromatographic method, the amount of CO released into the headspace gas of the incubation vial. There was time-dependent formation of endogenous CO in chorionic villi incubated at 37 degrees C during a 60-min time course. CO formation was found to be minimal in chorionic villi samples incubated at 4 degrees C and was increased relative to tissue weight. The data demonstrate that there is endogenous CO formation by chorionic villi of term human placenta.

YC-1 enhances the responsiveness of tolerant vascular smooth muscle to glyceryl trinitrate.

A major limitation of the use of organic nitrates in cardiovascular medicine is the development of tolerance, which has been attributed, in part, to a decrease in their metabolic activation in the vascular smooth muscle cell. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was shown to potentiate vascular smooth muscle responsiveness to glyceryl trinitrate (GTN), sodium nitroprusside, and the nitric oxide donor NOC 18, in organic nitrate-naive vascular smooth muscle. We used GTN-tolerant rabbit aortic rings (RARs) to test the hypothesis that a non-vasorelaxant concentration of YC-1 enhances the ability of the prototypical organic nitrate GTN to relax vascular smooth muscle and elevate intravascular cGMP under conditions of GTN tolerance. Treatment with YC-1 (3 microM) produced a left shift of the GTN concentration-response curve and decreased the EC50 value for GTN-induced relaxation in both GTN-tolerant and non-tolerant RARs (P < 0.05). Intravascular cGMP elevation induced by GTN was enhanced in the presence of YC-1 in GTN-tolerant and non-tolerant RARs (P < 0.05). These observations indicate that YC-1, or similarly acting drugs, may be useful in overcoming the tolerance that develops during sustained GTN therapy, and that its mechanism may involve enhanced cGMP formation.

Heme oxygenase activity in term human placenta.

Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P< 0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme-CO (HO) pathway plays a complementary role with the L -arginine-nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics.

Potentiation of carbon monoxide-induced relaxation of rat aorta by YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole].

The hypothesis that endogenous carbon monoxide (CO), produced during the oxidation of heme catalyzed by heme oxygenase (HO), plays a role similar to that of nitric oxide (NO) in the regulation of cardiovascular tone has been criticized because of the low potency of CO compared with NO in relaxing blood vessels and stimulating soluble guanylyl cyclase (sGC). This criticism has been muted by the demonstration that, in the presence of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole], CO has similar potency to NO in stimulating sGC activity. In this study, we determined that YC-1 potentiated CO-induced relaxation of rat aortic strips (RtAS) by approximately ten-fold. Furthermore, CO-induced relaxation of RtAS was shown to be mediated through stimulation of sGC because vasorelaxation was inhibited by ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), a selective sGC inhibitor, in the absence and presence of YC-1. A gas chromatographic-headspace method was used to measure CO concentration in Krebs' solution following the addition of CO-saturated saline solution to the tissue bath, in order to provide an accurate determination of RtAS exposure to CO. The tissue bath concentration of CO was shown to be approximately one-half of that calculated to be present. We conclude that should an endogenous compound exist with properties similar to that of YC-1, then the potency of CO as a vasorelaxant in the presence of this factor would be increased. As a consequence, CO could play a role in the regulation of cardiovascular tone, comparable to that of NO.

Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations.

Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selectively inhibit HO activity without inhibiting the activity of either nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). The objective of this study was to test a series of metalloporphyrins that have previously been shown to inhibit HO activity, for their ability to inhibit HO without inhibiting NOS or sGC activities. Measurement of activity of HO in rat brain microsomes and NOS in rat brain cytosol was made for samples incubated with metalloporphyrins (0.15-50 microM), including zinc protoporphyrin IX, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG), chromium mesoporphyrin IX (CrMP), tin protoporphyrin IX, and zinc N-methylprotoporphyrin IX. CrMP and ZnBG were found to be the most selective inhibitors of HO activity (i.e., caused the greatest inhibition of HO activity, 89 and 80%, respectively, without inhibition of NOS activity). Based on these results, sGC activity in rat lung cytosol incubated with CrMP or ZnBG (0.15-15 microM) was measured. ZnBG did not affect basal sGC activity but did potentiate S-nitroso-N-acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not affect either basal or SNAP-induced activity. It was concluded that of the five metalloporphyrins studied, CrMP, at a concentration of 5 microM, was a selective inhibitor of HO activity and was the most useful metalloporphyrin for the conditions tested. Thus, CrMP would appear to be a valuable chemical probe in elucidating the physiological role of HO.

Glyceryl trinitrate-induced vasodilation is inhibited by ultraviolet irradiation despite enhanced nitric oxide generation: evidence for formation of a nitric oxide conjugate.

Our objective was to determine whether a stabilized form of nitric oxide (NO) such as an S-nitrosothiol, rather than NO itself, is the vasoactive metabolite produced when glyceryl trinitrate (GTN) interacts with vascular smooth muscle. In a control study, NO formation was measured by a chemiluminescence-headspace gas method during the incubation of a prototype S-nitrosothiol, namely, S-nitroso-N-acetylpenicillamine (SNAP), in Krebs' solution. NO formation from SNAP was increased when the incubation was carried out in the presence of UV light, indicating that homolytic photolysis of the S-nitrosothiol had occurred. When GTN was incubated with bovine pulmonary artery (BPA) in the absence of UV light, NO was not measurable until 5 min of incubation. By contrast, in the presence of UV light, NO was measurable as early as 0.5 min, and by 5 min, it was higher than that observed in the absence of UV light. BPA rings were relaxed with SNAP and GTN in the absence of UV light, and EC50 values of 0.24 +/- 0.28 microM and 10 +/- 6 nM, respectively, were observed. In the presence of UV light, the vasodilator response of BPA to SNAP and GTN was attenuated, and EC50 values of 2.7 +/- 3.0 microM and 49 +/- 23 nM, respectively, were observed. Our results are consistent with the idea that GTN biotransformation by vascular smooth muscle results in the production of a stabilized form of NO, possibly an S-nitrosothiol, and that degradation of this metabolite by UV light results in NO formation accompanied by decreased vasodilation.

Analysis of nanomolar S-nitrosothiol concentrations in physiological media.

S-Nitrosothiols occur endogenously and are thought to function as storage forms and/or stable carriers of nitric oxide. Moreover, the S-nitrosothiols have been postulated to function as neurotransmitters and mediate the vasodilator action of glyceryl trinitrate. Because of the increasing pharmacological and physiological interest in S-nitrosothiols, a sensitive method for analysis of these substances is required. We describe a sensitive method based on adsorptive stripping voltammetry for measurement of two S-nitrosothiols, namely, S-nitroso-N-acetyl-D-penicillamine (SNAP) and S-nitrosoglutathione (SNOG), in Krebs' solution. The method is based on the irreversible electrochemical reduction of SNAP and SNOG at the hanging mercury drop electrode (HMDE). The analytes were adsorbed at the HMDE for 60 s at -0.100 V, then exposed to a cathodic linear potential scan of 100 mV s(-10) which resulted in the reduction of SNAP at -0.470 V and SNOG at -0.530 V. Under these conditions, 4 nM SNAP and 11 nM SNOG were readily quantified. Using the above method, we were able to confirm the rapid degradation of SNAP under UV irradiation. Reproducibility of the method as applied to the analysis of these S-nitrosothiols in Krebs' solution was demonstrated by the within-day and day-to-day coefficients of variation of 1.5% and 2.0%, respectively.

Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation.

Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cell-cell communication in many organ systems, including the cardiovascular system. Although the presence of NO synthase (NOS) in the placenta and its role in the regulation of fetoplacental and uteroplacental blood flow are well established, little is known about placental expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during gestation. The objectives of this study were to elucidate the localization of HO-1 and HO-2 isoforms relative to NOS III protein, and to determine the enzymatic activity of HO in the placenta of the guinea-pig during gestation. Placentae were obtained from pregnant guinea-pigs at gestational day (GD) 34, 50, 62 and full term (term, about GD 68). Immunohistochemical localization of HO-1, HO-2 and NOS III protein was conducted using selective polyclonal antibodies. HO activity was determined by using a gas chromatographic method to measure the rate of formation of CO from heme. Faint staining for HO-1 was observed in the adventitial layer of larger fetal blood vessels of the placenta at GD 34. The intensity of this staining was higher at GD 50 and GD 62, and decreased at full term. Similar areas in serial sections of placentae obtained at these selected times during gestation exhibited lower staining intensity when incubated with anti-HO-2 antiserum. Placental HO activity was significantly increased (P<0.05) at GD 62 compared with GD 34, GD 50 and full term. NOS III (endothelial constitutive NOS) staining was highest at GD 34, decreasing thereafter, and was localized mostly to trophoblast lining maternal channels. The data demonstrate that, in the guinea-pig, placental HO and NOS differ in tissue localization during the second half of gestation, with expression of HO protein and its catalytic activity being higher during near-term pregnancy. In a preliminary immunohistochemical investigation of the full-term human placenta, HO-1 protein was localized primarily in the adventitial region of fetal blood vessels of stem chorionic villi. In view of the vasodilator action of CO and NO, the HO and NOS systems may play key roles in the regulation of placental haemodynamics.

Thiol agents separate nitric oxide formation from vasodilation induced by glyceryl trinitrate.

The role of nitric oxide (NO) and thiol-containing compounds in glyceryl trinitrate (GTN)-induced vasodilation was investigated using the thiol-alkylating agent N-ethylmaleimide (NEM). Bovine pulmonary artery (BPA) rings were submaximally contracted with K+ and exposed to increasing concentrations of GTN after a 30-min incubation with 50 microM NEM. NEM decreased maximal relaxation (10 microM GTN) by 20%, compared with controls. Treatment with 5 mM L-cysteine for 30 min before incubation with 50 microM NEM (protection protocol) prevented this decrease in GTN-induced relaxation, but 5 mM D-cysteine did not. Treatment of BPA rings with 5 mM L-cysteine after NEM treatment (reversal protocol) did not reverse the effect of NEM to decrease relaxation inducible by GTN. NO production from 30 microM GTN (chemiluminescence-headspace gas method) in the presence of BPA strips was 46.7 +/- 19.4 pmol NO/g tissue after 10 min of incubation and 76.4 +/- 10.4 pmol NO/g tissue after 20 min. After a 30-min incubation with 50 microM NEM, NO was not detected at either time point. NO production from GTN by BPA strips, with either the protection or reversal protocol, was elevated approximately 2-fold at both time points, compared with controls. No increase in NO production from GTN was observed at either time point for tissues treated with 5 mM D-cysteine using the same protocols. These results are consistent with the concept that thiol compounds play a role in the mechanism of GTN-induced vasodilation, but they indicate that the mechanism of action of GTN and other organic nitrates is more complex than their acting as immediate prodrugs of NO.