Regulation of Nitric Oxide Synthase Activity by Tetrahydrobiopterin in Human Placentae from Normal and Pre-eclamptic Pregnancies

Placenta (2000), 21, 763–772 doi:10.1053/plac.2000.0584, available online at http://www.idealibrary.com on

Regulation of Nitric Oxide Synthase Activity by Tetrahydrobiopterin in Human Placentae from Normal and Pre-eclamptic Pregnancies Z. Kukor, S. Valenta and M. To´thb Department of Medical Chemistry, Molecular Biology and Pathobiochemistry and a 2nd Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary Paper accepted 20 June 2000

The possible regulatory role of tetrahydrobiopterin (BH4) in Type III nitric oxide synthase (NOS III) activity of human placentae from first trimester, term and pre-eclamptic pregnancies was investigated. In homogenates of first-trimester or term placentae, BH4 stimulated NOS III activity up to 2.5-fold in a concentration dependent manner from 20 n to 1
 BH4, and half-maximal stimulation (EC50) was observed at 100–110 n. No significant further stimulation was detectable over an extended concentration range from 1
 to 50
 BH4. NOS III present in microsomal and gel-filtered cytosol fractions exhibited similar BH4-activation patterns, with an identical EC50 value of 50 n. Remarkably, tissue concentrations of BH4 showed a marked decrease in term placentae (5723 n, means.d., n=26) relative to first-trimester placentae (18979 n, means.d., n=17), suggesting that alterations in cellular BH4 concentrations may play a more significant role in the regulation of NOS III activity in late pregnancy. Placental homogenates from 10 pre-eclamptic pregnancies exhibited two distinct types of response to BH4. In seven placental homogenates, addition of physiological concentrations of BH4 (20 n to 1
) elicited no increase whatsoever in basal NOS III activity, and only high BH4 concentrations (50
) caused notable stimulation (BH4 resistant group). In contrast, in three of 10 placental homogenates both physiological and 50
 BH4 concentrations stimulated NOS III to levels similar to that of normal placentae (BH4 responsive group). There were no appreciable differences in the clinical presentation of pre-eclampsia between the two groups. Importantly, BH4 concentrations in pre-eclamptic placentae were comparable with those of normal, control placentae. Taken together, the observations suggest that BH4 controls NOS III activity in the human placenta, and a defect in BH4 regulation of NOS III may contribute to the development of pre-eclampsia. A model implicating the malfunction of placental NOS III rather than its actual tissue level in the pathogenesis of pre-eclampsia is discussed.  2000 Harcourt Publishers Ltd Placenta (2000), 21, 763–772

INTRODUCTION Trophoblast cells of first-trimester human placenta express high levels of nitric oxide synthase (NOS) activity, and the predominant isoform is the constitutive, Ca2+ -dependent, endothelial or type III NOS (Kukor and To´th, 1994; Rutherford et al., 1995; To´th et al., 1995; Ramsay, Sooranna and Johnson, 1996). Placental NOS III activity appears to be regulated by a dual pathway, involving concentration changes of Ca2+ and tetrahydrobiopterin (BH4). In placental homogenates, the activating action of Ca2+ occurs within narrow concentration limits, between 70 and 400 n, with a halfmaximal stimulation at about 150 n both in the presence and absence of BH4. The presence of Ca2+ is indispensable for enzyme activity, whereas addition of BH4 to homogenates containing Ca2+ in 150 n or greater concentrations results in b

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0143–4004/00/080763+10 $35.00/0

a further two- to two and a half-fold activation (Kukor et al., 1996). Homodimerization and the presence of BH4 are absolute requirements of NO production by each NOS isoform (Baek et al., 1993; Tzeng et al., 1995; Venema et al., 1997; Leber et al., 1999; Reif et al., 1999). Isolated cytosolic fractions of placental homogenates contain predominantly dimeric NOS III, and addition of BH4 elicits an approximately twofold increase in activity without significantly influencing dimerization, indicating that BH4 in this fraction exerts its stimulatory effect on the already active, dimeric enzyme (Sahin-To´th, Kukor and To´th, 1997; To´th, Kukor and Sahin-To´th, 1998). These observations have led to the proposal that placental NOS III present in the cytosolic fraction, contains one firmly bound BH4 molecule responsible for maintenance of the dimeric structure and basal enzyme activity, whereas the relatively weaker binding of an additional BH4 to the second subunit results in the observed BH4-dependent stimulation (To´th, Kukor and Sahin-To´th, 1998). It was also proposed that changes in BH4 concentrations may serve as a  2000 Harcourt Publishers Ltd

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physiological regulatory mechanism of NOS activity via maintaining varying degrees of saturation with BH4 of the second subunit of the NOS III homodimer (To´ th, Kukor and Sahin-To´ th, 1998). The finding that pre-incubation of NOS III with low concentrations of sodium dodecyl sulphate (SDS) selectively eliminates the BH4-stimulated activity and leaves the basal activity unchanged (To´ th, Kukor and Sahin-To´ th, 1998), also supports this notion. A similar differential binding affinity of the two BH4 molecules to the enzyme has been recently proposed for the neuronal (Gorren et al., 1996; Riethmu¨ ller et al., 1999), as well as the inducible (Mayer et al., 1997), isoform of NOS. Although the structural basis of such a differential binding of BH4 to NOS III is unclear, crystal structures of the oxygenase domain of the bovine enzyme indicates that the BH4 molecules are positioned at the interface and form connecting bridges between the two subunits (Raman et al., 1998). Furthermore, comparison of the crystal structures of an inactive iNOS monomer and the BH4-bound dimer has led to the conclusion that BH4 binding is critical for dimer formation, and causes major conformational changes in the NOS II isoform (Crane et al., 1998). In contrast to the cytosolic fractions, microsomal preparations from first-trimester human placentae often contain primarily NOS III monomers. Addition of BH4 elicits a concentration-dependent, up to six-fold stimulation of NOS III activity and robust dimerization of the monomers (To´ th, Kukor and Sahin-To´ th, 1997; Sahin-To´ th, Kukor and To´ th, 1997). Dimerization can be demonstrated and quantitated conveniently by low-temperature SDS-polyacrylamide gel electrophoresis and immunoblot analysis, because BH4induced dimers are resistant to cold SDS (Klatt et al., 1995; Venema et al., 1997; Sahin-To´ th, Kukor and To´ th, 1997). The BH4 concentrations required for half-maximal stimulation of activity and dimerization of placental NOS III were comparable, 79 n and 148 n, respectively, suggesting that in microsomes rich in monomers two BH4 molecules are necessary—at least under in vitro experimental conditions—for associating the monomers into homodimers (To´ th, Kukor and SahinTo´ th, 1997). Therefore, the six-fold activity stimulation can be best explained as the sum of two BH4 effects: BH4-promoted dimer formation on the one hand, and saturation of the dimeric enzyme with BH4 on the other. Importantly, activity of microsomal preparations containing mainly dimers respond to BH4 with approx. twofold stimulation, exactly in the same fashion as the cytosolic enzyme. However, stable microsomal dimers dissociate readily when incubated at 37C with 0.02–0.5 per cent SDS in the absence of added BH4 and subjected to freezing and thawing. The monomers obtained in this way can be almost quantitatively re-associated by incubation with 1
 BH4 (To´ th, Kukor and Sahin-To´ th, 1998). Taken together, these observations strongly suggest a role of BH4 in the regulation of placental NOS III activity under physiological and possibly pathological conditions. The aim of the present study was to compare BH4 concentrations required for the activation of NOS III in normal first-trimester and term placentae with the tissue concentration of BH4, and to

determine whether or not BH4 can act as a physiological regulator of NOS III activity in these organs. In addition, tissue concentrations of BH4 and the effect of BH4 on NOS III activity were also studied in homogenates of term placentae obtained from 10 pre-eclamptic pregnancies. The results indicate a regulatory role of BH4 in NOS III activity under normal physiological conditions and an impaired response of NOS III activity to BH4 in seven of 10 pre-eclamptic placentae examined.

MATERIALS AND METHODS Materials -[2,3,4,5-3H]arginine–HCl (67 Ci/mmol; 2.37 TBq/nmol) and -[U-14C]arginine (298 mCi/mmol; 11 GBq/mmol) were purchased from Amersham International (Chalfont, Bucks, UK) and ICN (Costa Mesa, CA, USA), respectively. (6R)5,6,7,8-tetrahydro--biopterin (BH4) was obtained from Research Biochemicals International (Natick, MA, USA). -NG-nitroarginine methylester (NAME), ethyleneglycolbis(-aminoethylether)-N-tetraacetate (EGTA), NADPH, Dowex 50X8-400, dithiothreitol (DTT), citrulline, calmodulin, valine, leupeptin, pterin and -biopterin were from Sigma Chemical Co. (St. Louis, MO, USA). Phenylmethylsulphonylfluoride (PMSF) and HEPES were from Calbiochem (La Jolla, CA, USA), and aprotinin from Bayer Co. (Leverkusen, Germany). Soybean trypsin inhibitor, ethylene-diaminetetraacetate (EDTA) and other chemicals were from Reanal (Budapest, Hungary).

Tissue, homogenization and fractionation First-trimester human placentae from legal instrumental termination of 9–11 week old pregnancies, and term placentae from normal and clinically pre-eclamptic pregnancies were obtained from the second Department of Obstetrics and Gynecology, Semmelweis University, Budapest. Use of the placentae for these experiments has been approved by the Ethics Committee of the clinical department. Macroscopically isolated placental tissue mince was homogenized in 2 vol. of ice-cold homogenizing solution containing 0.3  sucrose, 40 m HEPES–Na, pH 7.4, 0.1 m EDTA, 1 m DTT, 1 m PMSF, 10
g/ml leupeptin, 10
g/ml soybean trypsin inhibitor and 0.2
g/ml aprotinin, using an UltraTurrax apparatus (IKA Werk, Staufen, Germany) at the three-quarter setting for 30 sec. The homogenate produced by the UltraTurrax apparatus was filtered through a nylon mesh and cell debris and tissue fragments were removed by a brief, 1 min centrifugation at 600 g using a Janetzky K-24 refrigerated centrifuge. This low-speed centrifugation step greatly improved reproducibility of individual measurements. In order to separate cytosol and microsomal fractions, heavy particulate material and mitochondria were sedimented first at 15 000 g for 30 min in a Beckman J-21 centrifuge. The supernatant was then

Kukor et al.: Tetrahydrobiopterin Regulation of Placental Nitric Oxide Synthase

centrifuged at 100 000 g for 60 min in a Beckman L2-65B ultracentrifuge to obtain the microsomal pellet and the cytosol fraction. The pellet was suspended in homogenizing solution whereas the cytosol was gel-filtered at 4C through commercial Sephadex G-25 M prepacked columns (PD-10, Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with homogenizing solution and used for incubations immediately.

Measurement of NOS activity Determination of NOS activity was performed by measuring the rate of conversion of radiolabelled arginine into radiolabelled citrulline as previously described (Sahin-To´ th, Kukor and To´ th, 1997; To´ th, Kukor and Sahin-To´ th, 1997). Briefly, 100
l tissue extract containing 1.75–4.74 mg protein was incubated with 0.4 m NADPH, 1 m citrulline, 1 m MgCl2, 16 m -valine, 25
 free Ca2+ , 3 IU calmodulin, 2
 BH4, 0.75
Ci [3H]arginine (47 n final concentration) or 0.15
Ci [14C]arginine (2
 final concentration), 20 m HEPES–Na, pH 7.4, in 250
l final volume for 10 min at 37C. Free Ca2+ concentrations of the incubates were adjusted with Ca2+ –EGTA buffers (Ba´ rtfai, 1979). Control incubates contained 1 m EGTA without Ca2+ added. In a separate control incubation, 1 m EGTA and 1 m -NAME were added to measure Ca2+ -independent citrulline formation, which proved to be insignificant in these experiments. Radiolabelled citrulline was separated from radiolabelled arginine using small columns of Dowex 50X8-400 cation exchange resin. When preparations from first-trimester pregnancies were studied, experiments were carried out three to four times and incubations in each experiment were run in triplicates. Radioactivities measured in d/min using a Packard Tri-Carb 2100 TR Liquid Scintillation Analyser were normalized for the disintegrations per minute value obtained as the mean of controls. NOS activity was finally calculated either as picomoles of citrulline per minute per milligram of protein (on the basis of an average arginine content of 542 nmol/g tissue; To´ th, Kukor and Sahin-To´ th, 1997) or as disintegrations per minute per milligram of protein.

Determination of NO synthase activity of pre-eclamptic and normal placentae NOS activity has been measured from homogenates prepared as described above. Removal of low molecular mass compounds by gel filtration was not applied due to considerable losses of activity during this procedure. Most of the placental NOS III activity is membrane-bound (To´ th et al., 1995) and membrane fragments and vesicles are retained by the gel. At the final seven and a half-fold dilution of tissue in the incubation mixture, endogenous BH4 concentrations of 6–8 n could be calculated from our data presented in this paper, which was negligible compared with the half-saturating 100 n BH4 concentration. Endogenous arginine concentrations of

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normal term placentae (280
) and a pre-eclamptic term placenta (560
) have been reported by Pearse and Sornson (1969). Km values of placental NOS III for arginine are in the range of 1–3
 as measured either in the presence or absence of 50
 BH4 (Conrad and Davies, 1995; To´ th, Kukor and Sahin-To´ th, 1997). Furthermore, no difference whatsoever was found between the Km values of placental NOS III for arginine in normal and pre-eclamptic term placentae (Conrad and Davies, 1995). From these data it could be calculated that in our incubation mixtures, either with normal or preeclamptic placentae, an excessive arginine concentration (37– 75
) was present which is amply sufficient to saturate the enzyme. In order to overcome the differential dilution of label (2
 final concentration of 14C-arginine) by endogenous arginine we regarded the basal activity (measured in the absence of exogenous BH4) of each placentae as 100 per cent and the stimulatory effect of BH4 was related to this value.

Definition of pre-eclampsia Pre-eclampsia was defined as a rise in blood pressure of >15 mm Hg diastolic or >30 mm Hg systolic from measurement in early pregnancy. If no early readings were available, >140/90 mm Hg at late pregnancy served as an indication of the disease. In addition, proteinuria >300 mg per 24 h was a requirement for the diagnosis (Roberts and Redman, 1993; Seligman et al., 1994; Cunningham et al., 1997).

Determination of tissue levels of tetrahydrobiopterin The procedure of Fukushima and Nixon (1980) was employed with the modification that BH4 was oxidized in alkaline medium and the resulting pterin was then determined after separation on an ODS-Anal 2504.6 mm analytical column and a JASCO PU-980 HPLC apparatus attached to a Hitachi F-4500 fluorescence spectrophotometer. We found that placental homogenates contain no measurable quantities of biopterin and dihydrobiopterin, and BH4 can be recovered quantitatively after alkaline iodine oxidation in the form of pterin. Quinonoid BH2 also appears as pterin in this procedure, but this pteridin is the immediate precursor of BH4, it is reduced rapidly to BH4 by pteridine reductase in the cell and its concentration is usually a small fraction of that of BH4. Therefore we regarded it as a BH4 equivalent. Briefly, 3 g placenta was homogenized with 9 ml of 0.1  ice-cold phosphoric acid, and 1.5 ml 2  NaOH was admixed, under continuous stirring with a glass rod. To this solution, 1 ml alkaline iodine solution (9 vol. of 1 per cent I2 +2 per cent KI and 1 vol. of 1  NaOH) was added and vortex-mixed. The mixture was incubated for 60 min at room temperature in the dark, with regular stirring at 10 min intervals. The solution was then acidified by mixing with 1.8 ml 2  TCA and centrifuged for 10 min at low speed to remove precipitate. The

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supernatant fluid was loaded on to a 800
l Dowex 50X8-400 minicolumn, in H + cycle. The column was washed with 5 ml water and pterines were eluted with 2.8 ml 1  NH3. The eluate was neutralized with 200
l concentrated acetic acid, and aliquots were analysed with HPLC, using 5 per cent methanol as eluent. A calibration line was prepared using increasing concentrations (0.2–2.0
) of pterin. To determine recovery, BH4 standard solutions were also analysed. BH4 concentrations determined were expressed as picomoles per gram of tissue, or more conveniently as nanomolar (n) concentrations.

Protein determination Protein was measured by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard.

Calculation of data Student’s t-test and Bonferroni’s analysis of variance (ANOVA) test were performed with the GraphPAD InStat, version 1.12 computer program. A difference was regarded statistically significant at P<0.05.

RESULTS First-trimester placentae Addition of BH4 to homogenates from first-trimester placentae stimulated NOS III activity up to approximately 2.5-fold in a concentration dependent manner from 20 n to 1
 [Figure 1(A)]. When an extended concentration range from 1
 to 50
 BH4 was tested, no significant additional stimulation was detected [Figure 1(B)], indicating that NOS III is fully saturated with BH4 around 1–2
. A double-reciprocal plot of data shown in Figure 1(A) revealed that half-maximal stimulation (EC50) was achieved at 110 n BH4 [Figure 1(C)]. Tissue concentrations of BH4 in early human placentae are 18979 n (Table 1; see also Kukor et al., 1996), and under the experimental conditions in Figure 1 the incubation mixtures contained an average of 25 n endogenous BH4. Therefore, the 110 n EC50 value is somewhat overestimated. In an effort to test the contribution of the endogenous BH4 to the observed activation pattern, BH4-depleted cytosolic and microsomal fractions were also prepared and tested. Microsomal vesicles were separated from cytosol by centrifugation, and cytosol was passed through a gel-filtration column. Under these conditions the soluble and loosely bound BH4 is removed from both preparations, and the strongly enzyme-bound BH4 is responsible for the remaining basal NOS III activity. Addition of exogenous BH4 to microsomes or cytosol resulted in about threefold and twofold activation, respectively, and Figure 2 demonstrates that BH4 activation characteristics of

Placenta (2000), Vol. 21

both microsomal and cytosolic fractions were highly similar, with EC50 values equally around 50 n. Importantly, placental concentrations of BH4 vary within the range of 58–330 n (Table 1), indicating that individual variations in endogenous BH4 levels may profoundly influence NOS III activity.

Term placentae from normal and pre-eclamptic pregnancies Activation kinetics by BH4 of NOS III in homogenates from normal term placentae were highly similar to those observed with first-trimester placentae (see Figure 3). Interestingly, however, significantly lower concentrations of BH4 were found in the villous tissue of term placentae (5723 n), relative to first-trimester placentae (Table 1). The observations indicate that NOS III is only partially saturated with BH4 under physiological conditions, and suggest a possible BH4dependent regulatory mechanism. Individual variations of BH4 concentrations (range: 15–115 n, Table 1) also lend support to such a function of BH4 in NOS III activity of term placentae. In further experiments the effect of BH4 on NOS III activity of homogenates prepared from the villous tissue of normal term placentae and from term placentae of pre-eclamptic pregnancies was compared. As shown in Figure 3, NOS III of normal term placentae (n=4) was activated by BH4 in a concentration-dependent manner over a concentration range from 20 n to 1
. The BH4 concentration that caused half-maximal activation was around 100 n and the extent of maximal activation was about twofold. The BH4 effect on normal placenta homogenates was tested in nine additional experiments. In all of the 13 normal placental homogenates studied NOS III activity responded to submicromolar concentrations (20 n–1
) of BH4 and in each case the activation exhibited similar dependence on various BH4 concentrations. The mean stimulation with 13 normal placentae and at 1
 BH4 concentration was 2.160.08-fold (mean value, n=13). Figure 3 summarizes only those experiments in which BH4 effect and BH4 concentration was determined using the same placenta. Homogenates of placentae from 10 patients having pre-eclampsia showed two distinct types of response to BH4. In seven placental homogenates, basal NOS III activity remained essentially unchanged upon addition of exogenous BH4 and no increase whatsoever was detectable over the entire BH4 concentration range tested (‘BH4-resistant’ group). In contrast, in three of 10 placental homogenates BH4 proved to be stimulatory (‘BH4-responsive’ group). The half-maximal activating concentration of BH4 was again around 100 n, but the extent of maximal stimulation over the baseline activity exceeded the stimulation observed with the normal placental enzyme. However, the difference was not significant. Clinically, the ‘BH4-resistant’ and ‘BH4-responsive’ groups showed no appreciable differences (Table 2), and both groups exhibited the same characteristic symptoms of pre-eclampsia: elevated systolic and diastolic blood pressure, proteinuria,

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767

Table 1. BH4 concentrations in the villous tissue of first trimester and normal term human placentae (A)

Tissue concentration Median 25–75 percentile Range

40

0.5

0

1.0

H-citrulline, dpm/mg protein/min × 10

BH4, µM

18979 (n=17) 175 168–225 58–330

5723 (n=26) 52 40–65 15–115

60

40

10

6

2

3

pmol citrulline/min/mg protein

(B)

20

0

0

0.1

0.3

0.5

BH4, µM

0

0

10

20

30

40

50

BH4, µM (C)

1/pmol citrulline × min × mg protein

Term

Values determined by the HPLC method of Fukushima and Nixon (1980) are given as pmol/g tissue, corresponding to approximately nanomolar (n BH4 concentrations. Mean valuess.d. are presented. BH4 concentrations in term placentae are significantly different from those of early pregnant placentae (P<0.001). An average tissue BH4 concentration of 20787 n from nine first trimester placentae was previously reported in Kukor et al., 1996.

20

0

First trimester

–3

pmol citrulline/min/mg protein

60

0.2

increased resistance of umbilical arteries and in some cases serious fetal retardation. Although there was no significant difference between the normal and the pre-eclamptic group with respect to the gestational age, the mean age of normal pregnancies (39 weeks) exceeded that of the pre-eclamptic group (34.4 weeks for the 10 pre-eclamptic patients).

0.1

0 –10

Figure 2. Effect of BH4 on NOS III activity of placental microsomes (— —) and gel-filtered cytosol (——) Enzyme activity was measured by the [3H]arginine<[3H]citrulline conversion assay, at 37C for 10 min using the standard assay mixture as described in Materials and Methods. Data from four experiments with triplicate incubations each are presented. Mean values (n=4) are given,  values are shown by error bars. The EC50 values are approximately 50 n with both type of preparations.

0

10

30

50

1/BH4, 1/µM

70

Figure 1. Dose-dependent activation of NOS III by BH4 in homogenates of primordial human placentae. Conversion of [3H]arginine into [3H]citrulline was measured at 37C for 10 min using the standard reaction mixture as described in Materials and Methods. Data from three experiments with triplicate incubations each are presented. (A) and (B) Activating effect of low concentrations (0.02–1
) and high concentrations (1–50
) of BH4, respectively. Mean values (n=3) are presented,  values are shown by error bars. (C) Double reciprocal plot of data shown in (A). The apparent Km and Vmax values are 110 n and 32 pmol citrulline/min/mg protein, respectively.

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pre-eclamptic placentae, although a decrease in arginine concentration with a consequent increase in specific radioactivity in pre-eclamptic incubation mixtures cannot be ruled out. In any event, it is important to note that kinetics of BH4 activation are not influenced by the absolute value of basal NOS III activity in the tissue examined. Furthermore, these data draw the attention to the possibility that malfunction of placental NOS III (that is the BH4 resistance of the enzyme) could be more important in the aetiology and pathogenesis of pre-eclampsia than the actual level of NOS III activity.

Percent of control (= 100 per cent)

300

200

100

0

0.2

0.4

0.6

0.8

1

BH4, µM Figure 3. Effect of BH4 on NOS III activity of term placental homogenates from normal and pre-eclamptic pregnancies. Homogenates were prepared from villous tissue of term placentae obtained from pregnancies characterized clinically in Table 2. Enzyme activity was measured by the [14C]arginine< [14C]citrulline conversion assay, at 37C for 10 min using the standard assay mixture as described in Materials and Methods. Homogenates were incubated in triplicates and data in each experiment were divided by the mean value of incubations performed in the absence of exogenous BH4 (=100 per cent). Mean values are presented,  values are shown by error bars. Values obtained from four ‘normal’ (— —), three pre-eclamptic, ‘BH4-responsive’ (—— ) and seven pre-eclamptic, ‘BH4-resistant’ (——) pregnancies are presented. EC50 values are equally around 100 n with the two BH4responsive homogenates. According to Bonferroni’s ANOVA test normal placenta values at and above 100 n BH4, BH4-responsive values at and above 50 n BH4 are significantly different from the respective normal values. There is no significant difference between the two pre-eclamptic groups.

It was of interest to examine whether or not NOS III in the ‘BH4-resistant’ placentae could be stimulated by high, pharmacological BH4 concentrations. As demonstrated in Figure 4, an approximately 1.7-fold activation of NOS III was elicited by 50
 BH4 in the ‘BH4-resistant’ group, while the physiological BH4 concentration (0.2
) gives no stimulation whatsoever. In sharp contrast, NOS III from normal and ‘BH4responsive’ placentae were activated by 0.2
 BH4 1.8-fold and 2.3-fold, respectively, and 50
 BH4 caused 2.3-fold and threefold stimulation, respectively, relative to basal (control) activity. Mean BH4 concentrations of the 10 pre-eclamptic placentae examined (41.8 n and 51.6 n for the ‘BH4-responsive’ and the ‘BH4-resistant’ groups, respectively, Table 3) were not significantly different from the value observed in the four control placentae (57.6 n) or in a large number of normal placentae (57 n, see Table 1). In separate experiments, determination of BH4 concentrations in the villous tissue of 17 other pre-eclamptic placentae revealed an average concentration of 57.628 n (means.d.), and a range between 29.5–149.8 n, which is also comparable with values found with normal and pre-eclamptic placentae in Table 3. Interestingly, basal NOS III activity of the normal (control) group was significantly (P<0.05) smaller relative to the preeclamptic group, whereas no statistical difference was found between the two groups of the pre-eclamptic placentae. The difference is probably due to elevations in NOS III content in

DISCUSSION The present study investigates the possible regulatory role of BH4 in NOS III activity in human placentae from firsttrimester, normal term, and pre-eclamptic pregnancies. The observations indicate that NOS III in homogenates from the villous tissue of both first-trimester and term placentae are activated by BH4 in a very similar manner. Thus, the extent of activation is 2–2.5-fold, and the half-maximal activating concentration (EC50) is 110 and 100 n BH4, respectively. A more-detailed characterization of NOS III in isolated microsomal and gel-filtrated cytosolic fractions of first-trimester placentae revealed an EC50 of 50 n BH4, suggesting that the EC50 value obtained in homogenates is slightly overestimated. Average concentrations and individual variations (see Table 1) of tissue BH4 levels determined in a large number of firsttrimester and term placentae support the notion that BH4 can act as a regulator of NOS III activity under physiological conditions. An additional remarkable finding is that BH4 levels significantly decrease in term placentae relative to firsttrimester placentae (Table 1), indicating that BH4-dependent regulation may be more important in late pregnancy. To gain insight into the possible pathophysiological role of BH4 in pre-eclampsia, BH4-dependent activation of NOS III in 10 placentae from pre-eclamptic pregnancies was also examined. Pre-eclampsia occurs in 6–8 per cent of all human gestations, and is one of the leading causes of several serious abnormalities of human pregnancies and parturitions, such as fetal growth retardation, premature birth, newborn morbidity and mortality, and maternal death. Despite continuous research efforts, the pathogenesis of this disease is still obscure and adequate treatment is lacking (Buhimschi et al., 1998; Dekker and Sibai, 1998). A number of studies have tried to correlate the leading symptoms—hypertension, proteinuria and an increased resistance of maternal and umbilical arteries—with impaired production of NO by placental homogenates and/or decreased nitrite–nitrate concentration of the blood serum of pre-eclamptic patients. The results are contradictory: increased NOS III activities relative to normotensive pregnancies (Rutherford et al., 1995; Di Iorio et al., 1998; Nasiell et al., 1998), decreased activities (Morris et al., 1995; Brennecke et al., 1997) or unchanged activity (Conrad and Davies, 1995; Boccardo et al., 1996; Poranen et al., 1998; Beinder et al., 1999) have been reported. Similarly, serum

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Table 2. Clinical evaluation of normal (control) and pre-eclamptic pregnancies studied

Maternal age (years) Range Duration of pregnancy (weeks) Range Birthweight (grams) Range Blood pressure (mmHg) Systolic Range Diastolic Range Urinary protein (grams/day) Range Pulsatility index Umbilical artery Range

Control (n=4)

BH4 responsive (n=3)

BH4 resistant (n=7)

24.23.0 22–27 39.01.0 38–40 3110400 2850–3700

28.03.5 24–30 32.06.2 27–39 16101465 700–3300

23.95.8 17–35 35.43.5 30–40 22251051 1180–4450

1156 110–120 735 70–80 0

15712 150–170 10712 100–120 2.452.3 1.0–5.1

15820 140–190 10111 90–120 4.373.7 0.4–9.1

0.910.13 0.7–1.1

2.03* 2.03

1.680.82† 1.08–2.85

Percent of control (=100 per cent)

Pre-eclamptic pregnancies were classified according to the BH4-sensitivity of placentae into BH4-responsive and BH4-resistant groups (see text for details). Mean valuess.d. are presented, *only one index has been measured, †four indices have been measured. Blood pressure values and daily protein discharge differ significantly (P<0.05) between the normal and the pre-eclamptic group. There is no significant difference between maternal ages, duration of pregnancies and birthweights.

400

a b b c B D

c

c d d B D

e e

f

A C

Table 3. Basal NOS III activities and BH4 concentrations of term placentae from control, BH4-responsive pre-eclamptic and BH4resistant pre-eclamptic pregnancies

300

NOS activity* Range BH4 concentration† Range

200

Control (n=4)

BH4 responsive (n=3)

BH4 resistant (n=7)

27678 135–492 57.69 47.7–67.3

918509 134–1867 41.85.7 30.9–49.9

692227 206–1143 51.64.6 29.5–91.2

100

0

Normal

BH4responsive

BH4resistant

Figure 4. Effects of 0.2 and 50
 concentrations of BH4 on NOS III activity of term placental homogenates from normal and pre-eclamptic pregnancies. Homogenates were prepared and NOS activity was measured as described in Figure 3. Enzyme assays were carried out in the absence of BH4 (open bars) and in the presence of 0.2
 (grey bars) and 50
 (black bars) BH4. Homogenates were incubated in triplicates, and data in each experiment were calculated relative to the mean value of incubations carried out in the absence of exogenous BH4 (=100 per cent). Mean values are presented,  values are shown by error bars. Values obtained from four normal, three pre-eclamptic, ‘BH4-responsive’ and seven pre-eclamptic, ‘BH4-resistant’ pregnancies are presented. Boferroni’s ANOVA test gives the following results (different letters indicate significant differences): (A) versus (B): P<0.05 (versus normal), P<0.01 (versus BH4-responsive) (C) versus (D): P<0.01; (a) versus (b): P<0.01; (c) versus (d): P<0.05; (e) versus (f): P<0.001.

nitrite–nitrate levels in pre-eclamptic pregnancies have been found to increase (Ranta et al., 1999; Bartha et al., 1999; Norris et al., 1999), decrease (Seligman et al., 1994; Mutlu-Turkoglu

Combined mean value of NOS activities and BH4 concentrations in the pre-eclamptic group (n=10) were 760140 dpm/min/mg protein and 48.74.3 pmol/g tissue, respectively. Bonferroni’s ANOVA test did not show significant differences between the groups. The control and the combined pre-eclamptic group showed significant difference in NOS activity (P<0.05). *dpm in 14C-citrulline/mg protein/min; mean value. †pmol/g tissue; mean values.d.

et al., 1999) or have shown no change (Silver et al., 1996; Hata et al., 1999; Di Iorio et al., 1998). In the present study, a defective response of NOS III of placental villous tissue to the stimulatory effect of BH4 was found in a surprisingly high number, seven out of 10, clinically typical pre-eclamptic pregnancies. Thus, in the seven placentae of the ‘BH4resistant’ group no stimulation was detectable with physiological concentrations of BH4, and only a high, pharmacological BH4 concentration (50
) was stimulatory. In sharp contrast, NOS III in three of 10 pre-eclamptic placentae (the ‘BH4responsive’ group) was readily activated by both concentrations of BH4 to levels that exceeded the stimulation observed in

770

normal term placentae. Importantly, tissue levels of BH4 in the ‘BH4-resistant’ and ‘BH4-responsive’ pre-eclamptic placentae were essentially identical to those of normal placentae. These data clearly indicate that changes of BH4 concentrations in the villous tissue cannot be a major pathogenic factor in preeclampsia, and provide evidence that impaired responsiveness of NOS III to BH4, rather than a change in BH4 concentration is involved in the pathomechanism. Although the number of cases studied here is relatively small, the high incidence of BH4-resistant placental NOS III deserves attention with respect to the pathogenesis of preeclampsia. On the basis of these and previous observations a speculative model emerges that attempts to explain the mechanism of the fetal and maternal endothelial and vascular dysfunctions, which are in the focus of this disorder. The lack of stimulation by BH4 of NOS III activity over the baseline level at low BH4 concentrations, and a detectable but decreased stimulation at a high, pharmacological concentration suggest that BH4 binding to NOS III is impaired, presumably due to a structurally altered binding site. Because the basal activity is maintained and activity of the enzyme requires a homodimeric structure, it is reasonable to propose that one of the subunits can bind BH4 firmly, whereas the BH4 binding site of the second subunit exhibits dysfunction. A similar phenomenon was demonstrated recently in vitro (To´ th, Kukor and Sahin-To´ th, 1998), where BH4-induced NOS III stimulation was selectively abolished by pre-incubation with low concentrations of SDS, while basal NOS III activity was unaffected. However, BH4 still promoted dimerization of NOS III monomers pre-incubated with SDS. A likely consequence of the markedly reduced BH4 binding is the increased production of superoxide anion radical by the BH4-free subunit. It is well documented that in the absence of BH4, NOS III catalyses Ca2+ /calmodulin dependent O2
formation, which can be suppressed in a concentration dependent fashion by the addition of BH4 (Xia et al., 1998; Cosentino and Lu¨ scher, 1998; Va´ squez-Vivar et al., 1998; Stroes et al., 1998). Remarkably, the concentration dependence of this suppression shows a pattern highly similar to the concentration dependent activation of placental NOS III by BH4 (see Figures 1 and 3). It has been suggested that an optimal BH4 concentration is critical for the proper cellular synthesis of NO and citrulline, and insufficient BH4 availability may switch NOS III from NO to O2
generation. Note that according to this suggestion, either decreased tissue concen-

Placenta (2000), Vol. 21

trations of BH4 or diminished affinity of NOS III for BH4 can lead to an increased rate of O2
formation at the expense of NO. A continuously uncoupled NOS III activity could be critical in the pathogenesis of pre-eclampsia for at least two reasons: (1) it can contribute to the formation of H2O2 via superoxide dismutase (SOD) activity. Subsequent heterolysis of H2O2 in the presence of O2
and Fe2+ ions may result in the formation of hydroxyl radical, a very aggressive oxidative agent. This process can lead to, and maintain, a steady state of oxidative stress (Davidge, 1998), which is thought to be characteristic of pre-eclamptic placentae. Superoxide itself was found to behave as a vasoconstricting agent capable of increasing vascular resistance (Cosentino, Sill and Katusic, 1994). Furthermore, it may lead to elevated lipid peroxide levels both in the placenta and in the maternal circulation with a consequent suppression of NO synthesis in the maternal vasculature (Hubel et al., 1989; Walsh, 1994; Mutlu-Turkoglu, 1999). (2) NO produced by the basal activity can react with O2
to furnish peroxynitrite. This reaction is three-times as rapid as the elimination of O2
by SOD (Cosentino and Lu¨ scher, 1998) and diverts NO away from its natural target, guanylate cyclase. This reaction may contribute to the NO deficiency believed to cause placental ischaemia, a putative pivotal factor in the development of pre-eclampsia. Recently, Leber et al. (1999) pointed out that recombinant human NOS III exhibits high NADPH oxidase activity in the absence of BH4, which is attended by significant O2
formation. As a corollary of these results, one may expect that in the absence of BH4 binding, the placental enzyme can produce abundant quantities of superoxide and peroxynitrite. One of the actions ascribed to peroxynitrite is the nitration of tyrosine residues of proteins (Tien et al., 1999). Increased peroxynitrite production in pre-eclamptic placentae was shown to result in increased nitrotyrosine immunostaining mainly in the villous vascular endothelium (Myatt et al., 1996) and in the maternal blood vessels (Roggensack, Zhang and Davidge, 1999). We may speculate that nitrated proteins and/or peptides raised in the pre-eclamptic placenta reach the maternal circulation and contribute to the pathogenesis of pre-eclampsia syndrome. In summary, the present findings suggest that at least in some of the pre-eclamptic pregnancies the malfunction of placental NOS III may have a role in the pathomechanism of this disease. Further efforts are needed to clarify a possible relationship between the anomalous behavior of placental NOS III and the pathogenesis of pre-eclampsia.

ACKNOWLEDGEMENTS The help and interest of Miklo´ s Sahin-To´ th and the technical assistance of Eszter Be´ rczi is greatly appreciated. This work was supported by Hungarian Research Fund (OTKA) grant T-29165, Health Research Council (ETT) grant 956/96, and Semmelweis University Research Support Grant 173/1999.

REFERENCES Baek KJ, Thiel BA, Lucas S & Stuehr DJ (1993) Macrophage nitric oxide synthase subunits. Purification, characterization and role of prostethic groups and substrate in regulating their association into a dimeric enzyme. J Biol Chem, 268, 21120–21129.

Ba´rtfai T (1979) Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes. In Advances in Cyclic Nucleotide Research, Vol. 10 (Eds) Brooker G, Greengard P & Robinson GA, pp. 219–242. New York: Raven Press. Bartha JL, Comino-Delgado R, Bedoya FJ, Barahona M, Lubian D & Garcia-Benasach F (1999) Maternal serum nitric oxide levels associated

Kukor et al.: Tetrahydrobiopterin Regulation of Placental Nitric Oxide Synthase with biochemical and clinical parameters in hypertension in pregnancy. Eur J Obstet Gynecol Reprod Biol, 82, 201–207. Beinder E, Mohaupt MG, Schlembach D, Fischer T, Sterzel RB, Lang N & Baylis C (1999) Nitric oxide synthase activity and Doppler parameters in the fetoplacental and uteroplacental circulation in preeclampsia. Hypertension Preg, 18, 115–127. Boccardo P, Soregaroli M, Aiello S, Noris M, Donadelli R, Lojacono A & Benigni A (1996) Systemic and fetal-maternal nitric oxide synthesis in normal pregnancy and preeclampsia. Br J Obstetr Gynaecol, 103, 879–886. Brennecke SP, Gude NM, Di Iulio JL & King RG (1997) Reduction of placental nitric oxide synthase activity in preeclampsia. Clin Sci (Colch), 93, 51–55. Buhimschi IA, Saade GR, Chwalisz K & Garfield RE (1998) The nitric oxide pathway in preeclampsia: pathophysiological implications. Hum Reprod Update, 4, 25–42. Conrad KP & Davies AK (1995) Nitric oxide synthase activity in placentae from women with preeclampsia. Placenta, 16, 691–699. Cosentino F & Lu¨scher TF (1998) Tetrahydrobiopterin and endothelial function. Eur Heart J, 19(Suppl. G), G3–G8. Cosentino F, Sill JC & Katusic ZS (1994) Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension, 23, 229–235. Cunningham FG, MacDonald PC, Gant NF, Leveno KJ, Gilstrap LC, Hankins GDV & Clark SL (1997) Williams Obstetrics. Stamford: Appleton and Lange, 694 pp. Crane BR, A u rvai AS, Ghosh DK, Wu CQ, Getzoff ED, Stuehr DJ & Tainer JA (1998) Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science, 279, 2121–2126. Davidge ST (1998) Oxidative stress and altered endothelial cell function in preeclampsia. Sem Reprod Endocrinol, 16, 65–73. Dekker GA & Sibai BM (1998) Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstetr Gynecol, 179, 1359–1375. Di Iorio R, Marinoni E, Emiliani S, Villaccio B & Cosmi EV (1998) Nitric oxide in preeclampsia: lack of evidence for decreased production. Eur J Obstet Gynecol Reprod Biol, 76, 65–70. Fukushima T & Nixon JC (1980) Analysis of reduced forms of biopterin in biological tissues and fluids. Analytical Biochem, 102, 176–188. Gorren ACF, List BM, Schrammel A, Pitters E, Hemmens B, Werner ER, Schmidt K & Mayer B (1996) Tetrahydrobiopterin-free neuronal nitric oxide synthase: evidence for two identical highly anticooperative pteridine binding sites. Biochemistry, 35, 16735–16745. Hata T, Hashimoto M, Kanenishi K, Akiyama M, Yanagihara T & Masumara S (1999) Maternal circulating nitrite levels are decreased in both normal normotensive pregnancies and pregnancies with preeclampsia. Gynecol Obstetr Invest, 48, 93–97. Hubel CA, Roberts JM, Taylor RN, Musci TJ, Rogers GM & McLaughlin MK (1989) Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstetr Gynecol, 161, 1025–1034. Klatt P, Schmidt K, Lehner D, Glatter O, Bachinger HP & Mayer B (1995) Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDSresistant dimer. EMBO J, 14, 3687–3695. Kukor Z & To´th M (1994) Ca2+ -dependent and Ca2+ -independent NO-synthesizing activities of human primordial placenta. Acta Physiol Hungarica, 82, 313–319. Kukor Z, Me´sza´ros G, Hertelendy F & To´th M (1996) Calcium-dependent nitric oxide synthase is potently stimulated by tetrahydrobiopterin in human primordial placenta. Placenta, 17, 69–73. Leber A, Hemmens B, Klo¨sch B, Goessler W, Raber G, Mayer B & Schmidt K (1999) Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris. J Biol Chem, 274, 37658–37664. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem, 143, 265–275. Mayer B, Wu C, Gorren ACF, Pfeiffer S, Schmidt K, Clark P, Stuehr DJ & Werner ER (1997) Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin. Biochemistry, 36, 8422–8427. Morris NH, Sooranna SR, Learmont JG, Poston L, Ramsay B, Pearson JD & Steer PJ (1995) Nitric oxide synthase activities in placental tissue from normotensive, preeclamptic and growth retarded pregnancies. Br J Obstetr Gynaecol, 102, 711–714.

771 Mutlu-Turkoglu U, Aykac-Toker G, Ibrahimoglu L, Ademoglu E & Uysal M (1999) Plasma nitric oxide metabolites and lipid peroxide levels in preeclamptic pregnant women before and after delivery. Gynecol Obstetr Invest, 48, 247–250. Myatt L, Rosenfield RB, Eis EL, Brockman DE, Greer I & Lyall F (1996) Nitrotyrosine residues in placenta. Evidence of peroxynitrite formation and action. Hypertension, 28, 488–493. Nasiell J, Nisell H, Blanck A, Lunell NO & Faxen M (1998) Placental expression of endothelial constitutive nitric oxide synthase mRNA in pregnancy complicated by preeclampsia. Acta Obstetr Gynecol Scand, 77, 492–496. Norris LA, Higgins JR, Darling MR, Walshe JJ & Bonnar J (1999) Nitric oxide in the uteroplacental, fetoplacental, and peripheral circulations in preeclampsia. Obstetr Gynecol, 93, 958–963. Pearse WH & Sornson H (1969) Free amino acids of normal and abnormal human placenta. Am J Obstetr Gynecol, 105, 696–701. Poranen AK, Aubry J, Kujari H & Ekblad U (1998) Expression of nitric oxide synthase in normal and preeclamptic placental tissue and effects of glyceryl trinitrate and shear stress on placental blood flow. Acta Obstetr Gynecol Scand, 77, 594–597. Raman CS, Li H, Marta´sek P, Kra´l V, Masters BSS & Poulos TL (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell, 95, 939–950. Ramsay B, Sooranna SR & Johnson MR (1996) Nitric oxide synthase activities in human myometrium and villous trophoblast throughout pregnancy. Obstetr Gynecol, 87, 249–253. Ranta V, Viinikka L, Halmesmaki E & Ylikorkala O (1999) Nitric oxide production with preeclampsia. Obstetr Gynecol, 93, 442–445. Reif A, Fro¨hlich LG, Kotsonis P, Frey A, Bo¨mmel HM, Wink DA, Pfleiderer W & Schmidt HHHW (1999) Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase. J Biol Chem, 274, 24921–24929. Riethmu¨ller C, Gorren ACF, Pitters E, Hemmens B, Habisch HJ, Heales SJR, Schmidt K, Werner ER & Mayer B (1999) Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin. Functional evidence against reductive oxygen activation by the pterin cofactor. J Biol Chem, 274, 16074–16051. Roberts JM & Redman CWG (1993) Pre-eclampsia: more than pregnancy induced hypertension. Lancet, 341, 1447–1454. Roggensack AM, Zhang Y & Davidge ST (1999) Evidence for peroxynitrite formation in the vasculature of women with preeclampsia. Hypertension, 33, 83–89. Rutherford RAD, McCarthy A, Sullivan MHF, Elder MG, Polak JM & Wharton J (1995) Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies. Br J Pharmacol, 116, 3099–3109. Sahin-To´th M, Kukor Z & To´th M (1997) Tetrahydrobiopterin preferentially stimulates activity and promotes subunit aggregation of membranebound calcium-dependent nitric oxide synthase in human placenta. Mol Hum Reprod, 3, 293–298. Seligman SP, Buyon JP, Clancy RM, Young BK & Abramson SB (1994) The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstetr Gynecol, 171, 944–948. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA & Caplan MS (1996) Evaluation of nitric oxide as a mediator of severe preeclampsia. Am J Obstetr Gynecol, 175, 1013–1017. Stroes E, Hijmering M, van Zandvoort R, Wever TJ, Rabelink TJ & van Faassen EE (1998) Origin of superoxide production by endothelial nitric oxide synthase. FEBS Letters, 438, 161–164. Tien M, Berlett BS, Levine RL, Chock PB & Stadtman ER (1999) Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration and methionine oxidation. Proc Nat Acad Sci USA, 96, 7809–7814. To´th M, Kukor Z, Romero R & Hertelendy F (1995) Nitric oxide synthase in first trimester human placenta: characterization and subcellular distribution. Hypertension Preg, 14, 287–300. To´th M, Kukor Z & Sahin-To´th M (1997) Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta. Placenta, 18, 189–196. To´th M, Kukor Z & Sahin-To´th M (1998) Differential response of basal and tetrahydrobiopterin-stimulated activities of placental type III nitric oxide synthase to sodium dodecyl sulphate: relation to dimeric structure. Mol Hum Endocrinol, 4, 1165–1172.

772 Tzeng E, Billiar TR, Robbins PD, Loftus M & Stuehr DJ (1995) Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer. Proc Natl Acad Sci USA, 92, 11771–11775. Va´ squez-Vivar J, Kalyanaraman B, Marta´ sek P, Hogg N, Masters BSS, Karou V, Tordo P & Pritchard KA (1998) Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci USA, 95, 9220–9225.

Placenta (2000), Vol. 21 Venema RC, Ju H, Zou R, Ryan JW & Venema VJ (1997) Subunit interactions of endothelial nitric oxide synthase. Comparisons to the neuronal and inducible nitric oxide synthase isoforms. J Biol Chem, 272, 1276–1282. Walsh SW (1994) Lipid peroxidation in pregnancy. Hypertension Preg, 13, 1–32. Xia Y, Tsai A, Berka V & Zweier JL (1998) Superoxide generation from endothelial nitric-oxide synthase. A Ca2+ /calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem, 273, 25804–25808.