Effect Of Tetrahydrobiopterin Research Articles

The effect of tetrahydrobiopterin on microvascular function with advancing age assessed by passive leg movement

Tetrahydrobiopterin (BH4) is a cofactor which recouples eNOS, potentially increasing nitric oxide (NO)‐bioavailability. Previously, BH4 has been documented to restore conduit artery vascular dysfunction, assessed by flow mediated vasodilation (FMD) in sedentary old subjects. This study aimed to determine if BH4 can restore microvascular function in the old, assessed by the, highly NO‐dependent, passive leg movement (PLM) test. Two approaches were employed, with the first recapitulating the experimental design of the prior FMD study: 1) Using a double‐blind, placebo controlled, crossover design, eight old subjects (74±8 yrs) ingested BH4 (100mg/kg) or placebo on two different days at least one week apart, with PLM assessed (blood flow ΔPeak and AUC) after both conditions. 2) On a single experimental day, PLM was assessed in an additional thirteen old subjects (74±8 yrs) prior to and ~ 4.5 hours after ingesting BH4 (100 mg/kg). With the first experimental approach, BH4 increased PLM ΔPeak and AUC by ≈20% (271±94 vs 326±158) and ≈29% (60±40 vs 77±51), respectively, compared to placebo, but did not achieve statistical significance. With the second approach, BH4 increased PLM ΔPeak and AUC ≈30% (323±182 vs 421±192; p=0.01) and ≈38% (64±62 vs 89±56; p=0.06), respectively. When the two data sets were combined, BH4 significantly increased both PLM ΔPeak ≈28%(305±156 vs 388±183; p=0.01) and AUC ≈43%(63±54 vs 85±53; p=0.02). Thus, improving eNOS‐recoupling with acute BH4 supplementation can improve microvascular function with advancing age and this is likely a result of increased NO‐bioavailability.

Protective effect of tetrahydrobiopterin on hepatic and renal damage after acute cadmium exposure in male rats

ABSTRACTCadmium (Cd) has been recognized as one of the most important environmental and industrial pollutants. This study investigated the impact of acute exposure to Cd on oxidative stress and the inflammatory marker interleukin-6 (IL-6) in the plasma of rats and the histological picture of liver and kidney, as well as to examine the potential protective effect of tetrahydrobiopterin (BH4). Methods: Rats were divided into control group, Cd group that received a single intraperitoneal (i.p.) dose of 4 mg/kg b.w. of CdCl2 and BH4+ Cd group that received a single dose of BH4 (20 mg/kg, i.p.) and subsequently exposed to a single dose of Cd 24 h after the BH4 treatment. Results: Cd increased the plasma levels of hepatic enzymes (ALT and AST), urea, creatinine, malondialdehyde (MDA), and IL-6 and decreased the superoxide dismutase (SOD) activity. Also, it induced histopathological alterations in the liver with severe degeneration, especially in centrilobular zones. Renal tubular epithelium showed vacuolated cytoplasm and dense nuclei. VEGF expression was mild. Ultrastuctural changes were seen in some renal tubules. The nuclei appeared distorted with electron dense chromatin. Mitochondria with destructed cristae were observed. BH4 pretreatment had protective effects, since it significantly reduced the levels of IL-6 and ameliorated the alteration in oxidative status biomarkers induced by Cd. Improvement of histopathological alterations was observed in Cd-groups. The nuclei were vesicular euchromatic, intact mitochondria and normal appearance of the filtration membrane. Moderate expression of VEGF was noted. Conclusion: This study has provided clear evidence for the protective efficacy of BH4 against experimental Cd toxicity.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

Effects of tetrahydrobiopterin on the angiogenesis in hepatocellular carcinoma

Objective: To investigate the effect and mechanism of tetrahydrobiopterin (BH4) on the angiogenesis in hepatocellular carcinoma (HCC). Methods: BALB/c-nu mice were subcutaneously injected with HepG-2 cells and randomly divided into control and BH4 groups. The BH4 group and control group received 20 mg/kg BH4 or saline by intraperitoneal injection daily for two weeks, respectively. The level of BH4 was measured by high performance liquid chromatography (HPLC), the level of nitric oxide (NO) was measured by Griess test array, the transcriptional level of K-ras was measured by quantitative RT-PCR, and the protein expressions of guanosine triphosphate cyclohydrolase Ⅰ(GTPCH), endothelial nitric oxide synthase (eNOS), phospho-Akt and Akt were determined by Western blot. Results: BH4 level in the tumor tissues of BH4 group was (0.24±0.02) μg/ml, significantly higher than the (0.17±0.01) μg/ml in the control group (P<0.01). The level of NO in the tumor tissues of BH4 group was (51.44±2.90) mmol/L, significantly higher than the (24.77±0.54) mmol/L in the control group (P<0.01). The tumor volume of BH4 group was (191.05±8.70) mm3, significantly higher than the (103.10±5.03) mm3 in the control group (P<0.01). The expressions of CD34, K-ras, phospho-eNOS, phospho-Akt and GTPCH were significantly up-regulated in the tumor tissues of BH4 group when compared with those of the control group (P<0.01). Conclusions: BH4 recognized as an essential cofactor of eNOS can increase tumor-produced NO by activating the wild-type Ras-PI3K/Akt pathway, thus induces angiogenesis. This might provide a novel and promising way to control the progression of hepatocellular carcinoma through targeting BH4 synthesis pathway and inhibiting angiogenesis.

Stabilizing Action of Tetrahydrobiopterin on the Activity of Neuronal Nitric Oxide Synthase

We studied the effect of tetrahydrobiopterin (BH4), one of the co-factors of NO synthase (NOS), on the process of inactivation of this enzyme in a cytoplasmic fraction obtained from rat cerebellar tissue. Tetrahydrobiopterin stimulated in a dose-dependent manner the activity of NOS measured according to accumulation of [3H]-citrulline. In the course of reaction at 37°С without the addition of BH4, the enzymatic activity rapidly decreased; in the presence of 10 μM BH4, the rate of inactivation of NOS was noticeably smaller. Preincubation (40 min long) under the same conditions but in the absence of substrates (arginine and NADPH) and BH4 also led to a decrease in the enzymatic activity. Preincubation in the presence of 1 mM BH4 not only slowed down inactivation of NOS but also provided an appreciable increase in its activity, as compared with the variant without preincubation. This fact is indicative of the presence of a BH4-free inactive form of the above-mentioned enzyme in the cytoplasmic fraction, which can be additionally activated. The obtained data show that BH4 stabilizes the activity of NOS due to a decrease in the rate of inactivation of this enzyme occurring independently of realization of the enzymatic reaction.

Effect of tetrahydrobiopterin and exercise training on endothelium-dependent vasorelaxation in SHR

We examined whether the improvement of impaired NO-dependent vasorelaxation by exercise training could be mediated through a BH4-dependent mechanism. Male spontaneously hypertensive rats (SHR, n = 20) and Wistar-Kyoto rats (WKY, n = 20) were trained (Tr) for 9 weeks on a treadmill and compared to age-matched sedentary animals (Sed). Endothelium-dependent vasorelaxation (EDV) was assessed with acetylcholine by measuring isometric tension in rings of femoral artery precontracted with 10(-5) M phenylephrine. EDV was impaired in SHR-Sed as compared to WKY-Sed (p = 0.02). Training alone improved EDV in both WKY (p = 0.01) and SHR (p = 0.0001). Moreover, EDV was not different in trained SHR than in trained WKY (p = 0.934). Pretreatment of rings with L-NAME (50 μM) cancelled the difference in ACh-induced relaxation between all groups, suggesting that NO pathway is involved in these differences. The presence of 10(-5) M BH4 in the organ bath significantly improved EDV for sedentary SHR (p = 0.030) but not WKY group (p = 0.815). Exercise training turned the beneficial effect of BH4 on SHR to impairment of ACh-induced vasorelaxation in both SHR-Tr (p = 0.01) and WKY-Tr groups (p = 0.04). These results suggest that beneficial effect of exercise training on endothelial function is due partly to a BH4-dependent mechanism in established hypertension.

Effect of tetrahydrobiopterin on Phe/Tyr ratios and variation in Phe levels in tetrahydrobiopterin responsive PKU patients

BackgroundWhilst a reduction in blood phenylalanine (Phe) levels is essential in patients with PKU, a decrease in Phe/Tyrosine (Tyr) ratio and fluctuations in blood Phe levels over time have been recently associated with improved neuropsychological outcome. The aim of this study was to identify if Tetrahydrobiopterin (BH4) offers additional benefit based on the assumption that these 2 factors are beneficial. MethodSince 2002, 9 patients identified through NBS as BH4 responsive (BH4 group) and 25 non-responsive patients (non-BH4 group) produced a total of 1384 and 4415 samples, respectively, for analysis. Statistical analysis was performed to compare mean and median Phe levels, Tyr levels and Phe/Tyr ratios in BH4 and non BH4 responsive patients. ResultsVariations in blood Phe levels were greater in the non-BH4 group (BH4: median 338μmol/L, 95% Confidence Interval (CI) 329–346, mean: 358μmol/L, CI 350–366; non-BH4: median 338μmol/L CI 332–344, mean: 370μmol/L CI 364–376). Variations in blood Tyr levels were slightly greater in the non-BH4 group: (BH4: median 59μmol/L CI 58–61, mean 67 CI 66–69; non-BH4: median 62μmol/L CI 61–63, mean 70 CI 69–71).The variation in Phe/Tyr ratios was greater in the non-BH4 group (mean 6.12, CI 5.9–6.3) than in the BH4 group (Mean 5.44, CI 5.3–5.6), particularly at blood Phe levels >600μmol/L. ConclusionBH4 responsive patients have smaller variations in blood Phe levels and tighter Phe/Tyr ratios than non-BH4 responsive patients, particularly at high blood Phe levels. If decreased fluctuations in Phe levels and a decreased Phe/Tyr ratio are indeed neuro-protective, then BH4 responsiveness is advantageous over diet alone in PKU. Neuropsychological testing in patients who have been treated with BH4 long term may be able to ascertain the clinical benefit of these biochemical findings.

Effect of tetrahydrobiopterin on endothelium function of the great saphenous vein from smokers

Objective To observe the effect of tetrahydrobiopterin ( BH4 ) on endothelium function of the great saphenous vein from smokers. Methods The rings of great saphenous vein were selected from 22 patients with coronary artery bypass grafting, which including smoking (experimental group,n = 12) and non-smoking (control group,n = 10) patients. After incubation in Krebs-Henseleit (KH), KH plus BH4 (20 μ mol/L) or N-nitro-L-arginine ( L-NNA, 0. 1 mol/L), the vasoconstriction and vasodilatation were induced by phenylephrine (1 × 10-5 mol/L) and non-receptor-mediated calcium ionophore (A23187,1 × 10-9-1 × 10 -5 mol/L) separately in an organ chamber. Results The maximum constriction in all groups had no significant difference ( P ＞ 0. 05 ). The maximum relaxation was significantly decreased in experimental group as compared with control group [(29.50 ± 6. 22 ) % vs (48.20 ± 7. 74 ) %, P ＜ 0. 01]after incubation in KH, while there was no significant difference (P ＞ 0. 05 ) after incubation in KH plus L-NNA [(24.50±5.72)% vs (28.8 ±5.64)%] or BH4[(46.40 ±6.32)% vs (50.80±6.91)%].Conclusion Smoking impairs the relaxation function mediated by endothelium-derived nitric oxide in great saphenous, which may be restored by BH4. Key words: Smoking; Great saphenous vein; Endothelium function; CABG

Effect of tetrahydrobiopterin on selective endothelial dysfunction of epicardial porcine coronary arteries induced by cardiopulmonary bypass

We hypothesized that cardiopulmonary bypass induces a selective alteration of the coronary arterial endothelial cell signal transduction which could be explained by a state of depletion and/or decreased activity of endogenous tetrahydrobiopterin (BH(4)). The aim of this study was to assess the effects of cardiopulmonary bypass and BH(4) on the endothelial function of epicardial coronary arteries in a swine model of cardiopulmonary bypass. Swine underwent 90 min of cardiopulmonary bypass alone (N=19) or in association with a brief cardioplegic arrest with (N = 6) or without (N = 5) in vivo BH(4) administration, followed by a 60-min period following weaning from cardiopulmonary bypass and were compared to a control group (N = 7). Endothelium-dependent relaxations of epicardial coronary artery rings were studied using standard organ chamber experiments in the presence or absence of in vitro BH(4) or superoxide dismutase (SOD) and catalase. Cardiopulmonary bypass caused a statistically significant reduction of endothelium-dependent relaxations to serotonin (p < 0.0001), bradykinin (p < 0.001), UK14304 (p < 0.0001) and calcium ionophore (p < 0.01) in epicardial porcine coronary arteries. In vitro and in vivo BH(4) supplementation improved endothelium-dependent relaxations to serotonin and bradykinin, which were left unchanged by SOD-catalase administration. Cardiopulmonary bypass was associated with a decrease in nitric oxide availability (p = 0.002) and increased oxidative stress (p < 0.001), which were both restored by in vivo BH(4) administration (p < 0.001). Treatment with BH(4) improves the endothelial dysfunction of porcine epicardial coronary arteries, restores nitric oxide availability and reduces the oxidative stress associated with cardiopulmonary bypass.

The effect of tetrahydrobiopterin (BH4) on γ-ray-induced micronucleus (MN) frequency

The effect of tetrahydrobiopterin (BH4) on γ-ray-induced micronucleus (MN) frequency

Effect of Tetrahydrobiopterin on Copper-Mediated DNA Oxidation: Its Prooxidant Property

Tetrahydrobiopterin (BH4) prevented an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA by the peroxynitrite (ONOO-) or Fe2+ + hydrogen peroxide (H2O2) system. But, the addition of BH4 to the Cu2+ + ascorbic acid (AsA) system caused a very large increase in base damage. Without the addition of AsA (in the presence of Cu2+ alone), BH4 could also induce 8-OHdG formation. Experiments utilizing the Cu+ chelator bathocuproine, catalase and superoxide dismutase revealed that Cu+ and H2O2 intermediates relate to Cu2+ + BH4-induced 8-OHdG formation. Electron spin resonance experiments also showed that the hydroxyl radical is not a major intermediate. These results suggest that although BH4 is known to prevent oxidative damage in proteins and lipids, it has the potential to cause oxidative DNA damage depending on the existence of copper ions.

Effect of tetrahydrobiopterin on nitric oxide synthase-containing cells in the rat hippocampus.

We have observed that tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS), acts as a self-protection factor against nitric oxide (NO) toxicity in PC12 cells. To further investigate the self-protection action of BH4 in vivo, the effect of deletion of endogenous BH4 on NO-producing cells was examined in the rat hippocampus. Following the peripheral infusion of 50 mM 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, using a miniosmotic pump for 14 days, BH4 content in the hippocampus decreased as compared with the control group administered with vehicle solution, which had no effect on brain BH4 content. When the rats were administered with 50 mM DAHP and 10 mM BH4, the DAHP-induced decrease in BH4 content was prevented. The extracellular concentration of NO metabolites remained unchanged following DAHP administration, suggesting that DAHP-induced decrease in BH4 content had no effect on NO production. The number of NOS-positive cells decreased following DAHP administration in the hippocampal regions, while the number of NOS-negative cells remained unchanged. The DAHP-induced decrease in the NOS-positive cell number was prevented by the administration of 10 mM BH4 and DAHP. These results suggest that endogenous BH4 may affect NOS-positive cell number in the rat hippocampus.

Effect of Tetrahydrobiopterin on Blood Pressure in Rats after Subtotal Nephrectomy

Background: Previous studies have shown that endothelial dysfunction after 5/6 nephrectomy (5/6 Nx) in rats is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. Blood pressure is significantly increased by day 10 after surgery. Tetrahydrobiopterin (BH4) is a key cofactor of NO synthase. Suboptimal levels of BH4 result in uncoupling of NO synthase, low NO synthesis and augmented production of superoxide anions. The aim of this study was to evaluate whether BH4 supplementation may improve NO production and prevent the increase of blood pressure after 5/6 Nx. Methods: Three groups were evaluated: 5/6 Nx (untreated rats), BH4 (5/6 Nx rats treated with BH4, 10 mg/day i.p. for 10 days) and L-ARG (5/6 Nx rats treated with L-arginine, 260 mg/kg BW, p.o for 10 days). Systolic blood pressure (SBP), urinary nitrate excretion (UNO₃) and creatinine clearance (CCR) were measured before surgery and on days 3 and 10 after surgery. Endothelial NO synthase (eNOS) protein content of mesenteric resistance vessels was measured at the end of the study. Results: SBP increased from 107 ± 2 to 127 ± 4 mm Hg in untreated 5/6 Nx rats (p < 0.01). By contrast, rats treated with BH4 or L-ARG remained normotensive. Ten days after 5/6 Nx, creatinine clearance decreased similarly in all groups. Both BH4 and L-ARG supplementation markedly increased UNO₃ excretion. The mesenteric vascular expression of eNOs protein was significantly higher in BH4 but not in L-ARG, compared with Nx rats. Conclusions: BH4 supplementation prevents the earlier increase in blood pressure observed in rats after 5/6 Nx, possibly by upregulating eNOS in resistance vessels.

Anti-inflammatory effects of tetrahydrobiopterin on early rejection in renal allografts: modulation of inducible nitric oxide synthase.

Oxidative stress contributes to the development of early transplant failure. As nitric oxide synthases (NOS) can act as sources of superoxide, we investigated the effect of the NOS cofactor tetrahydrobiopterin (BH4) on oxyradical production and early rejection in a rat kidney transplantation model. Allograft transplantation (Brown Norway to Lewis) showed more renal superoxide production and monocyte infiltration when compared with isografts (Lewis to Lewis). Administration of the stable BH4 precursor sepiapterin had no effect on superoxide production in the isografts (51+/-10 vs. 69+/-17 cps/10 mg protein), but led to a marked decrease in superoxide production in the allografts (116+/-11 vs. 60+/-6 cps/10 mg protein; P<0.05) and was accompanied by a reduction in periarterial macrophage infiltration (3.3+/-0.7 vs. 1.3+/-0.3 cells/vessel; P<0.05) and an increase in NO production (78+/-22 vs. 173+/-12 AU/g kidney) (P<0.01). In vitro experiments confirm that iNOS can produce superoxide mainly from the heme domain, whereas BH4 administration can reverse this superoxide production in the presence of adequate anti-oxidant defense. Our findings support the hypothesis that BH4 can be used to modulate the function of the inflammatory iNOS isoform and suggest a potential therapeutic role for sepiapterin in early allograft rejection.

Effect of tetrahydrobiopterin on coronary microcirculation in healthy and hypercholesterolemic subjects

Effect of tetrahydrobiopterin on coronary microcirculation in healthy and hypercholesterolemic subjects

Protective Effect of Tetrahydrobiopterin Against Reactive Oxygen Species- and Nitric Oxide-induced Vascular Endothelial Cell Injury

Abstract Vascular endothelial cells are one of the major biological targets of reactive oxygen species (ROS) in ischemiareperfusion, diabetes, hypercholesterolemia and inflammation. Accumulated evidence has shown that tetrahydrobiopterin (BH4), which is an essential cefactor for nitric oxide synthase (NOS) activity, protected the cells against ROS and nitric oxide (NO) toxicities. Although NOS releases NO, which regulates vascular tone and immune surveillance under normal conditions, NOS also produces superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of ROS under pathological conditions. Since BH4 is easily oxidized by ROS, oxidative stress may induce ROS release from NOS by the decomposition of BH4. BH4 also has an antioxidative activity and scavenging activity for ROS. In this review, the r o l e of BH4 in the function of' NOS, and the mechanisms underlying the protective effect of BH4 against ROS- and/or NO-induced endothelial cell injury will be discussed.

Involvement of reactive oxygen species and nitric oxide in gastric ischemia-reperfusion injury in rats: protective effect of tetrahydrobiopterin.

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), a cofactor of nitric oxide (NO) synthase, attenuates gastric ischemia-reperfusion injury induced by clamping of the celiac artery. Gastric injury was assessed by a formation of gastric mucosal erosions. The gastric injury was observed at 30 and 60 min after reperfusion following 30-min ischemia and was reduced by superoxide dismutase (SOD), catalase, or NO synthase inhibitors. Therefore, reactive oxygen species (ROS) and NO seem to be implicated in the ischemia-reperfusion injury. Treatment with BH4 reduced the ischemia-reperfusion injury. Pretreatment with sepiapterin, a precursor of BH4, also reduced the ischemia-reperfusion injury with an increase in BH4 content in serum and stomach. Both the increase in BH4 content and the protective effect of sepiapterin were prevented of pretreatment with N-acetylserotonin, an inhibitor of BH4 synthesis. These results suggest that the increase in BH4 content may protect against gastric ischemia-reperfusion injury via reduction of ROS and/or NO toxicity. BH4 might be useful as a therapeutic agent for gastric ischemia-reperfusion injury.

Characterization of the Protective Effect of Tetrahydrobiopterin Against S-Nitroso-N -Acetyl-D ,L-Penicillamine Induced Endothelial Cell Injury

The purpose of this study was to characterize the protective effect of tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, against NO-induced endothelial cell injury. The addition of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, to endothelial cells induced the release of lactate dehydrogenase (LDH), a marker for cell injury. The SNAP-induced endothelial cell injury was markedly reduced by pretreatment with sepiapterin, a precursor of BH4 synthesis. On the other hand, exogenous BH4 had little effect on the SNAP-induced endothelial cell injury. We recently found that NO-induced endothelial cell injury involves a part of H202 production, since the injury was blocked by the treatment with catalase. Although BH4 released reactive oxygen species (ROS) in cell-free conditions, the increase in intracellular BH4 by pretreatment with sepiapterin strongly reduced H202-induced intracellular oxidative stress. These findings suggest that the increase in intracellular BH4 content but not extracellular BH4, strongly attenuates NO-induced endothelial cell injury by at least one of the mechanisms by which BH4 reduces H202-induced oxidative stress. Intracellular BH4 seems mainly to play a role as an antioxidant or as a ROS-scavenger.

Effect of Tetrahydrobiopterin on Dopamine Release from PC12 Cells

Effect of Tetrahydrobiopterin on Dopamine Release from PC12 Cells

Tetrahydrobiopterin preferentially stimulates activity and promotes subunit aggregation of membrane-bound calcium-dependent nitric oxide synthase in human placenta.

Type III nitric oxide synthase (NOS III) is responsible for > 90% of nitric oxide (NO) synthesizing activity in first trimester placentae. Enzyme activity is distributed between cytosolic (30%) and membrane-bound forms (70%), with highest specific activity observed in microsomal fractions. In the present study, the effect of tetrahydrobiopterin (BH4) on subunit structure and activity of microsomal and cytosolic NOS III was compared. As revealed by immunoblot analysis, incubation of microsomal membranes with 50 microM final concentration BH4 for 10 min at 37 degrees C resulted in a striking conversion of monomeric NOS III into a protein having the characteristics (electrophoretic mobility, resistance to sodium dodecyl sulphate) of the homodimeric form. In contrast, BH4 induced significantly less marked changes in the NOS III dimer content of cytosolic fractions. Enzyme activity in microsomes is stimulated approximately 6-fold upon addition of 50 microM BH4, while only a 2-fold activation is detectable in cytosolic fractions. Taken together, the observations suggest that BH4 activates NOS III in the primordial human placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH4. Compartment-specific action of BH4 represents a novel mechanism which is implicated in the regulation of placental NOS activity.