Biochemical and Biophysical Research Communications
Regular ArticleThe Protective Effect of Tetrahydrobiopterin on the Nitric Oxide-Mediated Inhibition of Purified Nitric Oxide Synthase
Abstract
The nitric oxide synthases (NOS) are a class of enzymes responsible for the generation of NO via an oxygen and NADPH dependent oxidation of the amino acid arginine. These enzymes are ironheme proteins which contain FAD and FMN and, enigmatically, require tetrahydrobiopterin (BH4). NOS has recently been shown to be subject to inhibition by its product, NO. Preliminary data by us indicate that a possible role for BH4 is to prevent and/or reverse the NO-mediated inhibition of NOS. The objective of this study was to elucidate the mechanism by which BH4 protects NOS against NO inhibition. Protection of NOS from NO inhibition was observed by both BH4 and the BH4 regeneration system, dihydropteridine reductase (DHPR)/NADH. NO, rather than an oxidation product, appears to be the inhibitory species. Protection by BH4 is not likely due to a simple chemical reaction between BH4 and NO or its oxidation product, NO2. The results are consistent with a protective mechanism by which BH4 may act as a nonstoichiometric reducing agent for a redox active enzyme component, such as the ironheme, to prevent NO ligation.
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Factors influencing the soluble guanylate cyclase heme redox state in blood vessels
2022, Vascular PharmacologyCitation Excerpt :Tetrahydrobiopterin (BH4) is ubiquitously present in all tissues of higher organisms and is a cofactor required for the synthesis of several biomolecules [61]. In addition to its activity as a cofactor, BH4 serves as a reducing agent [62]. Schmidt et al. reported that treatment of porcine aortic endothelial cells with BH4 restores DEA/NO [NO donor]-stimulated cGMP production reduced in the presence of ODQ [63].
Soluble guanylate cyclase (sGC) plays an important role in maintaining vascular homeostasis, as an acceptor for the biological messenger nitric oxide (NO). However, only reduced sGC (with a ferrous heme) can be activated by NO; oxidized (ferric heme) and apo (absent heme) sGC cannot. In addition, the proportions of reduced, oxidized, and apo sGC change under pathological conditions. Although diseased blood vessels often show decreased NO bioavailability in the vascular wall, a shift of sGC heme redox balance in favor of the oxidized/apo forms can also occur. Therefore, sGC is of growing interest as a drug target for various cardiovascular diseases. Notably, the balance between NO-sensitive reduced sGC and NO-insensitive oxidized/apo sGC in the body is regulated in a reversible manner by various biological molecules and proteins. Many studies have attempted to identify endogenous factors and determinants that influence this redox state. For example, various reactive nitrogen and oxygen species are capable of inducing the oxidation of sGC heme. Conversely, a heme reductase and some antioxidants reduce the ferric heme in sGC to the ferrous state. This review summarizes the factors and mechanisms identified by these studies that operate to regulate the sGC heme redox state.
Hepatoprotective effect of Arazyme on CCl<inf>4</inf>-induced acute hepatic injury in SMP30 knock-out mice
2008, ToxicologyArazyme is a novel protease produced by the HY-3 strain of Aranicola proteolyticus, which is a Gram-negative aerobic bacterium that has been isolated from the intestine of the spider Nephila clavata. This study focused on the hepatoprotective effect of Arazyme on carbon tetrachloride (CCl4)-induced acute hepatic injury in senescence marker protein 30 (SMP30) knock-out (KO) mice and SMP30 wild-type (WT) mice. WT mice and SMP30 KO mice were divided into eight groups as follows: (i) two negative control groups (G1, G5) which were treated with a single intraperitoneal (i.p.) olive oil injection. (ii) Two positive control groups (G2, G6) which received a single i.p. CCl4 (0.4 mL/kg) injection. (iii) Two vitamin C-treated groups (G3, G7) which received a single oral administration of vitamin C (100 mg/kg) and were injected with a single i.p. CCl4 (0.4 mL/kg). (iv) Two Arazyme-treated groups (G4, G8) which received a single oral administration of Arazyme (500 mg/kg) and were injected with a single i.p. CCl4 (0.4 mL/kg). Through present study, we could find that Arazyme-treated groups showed decreased degree of liver injury, increased expression of SMP30, decreased expression of phospho-Smad3 (p-Smad3), elevated expression of antioxidant proteins including sorbitol dehydrogenase, dihydropteridine reductase (DHPR), dehydrofolate reductase (DHFR), NADH dehydrogenase, glutathione S-transferase kappa 1 (GSTK1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) compared with non-Arazyme-treated groups. Therefore, it is concluded that Arazyme plays a significant role in protecting injured hepatocytes by increasing the expression of SMP30, inhibiting the transforming growth factor-β (TGF-β)/Smad pathway and elevating the expression of antioxidant proteins.
Nitric oxide involvement in reperfusion injury of denervated muscle
2004, Journal of Hand SurgeryTo investigate whether inhibition of inducible nitric oxide synthase (iNOS) improves microcirculation in denervated and reperfused skeletal muscle.
The cremaster muscles of 52 rats received iNOS inhibitor 1400W (3 mg/kg) or phosphate buffered saline (PBS) and underwent either 3 hours of ischemia and 1.5 hours of reperfusion or a sham operation. During reperfusion the vessel diameters were measured by using intravital videomicroscopy and overall muscle blood flow was measured with laser Doppler flowmetry. The expression of NOS messenger RNA (mRNA) and protein was determined by using real-time reverse-transcription polymerase chain reaction and Western blot, respectively.
1400W treatment significantly increased the mean blood flow of the reperfused muscle compared with controls, and this was associated with significantly less vasospasm in 10 to 20 μm, 21 to 40 μm, and 41 to 70 μm arterioles. The expression of iNOS mRNA and protein in controls increased 23-fold and 6-fold from normal, respectively, but was reduced to only a 2-fold increase in the 1400W-treated muscles. The ischemia/reperfusion (I/R)-induced decrease of endothelial NOS (eNOS) and neuronal NOS (nNOS) expression in controls was not significantly changed after 1400W treatment.
Our data support a nitric oxide-mediated mechanism in reperfusion injury and show the importance of inhibition of iNOS in reducing reperfusion injury in denervated skeletal muscle. Our results suggest potential benefits via inhibition of iNOS to improve clinical outcomes not only for hand surgeons who work in the microsurgery field, but also for other physicians whose work involves ischemia/reperfusion injury.
Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical
2001, Free Radical Biology and MedicineIt has been shown that BH4 ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH4. To examine this possibility we determined the rate constant for the reaction between BH4 and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH4 and superoxide of 3.9 ± 0.2 × 105 M−1s−1 at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH4 is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH2 and pterin are the stable products generated from the reaction. The formation of BH4 cation radical (BH4•+) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH4•+ is mainly delocalized on the pyrazine ring of BH4. In parallel experiments, we investigated the effect of ascorbate on 7,8-BH2 reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH2 to BH4, nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH2. In conclusion, it is likely that BH4-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH4 in the vasculature.
Tetrahydrobiopterin attenuates modulation of platelet 12-lipoxygenase and cyclooxygenase activities by nitric oxide
2001, Nitric Oxide - Biology and ChemistryEndothelial cells secrete large amounts of 5,6,7,8-tetrahydrobiopterin (BH4) in septic conditions. BH4 is a cofactor for nitric oxide (NO) synthase and an essential regulator of its activity. We recently showed that NO can be a modulator of both platelet 12-lipoxygenase and cyclooxygenase activities. In the present study, we investigated the effect of BH4 on the activities of 12-lipoxygenase and cyclooxygenase in rabbit platelets. The influence of BH4 on NO-induced modulation of these enzyme activities was investigated. Exogenous BH4 did not affect platelet 12-lipoxygenase and cyclooxygenase activities. The modulatory effects of NO on the two enzymatic pathways were reversed by addition of BH4 but not by reduced glutathione. These results suggest that exogenous BH4 is not essential for NO synthase activity of platelets, but that it is an important regulator of the action of NO released from other sources on platelet 12-lipoxygenase and cyclooxygenase activities.
Chapter 1 Nitric oxide synthase and the production of nitric oxide
2000, Handbook of Chemical NeuroanatomyThis chapter discusses neuronal nitric oxide synthase (nNOS), the isoforms of NO synthase, and their protein structure. Most of the early biological research on NO focused on its effects on vascular smooth muscle, platelets, and cytosolic guanylate cyclase, whereas little or no attention was given to the possible influence of NO on neuronal cell function or as a neurotransmitter. In the peripheral nervous system, NO appears to function as a neurotransmitter mediating vascular and nonvascular smooth muscle relaxation. These tissues are innervated by nonadrenergic–noncholinergic (NANC) neurons. nNOS containing neurons exist in the myenteric plexus of the gastrointestinal tract, and neuronal stimulation causes NO release and nonvascular smooth muscle relaxation associated with the inhibition of peristalsis. NANC neurons exist also in the outer adventitial layers of large blood vessels and in arteries within erectile tissue. Functional, constitutive nNOS was recovered from rabbit corpus cavernosum and later localized to neurons innervating the erectile tissue. In the erectile tissue of the penis, NO is the NANC neurotransmitter that mediates penile erection by provoking both vascular and nonvascular smooth muscle relaxation, thereby allowing the trabecular and sinusoidal vascular beds to become engorged with blood.