Sapropterin is safe and effective in patients less than 4-years-old with BH4-responsive phenylalanine hydrolase deficiency.

OBJECTIVE: To assess the safety and efficacy of tetrahydrobiopterin therapy with sapropterin to treat tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency in children aged <4 years compared with those aged ≥4 years. STUDY DESIGN: We analyzed a longitudinal follow-up study conducted in all patients with BH4-responsive PAH deficiency throughout Japan. At the end of 2011, 43 patients were receiving sapropterin, of whom 21 were aged <4 years at the initiation of treatment. The starting dose of sapropterin was ≥10 mg/kg/day in 11 of these 21 patients. The duration of follow-up was ≥4 years in 6 of those 11 patients; 3 of these 6 were followed for ≥10 years. Nine patients were receiving sapropterin monotherapy at the end of 2011. RESULTS: Serum phenylalanine level was maintained within the recommended optimal control range in all 21 patients who started sapropterin treatment before age 4 years. Only 1 nonserious adverse drug reaction occurred, an elevated alanine aminotransferase level in 1 patient. No significant abnormal behavior related to nerve disorders was reported. CONCLUSION: Sapropterin therapy initiated before age 4 years was effective in maintaining serum phenylalanine level within the favorable range and was safe in Japanese patients with BH4-responsive PAH deficiency.

Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions.

Glucagon-like peptide-1 (GLP-1) is a neuropeptide released following meal ingestion that, among other effects, decreases gastric tone and motility. The central targets and mechanism of action of GLP-1 on gastric neurocircuits have not, however, been fully investigated. A high density of GLP-1 containing neurones and receptors are present in brainstem vagal circuits, suggesting that the gastroinhibition may be vagally mediated. We aimed to investigate: (1) the response of identified gastric-projecting neurones of the dorsal motor nucleus of the vagus (DMV) to GLP-1 and its analogues; (2) the effects of brainstem application of GLP-1 on gastric tone; and (3) the vagal pathway utilized by GLP-1 to induce gastroinhibition. We conducted our experiments using whole-cell recordings from identified gastric-projecting DMV neurones and microinjection in the dorsal vagal complex (DVC) of anaesthetized rats while monitoring gastric tone. Perfusion with GLP-1 induced a concentration-dependent excitation of a subpopulation of gastric-projecting DMV neurones. The GLP-1 effects were mimicked by exendin-4 and antagonized by exendin-9-39. In an anaesthetized rat preparation, application of exendin-4 to the DVC decreased gastric tone in a concentration-dependent manner. The gastroinhibitory effects of exendin-4 were unaffected by systemic pretreatment with the pro-motility muscarinic agonist bethanechol, but were abolished by systemic administration of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), or by bilateral vagotomy. Our data indicate that GLP-1 activates selective receptors to excite DMV neurones mainly and that the gastroinhibition observed following application of GLP-1 in the DVC is due to the activation of an inhibitory non-adrenergic, non-cholinergic input to the stomach.

A possible involvement of crosstalk between advanced glycation end products (AGEs) and asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor in accelerated atherosclerosis in diabetes.

Endothelial dysfunction due to reduced bioavailability of nitric oxide (NO) is an early step in the course of atherosclerotic cardiovascular disease (CVD). NO is synthesized from L-arginine via the action of NO synthase (NOS), which is known to be blocked by endogenous L-arginine analogues such as asymmetric dimethylarginine (ADMA). ADMA is a naturally occurring amino acid found in plasma and various types of tissues. Recently, it has been demonstrated that plasma levels of ADMA are elevated in patients with diabetes. It has also been reported that elevated plasma levels of ADMA are associated with increased risks of nonfatal stroke and myocardial infarction in patients with early diabetic nephropathy. These findings suggest that the elevated ADMA in diabetes could contribute to acceleration of atherosclerosis in this population. In diabetes mellitus, the formation and accumulation of advanced glycation end products (AGEs) progress. There is a growing body of evidence to show that AGEs are involved in the development and progression of atherosclerosis in patients with diabetes. Since ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase (DDAH), it is conceivable that the impairment of DDAH actions by AGEs could be one possible molecular mechanism of the ADMA elevation in diabetic patients. In this paper, we would like to propose the possible ways of testing our hypotheses. Does treatment with metformin, which has a potential effect on the inhibition of glycation reactions in vivo, decrease the levels of ADMA in diabetic patients? If the answer is yes, is this beneficial effect of metformin superior to that of other anti-diabetic agents with equihypoglycemic properties? Does treatment with pyridoxamine, a post-Amadori inhibitor (so-called Amadorins) of AGE formation, also reduce the levels of ADMA and subsequently improve endothelial dysfunction in diabetic patients? Are the ADMA-lowering effects of these agents associated with an increase of DDAH expression and/or activity in endothelial cells? These clinical studies could clarify whether AGEs are involved in the elevation of ADMA in patients with diabetes via suppression of DDAH expression and/or activity, thus providing a novel molecular mechanism for accelerated atherosclerosis in diabetes.

Effects of nitric oxide inhibition by methylene blue in cirrhotic patients with ascites.

Increased endogenous nitric oxide production has been proposed as an important mediator of the peripheral arterial vasodilation and the hyperdynamic circulation in cirrhosis, whereas a decreased intrahepatic production of nitric oxide has been implicated in the pathogenesis of portal hypertension. The present study investigated the possible beneficial effects of methylene blue, which is a potent inhibitor of guanylate cyclase and nitric oxide synthase, on hyperdynamic circulation and renal function in cirrhotic patients with ascites together with the effects on portal hemodynamics. Twenty patients were evaluated at baseline and during 2 consecutive 4-hr periods after the administration of methylene blue at a dose of 3 mg/kg (10 patients) or placebo (10 patients). Mean arterial pressure, heart rate, cardiac output, systemic vascular resistance, plasma active renin, plasma aldosterone, plasma antidiuretic hormone, serum urea, serum creatinine, serum sodium, urinary flow rate, glomerular filtration rate, effective renal plasma flow, portal flow volume, and portal vein velocity were not modified by methylene blue or placebo. Urinary sodium excretion, fractional sodium excretion and serum nitric oxide levels were significantly decreased 4 hr after methylene blue administration (P < 0.05), to return toward basal levels over a further 4-hr period. It is concluded that methylene blue, at the dose used in the present study, has no effect on systemic and portal hemodynamics in cirrhotic patients with ascites. The reduction in renal sodium excretion, in the absence of changes in renal function and hemodynamics, suggests, at least partly, a direct antinatriuretic effect of methylene blue.

Beneficial effects of aminoguanidine on the cardiovascular system of diabetic rats.

BACKGROUND: The study focused on investigating the effect of aminoguanidine on cardiovascular damages in diabetes and the possible mechanisms of its action. METHODS: Aminoguanidine (AMNG) was used to treat streptozotocin-induced diabetic rats, and the effects were compared to those obtained under insulin treatment. Blood metabolic parameters, *NO and ONOO- as well as protein carbonyl levels and cardiac hypertrophy were determined. RESULTS: Diabetic animals showed increased *NO levels and markedly increased ONOO- generation in the aorta, along with a significant hypertrophy and protein carbonylation in the cardiac tissue. Both AMNG and insulin treatment suppressed the levels of overproduced *NO or ONOO- in the vasculature, but only AMNG was able to prevent hypertrophic alterations and reduce protein carbonylation in the cardiac tissue. CONCLUSIONS: Oxidative protein modification, together with cardiac hypertrophy and high generation of *NO and ONOO-, are important early events in the development of cardiovascular complications in diabetes. Aminoguanidine could prevent hypertrophy through inhibition of production of nonenzymatic glycation products rather than via inhibition of *NO production.

L-arginine prevents impaired endothelium-dependent cerebral arteriolar dilatation during acute infusion of nicotine.

Exogenous treatment with L-arginine has been shown to restore impaired nitric oxide synthase (NOS)-dependent dilatation of peripheral blood vessels during disease states. We have shown that nicotine impairs NOS-dependent arteriolar dilatation in the cerebral circulation. However, the role of L-arginine in impaired responses of cerebral arterioles during infusion of nicotine has not been examined. Thus the goal of the present study was to examine the role of L-arginine in nicotine-induced impairment of cerebral arteriolar reactivity. We measured the diameter of pial arterioles in response to NOS-dependent (5'-adenosine diphosphate [ADP] and acetylcholine) and NOS-independent (nitroglycerin) agonists before and after infusion of vehicle or nicotine (2 microg/kg/min intravenously for 30 min followed by a maintenance dose of 0.35 microg/kg/min) in the absence or presence of L-arginine (10(-3) M). We found that topical application of L-arginine to cerebral microvessels during infusion of nicotine could prevent impaired NOS-dependent vasodilatation. We suggest that exogenous L-arginine may have a beneficial role in preventing cerebral microvascular dysfunction during exposure to nicotine.

[Molecular aspects of toxic effects of nitric oxide]. / Molekularne aspekty toksycznego dzialania tlenku azotu.

Although Nitric oxide (NO) is involved in many physiological processes of the body, its physico-chemical properties may, under some conditions, become dangerous to living cells. NO exposure may be of endogenous (e.g. local overproduction by activated inflammatory cells) or exogenous origin (mainly by inhalation route). Together with a short review of potential sources of NO exposure, the author presents some molecular aspects of its toxic effects: prooxidant effect, genotoxicity and mutagenicity, impact on the cell cycle and feedback inhibitory effect on NO synthase.

Tumor necrosis factor-alpha: a potentially neurodestructive cytokine produced by glia in the human glaucomatous optic nerve head.

Tumor necrosis factor-alpha (TNF-alpha) mediates a range of cellular responses, which have potentially detrimental consequences that affect multiple cell types. To determine whether TNF-alpha contributes to glaucomatous optic neuropathy, we have studied the expression of this cytokine and its receptor, tumor necrosis factor receptor-1 (TNF- R1), in human glaucomatous optic nerve heads from patients with different stages of disease using double labeling fluorescence immunohistochemistry. We have also investigated the ability of this cytokine to induce nitric oxide synthase (NOS-2) in cultured human optic nerve astrocytes by immunocytochemistry and immunoblot. Normal tissue showed constitutive expression of TNF-R1 in the vasculature of the optic nerve heads but no positive labeling for TNF-alpha. In the glaucomatous optic nerve heads, the expression of both TNF-alpha and TNF-R1 were apparently upregulated, primarily in glial fibrillary acidic protein (GFAP)-positive astrocytes, and appeared to parallel the progression of optic nerve degeneration. In eyes with severe glaucomatous damage, some HLA-DR positive microglia also contained TNF-alpha and TNF-R1. In the most severely damaged optic nerve heads, the axons of the retinal ganglion cells contained TNF-R1 and, therefore, are direct targets for neurodegeneration caused by TNF-alpha. In vitro astrocytes constitutively express TNF-R1 and TNF-alpha stimulation induces expression of NOS-2. We hypothesize that TNF-alpha contributes to the progression of optic nerve degeneration in glaucoma by both a direct effect on the axons of the retinal ganglion cells and by inducing NOS-2 in astrocytes.

[Inhibition of nitric oxide synthase has an analgesic effect in chronic pain]. / Haemning af nitrogenoxid-syntase har analgetisk effekt ved kronisk smerte.

The efficacy of the inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine hydrochloride (L-NMMA), was tested in 16 patients with chronic tension-type chronic headache. The study was designed as a randomized double-blind, crossover trial. Patients were assigned intravenous infusion of 6 mg/kg L-NMMA or placebo on two days separated by at least one week in a randomized order. Headache intensity was measured on a 100 mm visual analogue scale at baseline and at 30 min, 60 min, and 120 min after start of treatment. L-NMMA reduced pain intensity significantly more than placebo: 120 min after start of treatment, the mean pain score was decreased from 49 to 33 with L-NMMA and from 44 to 40 with placebo (p = 0.01). The present study demonstrates that inhibition of NOS has an analgesic effect in chronic tension-type headache.

Endometriosis and free radicals.

Recent studies have shown that synthase for nitric oxide or scavenger enzymes is distributed throughout the endometrium. We have reported that endothelial nitric oxide synthase, originally identified in vascular endothelial cells, is distributed in glandular epithelial cells in the endometrium, peaking in the midsecretory phase. In addition, it is known that superoxide dismutase is distributed throughout the endometrium, varying with the menstrual cycle. Yet it is not clear how these enzymes are committed in the reproductive processes. Endometriosis is often complicated by infertility and miscarriage. Of particular interest is that these enzymes are overexpressed in the disease throughout the menstrual cycle. These findings strongly suggest that excessive amounts of free radicals are produced in endometriosis. Copyrightz1999S.KargerAG,Basel

[L-nitroargininemethylester (L-NAME), a nitric oxide synthase inhibitor, increases the anesthetic potency of etomidate]. / L-Nitroargininmethylester (L-NAME), ein Stickoxidsynthaseinhibitor, erhöht die anästhetische Potenz von Etomidat.

OBJECTIVE: Comparable to other intravenous anaesthetics, etomidate is thought to mediate its anaesthetic effect through an action on gamma-amino-butyric-acid (GABA) receptors. Recently, there is evidence that general anaesthetics act on second messenger systems such as the nitric oxide (NO) metabolism too. This study was designed to evaluate the effects of the NO-synthase inhibitor nitro-L-arginine methyl ester (L-NAME) on the anaesthetic potency of the intravenous anaesthetic etomidate. METHODS: With approval of the local animal care committee, the effect of L-NAME on the anaesthetic potency of etomidate was studied in Xenopus laevis tadpoles. The animals were exposed to different concentrations of the anaesthetic etomidate or a combination of etomidate and 1 mM L-NAME for 120 min. Anaesthesia was defined as loss of righting reflex for more than 5 s. A concentration-response curve was fitted to the data according to the method of Waud for quantal biological data and half maximal effects (EC50) and slopes of the curves were calculated. RESULTS: In both groups, the fraction of anaesthetised animals increased with increasing etomidate concentrations. The calculated values were EC50 4.5 +/- 0.2 microM in the etomidate group with a slope of 2.6 +/- 0.3 (mean +/- SE). The etomidate plus L-NAME group exhibited a significantly different EC50 of 3.0 +/- 0.2 microM with a slope of 2.3 +/- 0.3. CONCLUSION: The NO-metabolism has been suggested to be involved in the anaesthetic action of volatile as well as intravenous anaesthetics. The reduction in EC50 of etomidate in presence of L-NAME is comparable to that observed for thiopental or halothane, and thus may indicate an additional mechanism of action of etomidate.

Nitric oxide synthase inhibitor influences prostaglandin and interleukin-1 production in experimental arthritic joints.

OBJECTIVE: To assess involvement of nitric oxide (NO) in the increase in eicosanoid and interleukin- 1 (IL-1) levels in the synovial fluid during antigen-induced arthritis (AIA) in rabbits treated with a competitive inhibitor of NO synthesis. SUBJECTS: Thirteen New Zealand White rabbits were sensitized with 5 mg of methylated bovine serum albumin (mBSA). Arthritis was induced in the knee joint by injecting 0.5 ml of a sterile solution of mBSA (2 mg/ml) into the intra-articular cavity. TREATMENT: Prior to the induction of arthritis, the animals received N-Omega-Nitro-L-Arginine Methyl Ester (LNAME) or N-Omega-Nitro-D-Arginine Methyl Ester (DNAME) for 2 weeks, both at a dose of 20 mg/kg/day mixed with drinking water. METHODS: Leukocyte efflux (total and differential white cell count), vascular permeability (Evans's blue method), synovial PMN cell infiltrate, and total nitrite (NO2.)/nitrate (NO3.) (HPLC), PGE2, TxB2, LTB4 (radioimmunoassay), and IL-1 beta (ELISA) levels were quantified in the synovial fluid. RESULTS: LNAME but not DNAME significantly suppressed leukocyte efflux and protein leakage into the articular cavity as well as synovial PMN cell infiltrate. Total NO2./NO3., PGE2 and IL-1 beta levels were significantly reduced in the synovial fluid of LNAME treated animals. TxB2 and LTB4 were not affected by LNAME treatment. CONCLUSION: These data clearly show NO involvement in the IL-1-induced PGE2 production in the synovial fluid of antigen-induced arthritis in rabbits.

Induction of neuronal nitric oxide after hypothermic circulatory arrest.

BACKGROUND: Although hypothermic circulatory arrest (HCA) has become routine practice in cardiac surgery, it is associated with substantial neurotoxicity. We tested the hypothesis that increased nitric oxide production during HCA participates in neuronal death. We previously described a canine survival model of HCA that produces a consistent neurologic deficit and histopathologic pattern of selective neuronal death. METHODS: Adult male hound dogs (n = 17) were subjected to 2 hours of HCA at a brain temperature of 18 degrees C and reperfused to normothermia; they were sacrificed at various intervals up to 74 hours. Using in vivo cerebral microdialysis, dogs (n = 5) were given a simultaneous infusion of artificial cerebrospinal fluid containing L-[14C]arginine or L-[14C]arginine and L-nitroarginine methyl ester (a nitric oxide synthase inhibitor) in contralateral hemispheres while undergoing 2 hours of HCA and reperfusion to normothermia. RESULTS: L-[14C]citrulline recovery, a coproduct of nitric oxide, significantly increased during HCA in the hemisphere without the inhibitor (at 300 minutes: control, 236 +/- 94 fmol/min versus L-nitroarginine methyl ester, 6 +/- 6 fmol/min; p < 0.05). Citrulline production in vitro from canine cortical homogenates in the presence of calcium (n = 12) was significantly greater 8 and 20 hours after reperfusion (5.11 +/- 0.54 x 10(-7) mmol.mg-1.min-1 and 7.52 +/- 0.59 x 10(-7) mmol.mg-1.min-1, respectively) than before HCA (1.51 +/- 0.09 x 10(-7) mmol.mg-1.min-1; p < 0.05). Nitric oxide metabolites in the serum were also increased significantly early after reperfusion (baseline, 6.72 +/- 0.95 mmol/L; at 4 hours, 17.58 +/- 1.46 mmol/L; p < 0.05). Immunocytochemical staining of the cortex with neuronal nitric oxide synthase-specific monoclonal antibodies (Transduction Labs) revealed increased neuronal nitric oxide synthase expression 6 to 18 hours after HCA. Darkfield analysis demonstrated neuronal nitric oxide synthase localization to neuronal processes with widespread formation of dense plexi of nitric oxide synthase fibers. CONCLUSIONS: We conclude that neurotoxicity after HCA involves a significant, early induction in neuronal nitric oxide synthase expression in neuronal processes leading to widespread augmented nitric oxide production in the brain.

Nitric oxide synthase pathway may mediate human natural killer cell cytotoxicity.

The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L-arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell-mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L-arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell-mediated killing in DM. When all amino acids except L-arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L-arginine enhanced the NK cell activity in a dose-dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N-monomethyl-L-arginine (L-NMMA) inhibited NK cytolytic activity in DM supplemented with L-arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L-arginine on human NK cell-mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L-NMMA gave a dose-dependent reduction in NO generation as well. The nitrite and citrulline production dose-dependently correlated with not only the concentration of L-arginine in the cultivation medium, but also the enhanced NK cell-mediated cytolysis. Taken together, these findings could define a L-arginine/NO-linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell-mediated target cell killing took place in a L-arginine-free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO-dependent and -independent processes.