Acetyl-L-carnitine modulates glucose metabolism and stimulates glycogen synthesis in rat brain

A rapid and accurate voltammetric method for the quantitative determination of 2-mercapto-5-phenylammino-1,3,4-thiadiazole (MPATD) with carbon paste electrodes (CPE) has been developed. The study was made by cyclic voltammetry between -0.4 and +0.6 V with 50 mV s(-1) sweep rate in aqueous solution. After successive oxidation/reduction cycles we found a total oxidation of MPATD at +0.45 V. As the compound is oxidated, the reduction current peak increases at +0.13 V, indicating an irreversible process. Following only the oxidation process in the -0.1 to +0.6 V range, we investigated the optimum scan rates at different current densities and pH values (realised with buffers, pH between 1.0 and 10.0) with CPE versus Ag/AgCl reference electrode using linear sweep voltammetry. We found a good linear relation between the current peak height and concentration in a 2.5 x 10(-9)-1.25 x 10(-7) mol ml(-1). This method allows the quantitative detection of the MPATD as it or from dosage forms and biological media.     

铁-偶氮氯膦-I-硫脲配合物的极谱吸附波及其应用

铁-偶氮氯膦-I-硫脲配合物在0.04 mol/L的Tris-HCl缓冲溶液中(pH9.0±0.1)有一灵敏的吸附波,峰电位在-0.712 V(vs. SCE)左右,该波的二阶导数峰电流I^″_p与铁浓度在1.0×10^-7～1.0×10^-9 mol/L范围内呈线性关系(R²= 0.999 1),检出限为5.0×10^-10 mol/L(S/N=3)。经多种电化学方法证明,该吸附波为配合物吸附波,其电极过程为不可逆过程,电极反应电子转移数为1。考察了多种离子对峰电流I^″_p的影响。所拟方法用于水样中微量铁的测定,测定结果的标准偏差为0.22%～2.7%,加标回收率在97.0% ~103.6%之间。     

Trace Determination of Scandium Using Adsorption Voltammetry of Mix-Polynuclear Complex of Scandium-Calcium-Alizarin Red S at Carbon Paste Electrode

Scandiumisatypicaldispersalelement,whichiswidelydispersedinnature .Ithasbeenwidelyusedinthefieldsofmetallurgicalindustry ,chemicalindustry ,electronicsandelectrotechnics ,energysources ,radia tionchemistry ,etc .Scandium ,likeyttriumandmanyotherrareearthelements ,cannotbeeasilydeterminedbyconventionalvoltammetry ,evenifatamercuryelectrodewithhighhydrogenoverpotentialandinalka linesolution ,becauseitsreductionpotentialisclosetothereductionpotentialofhydrogen[1] .Advancingofthepolarographiccompl…     

Determination of penicillamine or tiopronin in pharmaceutical preparations by flow injection analysis

Two flow injection analysis (FIA) methods, using spectrophotometric detection, are proposed for the determination of penicillamine or tiopronin [N-(2-mercaptopropionylglycine)]. The procedures are based on the formation of yellow complexes between these thiol-containing drugs and Pd(II), in a 1 M or 0.25 M HCl medium, respectively. With peak height as a quantitative parameter, penicillamine is determined over the range 1.0 x 10(-5)-7.0 x 10(-4) M; for tiopronin the range is 1.0 x 10(-5)-6.0 x 10(-4) M. The methods have been applied to the routine determination of the drugs in pharmaceutical preparations.     

Electrochemical studies of S-nitrosothiols

S-nitrosothiols (RSNO), such as SNAP, GSNO, and Glc-SNAP-1 exhibited a single, totally irreversible, diffusion controlled reduction peak at potentials of -0.97 V, -0.98 V and -0.91 V, respectively, vs. Ag/AgCl (3 M NaCl) reference electrode. This corresponded to the nitric oxide (NO) release from the S-nitrosothiols. A possible mechanism is proposed for the reduction process.     

Spectroscopic and electrochemical evaluation of a perfluorosulfonated ionomer and its gel as preconcentrating media for [ReI(DMPE)3]+, where DMPE = 1,2-bis(dimethylphosphino)ethane

The interaction of [ReI(DMPE)3]+, where DMPE = 1,2-bis(dimethylphosphino)ethane, a nonradioactive analogue of a heart imaging agent, with Nafion gel, which is Nafion plasticized with tri-n-butyl phosphate, has been evaluated spectroscopically and electrochemically. Thin-layer spectroelectrochemistry on the rhenium compound yields a linear Nernst plot with an n value of 0.99 and E degree' of 0.049 V vs Ag/AgCl. The electrochemistry is consistent with a reversible one-electron transfer between the mono- and dicationic forms of the complex. The UV-visible spectrum of electrogenerated [ReII(DMPE)3]2+ is identical to that obtained by air oxidation of [ReI(DMPE)3]+. Thin, free-standing films of Nafion gel and Nafion that were sufficiently clear to record visible spectra were cast. Spectroscopic measurement of the partitioning of [ReI-(DMPE)3]+ from aqueous solution into these films shows a more rapid uptake of the complex by the Nafion gel. Preconcentration factors into Nafion gel and Nafion were 350 and 50, respectively, after 4 h of soaking. Cyclic voltammetry of 1.0 x 10(-4)-1.0 x 10(-7) M (ReI(DMPE)3]+ in 0.15 M supporting electrolyte aqueous solution at bare gold and spectroscopic graphite electrodes suggests that the complex adsorbs to these electrodes. By comparison, the well-defined cyclic voltammograms at Nafion gel-modified electrodes exhibit diffusion-controlled behavior. The formal reduction potential at Nafion gel-modified electrodes is shifted positively compared to bare electrodes. A current enhancement of approximately 4 was observed at Nafion gel-modified spectroscopic graphite over a bare electrode. A calibration plot of peak current for differential pulse voltammetry vs concentration at Nafion gel-modified spectroscopic graphite was linear in the 10(-7)-10(-5) M concentration range, with a detectable signal down into the 10(-9) M range.     

Effect of nomegestrol acetate on estrone-sulfatase and 17beta-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E1S: 5 x 10(-9)M), nomegestrol acetate blocked very significantly the conversion of E1S to E2. In the MCF-7 cells, using concentrations of 5 x 10(-6)M and 5 x 10(-5) M of nomegestrol acetate, the decrease of E1S to E2 was, respectively, -43% and -77%. The values were, respectively, -60% and -71% for the T-47D cells. Using E1S at 2 x 10(-6) M and nomegestrol acetate at 10(-5) M, a direct inhibitory effect on the enzyme of -36% and -18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E1: 5 x 10(-9)M) this estrogen is converted in a great proportion to E2. Nomegestrol acetate inhibits this transformation by -35% and -85% at 5 x 10(-7)M and 5 x 10(-5)M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, -48%, at 5 x 10(-5)M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17beta-HSD activities which converts E1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E2 can open new clinical possibilities in breast cancer therapy.     

Ouabain-like factors in human urine: identification of a Na-K-ATPase inhibitor as vanadium-diascorbate adduct

We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of > 30g NaCl/day 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Sephadex G-10, one of three OLFs eluted in the early subfractions FIV/1-2 close to H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with Mr around 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF- (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 cross-reacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-K-ATPase with IC50 of around 1.5 x 10(-5) M and 1.5 x 10(-4) M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance (1H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium (V) diascorbates (Mr 403) with similar elution times from RP-HPLC as OLF-2. They inhibited the enzyme in its E2-configuration with IC50 of 9 x 10(-5) M and 2 x 10(-6) M for V(IV)- and V(V)-diascorbate, respectively. OLF-1, OLF-2 and V-diascorbate raise intracellular free calcium in inner medullary collecting duct and vascular smooth muscle cells which also contract in vitro. V-diascorbate was also natriuretic in a bioassay. We suggest that V-diascorbates represent one of several OLFs excreted in human urine.     

Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

Time course of antibody response to tetanus toxoid and pneumococcal capsular polysaccharides in patients infected with HIV

The methods of measuring the affinity constants of anti-HIV-1 p17 monoclonal antibodies (MAbs) using the double antibody methods in the liquid phase and the biomolecular interaction analysis by BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden) were compared. MAbs, HyHIV1-6, recognizing residues 12-29 (P12-29) of p17 and the naive protein, p17, were used. The kinetic association constants (KAs) obtained using the double antibody method were 2.40 x 10(7) - 1.40 x 10(8)M(-1) for P12-29, and 4.80 x 106 - 1.80 x 10(7) M(-1) for p17. In the BIAcore system where P12-29 or p17 was used as immobilized antigens onto the sensorchip, the KAs were 1.57 x 10(9) - 4.81 x 10(9) M(-1) for P12-29, and 1.52 x 10(9) - 1.21 x 10(10) M(-1) for p17. On the other hand, when MAbs were immobilized onto the sensorchip and P12-29 or rp17 was used as analyte, the KAs for P12-29 and p17 were in the region 3 x10(8) - 3 x 10(9), 1 x 10(8) - 3 x 10(9) M(-1), respectively. These data show that the KAs were higher than those obtained using the double antibody method, however, no significant difference could be observed. Moreover, the KAs obtained for p17 using MAbs as ligand were similar for BIAcore and the double antibody method except for HyHIV2. Therefore, the BIAcore system can be used for the affinity measurement instead of the double antibody method.     

Properties of methemoglobin reductase and kinetic study of methemoglobin reduction

A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0     

The cyano-NNO-azoxy function in the design of an irreversible label for alpha 1 adrenoreceptors

A potential alpha 1-adrenergic irreversible antagonist 6, containing the cyano-NNO-azoxy function was synthesized and tested. The effects of norepinephrine on rat thoracic aorta were irreversibly blocked by this compound at the concentration of 1 x 10(-5) M after 60 minutes. Binding studies showed that 6, at 1 x 10(-6) M, did not modify the KD of Prazosin and caused a 30% decrease of the Bmax. Substitution in 6 of the bis (2-chloroethyl)amino moiety for the cyano-NNO-azoxy function afforded 7 which behaves as an irreversible antagonist able to change KD of Prazosin without influencing Bmax.     

Health professionals'' responses to multiple sclerosis and motor neurone disease

The effect of vasopressin and oxytocin on the contractile activity of preparations isolated from the feline gastric corpus wall was investigated. Vasopressin (1.5 x 10(-9)-2.1 x 10(-7) M), but not oxytocin, evoked concentration-dependent tonic contractions only of longitudinal muscle strips. At the same time, vasopressin (1.5 x 10(-9)-2.1 x 10(-7) M) potentiated the magnitude of amplitudes, but not the frequency, of spontaneous contractions. Both the vasopressin V1 receptor antagonist d(CH2)5-(Me)2-Tyr-AVP and the predominantly vasopressin V2 receptor antagonist d(CH2)5, D-Ile2, Ile4-AVP, the non-selective muscarinic receptor antagonist, atropine, the predominantly selective muscarinic M1 receptor antagonist, pirenzepine, the predominantly selective muscarinic M2 antagonist, methoctramine, the predominantly selective muscarinic M3 receptor antagonist, para-fluoro-hexahydro-siladifenidol, and the calcium channel blocker, nifedipine, but not the ganglion blocking agent, mecamylamine, depressed or blocked the tonic contractions induced by vasopressin. Among the antagonists, only atropine and nifedipine inhibited the spontaneous contractions. On the other hand, the anticholinesterase, physostigmine, potentiated both the vasopressin-induced tonic and spontaneous contractions. With regard to the receptors, the vasopressin-induced tonic contractions are mediated at least in part through vasopressin V1 and V2 receptors, non-selective muscarinic and selective muscarinic M1, M2 and M3 receptors. The increase in amplitudes of spontaneous contractions is mediated only via-nonselective muscarinic receptors. Vasopressin receptors appear to be located mostly pre-synaptically, although the direct effect of vasopressin on post-synaptic receptors cannot be excluded. The pA2 values suggests rather V1a than V1b vasopressin receptor subtype involvement in tonic contractions vasopressin had produced. The tonic as well as spontaneous contractions are calcium-dependent. In addition, these results point to the existence of non-selective muscarinic receptors, which participate in the regulation of both tonic and spontaneous contractions, while muscarinic M1, M2 and M3 receptors subserve only the tonic contractions.     

Low-level laser therapy in osteoarticular diseases in geriatric patients]

The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of concurrent homologous interactions of heavy and light chain variable domains limits the heterologous interaction leading to productive formation of the Fv. To address the possible role of light chain dimerization in this phenomenon, the Gln38 residue at the dimer interface of an immunoglobulin light chain variable domain (VL) was replaced by charged amino acids. The effects of these mutations on VL homodimer formation were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL homodimerization was observed in three of the four mutants, Q38R, Q38D and Q38K. The association constants for the Q38R and Q38D homodimers were 1.2 x 10(4) and 3.2 x 10(3) M(-1), respectively. This corresponded to a 20-75-fold reduction in the homodimer association constant relative to the wild-type VL, which had an association constant of 2.4 x 10(5) M(-1). Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type VL. The potential for charged residues to facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R V(L)s, but with an association constant of 4.7 x 10(4) M(-1), approximately fivefold lower than that obtained for homodimerization of the native V(L). In addition, replacement of the neutral, solvent-accessible Gln38 residue with either Asp or Arg was found to be significantly destabilizing. These results suggest that charged residues could be introduced at immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless, these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of particular variable domains.     

Kinetic characterization of myosin head fragments with long-lived myosin.ATP states

We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.     

Contribution of PO2, P50, and Hb to changes in arteriovenous O2 content during exercise in heart failure

Arteriovenous O2 content (a-vCO2) differences increase during exercise in normal subjects through several mechanisms including PO2, O2 pressure at which hemoglobin (Hb) is half saturated with O2 (P50), and Hb concentration changes. The present study was undertaken to evaluate how much these biochemical changes are relevant to a-vCO2 difference through exercise in patients with heart failure. Twenty-seven patients with congestive heart failure [10 patients in functional class A (peak exercise O2 uptake >20 ml x kg-1 x min-1), 9 in class B (20-15 ml x kg-1 x min-1), and 8 in class C (15-10 ml x kg-1 x min-1)] underwent a cardiopulmonary exercise test with once-per-minute simultaneous blood sampling from the pulmonary and systemic arteries for determination of Hb, PO2, PCO2, pH, O2 content (CO2), Hb saturation and lactic acid (pulmonary artery only), and calculation of P50. Analysis of data was done at six exercise stages: the first at rest, the last at peak exercise, and the second to the fifth at one-, two-, three-, and four-fifths of O2 consumption increase. a-vCO2 difference at peak exercise was 14.3 +/- 2.1, 16.9 +/- 2.4, and 14.7 +/- 2.1 (SD) ml/dl in class A, B, and C patients, respectively. The contribution of Hb, P50, and PO2 changes to the increments of a-vCO2 difference during exercise was 21, 17, and 63%, respectively; the only interclass difference observed was for P50, which plays a greater role in a-vCO2 difference in class A. Hb changes act mainly at the arterial site, whereas P50 and PO2 act at the venous site. Hb increase was constant through the test, venous P50 increase was greater above anaerobic threshold, and venous PO2 reduction was most remarkable at the onset of exercise; in class C patients, no venous PO2 change was recorded in the second half of exercise. Thus a-vCO2 difference increase during exercise is notable in patients with heart failure but unrelated to the severity of the syndrome. Hb, P50, and, to the greatest degree, PO2 changes participate in the increment of a-vCO2 difference. In class C patients, the lack of PO2 reduction in the second half of exercise suggests the achievement of a "whole body critical venous PO2."     

钯-硫脲-碘酸钾体系极谱络合吸附波及其应用

钯(Ⅱ）-硫脲-碘酸钾络合物在pH8.20 NH3·H2O-NH4Cl缓冲溶液中有一灵敏的吸附波,峰电位在-0.42 V （vs. SCE）左右,该波的二阶导数峰峰电流与钯质量浓度在0.60～2.20 μg/L 范围内呈线性关系（r = 0.998 8）,检出限为0.30μg/L。经多种电化学方法证明,该吸附波为络合物吸附波,其电极过程为不可逆过程。考察了多种离子对峰电流Ip'的影响。当测定0.1μg/L钯溶液时除Mo（Ⅵ）、Pb2+、Cd2+、Mn2+离子的允许量（分别为0.01μg/L、0.08μg/L、0.1μg/L、0.2μg/L）较低外,其他被试验的阳离子允许量都较高;但是,当溶液中的Mo(Ⅵ)(Ⅵ)、Pb2+、Cd2+、Mn2+离子超过允许量时可以加入酒石酸和二巯基丙醇进行掩蔽以消除干扰。所拟方法用于铝合金中微量钯（Ⅱ）的测定,测定结果的相对标准偏差为2.1%, 加标回收率在95% ~ 102%之间。     

Selective anesthetic inhibition of brain nitric oxide synthase

BACKGROUND: It has been postulated that nitric oxide (NO) is a neurotransmitter involved in consciousness, analgesia, and anesthesia. Halothane has been shown to attenuate NO-mediated cyclic guanosine monophosphate accumulation in neurons, and a variety of anesthetic agents attenuate endothelium-mediated vasodilation, suggesting an interaction of anesthetic agents and the NO-cyclic guanosine monophosphate pathway. However, the exact site of anesthetic inhibitory action in this multistep pathway is unclear. The current study examines effects of volatile and intravenous anesthetic agents on the enzyme nitric oxide synthase (NOS) in brain. METHODS: NOS activity was determined by in vitro conversion of [14C]arginine to [14C]citrulline. Wistar rats were decapitated and cerebellum quickly harvested and homogenized. Brain extracts were then examined for NOS activity in the absence and presence of the volatile anesthetics halothane and isoflurane, and the intravenous agents fentanyl, midazolam, ketamine, and pentobarbital. Dose-response curves of NOS activity versus anesthetic concentration were constructed. Effects of anesthetics on NOS activity were evaluated by analysis of variance. RESULTS: Control activities were 57.5 +/- 4.5 pmol.mg protein-1.min-1 in the volatile anesthetic experiments and 51.5 +/- 6.5 pmol.mg protein-1.min-1 in the intravenous anesthetic experiments. NOS activity was not affected by ketamine (< or = 1 x 10(-4) M), pentobarbital (< or = 5 x 10(-5) M), fentanyl (< or = 1 x 10(-5) M), and midazolam (< or = 1 x 10(-5) M). Halothane decreased NOS activity to 36.7 +/- 2.5 (64% of control, P < 0.01 from control), 23.8 +/- 4.3 (41%, P < 0.01 from control and < 0.05 from 0.5% halothane), 25.2 +/- 3.8 (44%, P < 0.01 from control and < 0.05 from 0.5% halothane), and 19.7 +/- 2.8 (34%, P < 0.01 from control and < 0.05 from 0.5% halothane) pmol.mg protein-1.min-1 at 0.5, 1.0, 2.0, and 3.0% vapor. Isoflurane decreased NOS activity to 48.9 +/- 6.1 (85% of control), 46.0 +/- 3.2 (80%, P < 0.05 from control), 40.3 +/- 5.1 (70%, P < 0.05 from control), and 34.2 +/- 4.0 (60%, P < 0.05 from control and 0.5% and 1.0% isoflurane) pmol.mg protein-1.min-1 at 0.5, 1.0, 1.5, 2.0% vapor, respectively. CONCLUSIONS: Volatile anesthetics inhibit brain NOS activity in an in vitro system, but the intravenous agents examined have no effect at clinically relevant concentrations. This inhibition suggests a protein-anesthetic interaction between halothane, isoflurane, and NOS. In contrast, intravenous agents appear to have no direct effect on NOS activity. Whether intravenous agents alter signal transduction or regulatory pathways that activate NOS is unknown.