Flow injection chemiluminescence determination of 2-methoxyestradiol based on inhibition of luminol-potassium ferricyanide reaction.

A novel flow-injection chemiluminescence (FI-CL) method is described for the determination of 2-methoxyestradiol (2-ME). The method is based on the inhibitory effect of 2-ME on the CL reaction of luminol and potassium ferricyanide in alkaline solution. Under optimal conditions, net CL intensity was proportional to 2-ME concentration in synthetic and mouse plasma samples. Corresponding linear regression equations were 8.0 x 10(-9) -1.0 x 10(-7) g/mL for synthetic samples and 2.0 x 10(-9) -1.0 x 10(-7) g/mL for plasma samples. Detection limit for synthetic samples and limits for quantification of plasma samples were 8.4 x 10(-10) g/mL (3σ) for synthetic samples and 4.0 x 10(-9) g/mL for mouse samples. A complete analysis was performed for 60 s, including washing and sampling, resulting in a throughput of ≈ 60/h. The proposed method was applied for the determination of 2-ME in synthetic and mouse plasma samples. Percentage recoveries were 101.0-102.8% and 98.0-105.0%, respectively. A possible mechanism responsible for CL reaction is proposed.

Reactions of the flavin mononucleotide in complex I: a combined mechanism describes NADH oxidation coupled to the reduction of APAD+, ferricyanide, or molecular oxygen.

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory chain enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. Alternatively, NADH oxidation, by the flavin mononucleotide in complex I, can be coupled to the reduction of hydrophilic electron acceptors, in non-energy-transducing reactions. The reduction of molecular oxygen and hydrophilic quinones leads to the production of reactive oxygen species, the reduction of nicotinamide nucleotides leads to transhydrogenation, and "artificial" electron acceptors are widely used to study the mechanism of NADH oxidation. Here, we use a combined modeling strategy to accurately describe data from three flavin-linked electron acceptors (molecular oxygen, APAD(+), and ferricyanide), in the presence and absence of a competitive inhibitor, ADP-ribose. Our combined ping-pong (or ping-pong-pong) mechanism comprises the Michaelis-Menten equation for the reactions of NADH and APAD(+), simple dissociation constants for nonproductive nucleotide-enzyme complexes (defined for specific flavin oxidation states), and second-order rate constants for the reactions of ferricyanide and oxygen. The NADH-dependent parameters are independent of the identity of the electron acceptor. In contrast, a further flavin-linked acceptor, hexaammineruthenium(III), does not obey ping-pong-pong kinetics, and alternative sites for its reaction are discussed. Our analysis provides kinetic and thermodynamic information about the reactions of the flavin active site in complex I that is relevant to understanding the physiologically relevant mechanisms of NADH oxidation and superoxide formation.

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

Propranolol antagonizes hypotension induced by alpha-blockers but not by sodium nitroprusside or methacholine.

A pressor response has been observed with propranolol, a nonselective beta-adrenoceptor antagonist, in animals given a nonselective alpha-adrenoceptor antagonist. This study investigates whether a pressor response to propranolol occurs in conscious unrestrained rats following a hypotensive response induced by phentolamine (nonselective alpha-antagonist), prazosin (selective alpha 1-antagonist) and (or) rauwolscine (selective alpha 2-antagonist), sodium nitroprusside (smooth muscle relaxant), or methacholine (muscarinic agonist). The rats were subjected to a continuous infusion of a hypotensive agent or normal saline followed by i.v. injection of propranolol. The infusion of phentolamine significantly decreased mean arterial pressure (MAP). Subsequent injection of propranolol restored MAP to the control level. Prazosin and rauwolscine each caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a significant decrease in MAP. Subsequent injection of propranolol caused a large pressor response which increased MAP to 20% above control MAP prior to the administration of drugs. Nitroprusside or methacholine each caused a significant decrease in MAP, but the hypotension was not antagonized by propranolol. The concurrent infusions of a low dose of nitroprusside and prazosin caused a significant decrease in MAP which was reversed by propranolol. The infusion of saline did not alter MAP, and propranolol did not cause a pressor response. It is concluded that propranolol antagonizes the hypotensive effect of an alpha-blocker but not that of sodium nitroprusside or methacholine. Our results suggest the presence of a specific interaction between alpha- and beta-antagonists.

Sodium thiosulphate decreases blood cyanide concentrations after the infusion of sodium nitroprusside.

Plasma and red cell cyanide, and plasma thiocyanate, concentrations were measured in 30 patients undergoing elective nitroprusside-induced hypotension. One randomly selected group (n = 15), who received 0.21-0.70 mg kg-1 over periods of 50-160 min, were given a bolus of sodium thiosulphate 10.6-38.5 mg kg-1 immediately on cessation of the nitroprusside administration. The other group, who received infusions of 0.11-0.85 mg kg-1 for periods of 59-197 min, received no antidote. Cyanide concentrations, expressed as a percentage of the immediate post-infusion values, were significantly lower in the treated group in all subsequent blood samples (at 10, 30 and 60 min; plasma cyanide P less than 0.05; red cell cyanide P less than 0.001). Improved cyanide metabolism was further demonstrated by a sharp increase in mean plasma thiocyanate concentration (P less than 0.05) in the group receiving the antidote.

Propranolol prevents hemodynamic and humoral events after abrupt withdrawal of nitroprusside.

Hemodynamic and humoral events after intraoperative discontinuation of nitroprusside were studied in subjects without and with pretreatment with intravenous propranolol, 0.1 mg X kg-1. Nitroprusside-induced hypotension was associated with increases in heart rate, cardiac output, plasma renin activity (PRA), and catecholamine levels; these changes were prevented by propranolol. In subjects pretreated with propranolol, dose requirements of nitroprusside for hypotension of comparable degree and duration decreased 40%. On discontinuation of nitroprusside, mean systemic pressure rose to 100.2 mm Hg--a level higher than prehypotension and awake values--because of increased systemic vascular resistance. Hemodynamic events were associated with persistent elevations of PRA and catecholamine levels. These rebound changes were maximal 15 min after nitroprusside withdrawal and returned to control levels 30 to 60 min later. Pretreatment with propranolol completely prevented rebound hemodynamic events after nitroprusside. Persistent elevations of PRA and catecholamine levels after nitroprusside action subsided were responsible for the effects of withdrawal.

Effect of cyanide on nitrovasodilator-induced relaxation, cyclic GMP accumulation and guanylate cyclase activation in rat aorta.

The effects of sodium cyanide on relaxation, increases in cyclic GMP accumulation and guanylate cyclase activation induced by sodium nitroprusside and other nitrovasodilators were examined in rat thoracic aorta. Cyanide abolished nitroprusside-induced relaxation and the associated increase in cyclic GMP levels. Basal levels of cyclic GMP and cyclic AMP were also depressed. Reversal of nitroprusside-induced relaxation by cyanide was independent of the tissue level of cyclic GMP prior to addition of cyanide. Incubation of nitroprusside with cyanide prior to addition to aortic strips did not alter the relaxant effect of nitroprusside. Sodium azide-, hydroxylamine-, N-methyl-N'-nitro-N-nitrosoguanide-, nitroglycerin- and acetylcholine-induced relaxations and increased levels of cyclic GMP were also inhibited by cyanide. Relaxations induced by nitric oxide were also inhibited by cyanide, although the relaxation with the low concentration of nitric oxide employed was not accompanied by detectable increases in cyclic GMP. Relaxation to 8-bromo-cyclic GMP was essentially unaltered by cyanide; however, isoproterenol-induced relaxation was inhibited. Guanylate cyclase in soluble and particulate fractions of aorta homogenates was activated by nitroprusside and the activation was prevented by cyanide. The present results suggest that cyanide inhibits nitrovasodilator-induced relaxation through inhibition of guanylate cyclase activation; however, cyanide may also have nonspecific effects which inhibit relaxation.

Cyanide prevents the inhibition of platelet aggregation by nitroprusside, hydroxylamine and azide.

Sodium cyanide (CN-) in concentrations of 10 uM or more prevented the inhibition of epinephrine (2.5 uM) and of ADP (4.0 uM) induced primary and secondary aggregation brought about by 10 uM sodium nitroprusside (SNP). Cyanide alone in the same concentration had no effect on platelet aggregation induced by epinephrine or ADP. Even when the addition of CN- was delayed for as long as 9 min after epinephrine and SNP, it immediately reversed the SNP block and initiated a bimodal wave of aggregation. The effect of CN- on SNP inhibition of platelet aggregation appears to be competitive and reversible. Although they are less potent inhibitors of platelet aggregation than SNP, the effects of hydroxylamine (HA) and azide were also prevented by SNP. In our hands, sodium nitrite did not inhibit platelet aggregation consistently. The inhibitory effects of glyceryl trinitrate, papaverine and nitric oxide hemoglobin on platelet aggregation were not prevented by CN-. These interactions probably have no significance in vivo, but they indicate that SNP, HA and azide act on platelets and on vascular smooth muscle by similar or identical biochemical mechanisms. They also suggest that there are at least two sub-classes of so-called nitric oxide vasodilators. The effect of CN- may be mediated through an inhibition of the formation of nitric oxide from SNP, HA and azide.

Failure of cyanide to antagonize sodium nitroprusside in vivo in dogs.

The role of cyanide (CN) in the mechanism of action of sodium nitroprusside (SNP) has not been well defined. Some authors consider accumulation of CN in muscle to be the principal factor responsible for resistance to SNP when given to induce hypotension. To test this hypothesis, SNP was infused in 12 anesthetized dogs. Mean aortic blood pressure was reduced 50% and dogs were maintained at this pressure for 5 minutes. After recovery, eight of the dogs (group 1) received a potassium cyanide (KCN) infusion at a rate of 0.0125 mg/kg/min for 180 minutes; the other four dogs (group 2) were given saline solution and served as controls. After 180 minutes, all 12 dogs received a second infusion of SNP in the same dose as their first. No significant differences in blood pressure or heart rate were observed between groups. Left ventricular end-diastolic pressure (LVEDP) and dP/dt were elevated following KCN; however, after the second SNP infusion these parameters decreased in both groups. Cardiac output (Qt) decreased to 55% of baseline during the first SNP infusion and showed no significant changes during the second SNP infusion, but total peripheal resistance decreased significantly during both SNP infusions. Although whole-blood CN levels and gracilis muscle tissue CN concentrations were significantly greater (p < 0.05) in group 1 after KCN, the cardiovascular effects of SNP were not significantly altered by these increases in KCN, the cardiovascular effects of SNP were not significantly altered by these increases in CN. The authors conclude that resistance to SNP in induced hypotension is not the result of direct effects of CN on smooth muscle.

Sodium cyanide antagonism of the vasodilator action of sodium nitroprusside in theisolated rabbit aortic strip.

Resistance to sodium nitroprusside (SNP) is uncommon, but its occurrence has led to massive overdoses of SNP and sometimes death. To examine the mechanism responsible for resistance, aortic smooth muscle strips were prepared and dose-response curves for norepinephrine (NE) obtained. SNP alone caused a shift of the dose-response curve for NE to the right. However, this shift was less when the strips were exposed to both SNP and sodium cyanide (CN-). When CN- alone was added to the aortic strips, the response to NE was unchanged. In a further group of aortic muscle strips first contracted with NE and then relaxed with SNP, the addition of CN- caused the muscles to contract again. It is concluded that CN- antagonizes the action of SNP in vitro, and that this antagonism is specific for SNP.