Pelvic venous congestion induces lower urinary tract dysfunction in rats.

Pelvic venous congestion (PC) is thought to be related to several diseases of the lower urinary tract (LUT). We examined the characteristics of the LUT in rats with PC. To create PC, female rats were anesthetized with isoflurane, and the bilateral common iliac veins and bilateral uterine veins were ligated. At 1-8 weeks after either ligation or sham surgery, we performed cystometry with or without administration of carbazochrome sodium sulfonate hydrate or propiverine hydrochloride, histologic examination of the bladder, blood flow imaging, assessment of locomotor activity, measurement of urinary 8-hydroxydeoxyguanosine (8-OHdG) and nitric oxide metabolites (NOx), and the Evans blue dye extravasation test. PC elevated frequency of urination after 2-6 weeks, and caused a decrease of spontaneous locomotor activity. In addition, there was a decrease of bladder blood flow, an increase of bladder vascular permeability, an increase of urinary 8-OHdG, a decrease of urinary NOx, and mild inflammatory changes of the bladder. In rats with PC, frequency of urination was normalized by administration of propiverine or carbazochrome. Rats with PC may be used as a model of PC associated with high frequency of urination, and this model may be useful when developing treatment for LUT symptoms associated with PC.

Dynamic agent of an injectable and self-healing drug-loaded hydrogel for embolization therapy.

Embolic agents are crucial for trans-catheter arterial embolization (TAE) in the treatment of various unresectable malignant tumors. Although solid particles, liquid oils, and some polymeric hydrogels have proved their capacities for embolic therapies, the low efficiency, time sensitivity, and cytotoxicity are still considered as challenges. In this study, we developed a three-component dynamic self-healing hydrogel to overcome these limitations. With the help of the Schiff-base bonding, both glycol-chitosan and carbazochrome, containing amine groups, react with dibenzaldehyde-terminated poly(ethylene-glycol) (DF-PEG), forming the dynamic self-healing hydrogels under a mild condition within 200 s. 1H NMR and rheology test were used to characterize the Schiff-base formation and mechanical strength. Controlled-release of carbazochrome from different gelator concentrations of DF-PEG was also studied. Furthermore, in vivo evaluation of the embolization on rats showed the superior embolic effects of the injectable and self-healing hydrogel. Therefore, this new dynamic agent demonstrated the potential for application as a simple, inexpensive, and tunable embolic agent for cancer treatment and drug delivery system.

The chemical interaction between adrenochrome, three different classes of antipsychotic drugs and metabolites of the kynurenine pathway.

Two kynurenine metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid, are known to inhibit melanin polymer formation in in vitro reactions catalyzed by tyrosinase. The present study expands that finding to include inhibition of chlorpromazine-stimulated melanin formation from the endogenous melanin precursor adrenochrome. Several kynurenine pathway metabolites tested had no measurable effect on the reaction: tryptophan, kynurenine, kynurenic acid, quinolinic acid and nicotinic acid. However, at a concentration of 0.5mM in a pH 7.4 reaction mix, 3-hydroxykynurenine exerted~72% inhibition on product formation and the same concentration of 3-hydroxyanthranilic acid caused complete inhibition. Two other classes of antipsychotic drugs were evaluated in this paradigm, represented by olanzapine and minocycline. Although the adrenochrome reaction of both drugs was strongly inhibited by 3-hydroxyanthranilic acid, 3-hydroxykynurenine inhibited product formation from only the minocycline reaction. The results are discussed in terms of the well-studied kynurenine pathway upregulation in psychotic disorders and how such upregulation may either influence the efficacy of antipsychotic drug treatment or relate to the mechanism of action of these drugs.

High-dose tranilast administration to rats creates interstitial cystitis-like symptoms with increased vascular permeability.

AIM: We investigated whether the high-dose administration of tranilast could be used to create an animal model of interstitial cystitis (IC). Then, we used this model to assess the relationship between IC and changes in the vascular permeability of the bladder. MAIN METHODS: Female rats were divided into the following 4 groups: a control group, a tranilast group, a carbazochrome group and a combination (tranilast+carbazochrome) group. Continuous cystometry, bladder distension, and the Evans blue dye extravasation test were performed 4weeks after drug administration. Locomotor activity, the plasma TGF-ß1 level, and collagen fibers in the bladder wall were also examined in the control and tranilast groups. KEY FINDINGS: The interval between bladder contractions was shorter and the leakage of Evans blue dye into the bladder wall was greater in the tranilast group than in the control group. Glomerulations of the bladder wall after bladder distention and thinning of the collagen fiber layer in the bladder were observed in the tranilast group. Locomotor activity in darkness and the plasma TGF-ß1 level were both lower in the tranilast group than in the control group. In the combination group, the leakage of Evans blue dye was greater than in the control group; however, it was less prominent than in the tranilast group. SIGNIFICANCE: These results suggest that high-dose administration of tranilast to rats can create an IC-like rat model and that an increase in the vascular permeability of the bladder wall may be one cause of IC symptoms.

Carbazochrome sodium sulfonate (AC-17) reverses endothelial barrier dysfunction through inhibition of phosphatidylinositol hydrolysis in cultured porcine endothelial cells.

The effect of carbazochrome sodium sulfonate (AC-17), a hemostatic drug with capillary stabilising action, on the endothelial barrier dysfunction induced by a variety of vasoactive substances or agents that increase the vascular permeability was investigated in the monolayers of cultured porcine aortic endothelial cells (PAECs). The endothelial barrier function was determined by the transendothelial transport of albumin-conjugated Evans blue. AC-17 (0.1-1 M) reversed the barrier dysfunction induced by tryptase, thrombin and bradykinin without affecting the endothelial permeability enhanced by Ca(2+) ionophores such as ionomycin and A23187 or phorbol 12-myristate 13-acetate. Immunofluorescence analysis showed that AC-17 reversed the tryptase-induced formation of actin stress fibres and disruption of VE-cadherin in PAECs. On the other hand, AC-17 (0.1-10 M) reduced concentration-dependently the enhancement of [(3)H]inositol triphosphate formation from [(3)H]myo-inositol induced by bradykinin and thrombin.Therefore, it is suggested that AC-17 reduces the vascular hyperpermeability induced by a variety of vasoactive agents through inhibition of agonist-induced phosphoinositide hydrolysis.

The fallacy of using adrenochrome reaction for measurement of reactive oxygen species formed during cytochrome p450-mediated metabolism of xenobiotics.

The adrenochrome reaction (oxidation of epinephrine to adrenochrome) has been widely employed as a standard assay for reactive oxygen species, produced under a variety of conditions, including those produced during cytochrome P450 (CYP)-mediated oxidation of substrates such as cyclosporine. However, it has been reported that epinephrine and adrenochrome can be metabolized by hepatic microsomes and that adrenochrome can also be metabolized by NADPH-CYP reductase. Thus, in the present report, we provide evidence that measurement of adrenochrome cannot be used as an index of reactive oxygen species generated during CYP-mediated metabolism of xenobiotics because adrenochrome and its precursor, epinephrine, interact with the CYP enzyme system as substrates and inhibitors. Our results indicated that adrenochrome was moderately stable in phosphate buffer but degraded rapidly (over 50% consumed in less than 2 min) by (cloned and expressed) CYP3A4 and CYP reductase in the presence of NADPH. Furthermore, both epinephrine and adrenochrome were found to be inhibitors of CYP3A4-mediated oxidation of testosterone. Together, these results lead to the conclusion that the use of adrenochrome reaction for measurement of reactive oxygen species formed during CYP3A4-mediated metabolism of xenobiotics is inappropriate.

[Host-guest molecule interaction mechanism of hemostatics with liposomes and red blood cells studied with fluorescence polarimetric method].

The supermolecule compounds of adrenobazone, p-aminomethylbenzoic acid, vitamin K1, 6-amino caproic acid with liposomes and red blood cells were studied by fluorescence polarimetric method. The mechanisms of formation of the supermolecule compounds were examined by fluorescence probe of the link of 1,6-dipheny-1,3,5-hexatriene (DPH) with liposomes which were taken as a model of blood cells. The interaction mechanism of hemostatics with red blood cells was described according the quantitative relationship between polarization value (P) and the microviscosity [formula: see text]. The result showed that the acting force between hemostatics and liposomes or that between hemostatics and red blood cells were mainly supermolecular acting force. The acting force between vitamin K1 and cytomembrane is hydrophobic force and those between adrenobazone, p-aminomethylbenzoic acid, or 6-amino caproic acid and cytomembrane are hydrogen bond or electrostatic force. Under the same drug concentration, all of the four haemostatics can reduce the fluidity of the cell membrane, which benefits blood coagulation. The binding ways of hemostatics with red blood cells was also discussed.

Failure of carbazochrome sodium sulfonate (AC-17) to prevent dengue vascular permeability or shock: a randomized, controlled trial.

OBJECTIVE: We studied the ability of carbazochrome sodium sulfonate (AC-17) to prevent capillary permeability in dengue hemorrhagic fever/dengue shock syndrome. METHOD: A randomized, placebo-controlled trial in 95 children stratified by age and sex was conducted in two hospitals during 1992. AC-17 (n = 45 cases) or B vitamins as placebo (n = 50) were given as a bolus infusion and then as a continuous drip for 24 hours; a total of 300 mg of AC-17 was administered on the first 2 days and 150 mg on the third day. RESULTS: The two groups were comparable in age, sex, duration of illness, and clinical manifestations. No significant difference in shock or pleural effusion was noted between the two groups. Shock developed in 8.9% (4/45) of patients in the AC-17 group and 6% (3/50) in the placebo group (p = 0.44). Pleural effusion was found at 0, 24, 48, and 72 hours after admission in 4.4%, 20%, 31.1%, and 20% in the AC-17 group and 2%, 14%, 28%, and 14% in the placebo group, respectively. CONCLUSION: Administration of AC-17 does not prevent plasma leakage or shock in dengue hemorrhagic fever/dengue shock syndrome.

Substrate specificity for isomerase activity of macrophage migration inhibitory factor and its inhibition by indole derivatives.

Macrophage migration inhibitory factor (MIF) was discovered as a cytokine that inhibits random migration of macrophages and concentrates them at inflammatory loci. We recently reported the tertiary structure of MIF, and revealed its similarity to that of 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, MIF was found to have isomerase activity converting D-dopachrome, a stereoisomer of naturally-occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. In this study, we examined the effects of a series of compounds analogous to D-dopachrome on the enzyme activity to obtain vital information for identification of a natural substrate of MIF. Adrenochrome, lacking a carboxyl group at position 2 of the indolinequinone ring, could not be a substrate. Several indole-ring-containing compounds with a carboxyl group were inhibitory to D-dopachrome isomerase activity, of which indole-3-acrylic acid was the most potent inhibitor, with an inhibitor constant (Ki) of 2.8 mM. 2,3-Indolinedione, which lacks a complete indole ring or a carboxyl group but has carbonyl groups at positions 2 and 3, apparently inhibited the enzyme activity in a competitive or mixed manner with a Ki of 0.9 mM. Taken together, these facts suggest that the 2-carboxyl group of the substrate is essential for interaction with the active site of MIF.

Effects of antimigraine drugs on retinal spreading depression.

It has been suggested that spreading depression may play a role in triggering classical migraine. In this study the retinal spreading depression was used as a pharmacological tool to test the neuronal effects of several common antimigraine drugs. As the chicken retina is void of any blood vessels the observed effects must be of pure neuronal origin. It is shown that propranolol, sumatriptan, methysergide, paracetamol and acetylsalicyclic acid decrease the propagation velocity of retinal spreading depression waves, accelerate the recovery of the optical and electrical signal and reduce the amplitude of the negative potential shift, concomitant with the spreading depression. Barbiturate increases the spreading velocity, and the amplitude of the potential shift. Ergotamine, clonidine, lisuride and iprazochrome have no significant influence on retinal spreading depression.

Carbazochrome sodium sulphonate (AC-17) decreases the accumulation of tissue-type plasminogen activator in culture medium of human umbilical vein endothelial cells.

This study investigated the effect of carbazochrome sodium sulphonate (AC-17) on the accumulation of tissue-type plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) in culture medium of human umbilical vein endothelial cells (HUVEC). HUVEC were cultured in the presence of AC-17, and the concentration of t-PA:Ag and PAI-1:Ag in the medium was measured by an enzyme-linked immunoassay. AC-17, after 48 and 72 h incubation, significantly decreased the t-PA:Ag in the culture medium, while it had no significant effect on the PAI-1:Ag. The result of fibrin zymography was consistent with the result obtained by ELISA. It also revealed that all of the t-PA present in the culture medium formed complexes with PAI-1, indicating that AC-17 did not interfere with t-PA/PAI-1 complex formation. Furthermore, AC-17 did not inhibit the activities of t-PA and plasmin when measured by a fibrin plate method. These results suggest that AC-17 may modulate the fibrinolytic balance of blood through changing the function of endothelial cells, a new aspect of action of AC-17 as a haemostatic drug.

Interaction of serotonin- and dopamine-related neurotoxins with "serotonin binding proteins" in bovine frontal cortex.

Binding of [3H]serotonin and [3H]dopamine to serotonin-binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+. This group recently attributed this effect of Fe2+ to its ability to enhance the oxidation of [3H]serotonin and [3H]dopamine in the presence of dissolved molecular oxygen, and to the ability of the formed oxidation products to bind covalently to cysteine residues of SBP. In this study it is shown that the binding of both ligands is potently inhibited by dopamine as well as by several catecholamine-and serotonin-related neurotoxins: adrenochrome, 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 6-hydroxydopamine and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. In contrast, serotonin can only potently inhibit part (36%) of the [3H]dopamine binding, while 1,2,3,4-tetrahydroisoquinoline is only a weak competitor for both ligands. Potent inhibition by the toxins is associated with the presence of electrophilic centres at the aromatic ring, either of the products themselves (adrenochrome) or of their oxidation products (all other competitors). These findings suggest that "SBP" represent an important target for the Fe(2+)-mediated binding of [3H]-serotonin, [3H]dopamine and related neurotoxins.

Effects of combination of immunomodulators and an adrenochrome derivative on survival of irradiated mice.

PURPOSE: The combined effects of immunomodulators (lithium or OK432) and an adrenochrome derivative (AMM), an agent found to activate granulocyte-macrophage colony stimulating activity, on the survival of irradiated ddY mice is described. METHODS AND MATERIALS: ddY mice at 4-5 weeks old were whole body irradiated with X rays at 8.5 Gy. Sole injection and combined injection of AMM and/or one of the immunomodulators were performed before or after irradiation. Then, survival was monitored daily for 30 days after irradiation. RESULTS: Lithium at 60 mg/kg had no radioprotective effect; rather it accelerated radiation induced death. Sole treatment with AMM (100 mg/kg) had no effect on survival of irradiated mice. However, combination of both drugs caused a slight radioprotection. OK432 (25 KE/kg), which activates a variety of cellular effector cells had radioprotective effect. When combined with AMM, however, it totally lost radioprotective effect. CONCLUSION: Lithium chloride cannot be used as a radioprotector because of its adverse effect. Combination with AMM showed slight radioprotection, but the extent thereof may not be clinically useful. OK432 was proved to be a potent radioprotector. However, combination with AMM should be avoided, since the radioprotection was totally eliminated.

Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy.

PURPOSE: The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate+cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. METHODS AND MATERIALS: Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. Adrenochrome monoaminogluanidine methanesulfonate (AMM) (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard 51Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM+CCC, OK-432 or AMM+CCC+OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. RESULTS: Adrenochrome monoaminoguanidine methanesulfonate+cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. CONCLUSION: Adrenochrome monoaminoguanidine methanesulfonate+cytochrome C may have the potential as a differential modulator of radiosensitivity of normal tissues and of tumors.

Noradrenaline, propranolol and adrenochrome interactions with the dynamic of haemolymph lipids in worker honeybees.

Injection of worker honeybees with noradrenaline, in vivo, depresses the haemolymph levels of phospholipid, steroid and triacylglycerols. The major fatty acids fraction shows large amplitude variations which seem to be dependent on several distinct factors. Adrenochrome exhibits a tendency to induce global hypoglycemic effects without evident correlation with the other hormones, whereas propranolol counteracts the effects of noradrenaline, particularly for phospholipids, steroids and diacylglycerols. The results suggest that the major lipoproteic complex is partly under the control of low specificity adrenoceptors.

Effect of adrenochrome on adenine nucleotides and mitochondrial oxidative phosphorylation in rat heart.

Effects of adrenochrome, an oxidation product of epinephrine, on myocardial energy production were investigated by studying changes in adenine nucleotide content and mitochondrial oxidative phosphorylation activities in the isolated rat heart. Perfusion of the heart with 50 mg/L adrenochrome induced a marked decline in contractile force within 5 min and this was associated with a rapid decline in the myocardial ATP/AMP ratio. A significant decrease in ATP and ATP/ADP ratio as well as a significant increase in ADP and AMP content was observed at 10 min of perfusion with adrenochrome. Furthermore, mitochondrial oxidative phosphorylation activities were unchanged except that an increase in state 4 respiration and a decrease in RCI value were seen in the heart perfused with adrenochrome for 10 min. Autoradiography of the sections from hearts perfused with 14C-adrenochrome revealed the localization of a significant amount of radioactivity on mitochondria. Adrenochrome at concentrations of 20 mg/L or higher was found to inhibit the oxidative phosphorylation activities of heart mitochondria under in vitro conditions. The depressant effects of adrenochrome on mitochondrial oxidative phosphorylation were additive to those seen with calcium. These data suggest that adrenochrome in the presence of excess calcium in the myocardial cell may impair the process of energy production in mitochondria and this may result in contractile failure of hearts exposed to this cardiotoxic metabolite of epinephrine.

The time-dependent inactivation of human brain dihydropteridine reductase by the oxidation products of L-dopa.

Dihydropteridine reductase (DHPR) was irreversibly inactivated in a time-dependent way by incubation with 3,4-dihydroxyphenylalanine (L-dopa). The inactivation was oxygen-dependent; incubation under nitrogen gave partial protection. The inactivation was stimulated by the presence of horse-radish peroxidase/hydrogen peroxide. L-Dopa itself was not an inhibitor of DHPR although dopachrome, the aminochrome formed following oxidation of L-dopa, was a reversible inhibitor of DHPR with an I50 of 0.60 mM. The quinone products of oxidation of L-dopa were responsible for the time-dependent inactivation of DHPR. Adrenochrome also demonstrated a time-dependent inactivation of DHPR. Inactivation by adrenochrome demonstrated a saturation effect suggesting the reversible formation of a complex preceding inactivation. No radiolabel was incorporated into DHPR following inactivation by L-[14C]-dopa. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the presence of a dimer of DHPR. A mechanism of inactivation involving the oxidative coupling of essential thiol groups was proposed to explain inactivation.

[Pharmacological actions of iprazochrome on the vascular system].

The pharmacological actions of iprazochrome (IC) on the vascular system were studied, and the following results were obtained: No death nor abnormal behaviors were observed in acute toxicity tests conducted on male and female mice and rats despite the administration of large doses of IC (10,000 mg/kg, p.o. and 80 mg/kg, i.v., respectively). IC inhibited dose-dependently platelet aggregation in vitro induced by arachidonate and ADP, whereas no effect was observed on ADP-induced respiratory depression in mice, which is closely related to platelet aggregation in vivo. The antiserotonergic actions of IC on the isolated external carotid arteries and femoral arteries in dogs observed in a noncompetitive manner were found to be 1/24 to 1/65 that of methysergide. On the other hand, IC showed no inhibitory effect on the paw edema of rats in vivo induced by serotonin. The inhibitory effect of IC on peritoneal dye leakage in mice was less than half that of phenylbutazone. IC prevented apoplexy in stroke-prone SHR (SHRSP) without lowering the blood pressure. Histological changes in the cerebrum of SHRSP were ischemic changes such as swelling of the neurons and shrinkage of the nuclei, mainly in the cerebral cortex and corpus striatum area.