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K Nakatsu

Publications and source records attributed to K Nakatsu.

At least 37 records · Page 2Linked to original sources
PubMed1997

Carbon monoxide formation in the guinea pig hippocampus: ontogeny and effect of in vitro ethanol exposure.

Carbon monoxide (CO) is considered to be a novel neuronal messenger in the brain, similar to nitric oxide. The ontogeny of CO formation in transverse hippocampal slices of the guinea pig was elucidated at selected prenatal and postnatal ages, and the effect of in vitro ethanol exposure on hippocampal CO formation was determined. There was a higher rate of hippocampal CO formation in the fetus at gestational day (GD) 50 and GD 62 (term, about GD 68) compared with the adult. In vitro ethanol exposure (50 and 100 mM) decreased hippocampal CO formation in the GD 62 fetus, which was prevented by incubation with 500 microM L-glutamate.

Animals↗
PubMed1997

Electrospray mass-spectrometric, spectrophotometric and electrochemical methods do not provide evidence for the binding of nitric oxide by pyocyanine at pH 7.

In several recent publications on pyocyanine, its mechanism of action has been attributed to an ability to react with nitric oxide (NO), resulting in the formation of an adduct. We examined the chemical interaction of pyocyanine and NO using electrospray (ES) MS, spectrophotometry and voltammetry at neutral pH and with 10-100 microM pyocyanine. No binding of NO to pyocyanine was observed. Alternative mechanisms for the inhibition of NO-induced vasorelaxation by pyocyanine should be sought.

Binding Sites↗
PubMed1997

Heme oxygenase activity and immunohistochemical localization in bovine pulmonary artery and vein.

Recent studies suggest that carbon monoxide (CO) derived from heme oxygenase (HO)-catalyzed metabolism of heme plays a role in the regulation of cell function and communication. In blood vessels, CO may regulate vascular smooth-muscle tone through the activation of soluble guanylyl cyclase, in a manner similar to that of nitric oxide. The objective of this study was to determine the relation between HO enzymatic activity and localization of HO protein in bovine pulmonary blood vessels. HO enzymatic activity was determined by quantitating the rate of CO formation in the microsomal fraction of homogenates of bovine pulmonary artery (BPA) and vein (BPV). HO protein was localized by immunohistochemical analysis of paraformaldehyde-fixed tissue by using polyclonal antibodies to inducible HO (HO-1) and noninducible HO (HO-2). HO enzymatic activity was measured in BPA and BPV, which correlated with the presence of HO protein. In BPA, HO enzymatic activity was found in the adventitia and medial layer; HO protein was localized in the nerves and vasa vasorum of the adventitia and was found throughout the smooth-muscle cells in the medial layer. The data clearly demonstrate the presence of HO enzymatic activity for the formation of CO in blood vessels that contain HO protein.

Animals↗
PubMed1997

Inhibition of the action of nitric oxide prodrugs by pyocyanin: mechanistic studies.

Previously, we reported on the antagonism by pyocyanin (PYO) of the relaxant effects of nitrovasodilators such as glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1). The purpose of the present study was to elucidate the mechanism of this action of PYO by examining its effect on the steps considered to be necessary for nitrovasodilator-induced relaxation of blood vessels. PYO (10 microM) attenuated the accumulation of guanosine-3',5'-cyclic monophosphate (cGMP) in rabbit aorta induced by nitrovasodilators SIN-1, SNAP, and GTN, 65, 81, and 67%, respectively. Additionally, PYO (1 or 10 microM) interfered with in vitro activation of soluble guanylyl cyclase. PYO did not inhibit vascular relaxation induced by 8-bromo-cyclic guanosine monophosphate. PYO (10 microM) also decreased the quantity of nitric oxide measured in the headspace above intact vascular tissue incubated with glyceryl trinitrate in the presence of oxygen. These observations are consistent with the interpretation that PYO interfered with the nitrovasodilator action of glyceryl trinitrate by inactivation of NO or by inhibition of enzymatic biotransformation of GTN; this would result in decreased guanylyl cyclase activation and thus lowering cellular levels of cGMP. NO chemiluminescence studies with SIN-1 (10 microM) revealed that this NO donor produced NO in a time-dependent manner and PYO (10 microM) caused no inhibition of NO production, but in fact, potentiated NO release after 10 min of incubation (1395 +/- 179 pmol NO compared with 1088 +/- 154 pmol NO). NO production from 10 microM SNAP was similarly potentiated by PYO after 0.5, 2, 5, and 10 min of incubation. Therefore, it is likely that PYO acts as an inhibitor of guanylyl cyclase with respect to NO donors, SIN-1 and SNAP, but it also appears that PYO can exert additional inhibitory effects in the case of vascular relaxation by GTN. Such differences in relaxant effects may reflect inhibition of enzymatic biotransformation that is unique to GTN or that PYO may complex with an alternative redox form of NO (perhaps NO+) that is generated by vascular metabolic activation of GTN.

Animals↗
PubMed1997

Thiol agents potentiate glyceryl trinitrate mediated relaxation of rabbit taenia coli: evidence for thiol-dependent biotransformation.

In the present study, the role of thiols on glyceryl trinitrate (GTN) induced relaxation of rabbit taenia coli strips (RTCS) was investigated. This study was designed to test the hypothesis that a deficiency in thiols is responsible for RTCS insensitivity to GTN, and thus thiols play a key role in the enzymatic activation of GTN. Isolated RTCS bathed in normothermic, oxygenated Krebs solution were pretreated with the thiols L-cysteine (5 mM) and N-acetyl-L-cysteine (NAC, 5 mM) for 30 min and washed. The effects of GTN were determined by changes in isometric tension of K(+)-precontracted RTCS. Both L-cysteine and NAC resulted in increased relaxations to GTN (0.1 nM-10 microM) as the GTN relaxation EC50 decreased compared with that of the untreated RTCS (L-cysteine, 0.06 +/- 0.12 microM and NAC, 0.08 +/- 0.03 microM versus control 0.25 +/- 0.08 microM, n = 5, p < 0.05). In contrast, 5 mM D-cysteine had no significant effects on the RTCS GTN relaxation EC50 (0.16 +/- 0.13 microM, n = 5). Similarly, the thiol donor L-methionine significantly increased RTCS sensitivity to GTN, as the relaxation EC50 decreased from the control value of 0.25 +/- 0.08 microM to 10 +/- 4 nM (n = 5, p < 0.001), whereas the D-isomer did not. These results are consistent with the idea that thiols play a key stereospecific role in the metabolic activation of GTN in RTCS. However, RTCS treated with amino acids were still less sensitive to GTN compared with vascular tissue, and this suggests that RTCS may be deficient in some other enzyme(s) relative to vascular tissue that is (are) responsible for the activation of GTN.

Animals↗
PubMed1997

The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate.

Carbon monoxide (CO), a vasodilator, has been implicated as an activator of soluble guanylyl cyclase (sGC) to effect smooth muscle relaxation; however, this idea has not received universal support. The purpose of this study was to examine the effects of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) on relaxation of rabbit aortic rings (RARs) induced by CO. Administration of 10 microM ODQ completely abolished relaxation of RARs by CO (30 microM), whereas only a partial attenuation of NO-induced relaxation was achieved by the same concentration of ODQ. The results of this study suggest that CO-mediated relaxation of RARs is mediated by sGC and indicate that ODQ may serve as a useful tool in the investigation of the actions of CO. Furthermore, these observations support the idea that ODQ is less potent in inhibiting relaxations by NO, thereby implicating a component of NO-induced relaxation that is independent of sGC/cGMP.

Animals↗
PubMed1996

Ontogeny of heme oxygenase activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig.

Heme oxygenase (HO) catalyzes the oxidation of heme to produce carbon monoxide (CO), biliverdin and iron. CO is considered to function as a novel neuronal messenger in the brain analogous to nitric oxide. The ontogeny of microsomal HO activity in the hippocampus, frontal cerebral cortex, and cerebellum of the immature fetal, mature fetal and adult guinea pig was determined using an optimized assay which quantitated heme-derived CO formation by a gas chromatographic method. There was a distinct developmental profile of HO activity that was similar for all three brain regions. In particular, HO activity was maximal in the mature fetus compared with the immature fetus and the adult. These data demonstrate that HO activity is developmentally regulated and that there is similar ontogeny of HO activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig.

Aging↗
PubMed1996

Analysis of pyocyanin from Pseudomonas aeruginosa by adsorptive stripping voltammetry.

We have developed an adsorptive stripping voltammetric (AdSV) method that can be used for analysis of pyocyanin at the nanomolar level. The method employs a hanging mercury drop electrode (HMDE) as a working electrode in a three-electrode system in a 15-mL quartz cell. Preconcentration of pyocyanin in a 10-mM ammonia buffer (pH 8.0) on the HMDE is carried out by adsorption under conditions of controlled mass transfer for 60 sec, followed by scanning the electrode potential that results in reduction (cathodic scan) or oxidation (anodic scan) of the accumulated pyocyanin. For analysis of pyocyanin in samples containing a significant amount of surface-active species from a bacterial culture broth, a positive-going (anodic) potential scan must be applied after an adsorption at -0.50 V. Anodic scan of the adsorbed pyocyanin provided a well-defined oxidation peak at a potential of -0.17 V. The "anodic scan" variant of the method was tested for utility in Mueller-Hinton broth diluted from 20 to 200 times in a 10-mM ammonia buffer. Reproducibility of the method as applied to the analysis of pyocyanin produced by Pseudomonas aeruginosa (18.83 +/- 0.32 microM) in culture was demonstrated by the within-day and day-to-day coefficients of variation of 1.7% and 3.4%, respectively.

Adsorption↗
PubMed1996

Superoxide does not inhibit glyceryl trinitrate-rabbit aortic strip-mediated relaxation of rabbit Taenia coli. Evidence against a role for nitric oxide itself as the smooth muscle active drug metabolite?

This study was designed to test the hypothesis that nitric oxide (NO) is the relaxant metabolite produced by metabolic activation of glyceryl trinitrate (GTN) in rabbit aortic strip (RAS). Superoxide anion, an inactivator of NO, was included in a two-tissue bioassay in which rabbit Taenia coli strip (RTCS) relaxed to GTN in the presence of RAS. Superoxide as generated by xanthine (10 microM)/ xanthine oxidase (20 mU/ml) failed to attenuate relaxations of RTCS to GTN (0.1 nM-10 microM) and RAS, compared with the untreated control. In contrast, superoxide attenuated the relaxation of RTCS to both authentic NO gas and to SIN-1 (0.1 nM-10 microM), a known spontaneous releaser of NO; the attenuation of RTCS relaxation to NO gas was reversed by superoxide dismutase (100 units/ml). In addition, another drug that has been reported to scavenge NO, hydroquinone, did not attenuate the RTCS relaxation to GTN. These results suggest that biotransformation of GTN in vascular smooth muscle that leads to relaxation is caused by a NO-containing species (i.e. a S-nitrosothiol). Such a molecule would be less susceptible to inactivation by superoxide anion and hydroquinone.

Animals↗
PubMed1995

Heme oxygenase activity in the adult rat aorta and liver as measured by carbon monoxide formation.

Rat aorta was homogenized and the 13000 x g supernatant fraction was tested for heme oxygenase (HO) activity by using a sensitive gas chromatographic method to measure carbon monoxide (CO), one of the products of the HO reaction. The rate of NADPH-dependent CO formation, an index of HO activity, was 1.41 +/- 0.40 nmol CO.mg(-1)protein. h(-1) in the rat aorta supernatant fraction and 2.05 +/- 0.55 nmol CO.mg(-1) protein.h(-1) in the rat liver 13000 x g supernatant fraction, a tissue known to contain HO activity. Chromium mesoporphyrin (0.05 mM), an inhibitor of rat liver HO, significantly decreased HO activity by 26% in the aorta supernatant fraction and 50% in the liver supernatant fraction. On the basis of the results of this study, which demonstrated HO-catalyzed CO formation in aortic tissue, and previous observations that CO relaxes vascular smooth muscle, we suggest that a physiological role for CO in vascular smooth muscle relaxation should be further investigated.

Animals↗
PubMed1995

Sulfhydryl involvement in nitric oxide sequestration and nitric oxide induced guanylyl cyclase activation in vascular smooth muscle.

In the present study, the role of vascular smooth muscle sulhydryl groups was investigated with respect to sequestration of nitric oxide (NO) and activation of soluble guanylyl cyclase by NO. Vascular smooth muscle 100,000 x g supernatant (soluble) fraction was prepared in phosphate buffer, using the medial layer of bovine pulmonary artery. The soluble fraction was incubated with 100 pmol NO for 5 min in a sealed flask at 37 degree C under anerobic conditions in the presence or absence of the sulfhydryl reagent, N-ethylmaleimide (NEM, 5 mM). NO sequestration by the soluble fraction was measured as an indicator of NO binding. Total thiol content was measured in the soluble fraction with and without exposure to NEM. Guanylyl cyclase activity was measured in the soluble fraction with and without exposure to NO and a combination of NO and NEM. NEM decreased total thiol content in the soluble fraction from 103.59 nmol/mL to undetectable levels, and decreased guanylyl cyclase activity to below basal levels. The percentage of NO sequestered by the soluble fraction was inhibited by NEM by approximately 25% from a control value of 26.52 +/- 9.39 to 18.72 +/- 8.52, n = 13, p < 0.05. The data indicate that sulfhydryl groups are essential for guanylyl cyclase activation by NO, and are also involved in the sequestration of NO by the vascular smooth muscle soluble fraction.

Animals↗
PubMed1995

Time-dependent increase in nitric oxide formation concurrent with vasodilation induced by sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine but not by glyceryl trinitrate.

Nitrovasodilators is a term used to describe a diverse group of agents believed to exert their vasodilator effect by a common mechanism, namely formation of nitric oxide. The objective of the present study was to test this concept by comparing the magnitude of nitric oxide formation from four representative nitrovasodilators incubated in the presence of rabbit aortic strips (RAS) with the magnitude of nitrovasodilator-induced relaxation of RAS, over the time course of relaxation. The RAS were incubated in anaerobic condition with 100 microM either sodium nitroprusside, glyceryl trinitrate (GTN), 3-morpholinosydnonimine, or S-nitroso-N-acetylpenicillamine in Krebs' solution. Relaxation of RAS by each of the four drugs started at approximately 10 sec and increased with incubation time. Formation of nitric oxide was measurable using a chemiluminescence-headspace-gas method at 0.5 min with sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine, and formation increased concurrently with relaxation of RAS. In contrast, formation of nitric oxide from GTN was not measurable until 5 min, at which time GTN-induced relaxation of RAS was nearly complete. These data support the concept that sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine are nitrovasodilator drugs acting via formation of nitric oxide. However, the present study suggests that the mechanism of action for GTN-induced vasodilation may involve the formation of a nitric oxide-adduct, such as a nitrosothiol, rather than nitric oxide per se.

Animals↗
PubMed1994

Nitric oxide formation from glyceryl trinitrate by rabbit aortic strip: detection by rabbit taenia coli concurrent with vasorelaxation.

1. The purpose of the present study was to assay NO formation from GTN biotransformation by the rabbit aortic strip (RAS) at times concurrent with its vasorelaxation. Such an assay is an important test of the prodrug hypothesis where it is postulated that glyceryl trinitrate (GTN) is biotransformed to nitric oxide (NO), the active species that initiates vascular smooth muscle relaxation. To test such a hypothesis, we propose that a sample of smooth muscle, poorly responsive to GTN, yet sensitive to the effects of NO could be used to detect RAS production of NO from GTN. 2. Muscle strips of rabbit taenia coli (RTCS) and RAS in close apposition, were mounted in tissue baths, and muscle relaxation was recorded with isometric force transducers. Tissues were submaximally precontracted with 30-35 mM K+ depolarizing solution and exposed to increasing concentrations of GTN (0.1 nM-10 microM). 3. EC25 for GTN-induced relaxation of RTCS in the presence of RAS was significantly decreased to that for RTCS in the absence of RAS (5.9 +/- 3.0 x 10(-8) M and 5.5 +/- 3.7 x 10(-6) M, respectively). Mean maximal levels of GTN-induced relaxation of similarly precontracted RTCS also differed in the presence and absence of RAS, viz., 80.8 +/- 2.1% and 29.8 +/- 8.3% respectively. 4. RTCS was found to relax upon administration of NO gas bubbled through the incubation medium. Analysis of tissue bath medium revealed that the NO concentration to which RTCS was exposed attained a maximum of 33 nM. Relaxation of RTCS by NO gas was inhibited by 1 microM reduced haemoglobin. 5. For GTN-incubation with intestinal and vascular smooth muscle preparations, NO formation was greater with RAS compared to RTCS. Thus, in the two-issue bioassay, the RAS was the predominant source of NO formation from GTN. 6. Reduced deoxyhaemoglobin (1 microM), a potent extracellular NO scavenger, was found to decrease the augmented GTN-induced relaxation in the RTCS-RAS sandwich preparation from 17.3 +/- 1.8% to 8.0 +/- 0.8%. The augmented RTCS response was restored upon washout and subsequent addition of GTN, in the absence of reduced Hb.7 These data indicate that nitric oxide or a closely related NO-donor is produced by vascular biotransformation of GTN as seen by the increased sensitivity of RTCS to GTN when in the presence of RAS. The results of this bioassay thus support the GTN-NO prodrug hypothesis.

Animals↗
PubMed1994

Inhibition of nitrovasodilators by pyocyanin and methylene blue is dissociated from nitric oxide formation.

The phenazine pigment pyocyanin (Pyo), like methylene blue (MB), inhibits vascular relaxation induced by organic nitrates. These nitrovasodilators are pro-drugs that have in common the ability to generate nitric oxide (NO). In this study, we characterized responses of rabbit isolated aortic ring to 3-morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside, glyceryl trinitrate (GTN), and isosorbide dinitrate in the presence and absence of 10 microM Pyo. We also examined the effect of Pyo (1 and 10 microM) and MB (1 and 10 microM) on vasorelaxation induced by authentic NO, and finally we tested the effects of Pyo and MB on the tissue-independent formation of NO from SIN-1, SNAP, and sodium nitroprusside, using the chemiluminescence--headspace gas method. Pyo (10 microM) surmountably inhibited aortic responses to GTN, isosorbide dinitrate, SIN-1, and SNAP with a characteristic rightward shift of the dose-response curve; the apparent EC50 of these drugs for relaxation of phenylephrine-contracted aorta was increased 18-, 4-, 13-, and 15-fold, respectively. Pyo (1 and 10 microM) and MB (10 microM) inhibited NO-induced vasorelaxation at the EC50 of NO by 35, 72, and 56%. In contrast, Pyo did not inhibit sodium nitroprusside induced vasodilation. For a 10-min incubation, 10 microM Pyro or MB increased NO production from SNAP 1.8- and 2.9-fold, respectively, and increased NO production from SIN-1 by 3.8- and 7.1-fold, respectively. Neither Pyo nor MB enhanced NO formation from sodium nitroprusside. These data indicate that Pyo and MB inhibit nitrovasodilator-induced relaxation of aortic ring by interfering with the action of NO, subsequent to its formation.

Animals↗
PubMed1994

The effects of hypothermic and normothermic cardiopulmonary bypass on glyceryl trinitrate activity.

Glyceryl trinitrate (GTN) is used to control arterial blood pressure during cardiopulmonary bypass (CPB) procedures, but its effects are often decreased during the period of extracorporeal support. The plasma and urine concentrations of GTN and glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN) for male and female patients who received GTN during hypothermic CPB, and male and female patients who were given GTN during normothermic CPB, were measured by gas-liquid chromatography. During hypothermic CPB, the male and female subjects experienced significant decreases in GTN clearance (P < 0.05), 66% and 52%, respectively. Neither the males nor the females who underwent normothermic CPB experienced any significant change in GTN clearance. These results suggest that the lower core temperature during hypothermic CPB may decrease the biotransformation of GTN to GDNs and nitric oxide, thereby resulting in less dilation of blood vessels. Furthermore, the males in the hypothermic CPB group had significantly greater urinary concentrations of 1,3-GDN and 1,2-GDN than the females (P < 0.05), and the normothermic CPB males had a significantly greater urinary concentration of 1,2-GDN than the females in that group. The normothermic CPB males also had significantly higher plasma concentration of GTN at two time points, and 1,3-GDN at one time point, than the females. These data suggest that there may be a gender difference in GTN biotransformation.

Aged↗
PubMed1993

In vitro cytochrome P450 monooxygenase and prostaglandin H-synthase mediated aflatoxin B1 biotransformation in guinea pig tissues: effects of beta-naphthoflavone treatment.

In the present study, we examined the effects of treating guinea pigs with beta-naphthoflavone (BNF) on aflatoxin B1 (AFB1) metabolism by microsomal cytochrome P450 monooxygenase (P450) and prostaglandin H synthase (PHS) in liver, lung and kidney tissues. After BNF treatment, microsomal 7-ethoxyresorufin O-deethylase activity was induced 13-, 25- and 11-fold in lung, kidney and liver, respectively, confirming that the BNF treatment protocol was effective at inducing monooxygenase activity. Treatment of guinea pigs with BNF did not change [3H]AFB1-DNA binding catalyzed by microsomal PHS or P450 in lung, kidney or liver. In contrast, AFM1 formation by P450 was significantly increased in microsomes from all three organs. The data indicate that BNF-inducible P450 isozymes of the P4501A class are responsible for the biotransformation of AFB1 to non-toxic metabolites. Guinea pig kidney microsomes could also catalyze NADPH-dependent formation of aflatoxicol (AFL), a metabolite usually produced by a cytosolic steroid dehydrogenase. Renal microsomal AFL formation was not altered by prior BNF treatment. The results in the present study suggest that BNF may alter the bioactivation of AFB1 in guinea pig tissues by inducing P450 activity, leading to the formation of less reactive metabolite.

Aflatoxin B1↗