The use of enzyme-linked immunosorbent assays (ELISA) for the determination of pollutants in environmental and industrial wastes.

Twelve enzyme-linked immunosorbent assays (ELISA), for the determination of surfactants [linear alkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), and alkylphenol ethoxylates (APE)], endocrine disruptors [alkylphenol (AP), AP + APE, and bisphenol A (BPA)], estrogens [17beta-estradiol (E2), estrone (El), estrogen (ES: El + E2 + estriol (E3)), 1 7alfa-ethynylestradiol (EE2)], dioxins and polychlorinated biphenyls (PCBs), were validated on environmental water and industrial wastes. The lowest quantification limits of these ELISAs were 0.05 microg/L (BPA, E2, El, ES and EE2), 2 microg/L (AE), 3 microg/L (dioxins and PCBs), 5 microg/L (AP, AP + APE) and 20 microg/L (LAS and APE). To apply these ELISAs to environmental or industrial waste samples, simple and appropriate pre-treatment methods were also developed for each ELISA. With optimized pre-treatments, the values of ELISAs were well co-related, in all cases, to those of instrumental analytical methods such as liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and high-resolution gas chromatography mass spectrometry (HR-GC-MS), etc.

Long-term administration of L-arginine improves nitric oxide release from kidney in deoxycorticosterone acetate-salt hypertensive rats.

To examine the effects of L-arginine (L-Arg) on endothelial function, we administered 0.5 g/L L-Arg in drinking water to deoxycorticosterone acetate (DOCA)-salt rats for 8 weeks and then measured nitric oxide (NO) release from isolated kidneys using a newly developed real-time chemiluminescence method. Renal pathology was also analyzed. Acetylcholine caused much smaller declines in renal perfusion pressure (10(-7) mol/L acetylcholine: -24 +/- 2% [SEM] versus -50 +/- 2%, P < .001) and NO release in DOCA-salt rats (+3 +/- 1 versus +33 +/- 3 fmol/min per gram kidney weight, P < .001) compared with control rats. L-Arg did not influence the time course of systolic blood pressure elevation in DOCA-salt rats (211 +/- 5 versus 208 +/- 6 mmHg, DOCA versus L-Arg/DOCA, P = NS). However, oral administration of L-Arg improved acetylcholine-induced declines in renal perfusion pressure (10(-7) mol/L acetylcholine: L-Arg/DOCA, -39 +/- 3%, P < .01 versus DOCA). This change was associated with an increase in NO release by acetylcholine (10(-7) mol/L acetylcholine: L-Arg/DOCA, +10 +/- 1 fmol/min per gram kidney weight, P < .05 versus DOCA). However, morphological changes in renal vessels and glomeruli were similar between DOCA and L-Arg/DOCA rats. These results suggest that L-Arg administration partially reverses renal endothelial function with respect to vasorelaxation and NO release independent of blood pressure changes, indicating that hypertensive vessels seem to be depleted of L-Arg and/or have defects in the availability of L-Arg for NO synthesis.

Receptor subtype for vasopressin-induced release of nitric oxide from rat kidney.

The vasopressin receptor subtype that causes nitric oxide (NO) release remains controversial. To elucidate this receptor-ligand interaction, we examined the effects of vasopressin receptor antagonists on vasopressin-induced release of NO from isolated perfused rat kidneys by using a sensitive chemiluminescence assay. Vasopressin increased renal perfusion pressure and NO signals in the perfusate in a dose-dependent manner. N omega-Monomethyl-L-arginine abolished this increase in NO release; however, a similar increase in renal perfusion pressure induced by prostaglandin F2 alpha was not associated with the increase in NO release. OPC-21268, a V1 receptor antagonist, significantly reduced the vasopressin-evoked renal vasoconstriction and NO release, whereas OPC-31260, a V2 receptor antagonist, had no effects. Moreover, desmopressin, a selective V2 receptor agonist, did not increase the NO signal. NO release by vasopressin was markedly attenuated in deoxycorticosterone acetate (DOCA)-salt hypertensive rat kidneys compared with control kidneys (10(-10) mol/L vasopressin: +0.8 +/- 0.3 versus +6.9 +/- 1.4 fmol/min per gram kidney, DOCA versus control; P < .001). Histochemical analysis for renal NO synthase revealed a substantial attenuation of the staining of endothelial NO synthase in DOCA-salt rats. These results directly demonstrate that vasopressin stimulates NO release via the endothelial V1 receptor in the rat kidney.

Nitric oxide release from kidneys of hypertensive rats treated with imidapril.

To examine whether endothelial dysfunction in hypertension is reversible or not, we studied the effects of imidapril, an angiotensin-converting enzyme inhibitor, on nitric oxide release in stroke-prone spontaneously hypertensive rats (SHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 4-week treatment with imidapril (1 or 10 mg/d SC) or vehicle, acetylcholine-induced vasodilation and nitric oxide release in the isolated kidneys were determined. Nitric oxide release was measured by a chemiluminescense assay. Imidapril lowered blood pressure in stroke-prone SHR in a dose-dependent manner. Untreated stroke-prone SHR exhibited significantly attenuated responses to acetylcholine (10(-8) mol/L) of both renal perfusion pressure (stroke-prone SHE 42 +/- 4% versus Wistar-Kyoto rats [WKY] 58 +/- 4% [mean +/- SE], P < .01) and nitric oxide release (stroke-prone SHR +7.6 +/- 2.1 versus WKY +29.7 +/- 9.7 fmol/min per gram of kidney wt, P < .01). Imidapril at 10 mg/d significantly increased acetylcholine-induced renal vasodilation and nitric oxide release in stroke-prone SHR (renal perfusion pressure, 56 +/- 3%; nitric oxide release, +27.1 +/- 6.4 fmol/min per gram of kidney wt; both P < .01 versus stroke-prone SHR treated with vehicle). On the other hand, imidapril neither decreased blood pressure nor changed nitric oxide release induced by acetylcholine in DOCA-salt hypertensive rats. Staining for endothelial nitric oxide synthase and brain nitric oxide synthase was clearly detected in the kidneys of both stroke-prone SHR and WKY, whereas staining intensity was weaker in DOCA-salt hypertensive rats. Inducible nitric oxide synthase immunoreactivity was barely noticeable in any type of rat. Thus, imidapril restored endothelial damage by pressure-dependent mechanisms. Most of the nitric oxide detected in the perfusate seemed to be derived from constitutive nitric oxide synthase.

Effect of 4-phenyl-1,2,3,4-tetrahydroisoquinoline on ambulation induced by injection of methamphetamine into the nucleus accumbens in rats.

4-Phenyl-1,2,3,4-tetrahydroisoquinoline (4-PTIQ) has previously been shown to have antagonistic properties to methamphetamine in the spinal cord. Administration of 4-PTIQ (5 mg/kg, s.c.) reduced the ambulation induced by methamphetamine (0.5 mg/kg, s.c.) in rats. Methamphetamine (3 micrograms), injected unilaterally into the nucleus accumbens, increased ambulation. Alone, 4-PTIQ (10 micrograms) failed to elicit ambulation; however, it inhibited the methamphetamine-induced increase in ambulation. The alpha 1-antagonist prazosin (0.5 micrograms) or the beta-antagonist propranolol (3 micrograms) showed no effect on ambulation induced by methamphetamine. Haloperidol (5 ng), which possesses strong dopamine-blocking activity, abolished the ambulation induced by methamphetamine. The drug 4-PTIQ had weak affinity for dopamine D1 and D2 receptors. These results support the possibility that the inhibitory effects of 4-PTIQ on the ambulation-stimulating effects of methamphetamine, are due to blocking of the dopamine-releasing effect of methamphetamine but not due to dopamine blocking effects.

Inhibitory effect of 4-phenyltetrahydroisoquinoline on locomotion and dopamine release induced by micro-injection of methamphetamine into the nucleus accumbens of the rat.

The inhibitory effects of 4-phenyl-1,2,3,4-tetrahydroisoquinoline (4-PTIQ) on methamphetamine-induced increases in dopamine and locomotion were investigated. Methamphetamine hydrochloride (10 micrograms) microinjected into the nucleus accumbens increased both locomotor activity and extracellular dopamine levels, measured by the brain microdialysis method. 4-PTIQ hydrochloride (20 micrograms) co-injected with methamphetamine inhibited both the increase in dopamine and the locomotor activity. The uptake blocker cocaine hydrochloride (20 micrograms) co-injected with methamphetamine failed to inhibit the effect of methamphetamine. Thus 4-PTIQ but not cocaine inhibited the dopamine-releasing effect of methamphetamine, and 4-PTIQ is suggested to block methamphetamine-induced locomotion by inhibition of dopamine release.

[13N]-beta-phenethylamine ([13N]PEA): a prototype tracer for measurement of MAO-B activity in heart.

[13N]beta-Phenethylamine ([13N]PEA) was evaluated as a radio tracer for the measurement of mouse heart monoamine oxidase (MAO) activity in vivo. After intravenous administration, [13N]PEA was deaminated by MAO-B. 13NH3 formed thereby was taken up by amino acids and trapped in the heart. The relation between the radioactivity trapped in the heart and the enzyme activity was examined. The radioactivity in the heart 15 min after administration was reduced in a dose-dependent manner by pretreatment with a specific MAO-B inhibitor, l-deprenyl, but not with a specific MAO-A inhibitor, clorgyline. A linear correlation existed between the heart radioactivity level and the heart MAO-B activity (0-45%). [13N]1,1-d2-2-Phenethylamine (C6H5-CH2-CD2-13NH2, [13N]d2PEA), a modified tracer with less reactivity towards the enzyme, was tested similarly. This tracer possessed a higher sensitivity than [13N]PEA, and a wider range (0-85%) of MAO-B activity correlated linearly with the trapped radioactivity. These results indicate that [13N]PEA derivatives ([13N]PEA and [13N]d2PEA) can be useful radiotracers for noninvasive measurements of MAO-B activity in the human heart.

Deprenyl decreases an endogenous parkinsonism-inducing compound, 1-benzyl-1,2,3,4-tetrahydroisoquinoline in mice: in vivo and in vitro studies.

We examined the effect of deprenyl, a promising drug for the therapy of Parkinson's disease on the formation of a parkinsonism-inducing compound, 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ). The 1BnTIQ content was significantly decreased in the brain of deprenyl-treated mouse in vivo, and deprenyl also inhibited 1BnTIQ formation from phenethylamine by a mouse brain homogenate supernatant in vitro. In vivo, the content of a parkinsonism-preventing compound, 1-methyl-1,2,3, 4-tetrahydroisoquinoline (1MeTIQ) was slightly increased in mice injected with deprenyl. The marked decrease of the ratio of 1BnTIQ to 1MeTIQ might play a role in the clinical effect of deprenyl.

Amphetamine-antagonistic properties of 4-phenyl-1,2,3,4- tetrahydroisoquinoline: inhibition of spinal reflex-enhancing effects of methamphetamine, phenylethylamine and nomifensine.

The antagonistic effects of 4-phenyl-1,2,3,4-tetrahydroisoquinoline (4PTIQ) against S(+)-methamphetamine, phenylethylamine and nomifensine were studied by measurement of spinal monosynaptic reflex potential (MSR). S(+)-Methamphetamine, phenylethylamine and nomifensine enhanced the amplitude of MSR in C1-spinalized rats through release of noradrenaline from the terminals of descending fibers and consequent activation of alpha 1-adrenoceptors. Although 4PTIQ alone did not change the amplitude of the MSR, 4PTIQ inhibited the enhancement of MSR induced by S(+)-methamphetamine and related compounds. The MSR of rats with an intact spinal cord was enhanced by conditioning stimulation of the ipsilateral locus ceruleus. The MSR enhancement produced by the stimulation was blocked by prazosin but unaffected by 4PTIQ, showing that 4PTIQ does not have an alpha 1-blocking action. These results suggest that the antagonistic effects of 4PTIQ on MSR enhancement by S(+)-methamphetamine, phenylethylamine and nomifensine are due to its blocking of noradrenaline release produced by these amphetamine-like agents.

4-Phenyltetrahydroisoquinoline, but not nomifensine or cocaine, inhibits methamphetamine-induced dopamine release.

The inhibitory effect of 4-phenyltetrahydroisoquinoline (4-PTIQ) on methamphetamine-induced dopamine release in the rat nucleus accumbens was investigated using a brain microdialysis method. Methamphetamine (10(-6) M) infusion through a microdialysis probe induced the release of dopamine. Although the uptake inhibitors, cocaine (3 x 10(-6) M) and nomifensine (10(-6) M), failed to block dopamine release, 4-PTIQ (10(-6 M) inhibited the dopamine-releasing effect of methamphetamine. 4-PTIQ did not affect the elevation of the extracellular dopamine level induced by high concentrations of nomifensine (10(-5) M) and cocaine (3 x 10(-5) M). 4-PTIQ was the weakest inhibitor of [3H]dopamine uptake by rat striatal synaptosomes. These results suggest that 4-PTIQ is a selective antagonist against the dopamine-releasing effect of methamphetamine in the nucleus accumbens.

Methamphetamine antagonistic property of (+)- and (-)-4-phenyltetrahydroisoquinoline in rat anococcygeus muscle.

The antagonistic effects of (+)- and (-)-4-phenyl-1,2,3,4-tetrahydroisoquinoline (4PTIQ) on methamphetamine in the rat anococcygeus muscle were compared with those of cocaine and nomifensine. Methamphetamine contracted the anococcygeus muscle through the release of norepinephrine from noradrenergic nerve terminals. (+)-4PTIQ inhibited the methamphetamine-induced contractions more strongly than cocaine and nomifensine. (+)-4PTIQ had no potentiating effects on exogenous norepinephrine-induced contraction, which was considered to be an index of amine neuronal uptake blockade. On the other hand, (-)-4PTIQ, cocaine and nomifensine produced a significant leftward shift of the norepinephrine concentration-response curve, i.e. they showed a strong blocking effect on amine neuronal uptake. These results suggest that the inhibitory effects of (+)-4PTIQ on the action of methamphetamine are mediated by a mechanism other than inhibition of amine neuronal uptake.

Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

Real-time measurement of nitric oxide production in rat brain by the combination of luminol-H2O2 chemiluminescence and microdialysis.

A chemiluminescence method of detecting nitric oxide (NO) in combination with a microdialysis technique was employed for the real-time measurement of NO production in living rat brain. This method based on the luminol-H2O2 system has a detection limit of 1 nM, and is the most sensitive method currently available for measuring NO. We applied this new technique to rat cerebellum to record the increase of chemiluminescence intensity arising from NO production after the injection of N-methyl-D-aspartate or kainic acid around the microdialysis probe. This highly sensitive method should be useful for the direct clarification of the functions of NO in the central nervous system.

Chronic administration of 1-benzyl-1,2,3,4-tetrahydroisoquinoline, an endogenous amine in the brain, induces parkinsonism in a primate.

1-Benzyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (1Bn TIQHCl) (22 mg/kg per day) was subcutaneously injected into a monkey, Macaca fascicularis for 66 days to investigate its acute and chronic effects Parkinsonism like motor symptoms appeared, and the acute toxicity was stronger than the chronic toxicity. This result suggested that 1BnTIQ may induce parkinsonism in humans. 1BnTIQ content in various regions of the monkey brain was determine by the gas chromatography-selected ion-monitoring method, but no significant variation was found.

Selective release of serotonin by endogenous alkaloids, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines, (R)- and (S)salsolinol, in the rat striatum; in vivo microdialysis study.

Dopamine-derived 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines, (R)- and (S)salsolinol, released an enormous amount of serotonin in the rat striatum; the concentration of serotonin increased from undetectable level to 2.53 +/- 0.12 and 3.69 +/- 0.01 microM after perfusion of (R)- and (S)salsolinol, respectively. Salsolinols increased extracellular dopamine level, but to a much lesser degree than serotonin. Other naturally occurring isoquinolines with catechol structure released serotonin and dopamine, but salsolinols were the most potent and selective releaser of serotonin. Serotonin release by salsolinols may be involved in some psychiatric symptoms in L-DOPA therapy or alcoholism.

Presence of tetrahydroisoquinoline and 1-methyl-tetrahydro-isoquinoline in foods: compounds related to Parkinson's disease.

This is the first report confirming the presence of 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline(1MeTIQ) in a number of foods with a high 2-phenylethylamine content. These compounds were determined by gas chromatography-mass spectrometry. This study also confirmed that 1MeTIQ and TIQ can cross the blood-brain barrier in rat. Thus, these compounds, suspected to have relation to parkinson's disease, may accumulate in the brain from food sources.

Metabolism and brain accumulation of tetrahydroisoquinoline (TIQ) a possible parkinsonism inducing substance, in an animal model of a poor debrisoquine metabolizer.

4-Hydroxytetrahydroisoquinoline (4OH-TIQ) was detected as a metabolite of a possible parkinsonism-inducing substance, tetrahydroisoquinoline (TIQ), in rat liver microsomes and rat urine. Urinary excretion of 4OH-TIQ was significantly reduced in female DA rat, an animal model of a poor debrisoquine metabolizer. The female DA rat also showed significantly higher brain accumulation of TIQ. These results suggest that the metabolic detoxication process is depressed and TIQ accumulation in the brain is enhanced in a poor debrisoquine metabolizer, which may be one possible explanation for poor debrisoquine metabolizers being susceptible to Parkinson's disease.

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats.

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10(-7) M: SHR +37 +/- 2 [SE] vs. WKY +32 +/- 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K+ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.

Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450.

In order to examine the reactivity of active intermediate derived form iron porphyrins, competitive oxidations of alkane and alkene were carried out. It has been proposed that the first step of alkane hydroxylation is H atom abstraction and that of alkene is one-electron transfer. Therefore, it is expected that alkene-alkane competitive oxidation can be used as a probe for discrimination of differences in chemical properties among active species. Cytochrome P450 and SR complex, which is a stable thiolate-ligated iron porphyrin, mediated the oxidation of alkane much more preferentially than iron porphyrin coordinated by imidazole or chloride. These results indicate that thiolate coordination alters the reactivity of the two-electron-oxidized intermediate in a manner that is much more favorable to alkane hydroxylation than the case of chloride or imidazole coordination.

Determination of serum tulobuterol concentrations by mass fragmentography: comparison with an electron-capture gas chromatographic method.

A simple and sensitive method is reported for the quantitative determination of the bronchodilator tulobuterol in human serum. Tulobuterol and an internal standard were extracted from alkalinized serum with ether and then back-extracted into dilute hydrochloric acid. After alkalinization and extraction of the aqueous solution, the extract was evaporated to dryness. The residue was silylated and subjected to mass fragmentography.