A multicenter study of transient global amnesia for the better detection of magnetic resonance imaging abnormalities.

BACKGROUND AND PURPOSE: The detection rate of diffusion-weighted (DWI) hyperintense lesions varies widely in patients with transient global amnesia (TGA). The aim was to examine the association of hyperintense lesions on DWI magnetic resonance imaging (MRI) with patient characteristics, precipitating factors, clinical presentation and MRI settings in patients with TGA. METHODS: In this multicenter retrospective observational study, using the standardized diagnosis entry system of electronic health records of four tertiary medical centers in the Kansai district of Japan, TGA patients (n = 261) who underwent brain MRI within 28 days of onset were examined. When the onset time was unavailable, the discovery time was used. RESULTS: Diffusion-weighted hyperintense lesions were observed in 79 patients (30%). There were no significant differences in age, sex, vascular risk factors, precipitating factors or clinical presentation between patients with and without DWI lesions. The detection rate increased linearly 24 h after onset and then reached a plateau of 60%-80% by 84 h. After 84 h, the detection rate decreased rapidly. In a multivariate logistic regression model, MRI examination 24-84 h after onset (odds ratio 7.00, 95% confidence interval 3.50-13.99) and a thin-slice (≤3 mm) DWI sequence (odds ratio 7.59, 95% confidence interval 3.05-18.88) were independent predictors of DWI lesions. CONCLUSIONS: This study suggests that DWI hyperintense lesions in TGA are not associated with patient characteristics and clinical presentation. Brain MRI examination 24-84 h after onset and thin-slice DWI sequences enhance the detection of DWI lesions in TGA patients.

Carotid artery disease in post-stroke survivors and effects of enriched environment on stroke pathology in a mouse model of carotid artery stenosis.

AIMS: Carotid artery disease (CAD) is an important risk factor for stroke. We first evaluated CAD and stroke pathology in elderly post-stroke survivors. To simulate CAD, we assessed long-term consequences of bilateral common carotid artery stenosis (BCAS) in mice and exposed them to environmental enrichment (EE). METHODS: Histopathological methods were used to determine degrees of CAD (% area stenosis), brain infarct types, sizes and distribution in post-stroke survivors and BCAS mice. Adult male C57BL/6J mice after BCAS or sham surgery were randomly assigned to standard housing (Std) or limited (3 h) or full-time (Full) exposure to EE per day for 12 weeks. RESULTS: High frequencies of moderate carotid artery stenosis (51-75%) were evident in post-stroke survivors whereas those with severe CAD (>75% stenosis) exhibited greater numbers of cortical rather than subcortical infarcts and, were at higher risk of developing dementia. BCAS in mice reduced cerebral blood flow by 52% (P < 0.01) and thickened carotid artery walls, regardless of EE duration. Remarkably, the total and cortical infarcts declined by >50% in BCAS mice exposed to EE compared with BCAS-Std (P < 0.01). Frontal lobe and cortical strokes were associated with worsening working memory tested in a radial maze paradigm. Proteomic analysis revealed EE, both BCAS-3 h and BCAS-Full attenuated coagulation cascade factors including fibrinogen and von Willebrand factor, markers of blood-brain barrier damage. CONCLUSION: Small cortical and subcortical infarcts were evident in both post-stroke survivors with CAD and BCAS mice. Experimental evidence suggested that moderate exposure to EE is sufficient to reduce subsequent stroke lesions.

Relationship between Cnm-positive Streptococcus mutans and cerebral microbleeds in humans.

OBJECTIVE: Cerebral hemorrhage has been shown to occur in animals experimentally infected with Streptococcus mutans carrying the collagen-binding Cnm gene. However, the relationship between cerebral microbleeds and oral hygiene, with a focus on Cnm gene-positive S. mutans infection, remains unclear. MATERIAL AND METHODS: One hundred and thirty-nine subjects participated. The presence or absence of Cnm-positive S. mutans and its collagen-binding activity were investigated using saliva samples, and relationship with cerebral microbleeds detected on MRI investigated, including clinical information and oral parameters. RESULTS: Fifty-one subjects were identified as Cnm-positive S. mutans carriers (36.7%), with cerebral microbleeds being detected in 43 (30.9%). A significantly larger number of subjects carried Cnm-positive S. mutans in the cerebral microbleeds (+) group. S. mutans with Cnm collagen-binding ability was detected in 39 (28.1%) of all subjects, and the adjusted odds ratio for cerebral microbleeds in the Cnm-positive group was 14.4. Regarding the presence of cerebral microbleeds, no significant differences were noted in the number of remaining teeth, dental caries, or in classic arteriosclerosis risk factors. CONCLUSIONS: The occurrence of cerebral microbleeds was higher in subjects carrying Cnm-positive S. mutans, indicating that the presence of Cnm-positive S. mutans increases cerebral microbleeds, and is an independent risk for the development of cerebrovascular disorders.

Subinsular vascular lesions: an analysis of 119 consecutive autopsied brains.

The insula of Reil constitutes a functionally intriguing complex of the brain related to multifunctional activities. We examined the subinsular region in 119 consecutively autopsied patients, as T2 hyperintense lesions are frequently observed in magnetic resonance diagnosis of this region. The patients were admitted in neurology wards and were diagnosed as having cerebrovascular disease in 55 patients (46%), other neurological diseases in 57 patients (48%) and non-neurological diseases in seven patients (6%). Demyelination of the white matter was semi-quantified as a fiber density score (percent stained area/total area) with computer-assisted image analysis on Klüver-Barrera-stained sections. Astrogliosis was assessed by immunohistochemistry for glial fibrillary acidic protein. The lesion analysis showed a dilated perivascular space in 29 patients (24%), demyelination (fiber density score less than the mean - 1 SD) in 27 patients (23%), slit-shaped lesion in six patients (5%), lacunar infarction in one patient (1%) and cerebral hemorrhage in one patient (1%). A histologic-radiologic comparison in two patients with subcortical ischemic vascular dementia showed correspondence between subinsular hyperintensities, and demyelination, gliosis and a dilated perivascular space. These results indicate that subinsular lesions rarely signifies focal vascular lesions, and are consisted of demyelination, gliosis and a dilated perivascular space.

Improvement of virus removal by pilot-scale coagulation-ultrafiltration process for wastewater reclamation: Effect of optimization of pH in secondary effluent.

Reclaimed water (i.e., reused advanced-treated wastewater) offers an alternative water resource. To reduce the health risks associated with its use, efficient virus removal such as with advanced wastewater treatment processes is important. Virus removal by coagulation followed by ultrafiltration (UF) for the treatment of drinking water has been well examined. But its efficacy in wastewater reclamation purpose using secondary treated effluent (SE) from wastewater treatment plant (WWTP) as feed water is unclear. Here, we optimized the virus removal efficiency of coagulation-UF in pilot-scale wastewater reclamation plants using SE as feed water, using the F-specific RNA bacteriophage MS2 as a model virus, at two wastewater treatment plants in Japan. We investigated how using coagulation as a pretreatment for UF improved virus removal efficiency. The efficiency varied greatly between SEs. To reveal the cause of the variation, we conducted laboratory-scale batch coagulation experiments. The efficiency of viral coagulation was negatively correlated with the concentration of dissolved organic matter in the feed water. The optimum pH for coagulation differed between SEs, and the efficiency of coagulation could be dramatically improved by optimizing the pH. We confirmed that the virus removal efficiency in the pilot-scale facility actually could be improved by adjusting the pH. In addition, we confirmed that coagulation-sedimentation-UF with pH adjustment could operate stably for more than 30 days at the pilot scale, with a high virus removal rate. Thus, the wastewater reclamation process described here offers promise in terms of reduced health risks and practical operation.

Translesional synthesis on a DNA template containing N2-methyl-2'-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I.

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N2-methyl-2'-deoxyguanosine (N2-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N2-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N2-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N2-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3'-terminus of a dT:N2-Me-dG pair was only 2.1 times lower than from a dC:N2-Me-dG pair. We conclude that N2-Me-dG is a miscoding lesion capable of generating G-->A transition mutations.

Potent neuropeptide Y Y1 receptor antagonist, 1229U91: blockade of neuropeptide Y-induced and physiological food intake.

Neuropeptide Y (NPY) is thought to increase food intake through the action of Y1 (-like) receptors in the hypothalamus. To confirm the involvement of Y1 receptors in feeding behavior, selective and potent antagonists for Y1 receptors are required. In the present study, we showed that a peptide, 1229U91 [(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg, Tyr-NH2)2 cyclic (2,4'),(2',4)-diamide], is a potent and selective antagonist for Y1 receptors. 1229U91 displaced [125I]peptide YY (PYY) binding to membranes of human neuroblastoma-derived SK-N-MC cells that predominantly express Y1 receptors with a K1 value 0.10 nM and inhibited the NPY-induced increase in intracellular calcium levels(IC50 = 0.27 nM). In contrast, the K1 values for [125I]PYY binding to Y2 receptors in membranes of human neuroblastoma-derived SK-N-BE2 cells and rat hypothalamus were 700 nM and more than 1 microM, respectively. Although [125I]PYY could not detect Y1 receptors in the rat hypothalamic membranes, [125I]1229U91 revealed binding sites with a high affinity (Kd = 18 pM), indicating the presence of Y1 receptors in the hypothalamus. Intracerebroventricular injection of 1229U91 (30 micrograms) into male Sprague-Dawley rats completely inhibited NPY (5 micrograms)-induced food intake without any other behavioral change. Furthermore, intracerebroventricular injection of 1229U91 significantly suppressed physiological feeding behavior after overnight fasting. These results indicate that Y1 receptors in the rat hypothalamus mediate NPY-induced food intake, and that physiological feeding behavior after overnight fasting may be largely regulated by NPY via Y1 receptors. 1229U91 may be useful for further elucidating the pathophysiological roles of NPY in feeding behavior.

Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice.

Neuropeptide Y (NPY) increases food intake through the action of hypothalamic NPY receptors. At least six subtypes of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) receptors have been identified in mice. Although the involvement of Y1 and Y5 receptors in feeding regulation has been suggested, the relative importance of each of these NPY receptors and the participation of a novel feeding receptor are still unclear. To address this issue, we generated a Y1 receptor-deficient (Y1-/-) and a Y5 receptor-deficient (Y5-/-) mouse line in which we directly compared the orexigenic effects of NPY and its analogs after intracerebroventricular (icv) administration. The icv NPY-induced food intake was remarkably reduced in Y1-/- mice, but was not significantly altered by inactivation of the Y5 receptor. The Y1 receptor therefore plays a dominant role in NPY-induced feeding. Stimulation of feeding by moderately selective Y5 agonists [PYY-(3-36), human PP, and bovine PP] was reduced in Y5-/- mice, although food intake did not decrease to vehicle control levels. These results indicate that the Y5 receptor functions as one of the feeding receptors. In addition, the finding that Y5-preferring agonists still induce food intake in Y5-/- mice suggests a role for another NPY receptor(s), including the possibility of novel NPY receptors. Surprisingly, despite the limited efficacy of PYY-(3-36) and PPs at the Y1 receptor, food consumption induced by these agonists was significantly diminished in Y1-/- mice compared with that in wild-type controls. These observations suggest that the feeding stimulation induced by NPY and its analogs may be directly or indirectly modulated by the action of the Y1 receptor. We conclude that multiple NPY receptors, possibly including the novel feeding receptor, are involved in the feeding response evoked by NPY and its analogs. Among them, the Y1 receptor plays a key role in NPY-induced feeding in mice.

Decrease in cortical benzodiazepine receptors in symptomatic patients with leukoaraiosis: a positron emission tomography study.

BACKGROUND AND PURPOSE: [11C]flumazenil (FMZ), a ligand that selectively binds to the central benzodiazepine receptor in the neuronal membrane, is useful for evaluating neuronal viability in a positron emission tomography (PET) scan. Using this ligand, we investigated whether there was a correlation between neuronal integrity in various brain structures and dementia in patients with leukoaraiosis. METHODS: Twelve patients with extensive leukoaraiosis on magnetic resonance imaging were divided into groups of patients with or without dementia. Based on a 2-compartment, 2-parameter model that included metabolite-corrected arterial input and PET-measured cerebral radioactivity, the distribution volume of FMZ (FMZ-V(d)) was calculated in various regions of interest by nonlinear curve fitting. Additionally, tracer kinetic analysis was applied for voxel-by-voxel quantification of FMZ-V(d), and data analysis was performed by statistical parametric mapping. RESULTS: The presence of dementia was associated with a reduced FMZ-V(d) in widespread areas of the cerebral cortex, including the bilateral frontopolar and frontal/insular areas, the left temporo-occipital border areas, and the left marginal cortical areas. CONCLUSIONS: Differences in neuronal integrity in the cerebral cortex might determine whether patients with leukoaraiosis become symptomatic or not.

Increased chymase-dependent angiotensin II formation in human atherosclerotic aorta.

Locally formed angiotensin II (Ang II) and mast cells may participate in the development of atherosclerosis. Chymase, which originates from mast cells, is the major Ang II-forming enzyme in the human heart and aorta in vitro. The aim of the present study was to investigate aortic Ang II-forming activity (AIIFA) and the histochemical localization of each Ang II-forming enzyme in the atheromatous human aorta. Specimens of normal (n=9), atherosclerotic (n=8), and aneurysmal (n=6) human aortas were obtained at autopsy or cardiovascular surgery from 23 subjects (16 men, 7 women). The total, angiotensin-converting enzyme (ACE)-dependent, and chymase-dependent AIIFAs in aortic specimens were determined. The histologic and cellular localization of chymase and ACE were determined by immunocytochemistry. Total AIIFA was significantly higher in atherosclerotic and aneurysmal lesions than in normal aortas. Most of AIIFA in the human aorta in vitro was chymase-dependent in both normal (82%) and atherosclerotic aortas (90%). Immunocytochemical staining of the corresponding aortic sections with antichymase, antitryptase or anti-ACE antibodies showed that chymase-positive mast cells were located in the tunica adventitia of normal and atheromatous aortas, whereas ACE-positive cells were localized in endothelial cells of normal aorta and in macrophages of atheromatous neointima. The density of chymase- and tryptase-positive mast cells in the atherosclerotic lesions was slightly but not significantly higher than that in the normal aortas, and the number of activated mast cells in the aneurysmal lesions (18%) was significantly higher than in atherosclerotic (5%) and normal (1%) aortas. Our results suggest that local Ang II formation is increased in atherosclerotic lesions and that chymase is primarily responsible for this increase. The histologic localization and potential roles of chymase in the development of atherosclerotic lesions appear to be different from those of ACE.

Chronic cerebral hypoperfusion induces MMP-2 but not MMP-9 expression in the microglia and vascular endothelium of white matter.

White matter lesions are closely associated with cognitive impairment and motor dysfunction in the aged. To explore the pathophysiology of these lesions, the authors examined the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the white matter in a rat model of chronic cerebral hypoperfusion. After bilateral clipping of the common carotid arteries, myelin staining revealed demyelinating changes in the optic tract and the corpus callosum on day 7. Zymographic analyses indicated an increase in the level of MMP-2, but not MMP-9, after the hypoperfusion. Immunohistochemical analyses revealed the presence (most abundantly on day 3) of MMP-2-expressing activated microglia in the optic tract and corpus callosum. In contrast, the capillary endothelial cells expressed MMP-2 later. IgM-immunoreactive glial cells were absent in the sham-operated animals, but were present in the hypoperfused animals by day 3, reflecting the disrupted blood-brain barrier. These findings suggest that the main sources of the elevated MMP-2 were the microglia and the endothelium, and that these cells may contribute to the remodeling of the white matter myelin and microvascular beds in chronic cerebral hypoperfusion.

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.

Identification of human endomucin-1 and -2 as membrane-bound O-sialoglycoproteins with anti-adhesive activity.

Using a signal sequence trap method and database search, we identified a series of human cDNAs encoding two structurally related type I membrane proteins of approximately 25 kDa with multiple glycosylation motifs. These genes, termed endomucin-1/-2, are expressed in several human tissues including heart, kidney, and lung. Exogenously expressed human endomucin-1/-2 proteins were modified into 80-120 kDa glycoproteins, which were susceptible to O-sialoglycoprotein endopeptidase digestion. Transient overexpression of endomucin-1/-2 reduced the number of adhesion plaques and reduced cell attachment to the substrate. This phenotype was suppressed by laminin or the protein kinase inhibitor staurosporine. Our findings suggest that human endomucin-1/-2 negatively regulate cell adhesion to the extracellular matrix.

Cytochrome P450 2E1 polymorphism in gastric cancer in Brazil: case-control studies of Japanese Brazilians and non-Japanese Brazilians.

Cytochrome P450 2E1 (Cyp2E1) is involved in the metabolic oxidation of carcinogenic nitroso compounds, including N-nitrosoamines. There is an RsaI polymorphism in the transcriptional regulatory region of this gene, and in vitro evidence suggests that the variant type of this polymorphic site has higher transcriptional activity but less chlorzoxazone-metabolizing activity. Interindividual differences in the metabolic capacity of Cyp2E1 are assumed to be associated with cancer susceptibility, but the results of the previous studies on the relation between Cyp2E1 RsaI polymorphism and cancer susceptibility have been inconsistent. Two case-control studies of gastric cancer in Japanese Brazilians (96 cases, 192 controls) and Brazilians not of Japanese ancestry (non-Japanese Brazilians; 236 cases, 236 controls) in São Paulo were designed to clarify the role of the Cyp2E1 RsaI genotype in susceptibility to gastric cancer after considering multifactorial environmental influences. The subjects with variant RsaI genotypes amounted to 47% (28 of 59) and 48% (64 of 133), respectively, of the Japanese cases and controls, and 6% (11 of 187) and 10% (19 of 192), respectively, of the non-Japanese cases and controls. As expected, a difference in the distributions of the two groups was observed. The odds ratio of the RsaI variant genotype of Cyp2E1 was 0.46 (95% confidence interval, 0.21-1.04) in the non-Japanese Brazilian population and 0.98 (95% confidence interval, 0.50-1.90) in the Japanese Brazilian population after adjusting for sex, age, tobacco use, and meat consumption. Additional adjustment for potential confounding factors did not change the odds ratio substantially. No significant interactions were observed between the polymorphism and environmental factors. In regard to the histological type of gastric cancer, the variant genotype was significantly more prevalent than the common genotype in Japanese subjects with diffuse type gastric cancer. Our study suggests that the Cyp2E1 RsaI polymorphism is associated with a reduced risk of gastric cancer, although how the assumed increase in Cyp2E1 expression produced by this polymorphism is related to a reduced risk of cancer remains unclear. The observations in this study are consistent with the recent observations of esophageal cancer in endemic areas of China.

Neuroleptic malignant syndrome with prolonged catatonia in a dopa-responsive dystonia patient.

The authors describe a patient with dopa-responsive dystonia who developed neuroleptic malignant syndrome with prolonged catatonia following treatment with neuroleptic agents. Use of these agents probably expanded the patient's neuronal dysfunction beyond the nigrostriatal system to involve multiple dopaminergic systems. Electroconvulsive treatment alleviated the prolonged catatonia.

Synthesis and structure-activity relationships of 2-substituted D-tryptophan-containing peptidic endothelin receptor antagonists: importance of the C-2 substituent of the D-tryptophan residue for endothelin A and B receptor subtype selectivity.

Continuing studies on modifications of potent cyclic pentapeptide endothelin (ET) receptor antagonists, represented by BQ-123, and potent linear tripeptide derivative ET receptor antagonists, represented by BQ-788, are described herein. The introduction of D-tryptophan analogues with C-2 substituents in these peptidic ET antagonists resulted in potent ET receptor antagonists with various ETA/ETB subtype selectivity. Combined ETA/ETB receptor antagonists were found in both cyclic pentapeptide and linear tripeptide series with 2-halo- and 2-methyl-D-tryptophans. In contrast, compounds with 2-cyano-D-tryptophan were ETB receptor-selective antagonists. The C-2 substituent of the D-tryptophanyl residue appeared to be very important for the discrimination of ETA/ETB subtype selectivity of the antagonists. The potent ET receptor antagonists with various ETA/ETB subtype selectivity synthesized in this study may be useful tools for elucidating the physiological and pathophysiological roles of ET and ET receptors.

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.

Human big-endothelin-1 and endothelin-1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung.

Although ET1 and ET2 binding sites were found in rat lung membranes, a selective ET1 receptor antagonist, BQ-123 (10 microM), did not displace [125I]-endothelin-1 ([125I]ET-1) from ET2 sites, illustrating the selectivity of the angatonist for ET1 receptors. In rat perfused lungs, BQ-123 (1 microM) markedly reduced the prostacyclin (PGI2) releasing properties of endothelin-1 (ET-1: 5 nM) and human big-ET-1 (100 nM) suggesting that both peptides induce the release of PGI2 via the selective activation of ET1 receptors.

Necessity of dual blockade of endothelin ETA and ETB receptor subtypes for antagonism of endothelin-1-induced contraction in human bronchi.

1. Endothelin (ET)-1 has been postulated to be involved in the development of obstructive airway diseases in man. In the present study, we attempted to characterize ET receptor subtypes mediating ET-1-induced contraction in human isolated bronchi. The ET receptor antagonists used in the present study were BQ-123 (ETA receptor-selective), BQ-788 (ETB receptor-selective) and BQ-928 (ETA/ETB dual). Sarafotoxin S6c (S6c) was also used as an ETB receptor-selective agonist. 2. In human bronchi, ET-1 and S6c (10(-12)M to 10(-7) M) produced concentration-dependent contraction with almost equal potency (pD2: 8.88 +/- 0.16 for ET-1 and 9.42 +/- 0.15 for S6c). The contraction induced by S6c was competitively antagonized by BQ-788 alone (1 and 10 microM) with a pKB value of 7.49 +/- 0.21, suggesting that the stimulation of ETB receptors causes a contraction of human bronchi. However, contrary to expectation, the concentration-response curves for ET-1 were not affected by BQ-788. The ET-1- and S6c-induced contractions were not affected by BQ-123 (10 microM). Thus, ET-1-induced contraction of human bronchi is not antagonized by BQ-123 alone or by BQ-788 alone. 3. Combined treatment with 10 microM BQ-123 and 10 microM BQ-788 significantly antagonized the contraction induced by ET-1 with a dose-ratio of 11. BQ-928 also significantly antagonized ET-1-induced contraction with a pKB value of 6.32 +/- 0.24. 4. The specific binding of [125I]-ET-1 to human bronchial membrane preparations was inhibited by BQ-123 (100 pM to 1 microM) by approximately 40%. Combination treatment with BQ-788 (100 pM to 1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. 5. In conclusion, the present study demonstrates that BQ-788 alone cannot inhibit ET-1-induced contractions in human bronchi, although human bronchial ETB receptors are BQ-788-sensitive. Furthermore, it was shown that blockade of both receptor subtypes antagonizes ET-1-induced contraction, and that both receptor subtypes co-exist in human bronchial smooth muscles. These findings suggest that ETA receptors as well as ETB receptors are involved in ET-1-induced contraction in human bronchi. If ET-1 is involved in human airway diseases, dual blockade of ETA and ETB receptors may be necessary to treat the diseases.