Therapeutic potential for insulin on type 1 diabetes-associated periodontitis: Analysis of experimental periodontitis in streptozotocin-induced diabetic rats.

AIMS/INTRODUCTION: The association between diabetes and periodontal disease is considered to be bidirectional. However, there is still controversy surrounding the relationship between periodontal disease and type 1 diabetes. We investigated whether insulin improves periodontitis without any local treatments for periodontitis under type 1 diabetes conditions using the ligature-induced experimental periodontitis model. MATERIALS AND METHODS: Type 1 diabetic rats were induced by streptozotocin injection. Experimental periodontitis was induced by ligature in normal and diabetic rats. Half of the diabetic rats were treated with insulin. Two weeks after the ligature, periodontitis was evaluated. RESULTS: Insulin treatment significantly improved inflammatory cell infiltration and inflammatory cytokine gene expression, leading to suppression of alveolar bone loss, in the periodontitis of diabetic rats. Insulin also suppressed the periodontitis-increased nitric oxide synthase-positive cells in periodontal tissue of the diabetic rats. Even without induction of periodontitis, diabetic rats showed decreased gingival blood flow and an increased number of nitric oxide synthase-positive cells in the gingiva and alveolar bone loss compared with normal rats, all of which were ameliorated by insulin treatment. We further confirmed that insulin directly suppressed lipopolysaccharide-induced inflammatory cytokine expressions in THP-1 cells. CONCLUSIONS: There were abnormalities of periodontal tissue even without the induction of periodontitis in streptozotocin-induced diabetic rats. Insulin treatment significantly ameliorated periodontitis without local periodontitis treatment in diabetic rats. These data suggest the therapeutic impacts of insulin on periodontitis in type 1 diabetes.

Role of poly(ADP-ribose) polymerase activation in the pathogenesis of periodontitis in diabetes.

AIM: The aetiology of progressive periodontitis in diabetes has not yet been elucidated. We previously demonstrated that nitrosative stress is increased in diabetic rats with periodontitis. Nitrosative stress induces poly(ADP-ribose) polymerase (PARP) activation. Here, we demonstrated the involvement of PARP activation in diabetic periodontitis and detailed the therapeutic effects of PARP inhibitor. MATERIALS AND METHODS: Experimental periodontitis was induced by placing a nylon thread ligature. Half of the normal and diabetic rats received the PARP inhibitor, 1,5-isoquinolinediol, for 2 weeks. Gingival PARP activation was detected by immunostaining for poly(ADP-ribose). Periodontitis was evaluated by gingival inflammatory cell infiltration, inflammatory gene expressions and micro-CT analyses. RESULTS: Although both periodontitis and the presence of diabetes increased PARP activation in the gingiva, diabetic rats with periodontitis had the highest activation of PARP. Diabetic rats with periodontitis also showed significant increases in monocyte/macrophage invasion into the gingiva, inflammatory gene expressions, nitrotyrosine-positive cells in the gingiva and alveolar bone loss, all of which were suppressed by treatment with the PARP inhibitor. CONCLUSIONS: These results indicate the involvement of PARP activation in the pathogenesis and aggravation of periodontal disease in diabetes and suggest the therapeutic potential of PARP inhibition for treating periodontal disease, especially in patients with diabetes.

Anti-inflammatory role of glucose-dependent insulinotropic polypeptide in periodontitis.

AIMS/INTRODUCTION: The involvement of glucose-dependent insulinotropic polypeptide (GIP) on inflammation was explored in atherosclerosis and adipose tissue. Periodontal disease is a chronic inflammatory disease, and is considered one of the diabetic complications. In the present study, to examine the effect of GIP on periodontitis, we induced experimental periodontitis in glucose-dependent insulinotropic polypeptide receptor-knockout mice (GIPRKO). We also investigated the anti-inflammatory effect of GIP in a culture system. MATERIALS AND METHODS: Experimental periodontitis was induced by ligature wire in GIPRKO and C57BL/C mice. Two weeks after the ligature, immunohistological evaluation and inflammatory messenger ribonucleic acid expression in the gingiva was examined. To elucidate the role of GIP in inflammation, the effects of GIP on lipopolysaccharide-induced gene expressions in THP-1 cells were evaluated. RESULTS: Periodontitis increased inflammatory cell infiltration, macrophage accumulation and tumor necrosis factor-α and nitric oxide synthase gene expressions in the gingiva. Periodontitis in GIPRKO showed a marked increase of inflammatory cells in the gingivomucosal tissue. Mac-1-positive macrophages and the inflammatory gene expressions were significantly increased in periodontitis in GIPRKO compared with C57BL/C mice periodontitis. Immunohistochemical staining confirmed that GIP receptors were expressed in residual and infiltrated Mac-1-positive macrophages. The in vitro study showed that GIP suppressed lipopolysaccharide-induced tumor necrosis factor-α and nitric oxide synthase gene expression in a dose-dependent manner. Furthermore, the inhibitory effect of GIP on lipopolysaccharide-induced inflammatory gene expressions was at least partially through cyclic adenosine monophosphate/protein kinase A pathway. CONCLUSIONS: These results suggest the beneficial effects of GIP on periodontal disease. In diabetic patients, GIP is expected to have a direct anti-inflammatory effect on periodontitis in addition to its glucose-lowering effect.

Nerve innervations in the human adult and fetal parotid duct

The human adult parotid duct is the longest of all major salivary gland ducts, approximately 6-8 cm in length. Its unique structure extends over the masseter muscle, penetrates through the buccinator muscle and opens into the oral cavity. Salivary secretion is under basic control of the sympathetic and parasympathetic divisions of the autonomic nervous system. Scarce reporting on the parotid duct nerve distribution led us to this study; to investigate the nervous distribution in the human adult and fetal parotid ducts using an antibody against protein gene product 9.5 (PGP9.5), a molecular marker for nerve cells and fibers. In order to identify the nerve fibers distributed throughout the parotid duct and confirm them to be part of the autonomic nervous system, we stained adult parotid ducts with tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) for observation. PGP9.5 staining of the parotid duct’s inside wall where it traverses over the masseter prior to penetrating the buccinator revealed a dense concentration of nerve fibers in the area. Staining revealed both sympathetic and parasympathetic nerve fibers in the same area, with the majority of the sympathetic nerve fibers surrounding blood vessels. However, the section of the duct penetrating the buccinator showed less concentration of nerve fibers in both adult and fetal specimens. The difference in the nerve distribution of the parotid duct suggests its direct association with the salivary transport function of the duct. PGP9.5 expression in fetuses over five months of age further suggests that the nerve distribution in the human parotid duct is fully established at six months of gestation

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Increased expression of interleukin (IL)-35 and IL-17, but not IL-27, in gingival tissues with chronic periodontitis.

BACKGROUND: Interleukin (IL)-35 plays an important role in immune regulation through the suppression of effector T-cell populations, including T-helper 17 (Th17) cells. Although Th17 cells and IL-17 are involved in the pathogenesis of periodontitis, the level of IL-35 in inflamed periodontal tissues is unclear. Here, IL-35, IL-17, and IL-27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. METHODS: GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme-linked immunosorbent assay for IL-35 (periodontitis, n = 36; healthy, n = 30) and IL-17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus-induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. RESULTS: IL-35 and IL-17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL-35 and probing depth and clinical attachment level (CAL) as well as between IL-17 and CAL. EBI3, IL12A (components of IL-35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL-27 is not produced in large quantities in periodontal tissue. CONCLUSION: IL-35 and IL-17, but not IL-27, may play important roles in the pathogenesis of periodontitis.

Age-related changes of the ultrastrucure in the parathyroid gland of the golden hamster.

We qualitatively and quantitatively investigated the parathyroid glands of golden hamsters aged 6, 12, 18, 24 and 30 months. Percent area of rER in the parathyroid gland in golden hamsters at 24 months of age was significantly higher when compared to 6 and 12 months of age, and the percent area at 30 months of age was significantly higher when compared to 12 months of age, but there were no significant differences between 24 and 30 months of age. Percent area of the Golgi apparatus at 24 and 30 months of age was significantly higher when compared to 6, 12 and 18 months of age. Ultrastructurally, we believe that in the parathyroid gland of the golden hamster, synthesis and release of parathyroid hormone increase gradually from 6 to 24months of age and are maintained from 24 to 30 months of age.

Periodontitis-activated monocytes/macrophages cause aortic inflammation.

A relationship between periodontal disease and atherosclerosis has been suggested by epidemiological studies. Ligature-induced experimental periodontitis is an adequate model for clinical periodontitis, which starts from plaque accumulation, followed by inflammation in the periodontal tissue. Here we have demonstrated using a ligature-induced periodontitis model that periodontitis activates monocytes/macrophages, which subsequently circulate in the blood and adhere to vascular endothelial cells without altering the serum TNF-α concentration. Adherent monocytes/macrophages induced NF-κB activation and VCAM-1 expression in the endothelium and increased the expression of the TNF-α signaling cascade in the aorta. Peripheral blood-derived mononuclear cells from rats with experimental periodontitis showed enhanced adhesion and increased NF-κB/VCAM-1 in cultured vascular endothelial cells. Our results suggest that periodontitis triggers the initial pathogenesis of atherosclerosis, inflammation of the vasculature, through activating monocytes/macrophages.

Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis.

Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.

The effect of sevoflurane on developing A/J strain mouse embryos using a whole-embryo culture system--the incidence of cleft lip in culture embryos.

A/J strain mice have a high spontaneous incidence of cleft lip (ICL) and/or palate. The primary palate-related effects of sevoflurane on developing A/J strain mouse embryos (embryos) were studied using a whole-embryo culture (WEC) system. This system could separate the direct effects of sevoflurane from those that are maternally mediated. A total of 205 10.5-d embryos were cultured for 24 h in either a control group (control gas: 95% O2 and 5% CO2) or sevoflurane-administered groups (1/4, 1/2, and 1 minimum alveolar concentration (MAC) with control gas) for 8 h. After 16 h, 11.5-d culture embryos were examined in terms of crown-rump length, number of somites, and protein content. Crown-rump length in the 1 MAC was significantly shorter than in the control group (p < 0.05). Protein content in the 1/2 MAC (p < 0.05) and 1 MAC (p < 0.001) was significantly lower than in the control group. The ICL showed no significant differences between each group. (The ICL rose with an increase in the sevoflurane concentration, but this was not significant). The positive findings in this study indicate that a WEC system is useful for studying the mechanisms of ICL (teratogenicity) associated with sevoflurane.

Confocal laser scanning microscopic analysis of ectopic sublingual gland-like tissue inside the hamster submandibular gland.

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3'-diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO-positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous-like cells were found in resin-embedded semi-thin sections of the DAB-reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance-mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity-negative secretory granules and seromucous cells with PO activity-positive spot-like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron-dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron-dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland-like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct.

Involvement of nitrosative stress in experimental periodontitis in diabetic rats.

AIM: Periodontal disease is highly prevalent and severe in diabetic patients, and is considered one of the diabetic complications. To elucidate how periodontitis progresses in diabetes, we examined an animal model of periodontitis in diabetic rats. MATERIALS AND METHODS: Two weeks after the induction of diabetes by streptozotocin, surgical nylon thread was ligated around the cervical portion of the unilateral maxillary second molar to induce periodontitis. Periodontitis was evaluated 2 weeks after the ligation by gingival blood flow, mRNA expressions, Western blot analysis, histological examination and micro CT. RESULTS: Ligation-induced severe periodontitis in the diabetic rats, which was apparently shown by the increase of TNF-α and iNOS mRNA expressions and inflammatory cell infiltration in the gingiva and alveolar bone loss. The number of nitrotyrosine, a footprint of nitrosative stress, -positive cells was significantly higher in the periodontitis of the diabetic rats compared with that in the normal rats. Western blot analysis confirmed that the nitrotyrosine was increased in the periodontitis of the diabetic rats. CONCLUSIONS: This is the first study to confirm increased nitrosative stress due to periodontitis in diabetic rats. Nitrosative stress may play a crucial role in the exacerbation of periodontitis in diabetic patients.

EFTEM cytochemistry and sexual dimorphism of secretory granules in male and female hamster submandibular glands.

After glutaraldehyde fixation followed by osmium tetroxide postfixing, the secretory granules of acinar cells in male hamster submandibular glands (SGs) exhibit a characteristic bipartite substructure, with an electron-lucid rim and a more electron-dense central core. In female hamsters, the reverse is seen, with the larger portion of the granules forming an electron-lucid core and an outer electron-dense crescent rim. In the present study of endogenous peroxidase (PO) activity of male and female hamster SGs, secretory granules in the acinar cells were studied by DAB cytochemical technique. Individual granules showed bipartite substructure with the PO activity in a positive center core and unreacted lucid rim in both the male and the female acinar cells. Through isolation of granular fractions, the male and the female granules exhibited the same bipartite structure. We also examined the relation between the PO activity and counterstained areas in male and female hamster SGs, and the secretory granules of acinar cells by using EFTEM. In the male SG, the secretory granules exhibited the characteristic bipartite substructure to carry out parallel-EELS, nitrogen reflecting the presence of DAB moieties and uranium from counterstaing the presence the central core but not in the rim. On the other hand, the female bipartite secretory granules of the SG, exhibit the nitrogen reflecting the presence in the central core and uranium in the rim.

Light and electron microscopic histochemistry of the peroxidase activity in the secretory granules of the developing hamster submandibular gland.

On the basis of light and electron microscopic observations of the post natal development of the hamster submandibular gland, granules in the acinar cells showed considerably variations in size and shape, as well as electron density of the peroxidase-positive reaction. The present study shows that secretory granules of the hamster submandibular gland undergo changes of area and of intensity for peroxidase activity 6 months after birth.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. IT forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3'-diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Quantitative assessment of succinate dehydrogenase activity of rat lateral pterygoid muscle in undecalcified fresh-frozen section.

The quantitative assessment of enzyme activities in situ in single muscle fibres is essential for understanding the functions of skeketal muscles. The function of the lateral pterygoid muscle (LPM) is not fully understood because it is a deeply located masticatory muscle and cannot be dissected in an intact configuration. Here we report how to measure the activities of mitochondrial succinate dehydrogenase (SDH) in single muscle fibres in situ in the LPM in sections of rat heads. Unfixed head sections were incubated on gel films containing SDH substrate and nitroblue tetrazolium. During incubation, images of the section due to deposition of the final reaction products, formazans, were captured at intervals of 10 s using a real-time image analysis system for absorbance measurements. We found that the belly of the LPM consisted of four areas with different mean activities of SDH. The lateral and upper areas of the muscle showed similarly high SDH activities. Mean activity in the lower area was the lowest, about half of those of the lateral and upper areas. These results agree with the hypothesis that the superior head of the LPM participates in more continuous contraction and is more resistant to fatigue than the inferior head.

Relationships between BK virus lineages and human populations.

BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney, in which it can cause nephropathy in renal transplant patients. BKV isolates are classified into four subtypes (I-IV) using serological or genotyping methods, and subtype I is further divided into four subgroups, Ia, Ib-1, Ib-2, and Ic, based on DNA sequence variations. To clarify whether there is an association between BK virus lineages and human populations, we examined BKV-positive urine samples collected from immunocompetent individuals at various locations in Europe, Africa, and Asia. Partial BKV DNA sequences (n=299) in these samples were determined and subjected to phylogenetic and single nucleotide polymorphism analysis to classify BKV isolates around the world. The validity of the classification was confirmed by analyses based on complete BKV DNA sequences. Subtype I was the major subtype throughout the studied regions, and subtype IV was prevalent only in Asia and Europe. Subtype-I subgroups showed close relationships to major geographical areas. It has recently been shown that JC virus (a human polyomavirus closely related to BKV) co-evolved with human populations, and the present study thus suggests that host-linked evolution is the general mode of polyomavirus evolution. Additionally, our results indicate certain unique aspects of the relationship between BKV and humans.

Cytochemical detection of endogenous peroxidase in the intralobular ducts of hamster major salivary glands.

Light and electron microscopic cytochemical investigation of endogenous peroxidase activity in the intralobular ducts of hamster major salivary glands was carried out using the diaminobenzidine-hydrogen peroxidase method. The peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, the Golgi apparatus and secretory granules in both the intercalated duct cells and the striated duct light cells of all glands. These results suggest the ability of the intralobular duct cells to secrete peroxidase the same as that of acinar cells in hamster salivary glands.

Changes of brain endothelin levels and peripheral endothelin receptors by chronic cigarette smoke in spontaneously hypertensive rats.

The present study was conducted to evaluate the contribution of endothelin (ET) to the pharmacodynamic response to chronic cigarette smoke in spontaneously hypertensive rats (SHR). The contribution of ET was studied consequent to the hemodynamic response following 8 weeks of cigarette smoke by determining the changes in tissue ET-1 content and ET receptors. The blood pressure (BP) at the early phase of smoking and the heart rate (HR) 24 h later were apparently reduced in SHR, while the HR at the early phase was transiently elevated in normotensive Wistar Kyoto (WKY) rats. Tissue ET-1 levels in the hypothalamus, striatum, and cortex of SHR were higher than those in WKY rats, and these higher levels in SHR were reduced by exposure to chronic cigarette smoke. The ET-1 contents in the medulla oblongata and midbrain of both strains were clearly increased by smoke exposure, although the levels of SHR and WKY rats were not different. In addition, the immunoreactivity of the ET type A receptor in the adrenal glands and type B receptor in the kidneys of SHR showed a different response to smoke exposure as compared to WKY rats. Our present findings suggest that the changes of ETs may relate to the pharmacodynamic effects of chronic cigarette smoke.

Highly enhanced permeability of blood-brain barrier induced by repeated administration of endothelin-1 in dogs and rats.

The effects of intracisternal administration of endothelin-1 on blood-brain barrier permeability were examined in dogs and rats. Single doses of endothelin-1 elevated blood-brain barrier permeability about two times compared with control groups in both species. However, repeated dosing with endothelin-1 at a 24 hr intervals caused a highly enhanced disruption of blood-brain barrier permeability in dogs, but not in rats, whereas the repeated administration with a 48 hr interval markedly increased the blood-brain barrier permeability in both species of animals (dogs: 923%, rats: more than 661%). Moreover, this abnormally enhanced permeability of blood-brain barrier in dogs was completely blocked by pretreatment with the endothelin ET-A receptor selective antagonist, S-0139, administered prior to either the first or second dosing with endothelin-1. From these results, we conclude that a repeated attack of endothelin-1 from the adventitial site of brain blood vessels produces a severe disruption of blood-brain barrier permeability through an endothelin ET-A receptor-mediated process.