Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1

Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production.These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.

Aprotinin Inhibits Vascular Smooth Muscle Cell Inflammation and Proliferation via Induction of HO-1

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1beta plus TNF-alpha), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.

Heme Oxygenase-1 Induced by Aprotinin Inhibits Vascular Smooth Muscle Cell Proliferation Through Cell Cycle Arrest in Hypertensive Rats

Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.

Kinetic Changes of COX-2 Expression during Reperfusion Period after Ischemic Preconditioning Play a Role in Protection Against Ischemic Damage in Rat Brain

A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.

Evaluation of Curriculums of Medical Schools for Fostering Various Basic Qualities of Medical Professionalism / 한국의학교육

PURPOSE: Today, the social requirement of medicine emphasizes the importance of medical professionalism. This forces medical educators to introduce new contents and methods into the curriculum. This study aims to offer ideas for developing the curriculum through clarifying priorities on the basic qualities of medical professionalism and evaluating the current curriculums in medical schools in South Korea. METHODS: In April 2005, 46 respondents majoring in basic medical sciences, clinical medicine, and medical humanities/social sciences completed a structured questionnaire. The questionnaire consisted of 3 categories related to: 1) the basic qualities of medical professionalism/general education courses, 2) the process of developing the qualities of humanities and social sciences in medical education, and 3) the appropriate allocation of credits for each subject to premedical and medical courses. The analysis consisted of frequency, chi-square, and multiple responses using Korean Ed, SPSS 14.0 for Windows. RESULTS: The most important basic quality is basic medical knowledge. The credits for the general education are sufficient but it's contribution is lacking (44.44%). The most lacking element in the general education courses is socio-cultural comprehension (45.65%). The knowledge of humanities and social sciences is very important in developing the basic qualities (56.52%). The important learning experiences related to these is the introspection into human beings (32.16%). Credits for medical humanities classes in premedical and medical course are noticeably insufficient (45.65, 54.35%, respectively). The appropriate program of informal curriculum for fostering the qualities is meeting with various medical specialists (44.44%, 47.83%, respectively). There is almost no difference among the major groups under (p<.05.) CONCLUSION: To assist medical students to be equipped with the basic qualities of medical professionalism, the realm of medical humanities should be made mandatory; and the general education courses need to be reformed, especially in the premedical curriculum. In particular, continuous cooperation between faculties in medicine, liberal arts, and/or social sciences need to exist with the conglomeration of these into fewer medical humanities majors in Korea.

Effects of Antioxidants on the Gamma-Radiation Damage of the Cultured Vascular Smooth Mucle Cells of Rat Aorta

To study the protective effects of antioxidants on the radiation damages of the cells, vascular smooth muscle cells (VSMC) from thoracic aorta of Sprague-Dawley rats were cultured and irradiated with gamma-ray. Cell viability was measured by direct cell counting and MTT assay, and flow cytometry was performed to measure fractional distributions of the cells. Gamma-ray irradiation inhibited cell proliferations accompanied with decreased G1 phase and increased S- and G2/M phases, and the maximum effects were observed at 1500 or 2000 cGy. Submaximal concentrations of antioxidants, such as allopurinol, vitamin C, N-acetylcycteine (NAC), lipoic acid, dihydrolipoic acid and rebamipide tended to increase the cell viability suppressed by low dose of radiation (500 cGy), and enalapril and vitamin E increased it significantly. Allopurinol, vitamin E, NAC, lipoic acid, captopril and enalapril significantly increased G1 phase. Allopurinol and vitamin E tended to increase c-Myc expression, detected by Western blot, that was reduced by the radiation, and enalapril increased it significantly. The cell viability and c-Myc expression were highly correlated (r=0.97) with each other. These results suggest that antioxidants, especially enalapril and vitamin E, recover the viability of VSMC from gamma-radiation injury, through a mechanism which includes increase of c-Myc protein expression.

Nitric Oxide Donor, NOR-3, Increased Expression of Cyclooxygenase-2, but not of Cyclooxygenase-1 in Cultured VSMC

NO and cyclooxygenase-2 (COX-2) are contributes to vascular inflammation induced by various stimulation. The mechanism, which explains a linkage between NO and COX-2, could be of importance in promoting pathophysiological conditions of vessel. We investigated the effects of NO donors on the COX-1 and COX-2 mRNA/protein expression, as well as the nitrite production in culture medium of vascular smooth muscle cell (VSMC). VSMC was primarily cultured from thoracic aorta of rat. In this experiments, COX-1 and COX-2 mRNA/protein expressions were analysed and nitrite productions were investigated using Griess reagent. VSMC did not express COX-2 protein in basal condition (Non-lipopolysaccharide (LPS) stimulated). In LPS-stimulated experiments, after 3 hours of NO donor pretreatment, LPS 10 microgram/ml was treated for 24 hours. COX-1 protein expressions were unchanged by SNP and NOR-3. NOR-3 significantly increased COX-2 mRNA/protein expression under LPS stimulation. In contrast, SNP did not increase COX-2 mRNA/protein expression under LPS stimulation. Nitrite production was higher in NOR-3 treatment than SNP treatment under LPS stimulation. These results suggest that the expression of COX-2 in VSMC is regulated by NOR-3, COX-2 expressions were depending on the types of NO donor and LPS stimulation in VSMC.

Effects of Cyclooxygenase Inhibitors on Vascular Reactivity and Alterations of Cyclooxygenase Expression / 영남의대학술지

BACKGROUND: There is controversy regarding whether COX-2 specific inhibitors are associated with elevation of blood pressure. We compared the effects of aspirin, indomethacin, and celecoxib for vascular reactivity induced by phenylephrine. We also tested the effects of indomethacin and NO donor on COX-1 and COX-2 protein expression, as well as nitrite production in culture medium of vascular smooth muscle cells. MATERILAS AND METHODS: In this experiment, we used the isometric tension study for vascular reactivity. After 45 minutes of pretreatment with aspirin, indomethacin, celecoxib, and phenylephrine induced contractions were tested. COX-1 and COX-2 protein expressions were analyzed by Western blot and nitrite production by the Griess reaction. RESULTS: Although celecoxib pretreatment caused enhanced arterial contraction, aspirin pretreatment induced more potent arterial contraction than celecoxib in the isometric tension study of rabbit femoral artery. COX-1 protein expression was unchanged by indomethacin, SNP and NOR-3; COX-2 protein expression was increased by the addition of indomethacin, SNP, and NOR-3. Especially, NOR-3, a NO donor, significantly increased COX-2 protein expression with unstimulated conditions as well as LPS stimulation. Induction of nitrite production was higher with NOR-3 treatment than SNP treatment with LPS stimulation. CONCLUSION: These results suggest that aspirin caused more potent vascular contraction than celecoxib and indomethacin. COX-2 expression in VSMC depended on the types of NO donor and LPS stimulation.

Effect of Progesterone on COX-2 Expression and Proliferation of Prostate Stromal Cell / 영남의대학술지

BACKGROUND: Benign prostatic hyperplasia (BPH) is the most common benign tumor in older men; the etiology of this disease remains poorly understood. Testosterone and dihydrotestosterone (DHT) both act as androgen via a single androgen receptor. Testosterone is converted to DHT by 5alpha-reductase in prostatic stromal cells. Progesterone has been reported to inhibit DHT conversion; howevwe, its effect on prostatic stromal cells remains to be elucidated. MATERILAS AND METHODS: In this experiment, we investigated the effect of progesterone on androgen receptor expression induced by DHT. We also tested the effect of progesterone on cyclooxygenase-2 (COX-2) expression, as well as prostate stromal cell proliferation using the cell count kit-8. RESULTS: Progesterone did not cause an increase of prostate stromal cell proliferation. The mRNA expression of the androgen receptor and COX-2 were not changed by progesterone; the expressions of androgen receptor and COX-2 proteins were decreased by progesterone in prostate stromal cells. CONCLUSION: These results suggest that in prostate stromal cells, progesterone decreases androgen receptor protein expression, which results in decrement of COX-2 protein expression. This effect might be mediated by post-transcriptional regulation.

The Operating Status of Medical Education Management Units in Korea / 한국의학교육

PURPOSE: The purpose of this study was to estimate the operating status of existing medical education management units in medical colleges and to define the roles of these units to provide basic information to medical schools contemplating to establish similar management units. METHODS: A structured questionnaire survey was conducted via mail. The survey 41 medical colleges across the nation and was done during September 2003 and March 2005. The assessment instrument included six items assessing the organizational structure, composition, major activities, self-satisfaction with performance, administration and financial aspects and the need for further development in the medical education unit for medical colleges with a medical education management unit. There were only two items assessing demand for establishment of a medical education management unit, prerequisite conditions for establishment, the expected role of such a system for medical colleges without a medical education management unit. RESULTS: Of 41 medical colleges, 18 had a medical education management unit as of September 2003 and 32 as of March 2005. The major activities of these 18 management units included curriculum development(26.7%), faculty development(26.7%), support for PBL(16.0%) and OSCE(12.0%). Recently, these units have become involved in enhancing clinical clerkship as well as improving teaching skills. To have a medical education-related unit run smoothly, at least two tenured faculty members majoring in education and medical education were needed. And a compensation systems was required for those professors working in the unit as a second post but without pay. CONCLUSION: This study underscored the importance of professional faculty members, and administrative and financial supports in having a medical education management unit meet its objectives. The role of the dean of medical college seems crucial in deciding how the unit is operated.

Androgen Hormone Inhibits Expression of iNOS and COX-2 Protein in Rat Vascular Smooth Muscle Cell

We investigated the effects of testosterone and dihydrotestosterone on inflammatory response of iNOS and COX-2 expression in rat vascular smooth muscle cells. Rat vascular smooth muscle cells (VSMC) stimulated with bacterial lipopolysaccharide (LPS; 10microgram/ml) for 24 hours were incubated with increasing amounts of testosterone and dihydrotestosterone (1 and 100 nM). LPS was found to induce inflammatory response of iNOS and COX-2 mRNA and protein in VSMC. These processes were affected by male sex steroid hormones. For 3 hours, however, pretreatment of the cells with 100 nM each of testosterone and dihydrotestosterone suppressed LPS induced iNOS and COX-2 protein expression. RT-PCR analysis revealed that testosterone and dihydrotestosterone did not inhibit mRNA expression of iNOS and COX-2 stimulated by 24 hours of LPS incubation. Proliferation rate was slower in VSMC treated with testosterone and dihydrotestosterone. Testosterone enhanced androgen receptor expression, and LPS significantly reduced androgen receptor protein expression in VSMC. These results indicate that the expression of both iNOS and COX-2 proteins was suppressed by testosterone and dihydrotestosterone in LPS stimulated VSMC and leading to reduction of vascular inflammation.

Suppression of iNOS Expression by NSAIDs and Acetaminophen in Vascular Smooth Muscle Cells

PURPOSE: iNOS expression in vascular smooth muscle cells (VSMC) causes the development of septic shock, and multiple organ dysfunction syndrome (MODS). For the inhibition of iNOS expression, glucocorticoids are known to inhibit iNOS expression but immunosuppression decreases its clinical availability. Recently, aspirin was reported to inhibit iNOS expression, but the mechanism and effectiveness are still unclear. In this investigation, on aspirin, several non steroidal antiinflammatory drugs (NSAIDs) were applied to clarify the inhibitory mechanism of iNOS expression and NO production in lipopolysaccharide (LPS) treated VSMCs. METHOD: VSMCs were primarily cultured from rat aorta and confirmed by immunocytochemistry of anti-smooth muscle myosin antibody. LPS, an inducer of iNOS, and NSAIDs, such as aspirin, indomethacin, ketoprofen sodium salicylate and acetaminophen were used. The concentrations of nitrite in culture media following the addition of LPS with a 1-hour pretreatment of NSAIDs were measured by spectrophotometry with griess reaction. Western blot and RT-PCR for iNOS protein and iNOS mRNA, respectively, were performed. RESULT: Acetaminophen had no effect on the inhibition of nitrite production. NSAIDs, especially ketoprofen and sodium salicylate, showed a significant inhibitory effect on nitrite production. In their mechanism, all the NSAIDs in present study inhibited iNOS mRNA and protein expression. CONCLUSION: These results suggest that the inhibitory mechanism on iNOS expression of NSAIDs is due to the inhibition of iNOS mRNA expression and subsequent inhibition of iNOS protein expression.

Basic Concepts of Experiments with Isolated Smooth Muscle Strips

No abstract available.

Lipopolysaccharide inhibits proliferation of the cultured vascular smooth muscle cells by stimulating inducible nitric oxide synthase and subsequent activation of guanylate cyclase

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

Nitroxergic nerve relaxes rat gastric smooth muscle by NO-cGMP pathway

This study was undertaken to investigate an involvement of nitroxergic innervation in gastric smooth muscle of rat. Isometric tension study, the measurement of single cell length, NADPH diaphorase stain of smooth muscle layers and neuronal nitric oxide synthase (nNOS) western blotting were performed. Sodium nitroprusside (SNP), a nitric oxide donor, relaxed the muscle strips precontracted by acetylcholine (ACh) in a concentration-dependent manner. Pretreatment of L-arginine decreased the contraction induced by electric field stimulation (EFS). Pretreatment of NG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, increased the EFS-induced contractions. LY 83583, a guanylate cyclase (GC) inhibitor, reversed the inhibitory actions of L-arginine on the muscle contractions. The effects of L-Arginine, L-NAME and LY 83583 on ACh-induced contractions were not significant. L-arginine reduced the EFS-induced contraction in circular muscle, whereas L-NAME enhanced the EFS-induced contraction in longitudinal strips. By EFS, the phasic contractions appeared approximately 20~25 seconds later. L-NAME significantly shortened the delay time to about 2~3 seconds. In single cell study, ACh contracted gastric smooth muscle cells, SNP relaxed the cells, and the latter also inhibited the ACh-induced contraction. LY 83583 enhanced the ACh-induced contraction and antagonized SNP-induced relaxation. NADPH diaphorase activity was assessed by a histochemistry, nitroblue tetrazolium (NTB) staining. Positive staining was observed in both circular and longitudinal muscle layers. L-arginine increased the staining, while L-NAME decreased the staining. Western blotting for nNOS proved the presence of nNOS in rat gastric smooth muscle. EFS and additional Ca2+ increased nNOS protein expression. These results suggest that in rat stomach, both circular and longitudinal muscle layers are innervated with nitroxergic nerves which relax the gastric smooth muscle via NO-cGMP pathway.

Effect of imipramine on calcium utilization of single cells isolated from canine detruso

This study is to investigate the mechanism of inhibitory effect of imipramine on the calcium utilization in single cells isolated from canine detrusor. 2 mm thick smooth muscle chops were incubated in 0.12% collagenase solution at 36degreeC, and aerated with 95% O2/5% CO2, and then cell suspension was examined Acetylcholine (ACh) evoked a concentration-dependent contraction of the isolated detrusor cells in normal physiologic salt solution (PSS), and the ACh-induced contraction was significantly inhibited by imipramine. In Ca2+-free PSS, ACh-induced contraction was less than those in normal PSS and it was not affected by the pretreatment with imipramine. Ca2+-induced contraction in Ca2+-free PSS was supressed by imipramine, but addition of A 23187, a calcium ionophore, overcomed the inhibitory effect of imipramine. High potassium-depolarization (40 mM KCl) evoked cell contraction, which was inhibited by imipramine. Caffeine, a releasing agent of the stored Ca2+ from sarcoplasmic reticulum, evoked a contraction of the cells that was not blocked by the pretreatment with imipramine. These results suggest that imipramine inhibits the influx of calcium in the detrusor cells through both the receptor-operated- and voltage-gated-calcium channels, but does not affect the release of calcium from intracellular storage site.

Effect of NG-nitro-L-arginine methyl ester and Methylene Blue on the Endotoxin-induced Vascular Hyporesponsiveness / 영남의대학술지

The study was undertaken to examine the intensity of involvement of inducible nitric oxide synthase(iNOS) and cyclic GMP signal transduction pathway as one of the mechanisms of vaso-relaxative action of bacterial lipopolysaccharide (LPS) on the canine femoral artery strips. Canine femoral arteries were isolated and spiral strips of 10 mm long and 2 mm wide were made in the Tyroad solution of 0-4degrees C. The strips were prepared for isometric myography in Biancani's isolated muscle chamber contaning 1 ml of Tyrode solution, which was maintained with pH 7.4 by areation with 95% O2/5% CO2 at 37degrees C and nitric oxide (NO) production was measured simulltaneously with isolated nitric oxide mrter. LPS induced NO production, suppressed the phenylephrine (PE) induced contraction and enhanced the acetylcholine (ACh) induced relaxation. NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, methylene blue, a guanylyl cyclase inhibitor, potentiated PE induced contraction and suppressed ACh induced relaxation on the LPS treated strips. The inhibitory potency of methylene blue for LPS induced vascular hyporeponsiveness was stronger than that of L-NAME. These result suggest that in canine femoral artery, both iNOS and cyclic GMP signal transduction pathway are related with LPS indused vascular hyporeponsiveness, but in minor with iNOS and in major with cyclic GMP signal transduction pathway.

Changes of renal peripheral benzodiazepine receptor in the stress/anxiety response

Peripheral benzodiazepine receptor(PBR) has been identified in various peripheral tissues including kidney. The physiological and pharmacological functions of PBR are still uncertain, although it has been suggested that these are associated with the regulation of stress/anxiety response. Diazepam progeny, which were exposed to diazepam perinatally, was reported to be an animal model of chronic anxiety. However, PBR in the diazepam progenies are not known yet. In the present study, therefore, we examined the changes of PBR in the stress/anxiety response. Dams of rats were given injection of diazepam or vehicle during puerperium. Diazepam progenies showed increased level of anxiety on the performance of elevated plus maze, and increased Bmax of PBR. Saturation experiments followed by scatchard analysis of the results showed that the increase in the density of PBR and the affinity of the PBR remained unchanged. Forced swim stress increased anxiety on the plus maze in both groups of rats. In contrast to control, diazepam progenies did not show further upregulation of renal PBR immediately after swimming stress, but still higher than control. From the above results, it may be concluded that upregulation of renal PBR is associated with chronic anxiety as well as stress-induced response.

Effect of UV irradiation and rebamipide on the blood flow and viability of rabbit skin flap

This study was undertaken to examine the effects of ultraviolet light (UVL) and rebamipide on the cutaneous blood flow and tissue survival on rabbit skin flap. In a random bipedicle flap, Laser Doppler Flowmetry (LDF) was employed to measure the blood flow of flap (BFF). Wound Margin Strength (WMS) measured by force transducer and Light microscopy were used for evaluation of tissue viability. Single exposure to UVL increased the BFF gradually for more than 15 hours, and decreased the vasoconstrictor effect of intravenous phenylephrine. The UVL-induced increase in BFF regressed after 18 hours of irradiation, and this regression was tended to be enhanced by intradermal injection of L-NAME, a nitric oxide synthase (NOS) inhibitor, but the regression was significantly reversed by acetylcholine, an endothelial constitutive NOS (cNOS) activator and L-arginine, an NO precursor. Rebamipide, a novel antiulcer oxide synthase (NOS) inhibitor, but the regression was significantly reversed by acetylcholine, an endoagent known to scavenge the hydroxyl radical, abruptly reversed the spontaneous regression of the UVL-induced increase in BFF by the same manner as L-arginine. In ischemic skin flap, rebamipide increased the BFF abruptly by the same manner as sodium nitroprusside (SNP), an NO doner, while N-acetylcystein (NAC), a free radical scavenger, gradually increase the BFF. The rebamipide-induced increase in BFF was sustained at the level of the SNP-induced increase in BFF during the late period of experiment. Rebamipide increased the WMS of skin flaps and prevented the tissue necrosis in comparison with L-NAME. Based on these results, it is concluded that in rabbit skin, UVL irradiation increases the BFF by NO release, and rebamipide exerts a protective effect on the viability of ischemic skin flaps by either or both the increase in BFF by NO release and free radical scavenger effect.

Involvement of peripheral benzodiazepine receptor on contractility of canine trachealis muscle

Non-neuronal high affinity binding sites for benzodiazepines have been found in many peripheral tissues including cardiac muscle and vascular smooth muscle, and have been designated as 'peripheral benzodiazepine receptor'. Benzodiazepines have been shown to induce relaxation of the ileal, vesical, and uterine smooth muscles. However, it is still unclear about possible involvement of peripheral benzodiazepine receptor on the contractility of trachealis muscle. This study was performed to investigate the role of the peripheral benzodiazepine receptor on the contractility of canine trachealis muscle. Canine trachealis muscle strips of 15 mm long were suspended in an isolated organ bath containing 1 ml of physiological salt solution maintained at 37degreeC, and aerated with 95% O2/5% CO2. Isometric myography was performed, and the results of the experiments were as follows: Ro5-4684, FGIN-1-27 and clonazepam reduced a basal tone of isolated canine trachealis muscle strip concentration dependently, relaxant actions of Ro5-4684 and FGIN-1-27 were antagonized by PK11195, a peripheral benzodiazepine receptor antagonist. Flumazenil, a central type antagonist, did not antagonize the relaxant action of peripheral type agonists. Saturation binding assay of (3H)Ro5-4864 showed a high affinity (Kd = 5.33 +/- 1.27nM, Bmax = 867.3 +/- 147.2 fmol/mg protein) binding site on the canine trachealis muscle. Ro5-4684 suppressed the bethanechol-, 5-hydroxytryptamine- and histamine-induced contractions. Platelet activating factor (PAF) exerted strong and prolonged contraction in trachealis muscle strip. Strong tonic contraction by PAF was attenuated by Ro 5-4684, but not by WEB 2086, a PAF antagonist. Based on these results, it is concluded that the peripheral benzodiazepine receptor mediates the inhibitory regulation of contractility of canine trachealis muscle.