7alpha-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7alpha-Hydroxycholesterol (7alphaOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7alphaOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit alpha (p19) and the IL-12 subunit beta (p40). However, treatment with 7-ketocholesterol (7K) and 7beta-hydroxycholesterol (7betaOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7betaOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7alphaOHChol as well as secretion of IL-23 enhanced by 7alphaOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7alphaOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

FSL-1, a Toll-like Receptor 2/6 Agonist, Induces Expression of Interleukin-1alpha in the Presence of 27-hydroxycholesterol

We investigated the question of whether cholesterol catabolite can influence expression of inflammatory cytokines via Toll-like receptors (TLR) in monocytic cells. Treatment of THP-1 monocytic cells with 27-hydroxycholesterol (27OHChol) resulted in induction of gene transcription of TLR6 and elevated level of cell surface TLR6. Addition of FSL-1, a TLR6 agonist, to 27OHChol-treated cells resulted in transcription of the IL-1alpha gene and enhanced secretion of the corresponding gene product. However, cholesterol did not affect TLR6 expression, and addition of FSL-1 to cholesterol-treated cells did not induce expression of IL-1alpha . Using pharmacological inhibitors, we investigated molecular mechanisms underlying the expression of TLR6 and IL-1alpha. Treatment with Akt inhibitor IV or U0126 resulted in significantly attenuated expression of TLR6 and IL-1alpha induced by 27OHChol and 27OHChol plus FSL-1, respectively. In addition, treatment with LY294002, SB202190, or SP600125 resulted in significantly attenuated secretion of IL-1alpha . These results indicate that 27OHChol can induce inflammation by augmentation of TLR6-mediated production of IL-1alpha in monocytic cells via multiple signaling pathways.

The Cholesterol-Binding Antibiotic Nystatin Induces Expression of Macrophage Inflammatory Protein-1 in Macrophages

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1alpha and MIP-1beta, however, upon exposure to nystatin, significantly elevated expression of MIP-1alpha and MIP-1beta was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1alpha and MIP-1beta was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.

Multiple Signaling Molecules are Involved in Expression of CCL2 and IL-1beta in Response to FSL-1, a Toll-Like Receptor 6 Agonist, in Macrophages

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.

Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+ , an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

Angiotensin II-induced aortic ring constriction is mediated by phosphatidylinositol 3-kinase/L-type calcium channel signaling pathway

Angiotensin II (AngII) is a crucial hormone that affects vasoconstriction and exerts hypertrophic effects on vascular smooth muscle cells. Here, we showed that phosphatidylinositol 3-kinase-dependent calcium mobilization plays pivotal roles in AngII-induced vascular constriction. Stimulation of rat aortic vascular smooth muscle cell (RASMC)-embedded collagen gel with AngII rapidly induced contraction. AngII-induced collagen gel contraction was blocked by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) whereas ERK inhibitor (PD98059) was not effective. AngII-induced collagen gel contraction was significantly blocked by extracellular calcium depletion by EGTA or by nifedipine which is an L-type calcium channel blocker. In addition, AngII-induced calcium mobilization was also blocked by nifedipine and EGTA, whereas intracellular calcium store-depletion by thapsigargin was not effective. Finally, pretreatment of rat aortic ring with LY294002 and nifedipine significantly reduced AngII-induced constriction. Given these results, we suggest that PI3K-dependent activation of L-type calcium channels might be involved in AngII-induced vascular constriction.

Roles of ERK and NF-kappa B in Interleukin-8 Expression in Response to Heat Shock Protein 22 in Vascular Smooth Muscle Cells

Heat shock proteins (HSPs) serve as molecular chaperones and play a role in cell protection from damage in response to stress stimuli. The aim of this article is to investigate whether HSP22 affects IL-8 expression in vascular smooth muscle cells (VSMCs), and which cellular factors are involved in the HSP-mediated IL-8 induction in that cell type in terms of mitogen activated protein kinase (MAPK) and transcription element. Exposure of aortic smooth muscle cells (AoSMCs) to HSP22 not only enhanced IL-8 release but also induced IL-8 transcript via promoter activation. HSP22 activated ERK and p38 MAPK in AoSMCs. HSP22-induced IL-8 release was inhibited by U0126, but not by SB202190. A mutation in the IL-8 promoter region at the binding site of NF-kappa B, but not AP-1 or C/EBP, impaired promoter activation in response to HSP22. Delivery of I kappa B, but not dominant negative c-Jun, lowered HSP22-induced IL-8 release from AoSMCs. These results suggest that HSP22 induces IL-8 in VSMCs via ERK1/2, and that transcription factor NF-kB may be required for the HSP22-induced IL-8 up-regulation.

Synergistic Efficacy of Concurrent Treatment with Cilostazol and Probucol on the Suppression of Reactive Oxygen Species and Inflammatory Markers in Cultured Human Coronary Artery Endothelial Cells

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3~3micrometer) with probucol (0.03~0.3micrometer) significantly suppressed TNF-alpha-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-alpha release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3~3micrometer) with probucol (0.1~0.3micrometer) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.

Comparison of Students' Characteristics and Perceptions Before and After Introduction Into Graduate Medical School System / 한국의학교육

PURPOSE: This study was performed to compare the characteristics and perceptions of medical school students and professional graduate medical school students. METHODS: Study subjects were 131 medical students from a national university and 113 applicants of a professional graduate medical school. We developed a self-reported questionnaire asking about socio-demographic characteristics; the level of satisfaction of educational environment; perception of missions of medical education and career plan and student activities during school. RESULTS: Students from the professional graduate medical school were significantly different from medical students in socio-demographic characteristics. They also showed higher satisfaction with their education, were more supportive of student union activities and were more anxious about economic and health problems than medical students. However, there was no difference between the two groups regarding perception of missions of medical education and career plan after graduation. CONCLUSION: Based on the above results, it is necessary to consider the characteristics and perceptions of professional graduate medical students when developing educational policies for these older students. The limitation of this study includes a restricted sample, and generalization of results should be done carefully. Thus, more extensive, wide-ranging studies would be useful.

Development and Assessment of Multimedia Learning Tool on Clinical Microbiology for Undergraduate Medical Students / 한국의학교육

PURPOSE: Insufficient teaching of clinical microbiology, often caused by limited resources in medical schools, might be a reason for inaccurate diagnosis and treatment of infectious diseases by doctors. The purpose of this study is to develop and assess a multimedia self learning tool (MSLT) for clinical microbiology course. METHODS: We developed the MSLT based on existing self-directed learning tools. This tool was used by second- and third-year medical students. We randomly assigned 67 participating students to two groups: one (29) with lectures only and the other (38) with the MSLT only. We conducted pre- and post-tests. RESULTS: There are no differences in the pre- and post-test scores between the lecture group and the MSLT group in knowledge of bacterial classification, understanding of infectious diseases, proper use of laboratory tests, and proper selection of antimicrobials. However, post-test scores were significantly higher in both groups. CONCLUSION: The MSLT was found to be as equally effective as lectures, at least, test scorewise. Teachers could use either this tool alone or combined with conventional lectures to improve and enhance teaching in clinical microbiology. The results shed new insights into the possibility of introducing new teaching methods in clinical microbiology for future medical education.

Characterization of ETB Receptor-mediated Relaxation in Precontracted Mesenteric Artery from Streptozotocin-induced Diabetic Rats

Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive substances in various vasculature. In the present study, the authors have observed endothelin-B (ETB) receptor agonist-induced relaxation in precontracted mesenteric arterial segments from streptozotocin (STZ) -induced diabetic rats, which was not shown from control rats or in other arterial segments from diabetic rats. Accordingly, the goal of this study was to investigate in what way STZ-induced diabetes altered reactivity of the mesenteric arterial bed and to examine the causal relaxation, if any, between this ETB receptor-mediated relaxation and endothelial paracrine function, especially nitric oxide (NO) production. The relaxation induced by ETB agonists was not observed in mesenteric arteries without endothelium. The relaxation to ETB agonists was completely abolished by pretreatment with BQ788, but not by BQ610. N omega-nitro-L-arginine methyl ester and soluble guanylate cyclase inhibitors, methylene blue or LY83583 significantly attenuated the relaxant responses to ETB agonists, respectively. When the expression of eNOS and iNOS was evaluated on agarose gel stained with ethidium bromide, the expression of eNOS mRNA in diabetic rats was significantly decreased, but the expression of iNOS was increased compared with control rats. Furthermore, the iNOS-like immunostaining was densely detected in the endothelium and slightly in the arterial smooth muscle of diabetic rats, but not in control rats. These observations suggest that ETB receptor may not play a role in maintaining mesenteric vascular tone in normal situation. However, the alterations in ETB receptor sensitivity were found in diabetic rats and lead to the ETB agonist-induced vasorelaxation, which is closely related to NO production. In the state of increased vascular resistance of diabetic mesenteric vascular bed, enhanced NO production by activation of iNOS could lead to compensatory vasorelaxation to modulate adequate perfusion pressure to splanchnic area.

Conditioned Medium from Dying Smooth Muscle Cell Induced Apoptotic Death

In this study, the authors investigated whether death of vascular smooth muscle cell (VSMC) had a pathological pertinence. Conditioned media obtained from rat aorta smooth muscle cell (SMC) that were induced death by expressing FADD in the absence of tetracycline (FADD-SMC) triggered death of normal SMC. DNA fragmentation and caspase-3 activation were observed in dying SMC by conditioned media. FADD-SMC showed transcriptional activation of tumor necrosis factor (TNF)-alpha. Conditioned medium contained TNF-alpha, indicating secretion of the cytokine from dying FADD-SMC. It was investigated if secreted TNF-alpha was functional. Conditioned medium activated ERK and p38 MAPK pathways and induced MMP-9 expression, whereas depletion of the cytokine with its soluble receptor (sTNFR) remarkably inhibited induction of MMP-9 by conditioned medium. These findings suggest that TNF-alpha in conditioned medium seems to be active. Then, contribution of TNF-alpha on death-inducing activity of conditioned medium was examined. Depletion of TNF-alpha with soluble TNF-alpha receptor decreased the death activity of conditioned medium by 35%, suggesting that TNF-alpha play a partial role in the death activity. Boiling of medium almost completely abolished the death-inducing activity, suggesting that other heat labile death inducing proteins existed in conditioned medium. Taken together, these results indicate that SMC undergoing death could contribute to inflammation by expressing inflammatory cytokines and pathological complications by inducing death of neighboring cells.

Expression of Nitric Oxide Synthase and Endothelin-1 in Human Uterine Artery from Full-Term Pregnancies

The aim of this study was to determine the roles of ET-1 and NO on uterine blood flow in pregnancy. Uterine arteries were isolated from 17 nonpregnant and 12 pregnant women. Nonpregnant group included patients with median age of 48.6+/-2.3 years who underwent hysterectomy, because of myoma. Pregnant group included patients with median age of 31.3+/-1.4 years undergoing cesarean delivery. ET-1 and ET-2 induced concentration-dependent contraction in isolated nonpregnant and pregnant uterine arteries. The contractile response and maximal contraction were increased in pregnant uterine arteries. In nonpregnant uterine arteries, there was no contraction in response to ET-3, whereas pregnancy induced concentration-dependent contraction by ET-3. Tissue nitrite/nitrate level and immunohistochemical staining of eNOS and iNOS were increased in pregnant uterine arteries, compared with nonpregnant uterine arteries. In addition, the expressions of eNOS and iNOS mRNA were significantly increased in pregnancy. Moreover, contractions by ET isopeptides, including ET-1, were enhanced, and immunohistochemical staining of ET-1 and ET-1 mRNA expression was increased in pregnant uterine arteries. These results suggest that NO production by increased NOS activity, especially eNOS activity, is related to placental and uterine blood flow. Furthermore, ET-1 appears to play a pathophysiological role in pregnant complications such as hypertension.

A Study on Using Questions during Lectures in Medical School: Reality vs. Awareness of the Professors / 한국의학교육

PURPOSE: Questions are known to be an important teaching technique. And, waiting for the answers is essential in making questions effective and valuable. The objective of this study is to evaluate the status of the use of questions during lectures and to survey the level of awareness of the professors regarding the questioning method including waiting time in one medical college. METHODS: The study subjects were 42 medical college professors who have been lecturing to second year medical students from February to June 2004. The questionnaire consisted of 28 items on the questions used during their lectures. The lecturers were observes by an appointed student to get data on the characteristics of questions used. RESULTS: Most of the professors observed in this study used questions during lectures, predominantly questions requiring answers. The waiting time, however, for answers was too short than reported in the literature. About 50% of the professors answered that their usual waiting time is between 6 to 10 seconds but the results of the observation showed that the average waiting time was 0.6 second. CONCLUSION: There was significant discrepancy about waiting time between the results of the questionnaire and the data from the observation. Because the average waiting time was much shorter than expected, follow up studies after feedback and education would be recommended.

Changes in the endothelin-1-induced contraction of aorta in streptozotocin-induced diabetic rats

Vascular diseases are significant complications of diabetes mellitus (DM), and the endothelial cells may play a pivotal role in the development of vascular disease in DM. Endothelin-1 (ET-1) released from endothelium is a potent vasoconstrictor peptide and circulating level of ET-1 is increased in a variety of disease states. The purpose of this study was to determine the changes of responsiveness to ET-1 in DM, and we experimented on the changes in the ET-1-induced contraction, levels of nitrite and lipid peroxidation, and ET-1 immunoreactivity in aorta from streptozotocin-induced DM rats. DM was induced by single injection of streptozotocin (55 mg/kg, i.p.). The immunoreactive ET-1 levels in endothelial layer of thoracic aorta were much higher in DM rats than control rats. Nitrite in tissue homogenate was decreased and plasma nitrite was increased in DM rats. Malondialdehyde (MDA) was significantly increased in DM rats and cGMP was not significantly different between control and DM rats. ET-1 produced concentration-dependent contractile responses that are significantly attenuated in DM rats compared to controls. In the presence of selective ETA receptor antagonist BQ610, the maximum contraction was decreased and the concentration ratios for BQ610 yielded pA2 values of 7.3 (slope, 0.65) in control rats, whereas BQ610 had no antagonistic effect on ET-1-induced contraction in DM rats. However, pretreatment with BQ788, an ETB receptor antagonist, maximum response was decreased and the dose-response curves for ET-1 were shifted to the right in both groups and pA2 values were 7.9 and 7.7 (slope, 1.05 in control and DM rats), respectively. IRL 1620 and sarafotoxin S6c, ETB agonists, induced relaxation in control rats but not in DM rats. These results indicate that endothelial cell dysfunction and enhanced immunoreactivity of ET-1 have been found in DM rat and ET-1-induced contraction was attenuated in DM rat. These attenuated responses might be at least in part caused by the alteration of ETA receptor properties (e.g. desensitization), and partly related with an alteration in intracellular mechanism for contraction to ET-1.

Alteration in Endothelin Receptor-Mediated Responses in Femoral Arteries from Hyperlipidemic Rabbits / 대한정형외과학회잡지

OBJECTIVE: To investigate the roles of ET in the regulation of peripheral vascular tone, we studied the effect of hyperlipidemia on vascular responsiveness in femoral arteries from rabbits with control groups of rabbits and test groups receiving a hyperlipidemic diet. MATERIALS AND METHODS: New Zealand Whites were anesthetized with pentobabital and killed by exsanguination from the femoral arteries. Arteries which were suspended on muscle chambers at their optimal length for contractile properties, were examined. RESULTS: 1. After 14-16 weeks of cholesterol-rich diet, plasma cholestrol and HDL levels were significantly higher in the hyperlipidemic rabbits than in the control rabbits. There was no significant difference in the triglyceride levels between the two groups. 2. The contractions caused by 60 mM KCI in the femoral arterial strips were significantly augmented (P<0.01). The contractile responses to phenylephrine or angiotensin II were also augmented, whereas 5-hydroxytryptamine or U46619- induced contraction was not affected by the hyperlipidemic diet. 3. In control rabbits, ET-1 and ET- 2 contracted femoral arteries in a concentraction-dependent manner, whereas sarafotoxin S6c and IRL 1620 had no effect. 4. Contractions caused by ET-1 and ET-2 were significantly diminished by hyperlipidemia. 5. ET-1-induced concentration-response curves were inhibited by BQ-610, but not affected by BQ-788 in the femoral arterial strips from control and hyperlipidemic rabbits. CONCLUSIONS: These results suggest that ET is involved in the regulation of vascular tone in peripheral arteries and ETA receptor subtypes are mainly present in rabbit femoral arteries. Further more, ET-induced contraction is attenuated in hyperlipidemic rabbit, and the attenuated responses might be caused at least in part by the alteration of ET receptors (e.g. desensitization).

Contractile Responses to Endothelins in Isolated Arteries from Human Uterus / 대한산부인과학회잡지

OBJECTIVE: We examined the vasoconstricting poperties of endothelin (ET) on isolated arteries from pregnant as well as non-pregnant uterus. METHODS: Arteries of the uterus were obtained from both hysterectomized uterus and during pregnany hysterectomy for control group and cesarean section for pregnant group. Rings of uterine artery were suspended on muscle chambers at their optimal length for generating tension and contractile properties were examined. RESULTS: ET-1 and ET-2 induced concentration-dependent constriction of both isolated arterial strips from non-pregnant and pregnant uterus. The contraction to ET-1 and ET-2 were more enhanced in full-term pregnancy. Furthermore, in pregnant group, sarafotoxin S6c and IRL 1620, ET. agonists, induced a dose-dependent contraction, which was not shown in those from non-pregnant human. Pretreatment of human uterine arterial strips from pregnant uterus with BQ610, an ET. antagonist, for 10 min resulted in a dose-related rightward shift of ET-1 response curve with diminution of maximal response. Schild plot analysis yielded a pA value of 7.29 with a slope of 0.98. However, BQ788, an ET antagonist, did not produce any rightward shift. The contraction to lower concentration (10-8~3*10-7 M) of sarafotoxin S6c was not affected by BQ788, whereas that to higher concentration (10-s-8*10-7 M) was marked diminished. However, BQ610 did not exnt any efFect on sarafotoxin S6c-induced contraction in arterial staips from pregnant uterus. When the bath solution was replaced with Ca-free physiological salt solution (PSS) containing 1 mM EGTA for 10 min prior to adding sarafotoxin S6c, sarafotoxin S6c-induced contraction was completely abolished. Sarafotoxin S6c (10 nM)-induced contraction was prefetentially blocked by a protein kinase C antagonist, H-7, whereas it was less sensitive to a calmodulin antagonist, calmidazolium, CONCLUSION: Based on above results, we concluded that ET plays an important role in regulating uterine blood flow through the activation of ETa and ETB receptors. Furthermote, ETB receptors may predominantly contribute to the modulation of human uterine circulation in full-term pregnancy.

Pharmacological evidence that cromakalim inhibits Ca2+ release from intracellular stores in porcine coronary artery

In the present study, it was aimed to further identify the intracellular action mechanism of cromakalim and levcromakaliin in the porcine coronary artery. In intact porcine coronary arterial strips loaded with fura-2/AM, acetylcholine caused an increase in intracellular free Ca2+ ((Ca2+)-i) in association with a contraction in a concentration-dependent manner. Cromakalim (1 micrometer) caused a reduction in acetylcholine-induced increased (Ca2+)-i not only in the normal physiological salt solution (PSS) but also in Ca2+ -free PSS (containing 1mM EGTA). In the skinned strips prepared by exposure of tissue to 20 micrometer beta-escin, inositol 1,4,5-trisphosphate (IP-3) evoked an increase in (Ca2+)-i but it was without effect on the intact strips. The IP-3-induced increase in (Ca2+)-i was inhibited by cromakalim by 78% and levcromakalim by 59% (1 micrometer, each). Pretreatment with glibenclamide (a blocker of ATP-sensitive K+ channels, 10 micrometer and apamin (a blocker of small conductance Ca2+/-activated K+ channels, 1 micrometer strongly blocked the effect of cromakalim and levcromakalim. However, charybdotoxin (a blocker of large conductance Ca2+ -activated K+ channels, 1-micrometer) was without effect. In addition, cromakalim inhibited the GTP-gamma-S (100 micrometer, nonhydrolysable analogue of GTP)-induced increase in (Ca2+)-i. Based on these results, it is suggested that cromakalim and levcromakalim exert a potent vasorelaxation, in part, by acting on the K+ channels of the intracellular sites (e.g., sarcoplasmic reticulum membrane), thereby, resulting in decrease in release of Ca2+ from the intracellular storage site.

Regulatory role of cyclic nucleotides in non-adrenergic non-cholinergic relaxation of lower esophageal sphincter from dogs

The role of the lower esophageal sphincter (LES) is characterized by the ability to maintain tone and to relax allowing the passage of a bolus. It is known that LES relaxation during swallowing may be induced by the cessation of the tonic neural excitation and the activation of non-adrenergic, non-cholinergic (NANC) inhibitory neurons. Furthermore, it is generally accepted that the relaxation of the smooth muscle is mediated primarily by the elaboration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) via activation of adenylate cyclase and guanylate cyclase, respectively. It is thus possible that cyclic nucleotides might be a second messenger involved in neural stimulation-induced relaxation of LES, although a relationship between relaxation and changes in cyclic nucleotides after neural stimulation has not been established. The present study was performed to define the participation of cyclic nucleotides in the relaxation of LES of dog in response to neural stimulation. Electrical field stimulation (EFS) caused relaxation of the canine isolated LES strips in a frequency-dependent manner, which was eliminated by pretreatment with tetrodotoxin (1 micrometer), but not by atropine (100 micrometer), guanethidine (100 micrometer) and indomethacin (10 micrometer). The nitric oxide synthase inhibitors, N-G-nitro-L-arginine, N-G-nitro-L-arginine methyl ester and N-G-monomethyl-L-arginine inhibited EFS-induced relaxation. Additions of sodium nitroprusside, a nitrovasodilator and forskolin, a direct adenylate cyclase stimulant, caused a dose-dependent relaxation of LES smooth muscle. Effects of sodium nitroprusside and forskolin were selectively blocked by the corresponding inhibitors, methylene blue for guanylate cyclase and N-ethylmaleimide (NEM) for adenylate cyclase, respectively. Dibutyryl cyclic AMP and dibutyryl cyclic GMP caused a concentration-dependent relaxation of the LES smooth muscle tone, which was not blocked by NEM or methylene blue, respectively. However, both NEM and methylene blue caused significant antagonism of the relaxation in LES tone in response to EFS. EFS increased the tissue cyclic GMP content by 124%, whereas it did not affect the tissue level of cyclic AMP. Based on these results, it is suggested that one of the components of canine LES smooth muscle relaxation in response to neural stimulation is mediated by an increase of cyclic GMP via the activation of guanylate cyclase. Additionally, an activation of cyclic AMP generation system was, in part, involved in the EFS-induced relaxation.