Phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate underlies agmatine-induced suppression of N-type Ca2+ channel in rat celiac ganglion neurons.

Agmatine suppresses peripheral sympathetic tone by modulating Cav2.2 channels in peripheral sympathetic neurons. However, the detailed cellular signaling mechanism underlying the agmatine-induced Cav2.2 inhibition remains unclear. Therefore, in the present study, we investigated the electrophysiological mechanism for the agmatine-induced inhibition of Cav2.2 current (ICav2.2) in rat celiac ganglion (CG) neurons. Consistent with previous reports, agmatine inhibited ICav2.2 in a VI manner. The agmatine-induced inhibition of the ICav2.2 current was also almost completely hindered by the blockade of the imidazoline I2 receptor (IR2), and an IR2 agonist mimicked the inhibitory effect of agmatine on ICav2.2, implying involvement of IR2. The agmatine-induced ICav2.2 inhibition was significantly hampered by the blockade of G protein or phospholipase C (PLC), but not by the pretreatment with pertussis toxin. In addition, diC8-phosphatidylinositol 4,5-bisphosphate (PIP2) dialysis nearly completely hampered agmatine-induced inhibition, which became irreversible when PIP2 resynthesis was blocked. These results suggest that in rat peripheral sympathetic neurons, agmatine-induced IR2 activation suppresses Cav2.2 channel voltage-independently, and that the PLC-dependent PIP2 hydrolysis is responsible for the agmatine-induced suppression of the Cav2.2 channel.

Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension.

Suppression of peripheral sympathetic activity underlies protease-activated receptor 2-mediated hypotension.

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ω-conotoxin GVIA (CgTx), a selective N-type Ca(2+) channel (ICa-N) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ω-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of ICa-N which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

Transient receptor potential c4/5 like channel is involved in stretch-induced spontaneous uterine contraction of pregnant rat.

Spontaneous myometrial contraction (SMC) in pregnant uterus is greatly related with gestational age and growing in frequency and amplitude toward the end of gestation to initiate labor. But, an accurate mechanism has not been elucidated. In human and rat uterus, all TRPCs except TRPC2 are expressed in pregnant myometrium and among them, TRPC4 are predominant throughout gestation, suggesting a possible role in regulation of SMC. Therefore, we investigated whether the TRP channel may be involved SMC evoked by mechanical stretch in pregnant myometrial strips of rat using isometric tension measurement and patch-clamp technique. In the present results, hypoosmotic cell swelling activated a potent outward rectifying current in G protein-dependent manner in rat pregnant myocyte. The current was significantly potentiated by 1µM lanthanides (a potent TRPC4/5 stimulator) and suppressed by 10µM 2-APB (TRPC4-7 inhibitor). In addition, in isometric tension experiment, SMC which was evoked by passive stretch was greatly potentiated by lanthanide (1µM) and suppressed by 2-APB (10µM), suggesting a possible involvement of TRPC4/5 channel in regulation of SMC in pregnant myometrium. These results provide a possible cellular mechanism for regulation of SMC during pregnancy and provide basic information for developing a new agent for treatment of premature labor.

Increase of L-type Ca2+ current by protease-activated receptor 2 activation contributes to augmentation of spontaneous uterine contractility in pregnant rats.

We evaluated the effects of protease-activated receptor (PAR)-2 on spontaneous myometrial contraction (SMC) in isolated term pregnant myometrial strips of rat, and elucidated the cellular mechanisms of this effect using a conventional voltage-clamp method. In isometric tension measurements, trypsin and SL-NH(2), PAR-2 agonists, significantly augmented SMC in frequency and amplitude; however, boiled trypsin (BT) and LR-NH(2) had no effect on SMC. These stimulatory effects of PAR-2 agonists on SMC were nearly completely occluded by pre-application of Bay K 8644, an L-type voltage-gated Ca(2+) channel activator, thus showing the involvement of L-type voltage-gated Ca(2+) channels in PAR-2-induced augmentation of SMC. In addition, PAR-2 agonists significantly enhanced L-type voltage-gated Ca(2+) currents (I(Ca-L)), as measured by a conventional voltage-clamp method, and this increase was primarily mediated by activation of phospholipase C (PLC) and protein kinase C (PKC) via G-protein activation. Taken together, we have demonstrated that PAR-2 may actively regulate SMC during pregnancy by modulating Ca(2+) influx through L-type voltage-gated Ca(2+) channels, and that this increase of I(Ca-L) may be primarily mediated by PLC and PKC activation. These results suggest a cellular mechanism for the pathophysiological effects of PAR-2 activation on myometrial contractility during pregnancy and provide basic and theoretical information about developing new agents for the treatment of premature labor and other obstetric complications.

Epithelial Na+ channel proteins are mechanotransducers of myogenic constriction in rat posterior cerebral arteries.

It has been suggested that mechanosensitive ion channels initiate myogenic responses in vessels; however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although previous reports have suggested that epithelial Na(+) channel (ENaC) proteins are mechanotransducers in arteries, experimental evidence demonstrating that ENaC proteins are mechanotransducers are not fully elucidated. The goal of the present study was to determine whether the ENaC is a mechanotransducer for the myogenic response by providing supporting evidence in the rat posterior cerebral artery (PCA). We measured the effect of ENaC inhibition on the pressure-induced myogenic response, Ca(2+) concentration and 20 kDa myosin light chain (MLC(20)) phosphorylation. We detected expression of ßENaC and γENaC subunits in rat PCA by Western blots and immunofluorescence. Inhibition of ENaCs with amiloride, ethyl isopropyl amiloride or benzamil blocked the myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with ßENaC and γENaC small interfering RNA. The myogenic response was inhibited by elimination of external Na(+), which was replaced with N-methyl-d-glucamine. Amiloride and nifedipine inhibited the pressure-induced increase in Ca(2+) concentration. Finally, MLC(20) increased when the intraluminal pressure was raised, and the pressure-induced increase in MLC(20) phosphorylation was inhibited by pretreatment with amiloride, and in arteries transfected with ßENaC or γENaC small interfering RNA. Our results suggest that ENaCs may play an important role as mechanosensitive ion channels initiating pressure-induced myogenic responses in rat PCA.

Modulation of N-type Ca²âº currents by moxonidine via imidazoline I1 receptor activation in rat superior cervical ganglion neurons.

Moxonidine, an imidazoline deriviatives, suppress the vasopressor sympathetic outflow to produce hypotension. This effect has been known to be mediated in part by suppressing sympathetic outflow via acting imidazoline I(1) receptors (IR(1)) at postganglionic sympathetic neurons. But, the cellular mechanism of IR(1)-induced inhibition of noradrenaline (NA) release is still unknown. We therefore, investigated the effect of IR(1) activation on voltage-dependent Ca(2+) channels which is known to play an pivotal role in regulating NA in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. In the presence of rauwolscine (3 µΜ), which blocks α(2)-adrenoceptor (R(α2)), moxonidine inhibited voltage-dependent Ca(2+) current (I(Ca)) by about 30%. This moxonidine-induced inhibition was almost completely prevented by efaroxan (10 µΜ) which blocks IR(1) as well as R(α2). In addition, ω-conotoxin (CgTx) GVIA (1 µΜ) occluded moxonidine-induced inhibition of I(Ca), but, moxonidine-induced I(Ca) inhibition was not affected by pertussis toxin (PTX) nor shows any characteristics of voltage-dependent inhibition. These data suggest that moxonidine inhibit voltage-dependent N-type Ca(2+) current (I(Ca-N)) via activating IR(1). Finally, moxonidine significantly decreased the frequency of AP firing in a partially reversible manner. This inhibition of AP firing was almost completely occluded in the presence of ω-CgTx. Taken together, our results suggest that activation of IR(1) in SCG neurons reduced I(Ca-N) in a PTX-and voltage-insensitive pathway, and this inhibition attenuated repetitive AP firing in SCG neurons.

Intracellular acidification evoked by moderate extracellular acidosis attenuates transient receptor potential V1 (TRPV1) channel activity in rat dorsal root ganglion neurons.

Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pH(o)). Results from recent in vivo studies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pH(o) (6.0 < pH < 7.0) than predicted from the in vitro experiments. A clear explanation for this discrepancy between the in vitro and in vivo data has not yet been provided. We report here that intracellular acidification induced by a moderately low pH(o) (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin-evoked TRPV1 current (I(CAP)) in a reversible manner in whole-cell patch-clamp mode and shifted the concentration-response curve to the right. Likewise, the concentration-response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressed I(CAP) in inside-out patch mode. In cell-attached patch mode, the single-channel activity of i(CAP) was significantly attenuated by intracellular acidosis that was induced by a decrease in pH(o) (6.4). These results suggested that intracellular acidification induced by a low pH(o) inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37 °C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pH(o) (6.4) at a physiological temperature (37 °C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pH(o), and this phenomenon is due to attenuation of TRPV1 channel activity by low-pH(o)-induced intracellular acidification.

Modulation of N-type calcium currents by presynaptic imidazoline receptor activation in rat superior cervical ganglion neurons.

Presynaptic imidazoline receptors (R(i-pre)) are found in the sympathetic axon terminals of animal and human cardiovascular systems, and they regulate blood pressure by modulating the release of peripheral noradrenaline (NA). The cellular mechanism of R(i-pre)-induced inhibition of NA release is unknown. We, therefore, investigated the effect of R(i-pre) activation on voltage-dependent Ca(2+) channels in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. Cirazoline (30 µM), an R(i-pre) agonist as well as an α-adrenoceptor (R(α)) agonist, decreased Ca(2+) currents (I(Ca)) by about 50% in a voltage-dependent manner with prepulse facilitation. In the presence of low-dose rauwolscine (3 µM), which blocks the α(2)-adrenoceptor (R(α2)), cirazoline still inhibited I(Ca) by about 30%, but prepulse facilitation was significantly attenuated. This inhibitory action of cirazoline was almost completely prevented by high-dose rauwolscine (30 µM), which blocks R(i-pre) as well as R(α2). In addition, pretreatment with LY320135 (10 µM), another R(i-pre) antagonist, in combination with low-dose rauwolscine (3 µM), also blocked the R(α2)-resistant effect of cirazoline. Addition of guanosine-5-O-(2-thiodiphosphate) (2 mm) to the internal solutions significantly attenuated the action of cirazoline. However, pertussis toxin (500 ng ml(1)) did not significantly influence the inhibitory effect of cirazoline. Moreover, cirazoline (30 µM) suppressed M current in SCG neurons cultured overnight. Finally, omega-conotoxin (omega-CgTx) GVIA (1 µM) obstructed cirazoline-induced current inhibition, and cirazoline (30 µM) significantly decreased the frequency of action potential firing in a partly reversible manner. This cirazoline-induced inhibition of action potential firing was almost completely occluded in the presence of omega-CgTx. Taken together, our results suggest that activation of R(i-pre) in SCG neurons reduced N-type I(Ca) in a pertussis toxin- and voltage-insensitive pathway, and this inhibition attenuated repetitive action potential firing in SCG neurons.

Similarity of the cut score in test sets with different item amounts using the modified Angoff, modified Ebel, and Hofstee standard-setting methods for the Korean Medical Licensing Examination.

PURPOSE: The Korea Medical Licensing Exam (KMLE) typically contains a large number of items. The purpose of this study was to investigate whether there is a difference in the cut score between evaluating all items of the exam and evaluating only some items when conducting standard-setting. METHODS: We divided the item sets that appeared on 3 recent KMLEs for the past 3 years into 4 subsets of each year of 25% each based on their item content categories, discrimination index, and difficulty index. The entire panel of 15 members assessed all the items (360 items, 100%) of the year 2017. In split-half set 1, each item set contained 184 (51%) items of year 2018 and each set from split-half set 2 contained 182 (51%) items of the year 2019 using the same method. We used the modified Angoff, modified Ebel, and Hofstee methods in the standard-setting process. RESULTS: Less than a 1% cut score difference was observed when the same method was used to stratify item subsets containing 25%, 51%, or 100% of the entire set. When rating fewer items, higher rater reliability was observed. CONCLUSION: When the entire item set was divided into equivalent subsets, assessing the exam using a portion of the item set (90 out of 360 items) yielded similar cut scores to those derived using the entire item set. There was a higher correlation between panelists' individual assessments and the overall assessments.

Developing a Korean communication skills attitude scale: comparing attitudes between Korea and the West.

OBJECTIVES: We aimed to develop a Korean version of the Communication Skills Attitude Scale (CSAS) created by Rees et al. in order to elucidate the positive and negative aspects of Korean pre-medical and medical students' attitudes towards communication skills (CS) learning. METHODS: We performed two surveys. In the first of these, 325 pre-medical and medical students completed a translated version of the CSAS. In the second survey, 257 medical students and doctors-in-training answered five open-ended questions to obtain more qualitative data about their attitudes. Results Principal component analysis with direct oblimin rotation performed with the data from the first survey produced the following five factors: facilitation of interpersonal skills; doubts about the importance of CS learning in medicine; motivation; negative attitudes towards assessment, and overconfidence. RESULTS: from the second survey indicated that facilitation and importance within a medical context were two core attitudinal factors and suggested some modification to the CSAS to improve its fit for Korean pre-medical and medical students. CONCLUSIONS: Using a Korean version of the CSAS (CSAS-K), we determined five factors that revealed a somewhat complex attitude structure among students towards CS learning. The CSAS required some modification, possibly because CS teaching and learning in Korea are in the development stage. Finally, the educational implications of the results are discussed.

Comparison of standard-setting methods for the Korea Radiological technologist Licensing Examination : Angoff, Ebel, Bookmark, and Hofstee.

PURPOSE: This study aims to compare the various standard setting methods for the Korean Radiological Technologist Licensing Examination with the fixed cut score and suggest the most appropriate method. METHODS: Six radiological technology professors, set the standards of 250 items for Korean Radiological Technologist Licensing examination that were conducted on December 2016 by using Angoff, Ebel, bookmark, and Hofstee methods. RESULTS: With the maximum percentile score of 100, the cut score for the examination was 71.27 in Angoff method, 62.2 in Ebel method, 64.49 in bookmark method, and 62 in Hofstee. Based on the Hofstee's acceptable cut score, the acceptable cut score for the examination was between 52.83 and 70, but the cut score was 71.27 in Angoff method. CONCLUSION: Above results suggested that the best standard setting methods to determine the cut score was panel discussion with the modified Angoff or Ebel methods, and verification of the rated results by Hofstee method. Because there was still no adoption of standard setting in the Korean Radiological Technologist Licensing Examination, this study will be able to provide the practical guideline to introduce the standard setting.

Changes in stretch-induced tone induced by intracellular acidosis in rabbit basilar artery: effects on BKCa channel activity.

It is known that myogenic reactivity is a fundamental determinant of the relative constancy of blood flow through the cerebral artery. It is also known that acute alteration of pH significantly affects the cerebral circulation and, therefore, we investigated the effect of mechanism of action of intracellular acidosis on myogenic tone in rabbit basilar artery. Myogenic tone was developed by imposed stretch of basilar artery and intracellular acidosis induced by the bath application of 20 mmol/L sodium acetate. Sodium acetate caused a biphasic increase in myogenic tone. The initial component reached a peak quickly and then fell slowly to a lower steady-state significantly above basal tone. The sodium acetate-induced increase in myogenic tone was completely inhibited by elimination of external Ca2+, or treatment of nifedipine, but not with gadolinium or NPPB. TEA (5 mmol/L) and iberiotoxin (100 nmol/L) inhibited the sodium acetate-induced increase in myogenic tone. In inside-out patch-clamp recordings, decreasing pH of the mock intracellular solution from 7.4 to 6.9 markedly inhibited BKCa currents. Several inhibitors involved in Ca2+ sensitization pathways, 10(-6) mol/L Y-27632, 5 x 10(-7) mol/L calphostin C and 10(-5) mol/L PD98059 had no effect on the sodium acetate-induced increase in myogenic tone. These results suggest that intracellular acidosis increases stretch-induced myogenic tone in rabbit basilar artery. Furthermore, voltage-dependent Ca2+ influx plays a key role in intracellular acidosis-induced increase in myogenic tone and may be mediated, at least in part, by inhibition of BKCa.

Augmented sphingosylphosphorylcholine-induced Ca2+-sensitization of mesenteric artery contraction in spontaneously hypertensive rat.

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca2+ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC20) were measured. Ten microM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5 +/- 4.3%, n=9) than in those of the WKY (26.7 +/- 4.5%, n=10). In Ca2+-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K+ depolarization at a given Ca2+ ratio, and the Ca2+ ratio-tension curve induced by SPC was significantly shifted to the left compared with that induced by high K+ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC20 in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC(20) phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca2+ sensitization in the SHR.

The OSCE: a new challenge to the evaluation system in Korea.

Introduced in Korea in 1994, the number of medical schools using Objective Structured Clinical Examination (OSCE) and standardized patients (SPs) has been steadily increasing. Although OSCE is accepted as an effective teaching and evaluation tool in Korea, many challenges in applying OSCE remain. These problems stem mainly from differences in the educational environment and infrastructure in medical schools between different countries. To reduce trial and error cost inefficiencies and to help accelerate the adaptation process, sharing experiences at all levels of applying OSCE to non-Western settings would be valuable. The authors describe how and to what extent the OSCE has been applied to the Korean medical education system. Additionally, the elements that should be in place for OSCE to successfully transform the national licensure examination into a high stakes examination are described.

C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta: role of RhoA/Rho-kinase and intracellular Ca2+ concentration.

It is known that ceramide may play an important regulatory role in vascular tone although its effect on vascular tone and the mechanisms involved are controversial. The present study was designed to investigate the effects of ceramide and its key initial regulators, TNF-alpha and neutral sphingomyelinase (SMase), on vascular tone of isolated rat thoracic aortic rings and elucidate the mechanisms involved in the changes in vascular tone induced by ceramide. Contractile responses and Fura-2 Ca2+ signals were measured in rat thoracic aortic rings or strips. 10(-5) M C2-ceramide, 0.1 U/ml neutral sphingomyelinase (SMase), and 5x10(-7) g/ml TNF-alpha had no effect on resting tone in rat thoracic aortic rings. However, in phenylephrine-induced pre-contracted rings, treatment with ceramide, SMase, and TNF-alpha evoked a gradual but sustained vasodilation. Vasodilation effect in response to 10(-5) M C2-ceramide was not significantly changed by the absence or presence of endothelium, a cyclooxygenase pathway inhibitor (10(-6) M indomethacin), or PKC inhibitors (10(-5) M H-7 & 5x10(-7) M calphostin-C). Pretreatment with 1 microM Y-27632, a RhoA/Rho-kinase inhibitor, significantly inhibited the phenylephrine-induced contraction itself as well as the C2-ceramide-induced vasodilation. Pre-treatment with 10(-5) M C2-ceramide had no effect on phasic rise in [Ca2+]i and tension evoked by stimulation with 10(-8) M phenylephrine, but post-treatment of C2-ceramide significantly reduced the phenylephrine-induced secondary tonic [Ca2+]i and tension plateau. Our results indicate that C2-ceramide induces vasodilation in phenylephrine-induced pre-contracted rat thoracic aorta. Furthermore, inhibition of phenylephrine-induced activation of RhoA/Rho-kinase pathway as well as phenylephrine-induced elevations in [Ca2+]i are clearly a key factors in C2-ceramide-induced vasodilation.

Role of protein kinase C- or RhoA-induced Ca(2+) sensitization in stretch-induced myogenic tone.

OBJECTIVE: It has been suggested that Ca(2+) sensitization mechanisms might contribute to myogenic tone. However, specific mechanisms have yet to be fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced Ca(2+) sensitization in myogenic tone of the rabbit basilar vessel. METHODS: Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 Ca(2+) signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. RESULTS: Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) only in the presence of extracellular Ca(2+). Stretch evoked greater contraction than high K(+) at a given [Ca(2+)](i). The stretch-induced increase in [Ca(2+)](i) and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent Ca(2+) channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing [Ca(2+)](i). Immunoblotting using isoform-specific antibodies showed the presence of PKCalpha and PKCepsilon in the rabbit basilar artery. PKCalpha, but not PKCepsilon, and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. CONCLUSIONS: Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of PKCalpha and/or RhoA may be key mechanisms for the Ca(2+) sensitization associated myogenic tone in basilar vessels.

Stabilizing Morbidity and Predicting the Aesthetic Results of Radial Forearm Free Flap Donor Sites.

BACKGROUND: The radial forearm flap is a versatile, widely used flap. However, the possibility of donor site complications has led to concern over its use. Some surgeons prefer using other flaps whose donor sites can be closed primarily with less morbidity, including avoiding unpleasant scarring. However, in our experience, donor site stability of the radial forearm flap can be reliably achieved by using well-implemented specific procedures. Here, we present a collection of donor site cases of the radial forearm flap and investigate factors that affect the aesthetic results as the basis for a reference for selecting a radial forearm flap. METHODS: In this retrospective study, we reviewed 171 cases in which a radial forearm flap was used for free tissue transfer after resecting head and neck cancer. We focused on donor site morbidity rates. Each operation involved a detailed procedure designed to minimize donor site morbidity. Moreover, statistical investigations were conducted for 22 cases to determine factors affecting the scar appearance. RESULTS: Only one case developed total skin graft necrosis as a major complication. Scar-related aesthetic results were acceptable, and the body-mass index, body weight, diabetes, and cardiac problems were significant factors related to the appearance of scars. CONCLUSIONS: Performing the radial forearm flap using a well-implemented detailed technique helps achieve acceptable donor site morbidity results. The aesthetic results were more promising for patients without excess body weight, diabetes, or cardiac problems. Therefore, anxiety about donor site morbidity should not be a reason to avoid selecting the radial forearm flap in suitable patients.

Inhibitory mechanism of xestospongin-C on contraction and ion channels in the intestinal smooth muscle.

1. Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP(3) receptor inhibitor. In this study we examined the effects of this compound on smooth muscle from guinea-pig ileum. 2. In guinea-pig ileum permeabilized with alpha-toxin, xestospongin-C (3 microM) inhibited contractions induced by Ca(2+) mobilized from sarcoplasmic reticulum (SR) with IP(3) or carbachol with GTP, but not with caffeine. 3. In intact smooth muscle tissue, xestospongin-C (3-10 microM) inhibited carbachol- and high-K+-induced increases in [Ca(2+)](i) and contractions at sustained phase. 4. It also inhibited voltage-dependent inward Ba(2+) currents in a concentration-dependent manner with an IC(50) of 0.63 microM. Xestospongin-C (3-10 microM) had no effect on carbachol-induced inward Ca(2+) currents via non-selective cation channels; but it did reduce voltage-dependent K+ currents in a concentration-dependent manner with an IC(50) of 0.13 microM. 5. These results suggest that xestospongin-C inhibits the IP(3) receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca(2+) and K+ currents at a concentration range similar to that at which it inhibits the IP(3) receptor. Xestospongin-C is a selective blocker of the IP(3) receptor in permeabilised cells but not in cells with intact plasma membrane.

Curriculum design for problem-based learning on a volunteer basis: a Yonsei approach.

Innovative new medical programs such as Problem Based Learning (PBL) are being developed worldwide. An increasing number of medical schools are starting to introduce these programs into or even to replace the existing curriculum. At Yonsei University College of Medicine (YUCM), we developed our own PBL curriculum and evaluation method. In order to develop a program suitable for our school, we suggest that for trial purposes, a small number of student and teacher volunteers should be selected and that the tutors involved in the program be given adequate training.