Estudio multicéntrico argentino sobre la utilidad del panel BCID2 del Sistema FilmArrayTM en la detección de bacteriemias / Argentine multicenter study on the usefulness of the BCID2 panel of the FilmArray™ System in the detection of bacteremia

Resumen El panel BCID2 de BioFire® (BioFire, Salt Lake City, EE.UU.) utiliza un análisis de PCR múltiple a partir de hemocultivos positivos con resultados en una hora. El objetivo de este estudio fue determinar el desempeño del método a partir de hemocultivos positivos de pacientes sépticos en 5 hospitales de la Argentina. Se incluyeron 121 pacientes y 124 episodios. Con respecto a la identificación microbiana, la sensibilidad global y la correspondiente a los microorganismos incluidos en la base de datos fue del 94% y 97% respectivamente. La sensibilidad del BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B fue del 100% y la especificidad fue del 99% para NDM y VIM y del 100% para el resto. Esto llevó a cambios en el tratamiento antimicrobiano en 57/98 episodios (58%). El panel BCID2 es una herramienta importante para la adecuación del tratamiento antimicrobiano de pacientes con sepsis.

Abstract The BioFire® BCID2 panel (BioFire, Salt Lake City, UT) uses multiplex PCR analysis from positive blood cultures with results within one hour. The objective of this study was to determine the performance of the method from positive blood cultures of septic patients in 5 hospitals in Argentina. A total of 121 patients and 124 episodes were included. With regard to microbial identification, the global sensitivity and that corresponding to the microorganisms included in the database was 94% and 97%, respectively. The sensitivity of BCID2 to detect CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B was 100% and the specificity was 99% for NDM and VIM and 100% for the rest. This led to changes in antimicrobial treatment in 57/98 episodes (58%). The BCID2 panel is an important tool for the adequacy of antimicrobial treatment of patients with sepsis.

Resumo Estudo multicêntrico argentino sobre a utilidade do painel BCID2 do Sistema FilmArray™ na detecção de bacteremia O painel BCID2 de BioFire® B (BioFire, Salt Lake City, EUA) utiliza uma análise de PCR múltipla de hemoculturas positivas com resultados em uma hora. O objetivo deste estudo foi determinar o desempenho do método a partir de hemoculturas positivas de pacientes sépticos em 5 hospitais da Argentina. Cento e vinte e um pacientes e 124 episódios foram incluídos. No que se refere à identificação microbiana, a sensibilidade global e correspondente aos microrganismos incluídos na base de dados foi de 94% e 97%, respectivamente. A sensibilidade do BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B foi de 100% e a especificidade foi de 99% para NDM e VIM e 100% para o resto. Isso levou a mudanças no tratamento antimicrobiano em 57/98 episódios (58%). O painel BCID2 é uma ferramenta importante para a adequação do tratamento antimicrobiano de pacientes com sepse.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Fracturas vertebrales múltiples postsuspensión de denosumab: revisión del tema a partir de dos casos clínicos / Discontinuation of denosumab and multiple vertebral fractures: report of two cases and review of the literature

Los tratamientos para osteoporosis se indican por tiempo variable dependiendo del tipo de droga, anabólica o anticatabólica, y de la gravedad de la enfermedad. Denosumab es un anticuerpo monoclonal totalmente humano que inhibe a RANK-L evitando de esa manera la interacción entre RANKL-RANK, con la consiguiente inhibición de la formación de los osteoclastos, su activación y sobrevida. Disminuye la resorción ósea cortical y trabecular. Su administración subcutánea de 60 mg cada 6 meses al cabo de 3 años ha demostrado reducción de la resorción ósea, incremento de la densidad mineral ósea y disminución de las fracturas vertebrales, no vertebrales y de cadera. Está indicado para el tratamiento de la osteoporosis con alto riesgo de fractura. Su mecanismo de acción es reversible. Se han descripto pérdida de la DMO y elevación de los marcadores de remodelado óseo postsuspensión. Una situación clínica grave son las fracturas vertebrales múltiples postsuspensión. Este evento es infrecuente y se lo atribuye a un rebote del remodelado óseo, postulándose se postula una predisposición especial, probablemente relacionada con microRNA. Se escriben dos mujeres con osteoporosis que presentaron este cuadro. Las fracturas ocurrieron entre 7 y 10 meses posteriores a la última dosis de denosumab. Registraron elevación de C-telopéptidos y disminución de la DMO conjuntamente con las fracturas vertebrales agudas en cascada. (AU)

The duration of osteoporosis treatments depends on the drug type, anabolic or anticatabolic, and the severity of the disease. Denosumab is a fully human monoclonal antibody that inactivates RANK-L, inhibiting the RANKL-RANK interaction . This inhibits osteoclast formation, activation, and survival. It also reduces cortical and trabecular bone resorption. Subcutaneous administration of 60 mg every 6 months for 3 years has reduced bone resorption, increased bone mineral density (BMD) and decreased vertebral, non-vertebral and hip fractures. It is indicated for the treatment of osteoporosis with high risk of fracture. Denosumab mechanism of action is reversible. After discontinuation, loss of BMD and elevation of bone turnover markers have been observed. In addition, multiple vertebral fractures after the suspension of the drug have been reported. These rebound-associated vertebral fractures are rare. A special genetic predisposition related to miRNA has been proposed. Two women with this clinical presentation are described. Fractures occurred between 7 and 10 months respectively after the last dose of denosumab. They presented with an increase in circulating C-telopeptid levels and a decrease inBMD with acute multiple vertebral fractures. (AU)

Ryanodine Receptor-mediated Calcium Release Regulates Neuronal Excitability in Rat Spinal Substantia Gelatinosa Neurons

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium (BKca) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of Ca2+ that induces Ca2+-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective BKCa channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove Ca2+ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through BKca channels, which are activated by intracellular Ca2+ increase via activation of RyR of Ca2+ stores.

Research on the effect and mechanism of polypeptide extract from scorpion venom combined with 5-fluorouracil on vasculogenic mimicry of H22 hepatoma / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the inhibition effects of polypeptide extract from scorpion venom (PESV) combined 5-fluorouracil (5-Fu) on vasculogenic mimicry (VM) of H2 hepatoma carcinoma cells in mice and its possible mechanisms.</p><p><b>METHODS</b>The H22 carcinoma cell suspension was subcutaneously inoculated into 60 Kunming mice. Then tumor-bearing mice were randomly divided into three groups, i.e., the control group, the 5-Fu group, and the combination group (PESV +5-Fu), 20 in each group. The tumor volume was measured once every other day after 14 successive days of intervention. Then the tumor volume growth curve was drawn, and the tumor inhibitory rate was calculated. The morphological changes of the tumor tissue were observed by HE staining. The VM density of each tumor tissue were detected by immunohistochemical assay and periodic acid-schiff stain (PAS). The protein expression levels of hypoxia inducible factor-la (HIF-la) and matrix metalloproteinase-2 (MMP-2) were detected using immunohistochemical assay. The gray value was semi-quantitatively analyzed using LeicaQwinV3 Image Analysis Software.</p><p><b>RESULTS</b>The growth of H22 hepatoma transplantation tumor was inhibited more obviously in the combination group and the 5-Fu group than in the control group (P <0.05). There was statistical difference in the tumor weight and the tumor volume between the combination group and the 5-Fu group (P <0.05). Immunohistochemical assay and PAS showed that the VM density was obviously lower in the combination group than in the control group and the 5-Fu group (P <0.01). Compared with the control group, the protein expressions of HIF-la and MMP-2 significantly decreased in the combination group (P <0.01).</p><p><b>CONCLUSIONS</b>PESV combined 5-Fu could inhibit the generation of VM of H22 hepatoma transplantation tumor in mice. Its mechanisms might be associated with inhibiting the expressions of HIF-lalpha and MMP-2 in the microenvironment of tumors.</p>

Possible Involvement of Ca2+ Activated K+ Channels, SK Channel, in the Quercetin-Induced Vasodilatation

Effects of quercetin, a kind of flavonoids, on the vasodilating actions were investigated. Among the mechanisms for quercetin-induced vasodilatation in rat aorta, the involvement with the Ca2+ activated K+ (KCa) channel was examined. Pretreatment with NE (5 micrometer) or KCl (60 mM) was carried out and then, the modulation by quercetin of the constriction was examined using rat aorta ring strips (3 mm) at 36.5degrees C. Quercetin (0.1 to 100 micrometer) relaxed the NE-induced vasoconstrictions in a concentration-dependent manner. NO synthesis (NOS) inhibitor, NG-monomethyl-L-arginine acetate (L-NMMA), at 100 micrometer reduced the quercetin (100 micrometer)-induced vasodilatation from 97.8+/-3.7% (n=10) to 78.0+/-11.6% (n=5, p<0.05). Another NOS inhibitor, L-NG-nitro arginine methyl ester (L-NAME), at 100 micrometer also had the similar effect. In the presence of both 100 micrometer L-NMMA and 10 micrometer indomethacin, the quercetin-induced vasodilatation was further attenuated by 100 micrometer tetraethylammonium (TEA, a KCa channel inhibitor). Also TEA decreased the quercetin-induced vasodilatation in endothelium-denuded rat aorta. Used other KCa channel inhibitors, the quercetin-induced vasodilatation was attenuated by 0.3 micrometer apamin (a SK channel inhibitor), but not by 30 nM charybdotoxin (a BK and IK channel inhibitor). Quercetin caused a concentration-dependent vasodilatation, due to the endothelium-dependent and -independent actions. Also quercetin contributes to the vasodilatation selectively with SK channel on smooth muscle.

Emergencia de la resistencia antibiótica debida a las ß-lactamasas de espectro extendido (blee): detección, impacto clínico y epidemiología / Emergence of antimicrobial resistance to extended-spectrum ß-lactamases (ESBL): detection, clinic impact and epidemiology

RESUMEN La rápida emergencia de la resistencia antimicrobiana debida a las BLEE tiene un impacto significativo en la salud pública. En los últimos 24 años ha suscitado un gran interés el conocimiento acerca de las BLEE, esta explosión de publicaciones abarca a todos los continentes y más de 30 países, actualmente es motivo de preocupación y se considera un problema de salud pública. Las BLEE son enzimas que producen los gram negativos y confieren resistencia a las penicilinas, a todas las cefalosporinas y al aztreonam, pero no a los carbapenems ni a las cefamicinas y la mayoría son inhibidas por el acido clavulanico. En general las BLEE son derivadas de TEM-1, TEM-2 y SHV-1, difieren entre si de sus progenitoras por unos escasos aminoácidos por lo que su filogenia es cercana. Son comúnmente encontradas en E.coli, Klebsiella sp, y P.mirabilis, no obstante, existen otras BLEE que difieren filogenéticamente de TEM y SHV, como las CTX-M, las carbapenemasas tipo OXA y las metalo-β-lactamasas VIM e IMP, típicamente encontradas en especies de P. aeruginosa, Serratia sp and Enterobacter sp. La producción de BLEE en los patógenos de importancia clínica es un problema serio en los pacientes hospitalizados debido a las implicaciones clínicas, terapéuticas y económicas. Las técnicas para la detección de las BLEE van de lo simple con aspectos fenotipicos hasta las pruebas complejas moleculares de geno-detección específica. El objetivo de esta revisión es discutir el impacto clínico y epidemiológico de las BLEE más prevalentes así como las técnicas para su detección y su seguimiento nosocomial.

ABSTRACT The rapid emerge of antimicrobial resistance dueto ESBL has a significant impact in public health. In the last 24 years, the study of extendedspectrumβ-lactamases (ESBL) has created great interest. This has been documented by publications from all continents and more than 30 countries, and the extent of this problem is a public health concern. ESBLs produced by Gram negative bacilli are enzymes that confer resistance to penicillins, cepaholosporins and aztreonam, but not to carbapenems or cephamycins, and are usually inhibited by clavulanic acid. Most of the ESBLs are derived from TEM-1, TEM-2 and SHV-1, and differ from their progenitors by only a few amino-acids. Thus, their phylogeny is close. ESBLs are usually found in E. coli, Klebsiellasp, and Proteus mirabilis. However, there are some ESBL phylogenetic branches that differ from TEM and SHV, such as CTX-M, OXA carbapenemases, VIM and IMP metalo-β-lactamases, typically found in P. aeruginosa, Serratia sp and Enterobacter sp . ESBL production by different clinical pathogens imply an important clinical problem in nosocomial patients due to medical, therapeutic and economical impact. ESBL detection techniques include simple tests as well as complex detection system involving molecular genotyping. This review discusses the most prevalent ESBLs and their epidemiological and clinical impact. Also, it presents tools and strategies for ESBL detection and molecular tracking at the nosocomial level.

Mechanical Hyperalgesia Induced by Blocking Calcium-activated Potassium Channels on Capsaicin-sensitive Afferent Fiber

Small and large conductance Ca2+-activated K+ (SKCa and BKCa) channels are implicated in the modulation of neuronal excitability. We investigated how changes in peripheral KCa channel activity affect mechanical sensitivity as well as the afferent fiber type responsible for KCa channel-induced mechanical sensitivity. Blockade of SKCa and BKCa channels induced a sustained decrease of mechanical threshold which was significantly attenuated by topical application of capsaicin onto afferent fiber and intraplantar injection of 1-ethyl-2-benzimidazolinone. NS1619 selectively attenuated the decrease of mechanical threshold induced by charybdotoxin, but not by apamin. Spontaneous flinching and paw thickness were not significantly different after KCa channel blockade. These results suggest that mechanical sensitivity can be modulated by KCa channels on capsaicin-sensitive afferent fibers.

Efficacy of species specific anti-scorpion venom serum (AScVS) against severe, serious scorpion stings (Mesobuthus tamulus concanesis Pocock)--an experience from rural hospital in western Maharashtra.

BACKGROUND: Death caused by scorpion envenoming is a common event in the tropical and subtropical countries including many regions in India. Severe scorpion envenoming causes an autonomic storm producing multi-system organ-failure (MSOF) and death. OBJECTIVES: To determine the efficacy of Anti-scorpion venom serum (AScVS) in patients stung by scorpions (Mesobuthus tamulus concanesis Pocock--earlier called Buthus tamulus); to compare it with other modalities of therapy and to detect complications, if any, arising out of AScVS treatment. METHODS: Total 48 patients of severe, serious scorpion envenoming syndrome were studied during the period from 1992 to 2002. In 17 patients AScVS was the only mode of treatment. Others had received adjunctive therapy along with AScVS. RESULTS: 47 patients out of 48 scorpion sting victims recovered completely. Recovery period in patients given AScVS (10 hours) was faster than those who received alpha blockers (16-42 hours). No anaphylactic reaction with AScVS was observed. CONCLUSIONS: AScVS is effective and safe method of therapy in severe scorpion envenoming syndrome.

Aislamiento de plásmidos conjugativos en cepas de enterobacterias productoras de ß-lactamasas de espectro expandido / Isolation of conjugative plasmids in enterobacteriaceae strains producing beta-lactamases expanded sprectrum

Las ß-lactamasas de espectro expandido son enzimas capaces de hidrolizar el enlace amida de los oximino ß-lactámicos, (cefotaxime, ceflazidime y aztreonam). Las mismas son codificadas en plásmidos y por lo tanto pueden ser transferidas mediante conjugación a diversos géneros bacterianos. Diseminándose ampliamente en el ambiente hospitalario. En esta investigación se persigue, en cepas de enterobacterias aislar plásmidos que codifican para ß-lactamasas de espectro expandido y que sean capaces de transferirse mediante conjugación. Se estudio una población de 51 enterobacterias productoras de ß-lactamasas de espectro expandido aisladas de diferentes centros hospitalarios del área Metropolitana de Caracas. A las mismas se le determinó el perfil de resistencia a múltiples antibióticos mediante la metodología de Kirby-Bauer. Se detectaron las BLEE mediante dos ensayos fenotípicos basados en el efecto sinergístico con el ácido clavulánico y se tipificaron molecularmente por PCRIRFLP, seguidamente se transfirieron los plásmidos conjugativos en ensayos de conjugación en medio sólido y se aislaron los plásmidos de cepas donantes y transconjugautes, por el método de lisis alcalina. Los resultados fenotípicos indican una mayor proporción de BLEE con actividad ceflazidimasa y en menor grado actividad cefotaximasa. La tipificación molecular indicó que 60,8 por ciento de las cepas portan genes tipo SHV y 15,6 por ciento codifican ß-lactamasas de espectro expandido de la familia CTX-M. De 36 cepas conjugadas un 81 por ciento transfirió material plasmídico. El análisis de los aislamientos plasmídicos mostró la presencia en la totalidad de las transcojugantes de una banda de 25000 pb y en un 80 por ciento se evidenció una banda plasmídica mayor a 50000 pb. Se pudo constatar la cotransferencia de resistencia a otras familias de antibióticos.

Role of endothelium-derived hyperpolarizing factor in shear stress-induced endothelium-dependent relaxations of rats / 药学学报

<p><b>AIM</b>To investigate the role and mechanism of endothelium-derived hyperpolarizing factor (EDHF) in shear stress induced vasorelaxation of rat mesenteric artery.</p><p><b>METHODS</b>The changes in vessel diameter in response to variable flow (0-300 microL.min(-1)) were continuously examined. The contribution of prostacyclin (PGI2), NO and EDHF to shear stress induced relaxation were analyzed by inhibitory effects of indomethacin, N(G)-nitro-L-arginine (L-NA) and KCl. The nature and hyperpolarizing mechanism of EDHF were examined by the inhibitory effects of inhibitors of cytochrome P450 pathway and of various K+ channels.</p><p><b>RESULTS</b>The shear stress-induced relaxation were endothelium dependent and the contribution of NO was more prominent in large mesenteric arteries (400-500 microm) than that in resistance arteries (150-250 microm), whereas that of EDHF was noted in both-sized blood vessels. Tetrabutylammonium (a nonselective inhibitor of K channels) almost abolished, whereas the combination of charybdotoxin (an inhibitor of both large and intermediate-conductance Ca2+-activated K channels) and apamin (an inhibitor of small-conductance Ca2+-activated K channels) significantly inhibited the EDHF-mediated component of the shear stress-induced relaxations.</p><p><b>CONCLUSION</b>EDHF plays an important role in shear stress-induced endothelium-dependent relaxations, and K channels especially calcium-activated K channels appear to be involved.</p>

4-Aminopyridine Inhibits the Large-conductance Ca2+ -activated K+ Channel (BKCa) Currents in Rabbit Pulmonary Arterial Smooth Muscle Cells

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance Ca2+ -activated K+ channel (BKCa) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of BKCa. The BKCa currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single BKCa currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit BKCa currents in the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent K+ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.

Enhancement of ATP-induced Currents by Phospholipase D1 Overexpressed in PC12 Cells

Using phospholipase D1 (PLD1) -overexpressing PC12 (PLD1-PC12) cells, the regulatory roles of PLD1 on ATP-induced currents were investigated. In control and PLD1-PC12 cells, ATP increased PLD activity in an external Ca2+ dependent manner. PLD activity stimulated by ATP was substantially larger in PLD1-PC12 cells than in control cells. In whole-cell voltage-clamp mode, ATP induced transient inward and outward currents. The outward currents inhibited by TEA or charybdotoxin were significantly larger in PLD1-PC12 cells than in control cells. The inward currents known as Ca2+ permeable nonselective cation currents were also larger in PLD1-PC12 cells than in control cells. However, the difference between the two groups of cells disappeared in Ca2+ -free external solution, where ATP did not activate PLD. Finally, ATP-induced 45Ca uptakes were also larger in PLD1-PC12 cells than in control cells. These results suggest that PLD enhances ATP-induced Ca2+ influx via Ca2+ permeable nonselective cation channels and increases subsequent Ca2+ -activated K+ currents in PC12 cells.

Physiologic Role of K+ Channels in Relaxation of Rabbit Corpus Cavernosal Tissue / 대한남성과학회지

PURPOSE: Ion channels play key roles in determining smooth muscle tone by setting the membrane potential and allowing Ca2+ influx. Potassium channels may be important in modulating corporal smooth muscle tone. In this study, we investigated the effects of potassium channels in the rabbit corpus cavernosal smooth muscle by blocking them with various agents. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were prepared for mounting and isometric tension measurement in an organ bath. On cavernosal strips contracted with phenylephrine (PHE), sodium nitroprusside (SNP) was applied in increasing concentrations from 10(-7)M to 10(-4)M, causing dose-dependent relaxation. The effects of various potassium channel blockers on SNP-induced relaxation were then evaluated by measuring the tension of the cavernosal strips. The potassium channel blockers used were tetraethyl ammonium (TEA), charybdotoxin, gliben clamide, and apamin. RESULTS: The relaxation responses to SNP of the corporal preparations contracted in response to PHE were significantly attenuated by TEA (10(-2)M) and charybdotoxin (10(-7)M), with no significant difference observed between the two drugs. The SNP-induced relaxation responses were not significantly attenuated by glibenclamide (10(-5)M) or apamin (10(-5)M). CONCLUSIONS: These results suggest that maxi-K+ channels play an important role in corpus cavernosal relaxation. The KATP channel and small-conductance KCa channel are thought to be unrelated to corpus cavernosal smooth muscle relaxation.

Role of gap junctions in the endothelium-dependent hyperpolarization of vascular smooth muscle cells

Hyperpolarization of arterial smooth muscle by acetylcholine is considered to be produced by the release of an unidentified chemical substance, an endothelium-derived hyperpolarizing factor (EDHF). Several chemicals have been proposed as the candidate for EDHF. However, none of them fulfil completely the nature and property of EDHF. Ultrastructural observation with electron microscope reveals that in some arteries, gap junctions are formed between endothelial and smooth muscle cells. In small arterioles, injection of gap junction permeable dyes into an endothelial cell results in a distribution of the dye to surrounding cells including smooth muscle cells. These observations allow the speculation that myoendothelial gap junctions may have a functional significance. Simultaneous measurement of the electrical responses in both endothelial and smooth muscle cells using the double patch clamp method demonstrates that these two cell types are indeed electrically coupled, indicating that they behave as a functional syncytium. The EDHF-induced hyperpolarization is produced by an activation of Ca2+-sensitive K+-channels that are inhibited by charybdotoxin and apamin. Agonists that release EDHF increase (Ca2+)i in endothelial cells but not in smooth muscle cells. Inhibition of gap junctions with chemical agents abolishes the agonist-induced hyperpolarization in smooth muscle cells but not in endothelial cells. All these observations can be explained if EDHF is an electrotonic signal propagating from endothelium to smooth muscle cells through gap junctions.

Effects of PCLS-2 in Regulation of Rabbit Corpus Cavernosum Smooth Muscle Tone / 대한비뇨기과학회지

PURPOSE: Relaxation of the penile cavernosum smooth muscle is a critical event in erection. Artemisia iwaymogi(AI) is a perennial herb growing in Korea. The aerial parts have been used in folk medicine. Bioassay-guided fractionation of an H2O extract of AI has furnished an inhibitory substance (PCLS-2). We investigated compound extracted in the rabbit corporal cavernosum smooth muscle. MATERIALS AND METHODS: Bioassay-guided fractionation of an H2O extract was used. A strip of rabbit corpus cavernosum was mounted in an organ chamber to measure the isometric tension. PCLS-2 compound induced relaxations were evaluated by in vitro study using muscarinic receptor blocker atropine (ATR), cyclo-oxygenase inhibitor indomethacin, nitric oxide synthase (NOS) ihibitor Nitro-L Arginine-Methyl Ester (NAME), guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin 1-one (ODQ), intrinsic neurotransmission inhibitor tetrodotoxin (TTX), or/and potassium channel blockers. RESULTS: PCLS-2 showed relaxation in a dose-dependent manner. Atropine, Indomethacin, NAME, ODQ, TTX, glibenclamide, tetraethylammonium, 4-aminopyridine, charybdotoxin, or apamin did not inhibit the relaxation induced by PCLS-2 compound. CONCLUSIONS: The present results suggest that the PCL-2 compound has effect of relaxation of corpus cavernosum smooth muscle and the relaxation was not involved muscarinic receptor, nitric oxide, prostaglandin, potassium channels and intrinsic neurotransmission. Other mechanisms may by involved in the PCLS-2 induced relaxation in the rabbit corpus cavernosum smooth muscle.

Roles of Ca2+ activated K+ conductances on spontaneous firing patterns of isolated rat medial vestibular nucleus neurons

To investigate the contributions of intrinsic membrane properties to the spontaneous activity of medial vestibular nucleus (MVN) neurons, we assessed the effects of blocking large and small calcium-activated potassium channels by means of patch clamp recordings. Almost all the MVN neurons recorded in neonatal (P13~P17) rat were shown to have either a single deep after-hyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). Among the recorded MVN cells, immature action potential shapes were found. Immature type A cell showed single uniform AHP and immature B cell showed a lack of the early fast AHP, and the delayed AHP was separated from the repolarization phase of the spike by a period of isopotentiality. Application of apamin and charybdotoxin (CTX), which selectively block the small and large calcium-activated potassium channels, respectively, resulted in significant changes in spontaneous firings. In both type A and type B cells, CTX (20 nM) resulted in a significant increase in spike frequency but did not induce bursting activity. By contrast, apamin (300 nM) selectively abolished the delayed slow AHP and induced bursting activity in type B cells. Apamin had no effect on the spike frequency of type A cells. These data suggest that there are differential roles of apamin and CTX sensitive potassium conductances in spontaneous firing patterns of MVN neurons, and these conductances are important in regulating the intrinsic rhythmicity and excitability.

Alteration of 4-aminopyridine-sensitive, voltage-dependent K+-channel in arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rats

Using the patch-clamp technique, we investigated the alteration of 4-aminopyridine(4-AP)-sensitive, voltage-dependent K+ channel (KV) in the mesenteric arterial smooth muscle cell (MASMC) of renovascular hypertensive model, one-kidney one-clip Goldblatt hypertensive rat (GBH). To isolate KV current, internal pipette solution contained 5 mM ATP and 10 mM EGTA. Under these condition, MASMC was depolarized by 4-AP, but charybdotoxin did not affect membrane potential. Membrane potential of hypertensive cell (- 40.3 +/- 3.2 mV) was reduced when compared to that of normotensive cell (-59.5 +/- 2.8 mV). Outward K+ current of hypertensive cell was significantly reduced when compared to normotensive cell. At 60 mV, the outward currents were 19.10 +/- 1.91 and 14.06 +/- 1.05 pA/pF in normotensive cell and hypertensive cell respectively. 4-AP-sensitive K+ current was also smaller in hypertensive cell (4.28 +/- 0.38 pA/pF) than in normotensive cell (7.65 +/- 0.52 pA/pF). The values of half activation voltage (V1/2) and slope factor (k1) as well as the values of half inactivation voltage (V1/2) and slope factor (k1) were virtually similar between GBH and NTR. These results suggest that the decrease of 4-AP-sensitive K+ current contributes to a depolarization of membrane potential, which leads to development of vascular tone in GBH.

Decreased voltage dependent K+ currents in cerebral arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rat

The Kv channel activity in vascular smooth muscle cell plays an important role in the regulation of membrane potential and blood vessel tone. It was postulated that increased blood vessel tone in hypertension was associated with alteration of Kv channel and membrane potential. Therefore, using whole cell mode of patch-clamp technique, the membrane potential and the 4-AP-sensitive Kv current in cerebral arterial smooth muscle cells were compared between normotensive rat and one-kidney, one-clip Goldblatt hypertensive rat (1K,1C-GBH rat). Cell capacitance of hypertensive rat was similar to that of normotensive rat. Cell capacitance of normotensive rat and 1K,1C-GBH rat were 20.8+/-2.3 and 19.5+/-1.4 pF, respectively. The resting membrane potentials measured in current clamp mode from normotensive rat and 1K,1C-GBH rat were -45.9+/-1.7 and -38.5+/-1.6 mV, respectively. 4-AP (5 mM) caused the resting membrane potential hypopolarize but charybdotoxin (0.1 muM) did not cause any change of membrane potential. Component of 4-AP-sensitive Kv current was smaller in 1K,1C-GBH rat than in normotensive rat. The voltage dependence of steady-state activation and inactivation of Kv channel determined by using double-pulse protocol showed no significant difference. These results suggest that 4-AP-sensitive Kv channels play a major role in the regulation of membrane potential in cerebral arterial smooth muscle cells and alterations of 4-AP-sensitive Kv channels would contribute to hypopolarization of membrane potential in 1K,1C-GBH rat.

External pH effects on delayed rectifier K+ currents of small dorsal root ganglion neuron of rat

Under certain pathophysiological conditions, such as inflammation and ischemia, the concentration of H+ ion in the tissue surrounding neurons is changed. Variations in H+ concentration are known to alter the conduction and/of the gating properties of several types of ion channels. Several types of K+ channels are modulated by pH. In this study, the whole cell configuration of the patch clamp technique has been applied to the recording of the responses of change of external pH on the delayed rectifier K+ current of cultured DRG neurons of rat. Outward K+ currents were examined in DRG cells, and the Charybdotoxin and Mn2+ could eliminate Ca2+-dependent K+ currents from outward K+ currents. This outward K+ current was activated around -60 mV by step depolarizing pulses from holding potential -70 mV. Outward K+ currents were decreased by low external pH. Activation and steady-state inactivation curve were shifted to the right by acidification, while there was small change by alkalization. These results suggest that H+ could be alter the sensory modality by changing and modifying voltage-dependent K+ currents, which participated in repolarization.