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.
Animals ; Calcium* ; Charybdotoxin ; Endoplasmic Reticulum ; Humans ; Membranes ; Neurons* ; Nitric Oxide ; Nitroprusside ; Potassium ; Rats* ; Ryanodine Receptor Calcium Release Channel ; Ryanodine* ; Substantia Gelatinosa* ; Thapsigargin ; Tissue Donors

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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).

CONCLUSIONSPESV 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.

Animals ; Carcinoma, Hepatocellular ; blood supply ; Cell Line, Tumor ; Charybdotoxin ; pharmacology ; Fluorouracil ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Liver Neoplasms ; blood supply ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Mice ; Mice, Inbred Strains

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.
Animals ; Aorta ; Apamin ; Arginine ; Charybdotoxin ; Constriction ; Endothelium ; Flavonoids ; Indomethacin ; Muscle, Smooth ; omega-N-Methylarginine ; Potassium Channels, Calcium-Activated ; Quercetin ; Rats ; Tea ; Tetraethylammonium ; Vasoconstriction ; Vasodilation

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.
Apamin ; Capsaicin ; Charybdotoxin ; Hyperalgesia* ; Neurons ; Potassium Channels, Calcium-Activated*

AIMTo investigate the role and mechanism of endothelium-derived hyperpolarizing factor (EDHF) in shear stress induced vasorelaxation of rat mesenteric artery.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONEDHF plays an important role in shear stress-induced endothelium-dependent relaxations, and K channels especially calcium-activated K channels appear to be involved.

Animals ; Apamin ; pharmacology ; Biological Factors ; physiology ; Charybdotoxin ; pharmacology ; Cytochrome P-450 Enzyme Inhibitors ; Endothelium, Vascular ; drug effects ; physiology ; In Vitro Techniques ; Large-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; Male ; Mesenteric Arteries ; drug effects ; physiology ; Nitric Oxide ; physiology ; Potassium Channel Blockers ; pharmacology ; Proadifen ; pharmacology ; Quaternary Ammonium Compounds ; pharmacology ; Rats ; Rats, Wistar ; Small-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; Vasodilation ; drug effects

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.
4-Aminopyridine* ; Charybdotoxin ; Discrimination (Psychology) ; Ion Channels ; Membrane Potentials ; Muscle, Smooth* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle* ; Patch-Clamp Techniques ; Pulmonary Artery

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.
Adenosine Triphosphate ; Animals ; Charybdotoxin ; PC12 Cells* ; Phospholipases* ; Tea

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.
Ammonium Compounds ; Apamin ; Baths ; Charybdotoxin ; Glyburide ; Ion Channels ; Membrane Potentials ; Muscle, Smooth ; Nitric Oxide ; Nitroprusside ; Phenylephrine ; Potassium Channel Blockers ; Potassium Channels ; Relaxation* ; Tea

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.
4-Aminopyridine ; Apamin ; Artemisia ; Atropine ; Charybdotoxin ; Glyburide ; Guanylate Cyclase ; Indomethacin ; Korea ; Medicine, Traditional ; Muscle, Smooth* ; Nitric Oxide ; Nitric Oxide Synthase ; Potassium Channel Blockers ; Potassium Channels ; Prostaglandin-Endoperoxide Synthases ; Receptors, Muscarinic ; Relaxation ; Synaptic Transmission ; Tetraethylammonium ; Tetrodotoxin

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.
Acetylcholine ; Apamin ; Arteries ; Arterioles ; Calcium ; Charybdotoxin ; Coloring Agents ; Endothelial Cells ; Endothelium ; Gap Junctions* ; Giant Cells ; Muscle, Smooth ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Potassium Channels