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*

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

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.
Acetylcholine ; Apamin ; Charybdotoxin ; Coronary Vessels* ; Cromakalim* ; Escin ; Glyburide ; Inositol 1,4,5-Trisphosphate ; Sarcoplasmic Reticulum ; Skin ; Vasodilation

It is well known that nitric oxide (NO) mediates smooth muscle relaxation via an increase in cyclic GMP (cGMP) 1evels. Acetylcholine (ACh) and nitroprusside (SNP) are known to mediate relaxation of cavernous smooth muscle via increasing the levels of NO. In recent years, the role of K+ channels in the hyperpolarization induced by nitrates and ACh in smooth muscle have been investigated. In this study, we attempted to characterize the role of K+ channel in rabbit cavernous smooth muscle relaxation by ACh and SNP under organ bath. Changes in isometric tension of corporal strips were monitored. The results were as follows; 1. The relaxant effects of ACh and SNP on contracted smooth muscle induced by 80 mM K' were less than those by phenylephrine. The ACh-induced relaxation was almost abolished in rabbit cavernous smooth muscle which endothelium was denuded, but the that of SNP was not affected by removal of endothelium. 2. Ng-nitro-L-arginine(L-NOARG) (3x0.00001M) and methylene blue (3x0.00001M) significantly inhibited the relaxant effect of ACh in cavernous smooth muscle, but that of SNP was not influenced by these drugs. The inhibition of L-NOARG on the relaxant effect of Ach was reversed by the addition of L-arginine(3x0.0001M). 3. Charybdotoxin (ChTx, 0.0000001M), significantly inhibited the relaxant effects of ACh,SNP and 8-Br-cGMP, but glibenclamide (0.00001M) and apamin (0.00001M) did not influence those of ACh and SNP 4. ACh (0.0001M} and SNP (0.0001M) increased the tissue content of cGMP The effect of ACh on the tissue content of cGMP was significantly affected by L-NOARG (3x0.00001M) and methylene blue (3x0.0000lM), but that of SNP was not influenced by these drugs. ChTx (0.000000lM) did not attenuate the accumulation of cGMP induced by ACh and SNP. Above results suggest that the relaxing effect of Ach and SNP on the isolated rabbit cavernous smooth muscle is associated with an increase in the tissue content of cGMP. Furthermore, ChTx sensitive-K+ channel-mediated hyperpolarization by increased cGMP may play a part in the relaxation of rabbit cavernous smooth muscle by ACh and SNP
Acetylcholine* ; Apamin ; Baths ; Charybdotoxin ; Cyclic GMP ; Endothelium ; Glyburide ; Methylene Blue ; Muscle, Smooth* ; Nitrates ; Nitric Oxide ; Nitroprusside* ; Phenylephrine ; Relaxation*

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.
Animals ; Charybdotoxin ; Diagnosis-Related Groups ; Ganglia, Spinal* ; Hydrogen-Ion Concentration* ; Inflammation ; Ion Channels ; Ischemia ; Neurons ; Rats* ; Spinal Nerve Roots*

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.
Action Potentials ; Animals ; Apamin ; B-Lymphocytes ; Charybdotoxin ; Fires* ; Membranes ; Neurons* ; Periodicity ; Potassium ; Potassium Channels, Calcium-Activated ; Rats* ; Vestibular Nuclei*

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.
Animals ; Blood Vessels ; Charybdotoxin ; Hypertension ; Membrane Potentials ; Muscle, Smooth* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle* ; Patch-Clamp Techniques ; 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.
Adenosine Triphosphate ; Animals ; Charybdotoxin ; Egtazic Acid ; Membrane Potentials ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Patch-Clamp Techniques ; Rats*

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

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