Intracellular recordings of oscillatory firing (bursting activity) were obtained from Purkinje cells (PCs) in rat cerebellar slices. Apamin inhibited post-burst hyperpolarizations (PBHs) progressively and finally terminated oscillatory firing activity of PCs. Apamin did not affect the amplitude or duration of the after-hyperpolarization (AHP) between spikes within the burst. In the voltage clamp mode, apamin shifted the whole-cell, quasi-steady state I/V relationship in an inward direction and abolished the zero slope resistance (ZSR) region by blocking outward current. Nickel (Ni2+) terminated oscillatory activity and also abolished the ZSR region. However, Ni2+ did not have progressive blocking action on the post-burst hyperpolarization before it blocked oscillatory activity. Ni2+ blocked an inward current at potentials positive to approximately -65 mV, which was responsible for the ZSR region and outward current at more negative potentials. These data indicated that oscillatory activity of PCs is sustained by a balance between a slow Ni2+ -sensitive inward current and an apamin-sensitive outward current in the region of ZSR of the whole-cell I/V curve.
Animals ; Apamin* ; Fires ; Nickel* ; Purkinje Cells* ; Rats

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*

BACKGROUND: Bee venom (BV) has been widely investigated for potential medical uses. Recent inadvertent uses of BV based products have shown to mitigate signs of fungal infections. However, the component mediating the antifungal effect has not been identified. OBJECTIVE: This investigation compares bee venom in its whole and partial forms to evaluate the possible component responsible for the antifungal effect. METHODS: Forty-eight plates inoculated with Trichophyton rubrum were allocated into four groups. The groups were treated with raw BV (RBV), melittin, apamin and BV based mist (BBM) respectively and each group was further allocated accordingly to three different concentrations. The areas were measured every other day for 14 days to evaluate the kinetic changes of the colonies. RESULTS: The interactions of ratio differences over interval were confirmed in groups treated with RBV and BBM. In RBV, the level of differences were achieved in groups treated with 10 mg/100 µl (p=0.026) and 40 mg/100 µl (p=0.000). The mean difference of ratio in groups treated with RBV was evident in day 3 and day 5. The groups that were treated with melittin or apamin did not show any significant interaction. In BBM groups, the significant levels of ratio differences over time intervals were achieved in groups treated with 200 µl/100 µl (p=0.000) and 300 µl/100 µl (p=0.030). CONCLUSION: The the bee venom in its whole form delivered a significant level of inhibition and we concluded that the venom in separated forms are not effective. Moreover, BV based products may exert as potential antifungal therapeutics.
Antifungal Agents ; Apamin ; Bee Venoms* ; Bees* ; Melitten ; Negotiating ; Trichophyton* ; Venoms

The outward currents elicited in hamster eggs by depolarizing pulses were studied. The currents appeared to comprise at least two components, a transient outward component (Ito) and a steady-state outward component (Iinfin). Ito was transiently followed by the cessation of inward Ca2+ current (ICa), and its current-voltage (I-V) relation was a mirror image of that of ICa. Either blockade of ICa by Co2+ or replacement of Ca2+ with Sr2+ abolished Ito without change in Iinfin. Intracellular EGTA (10 mM) inhibited Ito but not Iinfin. suggesting strongly that generation of Ito requires intracellular Ca2+. Apamin (1 nM) abolished selectively Ito, indicating that Ito is Ca2+-dependent K+ current. On the other hand, Iinfin was Ca2+-independent. Both Ito and Iinfin were completely inhibited by internal Cs+ and external TEA. The estimated reversal potential of Ito was close to the theoretical EK. Taken together, both outward currents were carried by K+ channels. From these results, Ito is likely to be a current responsible for the hyperpolarizing responses seen in hamster eggs at fertilization.
Animals ; Apamin ; Cricetinae* ; Eggs ; Egtazic Acid ; Fertilization ; Hand ; Oocytes* ; Ovum ; Tea

The conductance change evoked by step depolarization was studied in primarily cultured rat adrenal chromaffin cells using patch-clamp and capacitance measurement techniques. When we applied a depolarizing pulse to a chromaffin cell, the inward calcium current was followed by an outward current and depolarization-induced exocytosis was accompanied by an increase in conductance trace. The slow inward tail current which has the same time course as the conductance change was observed in current recording. The activation of slow tail current was calcium-dependent. Reversal potentials agreed with Nernst equation assuming relative permeability of Cs+ to K+ is 0.095. The outward current and tail current were blocked by apamin (200 nM) and d-tubocurarine (2 mM). The conductance change was blocked by apamin and did not affect membrane capacitance recording. We confirmed that conductance change after depolarization comes from the activation of the SK channel and can be blocked by application of the SK channel blockers. Consequently, it is necessary to consider blocking of the SK channel during membrane capacitance recording.
Animals ; Apamin ; Calcium ; Chromaffin Cells* ; Exocytosis ; Membranes* ; Patch-Clamp Techniques ; Permeability ; Rats* ; Tubocurarine

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of Ca2+ -activated K+ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward K+ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of K+ current was increased by NS1619, a specific large-conductance Ca2+ -activated K+ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance Ca2+ -activated K+ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various Ca2+ -activated K+ channel modulators were added into culture media for 1~3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance Ca2+ -activated K+ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.
Apamin ; Cell Proliferation ; Clotrimazole ; Culture Media ; Fibroblasts* ; Hand ; Humans* ; Tea ; Wound Healing

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*

Medial vestibular nucleus (MVN) neurons are found to have spontaneous electrical activity in the absence of any detectable synaptic input. To investigate the contributions of intrinsic mechanisms to the spontaneous activity of type B MVN neurons, we examined the effects of various channel blockers on spontaneous firing by means of patch clamp recordings. Coronal slice (400 micrometer) of the vestibular nucleus region was sequentially treated with pronase 0.2 mg/ml and thermolysin 0.2 mg/ml, then single neurons were mechanically dissociated. MVN neurons recorded in neonatal rat were shown to have either a single deep afterhyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). In 300 nM TTX, spontaneous firing was blocked in type B cells tested. In 8 of 11 cells, underlying fluctuation or oscillations in membrane potential was not remained, and hyperpolarization did not produce rebound low-threshold calcium spikes. Although type B MVN neurons possessed hyperpolarization activated cation current (Ih), cesium had no effect on firing rates. The spike AHP is calcium dependent. When Ca2+ influx was blocked in external Ca2+ free solution, repetitive firing was abolished and the cell rested at depolarized membrane potentials. Application of apamin (300 nM) caused a profound reduction in the amplitude of the AHP and produced rhythmic burst firing. These findings suggest that the spontaneous activity of type B MVN neurons is regulated by interactions between the membrane depolarization mainly due to a persistent sodium conductances and hyperpolarization due to the calcium-activated potassium conductances.
Animals ; Apamin ; B-Lymphocytes ; Calcium ; Calcium Signaling ; Cesium ; Fires* ; Membrane Potentials ; Membranes ; Neurons* ; Potassium ; Pronase ; Rats ; Sodium ; Thermolysin ; Vestibular Nuclei*

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*