• The Korean Journal of Physiology and Pharmacology
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• v.15 no.5
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• pp.285-289
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• 2011

Shilajit, a medicine herb commonly used in Ayurveda, has been reported to contain at least 85 minerals in ionic form that act on a variety of chemical, biological, and physical stressors. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) are involved in orofacial nociceptive processing. Shilajit has been reported to be an injury and muscular pain reliever but there have been few functional studies of the effect of Shilajit on the SG neurons of the Vc. Therefore, whole cell and gramicidin-perfotrated patch clamp studies were performed to examine the action mechanism of Shilajit on the SG neurons of Vc from mouse brainstem slices. In the whole cell patch clamp mode, Shilajit induced short-lived and repeatable inward currents under the condition of a high chloride pipette solution on all the SG neurons tested. The Shilajit-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated $Na^+$ channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, and AP5, an NMDA receptor antagonist. The Shilajit-induced responses were partially suppressed by picrotoxin, a $GABA_A$ receptor antagonist, and totally blocked in the presence of strychnine, a glycine receptor antagonist, however not affected by mecamylamine hydrochloride (MCH), a nicotinic acetylcholine receptor antagonist. Under the potassium gluconate pipette solution at holding potential 0 mV, Shilajit induced repeatable outward current. These results show that Shilajit has inhibitory effects on the SG neurons of Vc through chloride ion channels by activation of the glycine receptor and $GABA_A$ receptor, indicating that Shilajit contains sedating ingredients for the central nervous system. These results also suggest that Shilajit may be a potential target for modulating orofacial pain processing.

• Toxicological Research
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• v.12 no.1
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• pp.69-79
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• 1996

Primary culture of cerebellar neuronal cells derived from 8-day old Long-Evans rats was used. Pure granule cells, astrocytes or mixed cells culture systems were prepared. These cells were differentiated and developed synaptic connections. And the astrocytes were identified by immunostaining with glial fibrillary acidic protein (GFAP). Methamphetamine (MAP), which acts on dopaminergic system and cadmium (Cd), a toxic heavy metal, were applied and biochemical assays and electrophysiological studies were performed. $LC_50$ values estimated by MTT assay of MAP and Cd were 3 mM and 2$\mu M$ respectively. Cells were treated with 1 mM or 2 mM MAP and 1$\mu M$ $CdCl_2$ for 48 hour, and the incubation media were analyzed for the content of released LDH. MAP (2 mM) and Cd significantly increased the LDH release. Cell viability was decreased in both groups and some cytopathological changes like cell swelling or vacuolization were seen. The cerebellar granule cells were used for measuring membrane currents using whole-cell clamp technique. Sodium and potassium currents were not affected by MAP neither Cd, but calcium current was significantly reduced by Cd but not affected by MAP. Therefore, in vitro neurotoxicity test system using neuronaI cells and astrocytes cultures were established and can be used in screening of potential neurotoxic chemicals.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.6
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• pp.445-453
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• 2000

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of $Ca^{2+}-activated$ potassium channel $(K_{Ca}\;channel)$ and is involved in the activation of $K_{Ca}$ channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide $(SIN-1,\;50\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate $(8-pCPT-cGMP,\;100\;{\mu}M),$ a membrane-permeable analogue of cGMP, increased the $K_{Ca}$ channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP $(100\;{\mu}M),$ cGMP $(100\;{\mu}M),$ ATP+cGMP $(100\;{\mu}M\;each),$ PKG $(5\;U/{\mu}l),$ ATP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l),$ or cGMP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ did not increase the channel activity. ATP $(100\;{\mu}M)+cGMP\;(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer $(Rp-pCPT-cGMP,\;100\;{\mu}M),$ a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The $K_{Ca}$ channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of $K_{Ca}$ channels, or some associated proteins in the membrane patch increase the activity of the $K_{Ca}$ channel, and the activation may be associated with the vasodilating action.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.2
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• pp.83-89
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• 2010

In this study, we studied whether hydrogen sulfide ($H_2S$) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of $H_2S$ on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular $Ca^{2+}$ analysis at $30^{\circ}C$ and RT-PCR in cultured mouse intestinal ICC. Exogenously applied sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide, caused a slight tonic inward current on pacemaker activity in ICC at low concentrations (50 and $100{\mu}m$), but at high concentration ($500{\mu}m$ and 1 mM) it seemed to cause light tonic inward currents and then inhibited pacemaker amplitude and pacemaker frequency, and also an increase in the resting currents in the outward direction. Glibenclamide or other potassium channel blockers (TEA, $BaCl_2$, apamin or 4-aminopydirine) did not have an effect on NaHS-induced action in ICC. The exogenous application of carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and thapsigargin also inhibited the pacemaker activity of ICC as NaHS. Also, we found NaHS inhibited the spontaneous intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) oscillations in cultured ICC. In doing an RT-PCR experiment, we found that ICC enriched population lacked mRNA for both CSE and CBS, but was prominently detected in unsorted muscle. In conclusion, $H_2S$ inhibited the pacemaker activity of ICC by modulating intracellular $Ca^{2+}$. These results can serve as evidence of the physiological action of $H_2S$ as acting on the ICC in gastrointestinal (GI) motility.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.3
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• pp.137-141
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• 2005

This study was undertaken to investigate the effects of [$D-Ala^2$, D-Leu^5$]-enkephalin (DADLE) on the spontaneous activity of medial vestibular nuclear neurons of the rat. Sprague-Dawley rats, aged 14 to 16 days, were anesthetized with ether and decapitated. After enzymatic digestion, the brain stem portion of medial vestibular nuclear neuron was obtained by micropunching. The dissociated neurons were transferred to a recording chamber mounted on an inverted microscope, and spontaneous action potentials were recorded by standard patch-clamp techniques. The spontaneous action potentials were increased by DADLE in 12 cells and decreased in 3 cells. The spike frequency and resting membrane potential of these cells were increased by DADLE. The depth of afterhyperpolarization was not affected by DADLE. The potassium currents were decreased in 20 cells and increased in 5 cells. These results suggest that DADLE increases the neuronal activity of the medial vestibular nuclear neurons by altering resting membrane potential.

• Animal cells and systems
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• v.2 no.4
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• pp.471-476
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• 1998

The G protein-activated inwardly rectifying $K^＋$ channel (GIRK1) was coex-pressed in Xenopus oocytes along with the $5-HT_{1A}$ receptor, a 7-helix receptor known to be coupled to $K^＋$ channels in many neural tissues. Thus, the activation of the $5-HT_{1A}$ receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5-HT in the presence of various external potassium concentrations $[K^＋]_0$. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. $K^{+}$ was the major ion carrier as evidenced by about 44㎷ voltage shift corresponding to a 10-fold external 〔$K^＋$〕 change. 5-HT induced a concentration-dependent inward $K^＋$ current ($EC_{50}{\equation omitted}10.7nM$) which was blocked by $Ba^{2＋}$. Pertussis toxin (PTX) pre-treatment reduced the $K^＋$ current by as much as about 70%, suggesting that PTX-sensitive G protein ($G_i or G_o$ type) are involved in the $5-HT_{1A}$ receptor-GIRK1 coupling in Xenopus oocytes.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.313-322
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• 2007

Extract of Japanese red pine needles has been used in Asia pacific regions since long periods believing its valuable properties as tonic and ability of curing diseases of unidentified symptoms. Some selective compounds present in the extract and their effects were analyzed. Carbohydrates and vitamin c were identified using HPLC; terpenoid compounds by GC-MS; anti-bacterial analysis by paper discs, plates count and gastrointestinal motility by whole cell patch clamp. The extract is a mixture of compounds therefore its diverse effect was expected. Self-fermentation in extract proceeds after spontaneous appearance of yeast strains without inoculation. Effects and composition of the extract vary with varying period of self-fermentation. Extract inhibits the growth of bacteria dose dependently exhibiting its antibacterial properties however effectiveness increases with increase in fermentation period. The extract also can modulate gastrointestinal motility in murine small intestine by modulating pace maker currents in ICC mediated through ATP sensitive potassium channel.

• Bulletin of the Korean Chemical Society
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• v.16 no.6
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• pp.511-515
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• 1995

Since much studies have been performed concerning the electrochemical behaviors and the practical applications of PB based devices, little has yet reported to investigate the best condition for the preparation for PB thin films. As considered some factors(peak shape, peak current, and peak separation) from the i-V curves, the optimal condition in the film growth were investigated under various immersion solutions. An electron-transfer processes of Fe2+/Fe3+ and Fe(CN)63-/4- redox couples were considered by measuring the observed currents as a function of the rotation velocity. The standard heterogeneous electron-transfer rates for these films and bare Au disc electrode in 10-3 M Fe2+/Fe3+ solution, applied at +0.65 V vs. SCE, were 6.14 × 10-3 and 7.78 × 10-3 cm/s, respectively, obtained using a rotating disc electrode. In case of the addition of potassium ion, the rate constants for these Fe2+/Fe3+ system on thin films of PB and bare electrode were given a little high values. The electron transfer rate for 10-3 M Fe(CN)63-/4- were 4.55 × 10-3 and 6.84 × 10-3 cm/s, respectively. The conductivity as directly determined during obtained the voltammogram, was 2.2 × 10-7 (Ω·cm)-1. This value is similar magnitude to that calculated from bulk sample.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.61-70
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• 2009

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.