• The Korean Journal of Physiology and Pharmacology
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• 제12권1호
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• pp.25-30
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• 2008

Although many studies show that thromboxane $A_2\;(TXA_2)$ has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of $TXA_2$ on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by $TXA_2$ in ICC with whole cell patch-clamp technique. Externally applied $TXA_2\;(5{\mu}M)$ produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by $TXA_2$ were inhibited by intracellular application of GDP-${\beta}$-S. The pretreatment of ICC with $Ca^{2+}$ free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the $TXA_2$-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the $TXA_2$-induced effects on pacemaker currents. These results suggest that $TXA_2$ can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by $TXA_2$ may occur by the activation of G protein and PKC independent pathway via extra and intracellular $Ca^{2+}$ modulation.

• The Korean Journal of Physiology and Pharmacology
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• 제21권1호
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• pp.75-82
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• 2017

The effects of acepromazine on human ether-$\grave{a}$-go-go-related gene (hERG) potassium channels were investigated using whole-cell voltage-clamp technique in human embryonic kidney (HEK293) cells transfected with hERG. The hERG currents were recorded with or without acepromazine, and the steady-state and peak tail currents were analyzed for the evaluating the drug effects. Acepromazine inhibited the hERG currents in a concentration-dependent manner with an $IC_{50}$ value of $1.5{\mu}M$ and Hill coefficient of 1.1. Acepromazine blocked hERG currents in a voltage-dependent manner between -40 and +10 mV. Before and after application of acepromazine, the half activation potentials of hERG currents changed to hyperpolarizing direction. Acepromazine blocked both the steady-state hERG currents by depolarizing pulse and the peak tail currents by repolarizing pulse; however, the extent of blocking by acepromazine in the repolarizing pulse was more profound than that in the depolarizing pulse, indicating that acepromazine has a high affinity for the open state of the channels, with a relatively lower affinity for the closed state of hERG channels. A fast application of acepromazine during the tail currents inhibited the open state of hERG channels in a concentration-dependent. The steady-state inactivation of hERG currents shifted to the hyperpolarized direction by acepromazine. These results suggest that acepromazine inhibits the hERG channels probably by an open- and inactivated-channel blocking mechanism. Regarding to the fact that the hERG channels are the potential target of drug-induced long QT syndrome, our results suggest that acepromazine can possibly induce a cardiac arrhythmia through the inhibition of hERG channels.

• The Korean Journal of Physiology and Pharmacology
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• 제23권5호
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• pp.419-426
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• 2019

Mosapride accelerates gastric emptying by acting on 5-hydroxytryptamine type 4 ($5-HT_4$) receptor and is frequently used in the treatment of gastrointestinal (GI) disorders requiring gastroprokinetic efficacy. We tested the effect of mosapride on 5-hydroxytryptamine type 3 ($5-HT_3$) receptor currents because the $5-HT_3$ receptors are also known to be expressed in the GI system and have an important role in the regulation of GI functions. Using the whole-cell voltage clamp method, we compared the currents of the $5-HT_3$ receptors when 5-HT was applied alone or was co-applied with mosapride in cultured NCB-20 cells known to express $5-HT_3$ receptors. The $5-HT_3$ receptor current amplitudes were inhibited by mosapride in a concentration-dependent manner. Mosapride blocked the peak currents evoked by the application of 5-HT in a competitive manner because the $EC_{50}$ shifted to the right without changing the maximal effect. The rise slopes of $5-HT_3$ receptor currents were decreased by mosapride. Pre-application of mosapride before co-application, augmented the inhibitory effect of mosapride, which suggests a closed channel blocking mechanism. Mosapride also blocked the opened $5-HT_3$ receptor because it inhibited the $5-HT_3$ receptor current in the middle of the application of 5-HT. It accelerated desensitization of the $5-HT_3$ receptor but did not change the recovery process from the receptor desensitization. There were no voltage-, or use-dependency in its blocking effects. These results suggest that mosapride inhibited the $5-HT_3$ receptor through a competitive blocking mechanism probably by binding to the receptor in closed state, which could be involved in the pharmacological effects of mosapride to treat GI disorders.

• The Korean Journal of Physiology and Pharmacology
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• 제13권6호
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• pp.461-467
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• 2009

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic $K^+$ channels by 4-aminopyridine, a $K^+$ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic $Ca^{2+}$ channels by $Cd^{2+}$, a general voltage-dependent $Ca^{2+}$ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic $Ca^{2+}$ channels in the presynaptic nerve terminals of A1 neurons.

• 약학회지
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• 제56권2호
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• pp.108-115
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• 2012

The hyperpolarization-activated current ($I_h$) is an inward cation current activated by hyperpolarization of the membrane potential and plays a role as an important modulator of action potential firing frequency in many excitable cells. In the present study we investigated the effects of extracellular divalent cations on $I_h$ in dorsal root ganglion (DRG) neurons using whole-cell voltage clamp technique. $I_h$ was slightly increased in $Ca^{2+}$-free bath solution. BAPTA-AM did not change the amplitudes of $I_h$. Amplitudes of $I_h$ were decreased by $Ca^{2+}$, $Mg^{2+}$ and $Ba^{2+}$ dose-dependently and voltage-independently. Inhibition magnitudes of $I_h$ by external divalent cations were partly reversed by the concomitant increase of extracellular $K^+$ concentration. Reversal potential of $I_h$ was significantly shifted by $Ba^{2+}$ and $V_{1/2}$ was significantly affected by the changes of extracellular $Ca^{2+}$ concentrations. These results suggest that $I_h$ is inhibited by extracellular divalent cations ($Ca^{2+}$, $Mg^{2+}$ and $Ba^{2+}$) by interfering ion influxes in cultured rat DRG neurons.

• The Korean Journal of Physiology and Pharmacology
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• 제4권2호
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• pp.81-90
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• 2000

The types of $K^+$ channel which determine the pattern of spontaneous action potential (SAP) were investigated using whole-cell variation of patch clamp techniques under current- and voltage-clamp recording conditions in rat clonal pituitary $GH_3$ cells. Heterogeneous pattern of SAP activities was changed into more regular mode with elongation of activity duration and afterhyperpolarization by treatment of TEA (10 mM). Under this condition, exposure of the class III antiarrhythmic agent E-4031 $(5\;{\mu}M)$ to $GH_3$ cells hardly affected SAP activities. On the other hand, the main $GH_3$ stimulator thyrotropin-releasing hormone (TRH) still produced its dual effects (transient hyperpolarization and later increase in SAP frequency) in the presence of TEA. However, addition of $BaCl_2$ (2 mM) in the presence of TEA completely blocked SAP repolarization process and produced membrane depolarization in all tested cells. This effect was observed even in TEA-untreated cells and was not mimicked by higher concentration of TEA (30 mM). Also this barium-induced membrane depolarization effect was still observed after L-type $Ca^{2+}$ channel was blocked by nicardipine $(10\;{\mu}M).$ These results suggest that barium-sensitive current is important in SAP repolarization process and barium itself may have some depolarizing effect in $GH_3$ cells.

• The Korean Journal of Physiology and Pharmacology
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• 제3권6호
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• pp.615-621
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• 1999

In spite many evidences has supported the cardioprotective effect of bradykinin, its direct effects at the cell level are still under question. We investigated the both effects of bradykinin (BK) on $Ca^{2+}-related$ ionic currents using whole cell voltage clamp technique in rabbit cardiomyocytes and on the intracellular $Ca^{2+}$ transient using calcium sensitive fluorescence dye, indo-1AM. Simultaneously with recording intracellular $Ca^{2+}$ transients, cell contractility was estimated from the changes in length of the electrical stimulated rat cardiac myocytes. L-type $Ca^{2+}$ current decreased by bradykinin at the entire voltage range. Inward tail current increased initially up to its maximum about 4 min after exposing myocytes to BK, and then gradually decreased again by further exposure to BK. This tail current decreased remarkably at washing BK off but slowly recovered ca. 20 min later. The change in cell contractility was similar to that in tail current showing initial increase followed by gradual decrease. Removal of BK brought remarkable decrease in contractility, which was recovered $15{\sim}20$ min after cessation of electrical stimulation. Bradykinin increased $Ca^{2+}$ transient initially but after some time $Ca^{2+}$ transient also decreased coincidentally with contractility. From these results, it is suggested that bradykinin exerts directly its cardioprotective effect on the single myocytes by decreasing the intracellular $Ca^{2+}$ level followed by an initial increase in $Ca^{2+}$ transient.

• The Korean Journal of Physiology and Pharmacology
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• 제1권3호
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• pp.233-240
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• 1997

Neurons in the nucleus raphe magnus are involved in descending modulation of nociceptive transmission. In this study, we attempted to investigate electrophysiological properties of the NRM neurons dissociated from the postnatal rat medulla. The NRM neurons in the coronal slices of and the dissociated neurons from the postnatal rat medullae were immunohistochemically identified using antibody against serotonin. Relatively small number of neurons were positively stained in both preparations. The positively stained neurons displayed large cell body with double or multiple neurites. Using whole-cell patch clamp configuration ionic currents were recorded from the dissociated NRM-like neurons selected by criteria such as size and shape of cell body and cell population. Two types, high- and low-threshold, of voltage-dependent calcium currents were recorded from the dissociated NRM-like neurons. Some neurons displayed both types of calcium currents, whereas others displayed only high-threshold calcium current. Voltage-dependent potassium currents were also recorded from the dissociated NRM neurons. Some neurons displayed both transient outward and delayed rectifier currents but others showed only delayed rectifier current. These results suggest that there are at least two types of calcium currents and two types of potassium currents in the dissociated NRM neurons.

• The Korean Journal of Physiology and Pharmacology
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• 제2권6호
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• pp.733-742
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• 1998

Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

• The Korean Journal of Physiology
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• 제26권2호
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• pp.99-111
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• 1992

Permissive action of thyroid hormone at the level of Ca channel and responsible mechanisms underlying thyroid hormone-induced change in myocardial contractile state and $T_3-induced$ arrhythmias were investigated in rabbit ventricular or atrial myocytes using whole cell patch clamp technique. Single cells were isolated by Langendorff perfusion with collagenase. Cardiac myocytes were incubated in $low-Cl^-,$, $high-K^+$ medium containing $1_{\mu}M\;L-triiodothyronine\;(T_3)$ at $4^{\circ}C$ for 2.10 hours. The calcium currrent $(I_{Ca})$ was increased in $T_3$ loaded cells, however, the shape of current voltage curve and reverse potential did not altered. Cyclic AMP, cyclic GMP, isoprenaline and 3-isobutyl-1-methyl-xanthine increased $I_{Ca}$ in euthyroid and hyperthyroid conditions, and acetylcholine blocked the increase of $I_{Ca}\;in\;T_3$ loaded cells. The amplitude of $I_{Ca}$ was much larger after perfusing cGMP than cGMP in both conditions, whereas the degree of increase of $I_{Ca}$ was greater after perfusing cAMP than cGMP in $T_3$ loaded cells. The degree of increase of $I_{Ca}$ after perfusing isoprenaline or IBMX also was greater in $T_3$ loaded cells than in control cells. Background current induced by isoprenaline also increased in $T_3$ loaded cells. The Ca release dependent inward current was increased in amplitude but its activation and inactivation time course was not changed in $T_3$ loaded cells. Activation of Na pump current was not changed in $T_3$ loaded cells. From the above results it is suggested that thyroid hormone induced increase in the contractile state of cardiac myocytes are accompanied by augmented $I_{Ca}$ and the increase of Ca release from sarcoplasmic reticulum and the permissive action of thyroid hormone to catecholamines could induce arrhythmias through the increase of $I_{Ca}$ and background current.