• The Korean Journal of Physiology and Pharmacology
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• v.4 no.3
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• pp.227-234
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• 2000

Many reports suggest that neurotensin (NT) in the gastrointestinal tract may play a possible role as a neurotransmitter, a circulating hormone, or a modulator of motor activity. NT exerts various actions in the intestine; it produces contractile and relaxant responses in intestinal smooth muscle. This study was designed to investigate the effect of NT on motility of antral circular muscle strips in guinea-pig stomach. To assess the role of $Ca^{2+}$ influx in underlying mechanism, slow waves were simultaneously recorded with spontaneous contractions using conventional intracellular microelectrode technique. At the concentration of $10^{-7}$ M, where NT showed maximum response, NT enhanced the magnitude $(863{\pm}198%,\;mean\;SEM,\;n=13)$ and the frequency $(154{\pm}10.3%,\;n=11)$ of spontaneous contractions. NT evoked a slight hyperpolarization of membrane potential, tall and steep slow waves with abortive spikes $(278{\pm}50%,\;n=4).$ These effects were not affected by atropine $(2\;{\mu}M),$ guanethidine $(2\;{\mu}M)$ and tetrodotoxin (0.2μM). NT-induced contractile responses were abolished in $Ca^{2+}-free$ solution and reduced greatly to near abolition by $10\;{\mu}M$ of verapamil or 0.2 mM of $CdCl_2.$ Verapamil attenuated the effects of NT on frequency and amplitude of the slow waves. Taken together, these results indicate that NT enhances contractility in guinea-pig gastric antral circular muscle and $Ca^{2+}$ influx through the voltage-operated $Ca^{2+}$ channel appears to play an important role in the NT-induced contractile mechanism.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.2
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• pp.113-120
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• 2000

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular $Ca^{2+}$ stores, we measured isometric contraction and $^{45}Ca^{2+}$ influx. $CaCl_2$ increased $Ca^{2+}$ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the $Ca^{2+}$ store emptying-induced contraction. The contraction was inhibited by voltage-dependent $Ca^{2+}$ channel antagonists dose dependently, but not by TMB-8 (intracellular $Ca^{2+}$ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In $Ca^{2+}$ store-emptied condition, $^{45}Ca^{2+}$ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular $Ca^{2+}$ stores was mediated by influx of extracellular $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channel, also protein kinase C and/or tyrosine kinase pathway modulates the $Ca^{2+}$ sensitivity of contractile protein.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.1
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• pp.35-45
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• 1999

Excitatory amino acid (EAA) and substance P (SP) have been known to be primary candidates for nociceptive neurotransmitter in the spinal cord, and calcium ions are implicated in processing of the sensory informations mediated by EAA and SP in the spinal cord. In this study, we examined how $Ca^{2+}$ modified the responses of dorsal horn neurons to single or combined iontophoretical application of EAA and SP in the rat. All the LT cells tested responded to kainate, whereas about 55% of low threshold (LT) cells responded to iontophoretically applied NMDA. NMDA and kainate excited almost all wide dynamic range (WDR) cells. These NMDA- and kainate-induced WDR cell responses were augmented by iontophoretically applied EGTA, but suppressed by $Ca^{2+},\;Mn^{2+},$ verapamil and ${\omega}-conotoxin$ EVTA, effect of verapamil being more prominent and well sustained. $Ca^{2+}$ and $Mn^{2+}$ antagonized the augmenting effect of EGTA. On the other hand, prolonged spinal application of EGTA suppressed the response of WDR cell to NMDA. SP had triple effects on the spontaneous activity as well as NMDA-induced responses of WDR cells: excitation, inhibition and no change. EGTA augmented, but $Ca^{2+},\;Mn^{2+}$ and verapamil suppressed the increase in the NMDA-induced responses and spontaneous activities of WDR cells following iontophoretical application of SP. These results suggest that in the spinal cord, sensory informations mediated by single or combined action of EAA and SP can be modified by the change in calcium ion concentration.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.377-384
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• 1998

Gold compounds depress phagocytic cell responses, including chemotaxis, and respiratory burst. However, the effects of gold compounds on the function of phagocytic cells are variable according to the preparation of medicine. In this study, effect of tetrachloroauric acid on activated neutrophil responses, including respiratory burst, lysosomal enzyme release and change of intracellular $Ca^{2+}$ level and on the synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by macrophages was studied. This study further examines how gold compounds affect the activation processes. The respiratory burst stimulated by complement C5a, degraded IgG and PMA in neutrophils was inhibited by tetrachloroauric acid. In contrast to C5a and degraded IgG, PMA-stimulated superoxide production was weakly inhibited by tetrachloroauric acid. Staurosporine, genistein, EGTA and verapamil inhibited superoxide and $H_2O_2$ production caused by C5a and degraded IgG. PMA-stimulated superoxide production was inhibited by staurosporine but was not affected by genistein. Tetrachloroauric acid, genistein, EGTA and verapamil inhibited the release of acid phosphatase and myeloperoxidase, while the effect of staurosporine was not detected. The synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by $interleukin-1{\beta}$ in macrophages was inhibited by tetrachloroauric acid. Preincubation with tetrachloroauric acid, genistein, EGTA and verapamil, the elevation of [$Ca^{2+}_i$] evoked by C5a was inhibited. Store-regulated $Ca^{2+}$ entry in thapsigargin-pretreated neutrophils was decreased by the addition of tetrachloroauric acid and genistein. The effect of staurosporine on intracellular $Ca^{2+}$ mobilization was not observed. In conclusion, tetrachloroauric acid may suppress neutrophil responses through its inhibitory action on elevation of intracellular $Ca^{2+}$ level and protein kinase C. It might exhibit an inhibitory effect on the action of protein tyrosine kinase. Tetrachloroauric acid depresses cytokine production by macrophages.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.5
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• pp.255-261
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• 2006

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin $(30\;{\mu}g/paw)$ into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or $12\;{\mu}g$; i.pl.,100 & $200\;{\mu}g$; i.p., 10 or 30 mg], N-type calcium channel blocker, ${\omega}-conotoxin$ GVIA (i.t., 0.1 or $0.5\;{\mu}g$; i.pl., $5\;{\mu}g$) and P-type calcium channel antagonist, ${\omega}-agatoxin$ IVA (i.t., $0.5\;{\mu}g$; i.pl., $5\;{\mu}g$) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and ${\omega}-conotoxin$ GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, ${\omega}-agatoxin$ IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and ${\omega}-conotoxin$ GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.

• Journal of Environmental Analysis, Health and Toxicology
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• v.21 no.4
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• pp.243-251
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• 2018

Pharmaceuticals in wastewater effluents have been recognized as emerging pollutants threatening freshwater organisms. To extend understanding for bioaccumulation and toxicity in those organisms, information on biotransformation products (or metabolites) and their metabolic pathway are crucial. The aim of the present study is to identify and elucidate metabolites of pharmaceuticals formed in exposed organisms using suspect and nontarget screening approach using LC-HRMS. As the target pharmaceuticals, carbamazepine, ketoprofen, metoprolol, propranolol, and verapamil were selected whereas Daphnia magna and Gammarus pulex were used as test organisms. After 24h exposure, metabolites formed in the organisms were identified using LC-HRMS. The structures of metabolites were elucidated via analysis of MS/MS fragment pattern and the comparison with fragment database. As the results, a total of 10 metabolites were identified for 5 parent compounds (C253/C356 for carbamazepine, K211 for ketoprofen, M256 for metoprolol, P218/P276/P306 for propranolol, V196/V291/V441 for verapamil). Among them, the presence of C253 and V291 was confirmed using standard materials. Most of the identified metabolites were formed through oxidative reactions such as hydroxylation, N-demethylation, and dealkylation. Cysteine conjugation (phase II reaction) metabolite (C356) for carbamazepine was found in daphnia. The metabolic pathway of verapamil showed similar metabolic pathways and metabolic pathways for both species. Although the toxicological information on the identified metabolites could not be confirmed, the molecular structure information of the proposed metabolites can be used for future evaluation and prediction of toxicity.

• The Korean Journal of Physiology
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• v.21 no.2
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• pp.241-257
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• 1987

The effects of external $Ca^{2+}$ and $Ca^{2+}-antagonists$ on the spontaneous contractions and electrical activities were investigated in guinea-pig stomach in order to clarify the mechanism for the generation of slow waves. Electrical responses of circular smooth muscle cells were recorded using glass capillary microelectrodes filled with 3 M KCl. All experiments were performed in tris-buffered Tyrode solution which was aerated with 100% $O_2$ and kept at $35^{\circ}C$. The results obtained were as follows: 1) The amplitude of spontaneous contractions was maximal at around 2-4 mM $Ca^{2+}$, whereas their frequency was inversely related with external $Ca^{2+}$ within the range of 0.5 to 16 mM $Ca^{2+}$. 2) Verapamil suppressed the amplitude of spontaneous contraction in a dose-dependent manner, while the frequency of spontaneous contractions was almost not changed over the whole concentration of verapamil $(0.01{\sim}5\;mg/l)$. 3) Manganese increased both the amplitude and the frequency of spontaneous contractions dose-dependently in low $Mn^{2+}$ (below 0.05 mM $Mn^{2+}$), while their amplitude and frequency were decreased in high $Mn^{2+}$ (above 0.1 mM $Mn^{2+}$). 4) The ampltude and maximum rate of rise of slow waves were incrased in high $Ca^{2+}$ solution. In $Ca^{2+}-free$ solution, the spontaneous contractions recorded simultaneously with slow waves ceased and tonic contraction ($Ca^{2+}-free$ contracture) was developed in parallel with membrane depolarization and the disappearance of slow waves. 5) Verapamil (1 mg/1) decreased the amplitude and maximum rate of rise of slow waves and it depolarized the membrane by about 6 mV, whereas the frequency of slow waves was not affected by verapamil. 6) Manganese showed different characteristic effects between low and high $Mn^{2+}$ on the slow waves: In low $Mn^{2+}$ (0.05 mM $Mn^{2+}$), the initial rapid increases and the subsequent gradual decreases in three parameters of slow waves (amplitude, rate of rise, and frequency of slow waves) till a new steady state were observed. However, in high $Mn^{2+}$ (0.5 mM $Mn^{2+}$) slow waves disappeared and membrane was depolarized. From the above results, the following conclusions could be made: 1) $Ca^{2+}$ is necessary for a generation of the slow waves, even though it is small amount. 2) Verapamil suppresses the spontaneous contractions of gastric antral strip by the decreases in amplitude and maximum rate of rise of slow waves, while this drug does not block the $Ca^{2+}-channel$ involved in the generation of slow waves. 3) Manganese has dual actions on the $Ca^{2+}-channels$; the $Ca^{2+}-channel$ involved in the generation of slow waves (or Na-Ca exchange system) or the channel for the generation of spike potentials are stimulated by a low concentration of $Mn^{2+}$, while both the $Ca^{2+}$. Channels are blocked by high concentration of $Mn^{2+}$.

• The Korean Journal of Physiology
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• v.21 no.2
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• pp.259-272
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• 1987

This was study carried out to investigate the effect of calcium on spontaneous contraction and electrical activity induced by ethanol in gastric smooth muscle. After peeling off the mucous membrane from the isolated whole stomach of 102 cats, two kinds of small muscle preparations $(2.0{\times}0.2\;cm)$, one longitudinal and the other circular, were excised from the fundus, the corpus and the antrum portion of each whole stomach specimen. The isometric contraction of the small muscle preparation was measured in a cylinder-shaped chamber filled with Krebs-Ringer-dextrose solution (pH 7.4, temperature $36{\pm}0.5^{\circ}C$) bubbling with 5% $CO_2$ in $O_2$. A large muscle preparation $(5.0{\times}1.2\;cm)$ was excised from the anterior wall of the corpus-antrum portion of the same specimen in 72 of 102 cats. The gastric electrical activity (slow wave and spike potential) was monopolarly recorded by four capillary electrodes (Ag-AgCl), of which two were placed on the corpus and two on the antrum, in a muscle chamber filled with the same solution as described above. Changes in the amplitude of the contraction, frequency of the gastric slow wave and the production of the spike potential were observed after adding ethanol and/or under the treatments with verapamil, $CaCl_2$ and Ca-free Krebs-Ringer-dextrose solution. The results were as follows: 1) After adding ethanol, the spontaneous phasic contraction of the corpus was reduced dose-dependently (0.125-2.0%), which was totally abolished by higher concentrations (2.0-8.0%) of ethanol. 2) The corporal phasic contraction was also completely abolished by verapamil $(3{\times}10^{-5}\;M)$ or Ca-free Krebs-Ringer-dextrose solution. The contraction was increased by $CaCl_2\;(1.8{\times}10^{-3}\;M)$, but the inhibitory effect of ethanol on the contraction persisted even under the treatment with $CaCl_2$. 3) At higher concentrations, ethanol caused tonic contraction of both preparations from the fundus, the corpus and the antrum in a dose-dependent manner. The tonic contraction of the fundus produced by ethanol was not influenced by $CaCl_2$ or verapamil, whereas the tonic contraction was not produced by ethanol in tile Ca-free solution. 4) Frequency of gastric slow wave was decreased dose-dependently by the addition of ethanol (0.25-1.0%), and tile slow wave was not produced by higher concentration of ethanol (2.0%). 5) The frequency of slow wave was significantly reduced by verapamil only and the inhibitory influence of ethanol on the slow wave frequency was reinforced by verapamil. 6) The treatment of $CaCl_2$ increased significantly the slow wave frequency, and attenuated the inhibitory effect of ethanol on the frequency. It is therefore suggested that ethanol regulates the phasic contraction and the production of slow wave by interfering with the transport of calcium in the stomach muscle of the cat.

• The Journal of Korean Medicine
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• v.19 no.2
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• pp.450-467
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• 1998

To research the characteristics of ion currents induced by inhibitory and excitatory herbs of oriental medicine, we used nystatin-perforated patch clamp technique under voltage clamp condition in periaqueductal gray neuron dissociated from Sprauge-Dawley rat, 10-15 days old. The results are as follows. 1. Ion current induced by $10mg/m{\ell}$ of Bupleuri Radix was inhibited $59.50{\pm}4.29%$ by $10^{-4}M$ bicuculline(p>0.01) but inhibition of $10.75{\pm}4.77%$ by $10^{-4}M$ tubocurarine and $4.75{\pm}4.23%$ by $10^{-4}M$ verapamil had no statistical significance(p>0.05). So ion current induced by Bupleuri Radix revealed only GABA induced $Cl^-$ current, not acetylcholine and $Ca^{2+}$ current. 2. Ion current induced by $20mg/m{\ell}$ of Coptidis Rhizoma was inhibited $47.20{\pm}7.88%$ by $10^{-4}M$ bicuculline(p<0.01) but $3.20{\pm}2.33%$ inhibition by $10^{-4}M$ tubocurarine and $1.00{\pm}1.00%$ inhibition by $10^{-4}M$ verapamil had no significance(p>0.05). So ion current induced by Coptidis Rhizoma revealed only GABA induced $Cl^-$ current, not acetylcholine and $Ca^{2+}$ current. 3. Ion current induced by $20mg/m{\ell}$ of Ecliptae Herba was inhibited $55.00{\pm}4.92%$ by $10^{-4}M$ bicuculline (p<0.01), and also inhibited $15.00{\pm}4.26%$ by $10^{-4}M$ tubocurarine(p<0.05), but inhibition of $6.00{\pm}3.03%$ by $10^{-4}M$ verapamil had no significance(p>0.05). So ion current induced by Ecliptae Herba showed GABA activated $Cl^-$ current and acetylcholine activated cation current, not $Ca^{2+}$ current 4. Ion current induced by $5mg/m{\ell}$ of Liriopis Tuber was inhibited $15.20{\pm}4.57%$ by $10^{-4}M$ bicuculline<0.05) and also inhibited $14.00{\pm}3.00%$ by $10^{-4}M$ tubocurarine(p<0.05), but inhibition of $5.20{\pm}4.80%$ by $10^{-4}M$ verapamil had no significance(p>0.05). So ion current induced by Liriopis Tuber showed GABA. activated $Cl^-$ current and acetylcholine activated cation current, not $Ca^{2+}$ current. 5. Ion current induced by $5mg/m{\ell}$ of Aconiti Tuber was inhibited $97.00{\pm}1.34%$ by $10^{-4}M$ bicuculline(p<0.01), $80.00{\pm}9.83%$ by $10^{-4}M$ tubocurarine(p<0.01), and $24.00{\pm}6.18%$ by $10^{-4}M$ verapamil(p<0.05). So ion current induced by Aconiti Tuber revealed GABA activated $Cl^-$ current and acetylcholine activated cation current and $Ca^{2+}$ current. 6. Ion current induced by $10mg/m{\ell}$ of Zingiberis Rhizoma was inhibited $33.00{\pm}7.43%$ by $10^{-4}$ bicuculline(p<0.05), $10.20{\pm}1.83%$ by $10-^{-4}M$ tubocurarine(p<0.01), and $14.00{\pm}2.16%$ by $10^{-4}M$ verapamil(p<0.01) So ion current induced by Zingiberis Rhizoma revealed GABA activated $Cl^-$ current and acetylcholine activated cation outtent and $Ca^{2+}$ current. 7. Ion current induced by $10mg/m{\ell}$ of Boshniakiae Herba was inhibited $65.00{\pm}13.75%$ by $10^{-4}M$ bicuculline(p<0.05), $38.00{\pm}9.24%$ by $10^{-4}M$ tubocurarine(p<0.05), and $33.25{\pm}7.42%$ by $10^{-4}M$ verapamiHp<0.05). So ion current induced by Bpshniakiae Herba revealed GABA activated $Cl^-$ current and acetylcholine activated cation current and $Ca^{2+}$ current. These results suggest that a point of difference between inhibitory and excitatory herbs is existence of$Ca^{2+}$ current.

• The Korean Journal of Physiology
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• v.23 no.2
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• pp.329-337
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• 1989

Cardiac muscles show stimulation frequency-dependent tension changes i.e. Bowditch phenomenon and Woodworth phenomenon, the former is an increase of tension with the increase of stimulation frequency, whereas the latter is an increase of tension with a decrease of stimulation frequency. Bowditch phenomenon is seen in the range of frequency 1.0 cps and above, and Woodworth phenomenon below the frequency 1.0 cps in the most of mammalian cardiac atrium. To throw some light on the possible mechanism of both phenomena in rat atrium, influences of drugs affecting $Ca^{2+}$ influx through the plasma membrane $(verapamil,\;La^{3+},\;norepinephrine)$ and $Ca^{2+}$ release from sarcoplasmic reticulum (SR) on frequency-tension curve were studied. The results obtained are summarized as follows: 1) At low temperature $(27.5^{\circ}C)$, both Bowditch and Woodworth phenomenon were demonstrated. But Bowditch phenomenon disappeared at the temperature above $(32.5^{\circ}C)$. 2) At $(27.5^{\circ}C)$, in the presence of verapamil, a $Ca^{2+}$ channel blocker, a time course of change in the frequency-tension was studied. It was found that Bowditch phenomenon was affected before the Woodworth phenomenon, then the former was completely disappeared. At $(32.5^{\circ}C)$, where no Bow-ditch is seen in normal atrial muscle, Bowditch phenomenon was reappeared by an administration of norepinephrine suggesting again that slow inward current of such as $Ca^{2+}$ channel is closely related to Bowditch phenomenon. 3) At $27.5^{\circ}C$, in the presence of $La^{3+}$, although tensions were decreased at all stimulation frequencies, Bowditch and Woodworth phenomenon were still demonstrated. However in the presence of both $La^{3+}$ and verapamil, Bowditch phenomena was disappeared suggesting that $La^{3+}$ is less effective in blocking $Ca^{2+}$ channel than verapamil. 4) At $27.5^{\circ}C$, in the presence of ryanodine, an inhibitor of calcium release from SR, Woodworth phenomenon was disappeared, which was consistent with previous reports of others, suggesting that $Ca^{2+}$ release from SR is closely related to Woodworth phenomenon. From the above findings, it may be concluded that Bowditch phenomenon is dependent on the magnitude of $Ca^{2+}$ influx through slow channel and Woodworth phenomenon is dependent on the amount of $Ca^{2+}$ stored in SR.