• 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.

• Archives of Pharmacal Research
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• v.28 no.1
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• pp.73-80
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• 2005

The effect of capsaicin on apoptotic cell death was investigated in HepG2 human hepatoma cells. Capsaicin induced apoptosis in time- and dose-dependent manners. Capsaicin induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration, and BAPTA, an intracellular $Ca^{2+}$ chelator, significantly inhibited capsaicin-induced apoptosis. The capsaicin-induced increase in the intracellular $Ca^{2+}$ and apoptosis were not significantly affected by the extracellular $Ca^{2+}$ chelation with EGTA, whereas blockers of intracellular $Ca^{2+}$ release (dantrolene) and phospholipase C inhibitors, U-73122 and manoalide, profoundly reduced the capsaicin effects. Interestingly, treatment with the vanilloid receptor antagonist, capsazepine, did not inhibit either the increased capsaicin-induced $Ca^{2+}$ or apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the HepG2 cells may result from the activation of a PLC-dependent intracellular $Ca^{2+}$ release pathway, and it is further suggested that capsaicin may be valuable for the therapeutic intervention of human hepatomas.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.25-30
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• 2006

Lysophosphatidylcholine (LPC), which accumulates in atherosclerotic arteries, has been reported to inhibit endothelium-dependent relaxation (EDR) in many different species. However, the underlying mechanism of LPC-induced inhibition of EDR is still uncertain. In the present study, we measured simultaneously both isometric tension and cytosolic free $Ca^{2+}$ ($[Ca^{2+}]_i$) in rabbit carotid strips, and examined the effect of LPC on tension and $[Ca^{2+}]_i$. In carotid strips with intact-endothelium, high $K^+$ (70 mM) increased both tension and $[Ca^{2+}]_i$, and cumulative addition of acetylcholine (ACh) from 0.1 to $10{\mu}M$ induced dose dependent increase of $[Ca^{2+}]_i$ with concomitant relaxation. In the presence of L-NAME (0.1 mM), ACh increased $[Ca^{2+}]_i$ without affecting the amplitude of high $K^+-induced$ tension. These ACh-induced change of $[Ca^{2+}]_i$ and tension was abolished by removal of endothelium or 10 nM 4-DAMP (muscarinic receptor antagonist) pretreatment. Pretreatment of LPC ($10{\mu}M$) inhibited ACh ($10{\mu}M$)-induced change of tension and $[Ca^{2+}]_i$ in endothelium-intact carotid artery. On the other hand, LPC had no effect on ACh-induced change of tension and $[Ca^{2+}]_i$ in endothelium denuded artery. In $Ca^{2+}$-free external solution, ACh transiently increased $[Ca^{2+}]_i$, and pretreatment of LPC significantly inhibited ACh-induced transient $[Ca^{2+}]_i$ change. Based on the above results, it may be concluded that LPC inhibits the ACh-induced $[Ca^{2+}]_i$ change through inhibition of $Ca^{2+}$ mobilization in vascular endothelial cells, resulting in decreased production of NO and concomitant inhibition of endotheliumdependent vascular relaxation.

• The Journal of Natural Sciences
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• v.9 no.1
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• pp.1-7
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• 1997

Calcium/calmodulin-dependent protein kinase II (CaMK-II) is responsible for the phosphorylation of proteins involved in various cellular functions. Since the level of intracellular calcium ($Ca_2+$) oscillate during the cell cycle, it is expected that the activity of CaMK-II is also dependent on the cell cycle. The kinase activity in NIH3T3 cells which were arrested at or released from certain phase of the cell cycle was measured and compared to that in the normally growing asynchronous control cells to investigate whether the activity of this kinase is cell cycle-dependent. Cells were arrested at G0, G1, G1/S, G2/M and M phase, respectively by use of various drugs which do not have any effect on the kinase activity of CaMK-II at G0, G1, G1/s and G2/M phase was similar to that of the control cells, whereas lower at M. Calcium-independent activity of CaMK_II by autophosphorylation was higher at M and, thus, higher autonomy at M, which represented the physiologically relevant activity of CaMK-II. A similar pattern of activity change of the kinase was demonstrated during the cell cycle of synchronized cells which were released from G1 arrest. These results indicate that the activity of CaMK-11 is cell cycle-dependent and is activity during the mitosis.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.5
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• pp.449-457
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• 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though $Ca^{2+}$ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ($[Ca^{2+}]_i$) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on $[Ca^{2+}]_i$ in human neutrophils. We observed that NAC ($1{\mu}M{\sim}1mM$) and cysteine ($10{\mu}M{\sim}1mM$) increased $[Ca^{2+}]_i$ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in $[Ca^{2+}]_i$ in human neutrophils was observed. In $Ca^{2+}$-free buffer, NAC- and cysteine-induced $[Ca^{2+}]_i$ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in $[Ca^{2+}]_i$ in human neutrophils occur through $Ca^{2+}$ influx. NAC- and cysteine-induced $[Ca^{2+}]_i$ increase was effectively inhibited by calcium channel inhibitors SKF96365 ($10{\mu}m$) and ruthenium red ($20{\mu}m$). In $Na^+$-free HEPES, both NAC and cysteine induced a marked increase in $[Ca^{2+}]_i$ in human neutrophils, arguing against the possibility that $Na^+$-dependent intracellular uptake of NAC and cysteine is necessary for their $[Ca^{2+}]_i$ increasing activity. Our results show that NAC and cysteine induce $[Ca^{2+}]_i$ increase through $Ca^{2+}$ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

• Archives of Pharmacal Research
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• v.26 no.1
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• pp.64-69
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• 2003

To analyze vasopressin-induced $Ca^{2+}$ increase in liver cells, rat hepatocytes were isolated and attached to collagen-coated cover slips. Using fura-2, a $Ca^{2+}$-sensing dye, changes in intracellular $Ca^{2+}$ concentration by vasopressin were monitored. Results in this communication suggested that vasopressin-induced $Ca^{2+}$ increase were composed of both $Ca^{2+}$ release from internal $Ca^{2+}$ stores and influx from the plasma membrane. The $Ca^{2+}$ influx consisted of two distinguishable components. One was dependent on the presence of vasopressin and the other was not. SK＆F96365 blocked vasopressin-induced $Ca^{2+}$ influx in a dose-dependent manner. Vasopressin-induced $Ca^{2+}$ release from internal stores diminished in a primary culture of hepatocytes according to the culture time. However, changes in vasopressin-induced $Ca^{2+}$ influx across the plasma membrane differed from changes in the $Ca^{2+}$ release from internal stores, suggesting two separate signalings from receptor activation to internal stores and to the plasma membrane.

• The Korean Journal of Physiology
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• v.24 no.1
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• pp.91-102
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• 1990

The effects of extracellular $Ca^{2+}$ and various $Ca^{2+}$ antagonists on endothelium-dependent relaxation to acetylcholine were studied in the isolated rabbit thoracic aorta in order to elucidate the control mechanism of endothelium derived relaxing factor (EDRF) release. Endothelium was removed from aortic strips by gentle rubbing with cotton ball. The effect of hemoglobin on basal tension was also observed with hemolysate. The results obtained were as follows: 1) Endothelium-dependent relaxation (EDR) to acetylcholine (ACh) showed biphasic pattern; the initial rapid relaxation phase and the late slow relaxation phase. 2) With the depletion of the extracellular $Ca^{2+}$, EDR was gradually suppressed, especially the late slow relaxation. 3) Verapamil, nifedipine, $Mn^{2+}$ and $Cd^{2+}$ had not any effect on EDR, while $La^{3+}$ and $Co^{2+}$ suppressed EDR completely. 4) The resting tension of the strips with rubbed endothelium was not altered by the addition of hemoglobin. That of the strips with intact endothelium, however, was enhanced and EDR to ACh was completely blocked From these results, we suggest that extracellular $Ca^{2+}$ is necessary for ACh-induced slow relaxation while $Ca^{2+}$ antagonists have not any effect on EDR.

• The Korean Journal of Physiology
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• v.28 no.2
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• pp.159-167
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• 1994

The contraction of rabbit basilar artery was examined as a function of changes in the $Na^+$ electrochemical gradient in order to determine the contribution of $Na^+/Ca^{2+}$ exchange to the modulation of contractility. Ouabain $(10^{-5}\;M)$ or $K^+-free$ Tyrode solution caused an increase in tonic tension even in the presence of a $Ca^{2+}$ channel blocker $(10^{-6}\;M\;verapamil)$ and an ${\alpha}-receptor$ blocker $(10^{-5}\;M\;phentolamine)$. After treatment with ouabain $(10^{-5}\;M)$, contractions were augmented by reduction of external $Na^+$ concentration. The longer the treatment with ouabain $(10^{-5}\;M)$ was, the larger the amplitude of $Na^+-free$ contracture was. $Na^+-free$ contracture wag induced by either substitution of equimolar Tris for $Na^+$ or substitution of equimolar $Li^+\;for\;Na^+$. The competition between $Na^+\;and\;Ca^{2+}$ for the $Na^+/Ca^{2+}$ exchange carrier would exist, because it was observed that contractility was dependent on the $Na^+$ electrochemical gradient or the extracellular $Ca^{2+}$ concentration (2 mM, 4 mM). Ryanodine $(10^{-7}\;M)$, the blocker of intracellular $Ca^{2+}$ release from the sarcoplasmic reticulum, did not suppress the development of $Na^+-free$ contracture. The contractile response to norepinephrine $(10^{-6}\;M)$ was augmented by reducing the extracellular $Na^+$ concentration. The relaxation rate from caffeine-induced contraction was dependent on the extracellular $Na^+$ concentration (0 mM, 140 mM). From the above results, it could be suggested that $Na^+/Ca^{2+}$ exchange can move $Ca^{2+}$ either into or out of rabbit basilar arterial smooth muscle. $Ca^{2+}$ entry or extrusion is dependent upon the $Na^+$ electrochemical gradient. $Na^+/Ca^{2+}$ exchange plays a significant role in the regulation of contractility in rabbit basilar arterial smooth muscle.

• Archives of Pharmacal Research
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• v.21 no.6
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• pp.664-670
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• 1998

The role of intracellular $Ca^{2+}$, in the regulation of tumor cell proliferation by products of arachidonic acid (AA) metabolism was investigated using U-373 MG human as trocytoma cells. Treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase (LOX) inhibitor, or caffeic acid (CA), a specific 5-LOX inhibitor, suppressed proliferation of the tumor cells in a dose-dependent manner. However, indomethacin (indo), a cyclooxygenase (COX) inhibitor, did not significantly alter proliferation of the tumor cells. At anti-proliferative concentrations, NDGA and CA significantly inhibited intracellular $Ca^{2+}$ release induced by carbachol, a known intracelluar $Ca^{2+}$ agonist in the tumor cells. Exogenous administration of leukotriene $B_4(LTB_4)$, an AA metabolite of LOX pathway, enhanced proliferation of the tumor cells in a concentration-dependent fashion. In addition, $LTB_4$, induced intracelluar $Ca^{2+}$ release. Intracellular $Ca^{2+}$-inhibitors, such as an intracellular $Ca^{2+}$ chelator (BAPTA) and intracellular $Ca^{2+}$-release inhibitors (dantrolene and TMB-8), significantly blocked the LTB4-induced enhancement of cell proliferation and intracellular $Ca^{2+}$ release. These results suggest that LOX activity may be critical for cell proliferation of the human astrocytoma cells and that intracelluar $Ca^{2+}$ may play a major role in the mechanism of action of LOX.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.6
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• pp.715-724
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• 1998

Frequency-force relationships (FFR) were studied in electrically field stimulated rat left atria (LA) by reducing the stimulation frequency from resting 3 Hz to test frequencies (0.1-1 Hz) for 5 minutes. The twitch amplitudes of LA elicited the typical negative staircases with 3-phased changes: the initial rapid increase, the second decrease and the following plateau at test frequencies. Verapamil $(3{\times}10^{-5}\;M)$ pretreatment elicited frequency-dependent suppression of the twitch amplitudes, exaggerating the negative staircase. Monensin pretreatment enhanced not the peak but the plateau amplitudes in a concentration-dependent manner. When the $Na^+-Ca^{2+}$ exchange was blocked by $Na^+\;and\;Ca^{2+}$ depletion in the Krebs Hensleit buffer (0 $Na^+-0\;Ca^{2+}$ KHB), the twitch amplitudes increased in a frequency-dependent manner, changing the negtive staircase into the positve one. Meanwhile, the 0 $Na^+-0\;Ca^{2+}$ KHB applicationinduced enhancement was strongly suppressed by caffeine (5 mM) pretreatment. Only dibucaine among the local anesthetics increased the basal tone during frequency reduciton. There were no differences in $^{45}Ca$ uptakes between 0.3 Hz and 3 Hz stimulation except at 1 min when it was significantly low at 0.3 Hz than 3 Hz, illustrating net $Ca^{2+}$ losses. Monensin pretreatment enhanced the rate of this $Ca^{2+}$ loss. Taken together, it is concluded that $Na^+-Ca^{2+}$ exchange extrudes more SR released $Ca^{2+}$ out of the cell in proportion to the frequency, resulting in the negative rate staircase in the rat LA.