• Journal of Chest Surgery
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• v.22 no.6
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• pp.901-913
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• 1989

This study was investigated under the postulation that activation of intracellular calcium- calmodulin complex during ischemia-reperfusion leads to myocardial injury. The protective effects of calcium channel blocker, diltiazem and calmodulin inhibitors, trifluoperazine, flunarizine and calmidazolium from ischemic injury in rat hearts were observed by using Langendorff apparatus when the antagonists were infused for 3 min in the beginning of ischemia. Thereby, an increase in resting tension developed during 30-min ischemia was analyzed with regard to [1] the degree of cardiac functional recovery following 60-min reperfusion, [2] changes in biochemical variables evoked during 30-min ischemia. The results obtained were as follows: l. In the ischemic group, the resting tension was increased by 4.1*0.2 g at 30-min ischemia. However, the increase in resting tension was markedly reduced not only by pretreatment with diltiazem [3.3 p M] but also with calmodulin inhibitors, trifluoperazine [3.3 p M], flunarizine [0.5 p M] and calmidazolium [0.5 p M], respectively. 2. Recovery of myocardial contractility, +dF /dt and coronary flow were much reduced when evoked by reperfusion in the ischemic group. These variables were significantly improved either by pretreatment with diltiazem or with calmodulin inhibitors. 3. The resting tension increment evoked during ischemia was significantly inversely correlated with the degree of cardiac function recovered during reperfusion. 4. Following 30-min ischemia, the production of malondialdehyde and release of lysosomal enzyme were much increased in association with a decrease in creatine kinase activity. 5. The increases in malondialdehyde production and release of free lysosomal enzyme were suppressed by pretreatment with calmodulin inhibitors as well as diltiazem. Likewise, the decrease of creatine kinase activities was prevented by these calcium antagonists. With these results, it is indicated that a increase in resting tension observed during ischemia has an inverse relationship to the cardiac function recovered following reperfusion, and further, the later may be significantly dependent on the degree of biochemical alterations occurred during ischemia such as decrease in creatine kinase activity, increased production of malondialdehyde and increased release of free lysosomal enzyme. Thus it is concluded that calmodulin plays a pivotal role in the process of ischemic injury.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.1
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• pp.109-117
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• 1998

Role of $Ca^{2+}$/calmodulin complex in intracellular $Ca^{2+}$ mobilization in neutrophils has not been clearly elucidated. In this study, effects of chlorpromazine, trifluoperazine and imipramine on the intracellular $Ca^{2+}$ mobilization, including $Ca^{2+}$ influx, in C5a-activated neutrophils were investigated. Complement C5a- stimulated superoxide production and myeloperoxidase release in neutrophils were inhibited by chlorpromazine, trifluoperazine and imipramine, except no effect of imipramine on myeloperoxidase release. A C5a-elicited elevation of [$Ca^{2+}$]i in neutrophils was inhibited by chlopromazine, trifluoperazine, imipramine, staurosporine, genistein, EGTA, and verapamil but not affected by pertussis toxin. The intracellular $Ca^{2+}$ release in C5a-activated neutrophils was not affected by chlorpromazine and imipramine. Chlorpromazine and imipramine inhibited $Mn^{2+}$ influx by C5a-activated neutrophils. Thapsigargin-evoked $Ca^{2+}$ entry was inhibited by chlorpromazine, trifluoperazine, imipramine, genistein, EGTA and verapamil, while the effect of staurosporine was not detected. The results suggest that $Ca^{2+}$/calmodulin complex is involved in the activation process of neutrophils. The depressive action of calmodulin inhibitors on the elevation of cytosolic $Ca^{2+}$ level in C5a-activated neutrophils appears to be accomplished by inhibition of $Ca^{2+}$ influx from the extracellular medium.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.4
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• pp.187-191
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• 2002

Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular $Ca^{2+}$ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular $Ca^{2+}$ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a $Ca^{2+}$ activated protein, and $Ca^{2+}$/calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of $Ca^{2+}$/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular $Ca^{2+}$ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and $Ca^{2+}$/calmodulin-dependent protein kinase II in HepG2 cells.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.159-163
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• 2005

The effects of sedative peptide alkaloids from Zizyphus species on calmodulin- dependent protein kinase II were investigated. Protein kinase II activity was assayed on the basis of its ability to activate tryptophan 5-monooxygenase as its substrate in the presence of calmodulin. All thirteen alkaloids tested were stronger inhibitors than chlorpromazine ($IC_50,\;98{\mu}M$) on calmodulin-dependent protein kinase II. Among them, the most potent inhibitor was daechuine S27 ($IC_{50},\;2.95{\mu}M$), which was stronger than pimozide ($IC_{50},\;15.0{\mu}M$).

• YAKHAK HOEJI
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• v.43 no.5
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• pp.659-664
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• 1999

To investigate the difference of contractile mechanism between KCI and phenylephrine-induced contraction, we observed effects of $Ca^{2+}$ antagonists and protein kinase inhibitors on aorta contraction of rats. Verapamil dose-dependently inhibited the contraction induced by KCI and phenylephrine, the inhibitory effect of verapamil was more potent in KCI-induced contraction than phenylephrine-induced contraction. Econazole and TMB-8 significantly inhibited CKI-induced contraction but did not inhibit phenylephrine-induced contraction. Staurosporine dose-dependently inhibited both KCI and phenylephrine-induced contraction. Genistein and calmodulin antagonists (W-7 and trifluoperazine) also inhibited both contraction in a dose dependent manner. However, the inhibitory effects of genistein and calmodulin antagonists were more potent in phenylephrine-induced contraction than KCI-induced contraction. These results suggest that involvements of $Ca^{2+}$ channel and protein kinase in rat aorta contraction were dependent on agonist causing aorta smooth muscle contraction.

• BMB Reports
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• v.34 no.3
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• pp.212-218
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• 2001

Although the blockage of a cell cycle by specific inhibitors of $Ca^{2+}/$calmodulin-dependent protein kinase II (CaMK-II) is well known, the activity profile of CaMK-II during the cell cycle in the absence of any direct effectors of the enzyme is unclear. The activity of native CaMK-II in NIH 3T3 cells was examined by the use of cell cycle-specific arresting and synchronizing methods. The total catalytic activity of CaMK-II in arrested cells was decreased about 30% in the M phase, whereas the $Ca^{2+}$-independent autonomous activity increased about 1.5-fold in the M phase and decreased about 50% at the G1/S transition. The in vivo phosphorylation level of CaMK-II was lowest at G1/S and highest in M. The CaMK-II protein level was unchanged during the cell cycle. When the cells were synchronized, the autonomous activity was increased only in M. These results indicate that the physiologically relevant portion of CaMK-II is activated only in M, and that the net activation of CaMK-II is required in mitosis.

• The Korean Journal of Zoology
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• v.37 no.1
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• pp.130-136
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• 1994

Although alteration in protein phosphorylation by specific protein kinases is of importance in transducing cellular signals in a variety of neural/endocrine systems, little is known about protein phosphorylation in the hvpothalamus. The present study aims to explore whether activation of the second messenger-dependent protein kinases affects phosphorylation of specific proteins using a cell free phosphorylation system followed by SDS-polvacrylamide gel electrophoresis. Cytoplasmic fractions derived from hvpothalami of immature rats were used as substrates and several activators and/or inhibitors of CAMP-, phosphatidylinositol- and Ca2+-calmodulin-dependent protein kinases were assessed. Many endogenous proteins were extensively phosphorylated and depending on the signal transduction pathways, phosphorvlation profiles were markedly different. The present data indicate that extracellular signals may affect cellular events through protein phosphorylation by second messengers-protein kinases in the rat hypothalamus.

• Korean Journal of Veterinary Research
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• v.43 no.4
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• pp.615-624
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• 2003

Vascular smooth muscle relaxation is modulated by an increase in cGMP subsequent to nitric oxide (NO) production by endothelial cells. The effects of cAMP and cGMP phosphodiesterase (PDE) inhibitors were investigated in phenylephrine-precontracted rat aorta rings by using the specific inhibitors of PDE I, III, IV and V as relaxing agents (calmodulin-activated PDE inhibitors, IBMX and $W_7$, type I; cAMP-specific PDE inhibitors, milrinone, type IV; Ro 20-1724, type III and cGMP-specific PDE inhibitor, zaprinast, type V). All the PDE inhibitors produced a concentration-dependent relaxation in the ring with intact endothelium (+E). Except for milrinone, all the PDE inhibitors-induced relaxations were inhibited by removal of extracellular $Ca^{2+}$, $N^G$-nitro-L-arginine, $N^G$-nitro-L-arginine methyl ester, methylene blue (MS) or nifedipine. The specific PDE I and PDE IV inhibitors both produced endothelium-independent relaxations which were inhibited by MS in -E rings. However, zaprinast had no effect in -E rings. Except for milrinone, sodium nitroprusside (a NO donor)-induced relaxation was significantly augmented by all PDE inhibitors in +E rings. The results suggest that I) the vasorelaxant properties of IBMX, $W_7$, Ro 20-1724 and zaprinast are dependent on endothelium or on interaction with $Ca^{2+}$ regulation, 2) each PDE is differently distributed in vascular tissues (endothelial and smooth muscle cells), 3) the vasodilations of PDE inhibitors are due to the increase of cAMP and cGMP formation through inhibition of cAMP- and cGMP-PDE and 4) the vasodilation action of milrinone does not involve in endothelial-cyclic nucleotide system.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.456-465
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• 2004

$K^+$ efflux through a certain type of $K^+$ channels causes the change of membrane potential and leads to cAMP synthesis in the transmembrane cell signaling for the initiation of sperm motility in the salmonid fishes. The addition of $Ca^{2+}$ conferred motility to the trout sperm that were immobilized by external $K^+$ and other alkaline metals, $Rb^+$ and $Cs^{2+}$, suggesting the participation of external $Ca^{2+}$ in the initiation of sperm motility. L-type $Ca^{2+}$ channel blockers such as nifedipine, nimodipine, and FS-2 inhibited the motility, but N-type $Ca^{2+}$ channel blocker, w-conotoxin MvIIA, did not. On the other hand, the membrane hyperpolarization and cAMP synthesis were suppressed by $Ca^{2+}$ channel blockers, nifedipine, and trifluoroperazine. Furthermore, these suppressions were relieved by the addition of $K^+$ ionophore, valinomycin. Inhibitors of calmodulin, such as W-7, trifluoperazine, and calrnidazol-C1, inhibited the sperm motility, membrane hyperpolarization, and cAMP synthesis. The results suggest that $Ca^{2+}$ influx through $Ca^{2+}$ channels that are sensitive to specific $Ca^{2+}$ channel blockers and calmodulin participate in the changes of membrane potential, leading to synthesis of cAMP in the cell signaling for the initiation of trout sperm motility.

• Molecules and Cells
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• v.28 no.3
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• pp.167-174
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• 2009

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energygenerating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or $Ca^{2+}$ channel opener Bay K8644. Genistein at a concentration of $50{\mu}M$ showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of $Ca^{2+}$/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of $[Ca^{2+}]_i$ and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.