• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.54-54
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• 2003

Prostate gland contains neuroendocrine cells (PNECs) are playing important roles in physiological and pathophysiological processes of the prostate gland. Here, we investigated the role of purinoceptors in PNECs freshly isolated from rat ventral prostate (RPNECs) that show immunoreactivity to chromogranin A. Fura-2 ratiometry revealed that ATP evokes both fast Ca$\^$2+/ influx and store Ca$\^$2+/ release in RPNECs. A whole-cell patch clamp study demonstrated fast inactivating cationic current activated by ATP or by ${\alpha}$,${\beta}$-MeATP, which was blocked by ATP-TNP. The activation of P2X inward current was tightly associated with a sharp increase in [Ca$\^$2+/]$\sub$c/. The presence of P2X1/3 subtypes were proved by RT-PCR analysis. For the stored Ca$\^$2+/ release, ATP and UTP showed similar effects, suggesting the dominant role or P2Y2 subtypes, also confirmed by RT-PCR. Both P2X (${\alpha}$,${\beta}$-MeATP) and P2Y (UTP) stimulation induced changes in the cell morphology (initial shrinkage and blob formation on the surface) reversibly. Exocytotic membrane trafficking events were monitored with the membrane-bound fluorescent dye, FM1-43 using confocal microscopy. In spite of the similar Ca$\^$2+/ responses, UTP was far less effective in triggering exocytosis than ${\alpha}$,${\beta}$ -MeATP. Since serotonin is reportedly stored in the secretory granule of PNECs, we directly examined whether the aforementioned agonists elicit release of serotonin using carbon fiber electrode-amperometry. In accordance with the results of FM1 -43 experiments, ${\alpha}$,${\beta}$-MeATP efficiently evoke serotonin secretion while not with UTP. In summary, the P2X-mediated Ca$\^$2+/ influx plays crucial roles in the exocytosis of RPNECs. Although a global increase in [Ca$\^$2+]$\sub$c/ might be related with the morphological changes, a sharp rise of [Ca$\^$2+/]$\sub$c/ in the putative sub-plasmalemmal ‘microdomains’ might be a decisive factor for the exocytosis.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.2
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• pp.43-49
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• 2008

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin ($1{\mu}M$ to $100{\mu}M$) for 5 min inhibited glutamate ($100{\mu}M$, 1 min) induced $[Ca^{2+}]_i$ increase, concentration-dependently. Pretreatment with apigenin ($30{\mu}M$) for 5 min significantly decreased the $[Ca^{2+}]_i$ responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, $10{\mu}M$, 1 min) and N-methyl-D-aspartate (NMDA, $100{\mu}M$, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the $[Ca^{2+}]_i$ response induced by 50 mM KCl solution, decreased the $[Ca^{2+}]_i$ responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxy-phenylglycine (DHPG, 100 $[Ca^{2+}]_i$, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced $[Ca^{2+}]_i$ responses. Furthermore, treatment with apigenin ($30{\mu}M$) significantly inhibited the amplitude and frequency of 0.1 mM $[Mg^{2+}]_o$-induced $[Ca^{2+}]_i$ spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.29 no.1
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• pp.105-117
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• 1999

Purpose: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca/sup 2+/] can lead to DNA fragmentation by Ca/sup 2+/-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca/sup 2+/] was measured only by minutes or hours respectively. We need to evaluate [Ca/sup 2+/] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells. Materials and Methods: We have measured [Ca/sup 2+/] in human gingival epitheloid cancer cell with 10Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca/sup 2+/ indicator dye, fura-2. In order to find out that the transient rise in [Ca/sup 2+/] could induced apoptosis, cells were incubated for 1 hour at 37℃ with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells. Results: After irradiation, the transient and temporal increasing of [Ca/sup 2+/] in the KB cells was founded. Though, there was no change in the intracellular [Ca/sup 2+/] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group. Conclusion: In KB cells there were incipient and temporal increasing of the [Ca/sup 2+/] with 10Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.1
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• pp.33-39
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• 2002

It has been suggested that $Ca^{2+}$ sensitization mechanisms might contribute to myogenic tone, however, specific mechanisms have not yet been fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced $Ca^{2+}$ sensitization in myogenic tone of the rabbit basilar vessel. Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 $Ca^{2+}$ signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ only in the presence of extracellular $Ca^{2+}$. Stretch evoked greater contraction than high $K^+$ at a given $[Ca^{2+}]_i.$ The stretch-induced increase in $[Ca^{2+}]_i$ and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent $Ca^{2+}$ channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing $[Ca^{2+}]_i.$ Immunoblotting using isoform-specific antibodies showed the presence of $PKC_{\alpha}$ and $PKC_{\varepsilon}$ in the rabbit basilar artery. $PKC_{\alpha},$ but not $PKC_{\varepsilon},$ and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of $PKC_{\alpha}$ and/or RhoA may be key mechanisms for the $Ca^{2+}$ sensitization associated with myogenic tone in basilar vessels.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.27-34
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• 1997

In the present study, it was aimed to further indentify the intracellular action mechansm of cromakalim and levcromakalim in the porcine coronary artery. In intact porcine coronary arterial strips loaded with fura-2/AM, acetylcholine caused an increase in intracellular free $Ca^{2+}$ $([Ca^{2+}]_i)$ in association with a contraction in a concentration-dependent manner. Cromakalim (1 ${\mu}M$) caused a reduction in acetylcholine-induced increased $[Ca^{2+}]_i$ not only in the mormal physiological salt solution (PSS) but also in $Ca^{2+}$-free PSS (containing 1 mM EGTA). In the skinned strips prepared by exposure of tissue to 20 .${\mu}M$ B-escin, inositol 1,4,5-trisphosphate ($IP_3$) evoked an increase in $[Ca^{2+}]_i$, but it was without effect on the intact strips. The $IP_3$-induced increase in $[Ca^{2+}]_i$ was inhibited by cromakalim by 78% and levcromakalim by 59% (1 .${\mu}M$, each). Pretreatment with glibenclamide (a blocker of ATP-sensitive $K^+$ channels, 10 .${\mu}M$) and apamin (a blocker of small conductance $Ca^{2+}$-activated $K^+$ channels, 1 .${\mu}M$) strongly blocked the effect of cromakalim and levcromakalim. However, charybdotoxin (a blocker of large conductance $Ca^{2+}$-activated $K^+$ channels, 1 .${\mu}M$) was without effect. In addition, cromakalim inhibited the $GTP{\gamma}S$ (100 .${\mu}M$, non-hydrolysable analogue of GTP)-induced increase in $[Ca^{2+}]_i$. Based on these results, it is suggested that cromakalim and levcromakalim exert a potent vasorelaxation, in part, by acting on the $K^+$ channels of the intracellular sites (e.g., sarcoplasmic reticulum membrane), thereby, resulting in decrease in release of $Ca^{2+}$ from the intracellular storage site.

• The Korean Journal of Pharmacology
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• v.29 no.2
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• pp.165-174
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• 1993

$Ca^{2+}$ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these $Ca^{2+}-dependent$ effects is unclear. Since cytoskeletal proteins are rapidly degraded by $Ca^{2+}-dependent $ proteinases (calpains) in vitro and in vivo, we investigated whether $Ca^{2+}-induced$ pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors EST (etyl (+)-(2S,3S)-3-[(S)-methyl-1-(3-methlbutylcarbamoyl)-butylcarbamoyl]2-oxiranecarboxylate) and MDL28170 (carbobenzoxyl-Val-Phe-H) to block the $Ca^{2+}$ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a signiciant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST $(5\;or\;20{\mu}M)$ to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 $(20\;{\mu}M)$ showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 nM and 300 nM concentrations, respectively, of A23187. EST $(20\;{\mu}M)$ did not block the A23187-induced rise of $[Ca^{2+}]_{i}$ as measured with fura-2. These results show that $Ca^{2+}-induced$ pruning of neurites in isolated hippocampal pyramidal neurons is mediated by calpains.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.2
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• pp.173-182
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• 2018

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by $d(CH_2)_5[Tyr(Me)^2,Dab^5]$ AVP, a vasopressin-1a (V1a) receptor antagonist, but not by $desGly-NH_2-d(CH_2)_5[D-Tyr^2,Thr^4]OVT$, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.303-310
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• 1997

소 난포란의 체외성숙시 성숙배지에 FSH 및 LH의 첨가가 체외성숙난자의 calcium 반응과 체외수정란의 발달에 미치는 영향을 조사하였다. 난포란의 체외성숙은 TCM199을 기초로 한 4가지의 배양조건 하에서 : 1) 0.5$\mu\textrm{g}$/ml FSH＋5$\mu\textrm{g}$/ml LH, 2) 0.5$\mu\textrm{g}$/ml FSH, 3) 5$\mu\textrm{g}$/ml LH 및 4) 무 호르몬 첨가구로서 5% CO2에 24시간 동안 체외성숙을 유도하였다. 체외성숙 24기간째에 난포란의 과립막세포는 1ml PB1＋에서 4분 동안 vortexing을 하여 완전히 제거하였다. 세포 내 calcium 반응을 측정하기 위하여 2mM Fura-2 AM ester 및 0.02% Pluronic F-127가 첨가된 PB1-용액에 39$^{\circ}C$ in cubator에서 40분 동안 배양하였다. 30${\mu}\ell$ M2 medium drop을 30mm plastic dish에 만들어 20$\times$ 형광대물렌즈가 장착된 Nikon Diaphot 현미경의 장착된 Nikon Diaphot 현미경의 warm stage에 설치하였다. 세포 내 calcium 방출을 자극하기 위하여 난자에 25mM inositol 1, 4, 5-trasphophate(IP3)로 1.21kV/cm의 전기자극 또는 20mM ryanodine으로 미세주입을 실시하였다. 이러한 처리를 하지 않은 난자는 체외수정 후 CR1aa와 BRL monolayers의 공배양조건 하에서 체외발달을 유도하였다. 분할율(Day 2)과 배반포기발달율(Day 9)을 조사하였다. FSH와 LH의 처리구에서 IP3 또는 ryanodine으로 자극된 난자(1.79$\pm$0.05, 1.66$\pm$0.06)는 FSH, LH 및 무 호르몬처리구에 비하여 유의적으로 높은 calcium 반응을 보였다(1.00$\pm$0.03, 1.28$\pm$0.04, and 0.53$\pm$0.02 in IP3 elctroporation; 0.68$\pm$0.05, 1.03$\pm$0.05, and 0.47$\pm$0.04 in ryanodine microinjection). FSH와 LH, FSH, LH처리구에서 분할율(87.9, 71.5 및 75.6%)은 무 호르몬처리구(60.7%)(P<0.05)에 비하여 유의적으로 높았으며, FSH와 LH처리구(29.3%)에서의 배반포기 발달율은 FSH, LH 처리구뿐만 아니라 무 호르몬처리구보다 유의적으로 높았다(16.5, 19.0 and 9.8%)(P<0.05). Bovine FSH 및 Ovine FSH의 처리구에서의 calcium 반응은 유의적인 차이가 없었다(1.72$\pm$0.05, 1.61$\pm$0.06). 또한 분할율(82.2 and 84.0%) 및 배반포기(27.8 and 27.1%) 발달율도 bovine 및 ovine FSH처리구간에는 유의적인 차이가 없었다. 이상의 결과에서 전기자극에 의한 세포 내 calcium 반응은 체외성숙배지에 첨가하는 호르몬의 처리에 따라서 유의적인 변화를 보였다. 비록 분할율은 처리구간에 유의적인 차이가 없었지만 배반포기 발달율은 FSH와 LH 공동처리구에서 FSH, LH 단독처리구 및 무 호르몬처리구에 비하여 유의적으로 높은 발달율을 보였다. 체외성숙기간에 FSH와 LH의 공동첨가는 체외성숙 및 체외발달의 생리적인 교정을 위하여 요구되는 것으로 사려된다.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.11-16
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• 1988

Various Benzylacyclouridine (BAU, HM-BAU, suc-BAU, BBAU, HMBBAU, suc-BBAU, and BBBAU) developed as specifc inhibitors of uridine phosphorylase (UrdPase), inhibit transport (zero-trans influx) of ridine (Urd) in human erythrocytes. The inhibition pattern of these compounds is competitive, though suc-BBAU and BBBAU show a slight noncompetivieness. The order of potency as an inhibitor of the nucleoside transport system is BBAU ${\sim}$ HM-BBAU ${\sim}$ suc-BBAU > BBBAU > BAU ${\sim}$ suc-BAU ${\sim}$ HM-BAU ($K_1$ values of 19, 23, 38, 112, 124, 174 and 176 ${\mu}M$, respectively). These data indicate that there is a differnece in potency of Urd transport inhibition between the analogs of BAU and BBAU. Further, the potency correlates with the hydrophobicity of the compound, but it has a limit in the size of $C_5$ substitution. Abbreviation: BAU (5-benzylacyclouridine), 5-benzyl-1-(2'-hydroxyethoxymethyl) uracil; HM-BAU (3'-hydroxymethyl-BAU), 5-benzyl-1- [(1',3'-dihydroxy-2-propoxy) methyl]uracil; suc-BAU, 3'-succinyl-BAU; BBAU (benzyloxybenzylacyclouridine), 5-( m-benzyloxybenByl)-1-(2'-hydroxyethoxymethyl)uracil; HM-BBAU (3'-hydroxymethyl-BBAU), 5-(m-benzyloxybenzyl)-1- [(1'3'-dihyd.oxy-2-p.epoxy)methyll u.acil; suc-BAU, 3'-succinyl-BBAU; BBBAU, 5- f 3-(4-benzyloxyben-Eyl)benzyll -1-(2'-hydroxyethoxymethyl)uracil; Urdpase, uridine phosphorylase; Urd, Uridine; Fd-Urd, 5-fluoro-2'-deoxyuridine; AcThd, aoyclothyrnidine; AcUrd, acyclouridine; dThd, thymidine; NBMPR, nitrobenzylthioisone; FUra, 5-fluorouracil.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.