• The Korean Journal of Physiology and Pharmacology
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• 제14권1호
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• pp.37-43
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• 2010

The serine/threonine kinase Akt has been shown to play a role of multiple cellular signaling pathways and act as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI3K). It has been reported that phosphorylated Akt activates eNDS resulting in the production of NO and that NO stimulates soluble guanylate cyclase (sGC), which results in accumulation of cGMP and subsequent activation of the protein kinase G (PKG). It has been also reported that PKG activates PI3K/Akt signaling. Therefore, it is possible that PI3K, Akt, eNOS, sGC, and PKG form a loop to exert enhanced and sustained activation of Akt. However, the existence of this loop in eNOS-expressing cells, such as endothelial cells or astrocytes, has not been reported. Thus, we examined a possibility that Akt phosphorylation might be enhanced via eNOS/sGC/PKG/PI3K pathway in astrocytes in vivo and in vitro. Phosphorylation of Akt was detected in astrocytes after KA treatment and was maintained up to 72 h in mouse hippocampus. 2 weeks after KA treatment, astrocytic Akt phosphorylation was normalized to control. The inhibition of eNOS, sGC, and PKG significantly decreased Akt and eNDS phosphorylation induced by KA in astrocytes. In contrast, the decreased phosphorylation of Akt and eNDS by eNDS inhibition was significantly reversed with PKG activation. The above findings in mouse hippocampus were also observed in primary astrocytes. These data suggest that Akt/eNOS/sGC/PKG/PI3K pathway may constitute a loop, resulting in enhanced and sustained Akt activation in astrocytes.

• 동의생리병리학회지
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• 제27권4호
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• pp.400-408
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• 2013

This study aimed to investigate the relaxation effects and its underlying mechanisms of Rubus coreanus(RC) extract in contracted rabbit corpus cavernous tissues by phenylephrine(PE) $1{\mu}M$. In order to define the relaxation effects of RC, rabbit corpus cavernous tissues were prepared in $2{\times}2{\times}6mm$ sized strip. The dose-dependent relaxation responses of RC at 0.01-3.0 $mg/m{\ell}$ in contracted strips induced by PE were measured and also observed after endothelial denudation. To analyze the underlying mechanisms of RC-induced relaxation, indomethacin(IM), tetraethylammonium chloride(TEA), $N{\omega}$-nitro-L-arginine (L-NNA), methylene blue(MB) were treated before RC extract infused into precontracted strips induced by PE. To study the effect of RC extract on influx of extracellular $Ca^{2+}$ in corpus cavernous strips, calcium chloride(Ca) 1 mM infused into precontracted strips induced by PE after pretreatment of RC extract in $Ca^{2+}$-free krebs-ringer solution. To investigate cytotoxic activity and nitric oxide(NO) concentration of RC extract on human umbilical vein endothelial cell(HUVEC), cell viability on HUVEC was measured by MTT assay, and NO concentration was measured by Griess reagent system. The cavernous strips were significantly relaxed by RC extract at 1.0 $mg/m{\ell}$, 3.0 $mg/m{\ell}$ and the relaxation responses to RC were inhibited significantly by endothelial disruption. The pretreatment of IM, TEA didn't affect RC extract-induced endothelium-dependent relaxation, but the pretreatment of L-NNA, MB reduced RC extract-induced endothelium-dependent relaxation. When $Ca^{2+}$ was supplied the cavernous strips which were precontracted by PE in a $Ca^{2+}$-free krebs-ringer solution, contraction of strips was increased, but pretreatment of RC inhibited contractile response to $Ca^{2+}$. When RC extract was applicated on HUVEC, NO concentration was increased. Our findings show that RC extract exerts a relaxing effect on corpus cavernosum in part by suppressing influx of extracellular $Ca^{2+}$ through activating the NO-cGMP system.

• The Korean Journal of Physiology and Pharmacology
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• 제20권3호
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• pp.315-324
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• 2016

Human cardiac fibroblasts (HCFs) have various voltage-dependent $K^+$ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide ($H_2O_2$) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether $H_2O_2$ could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of $H_2O_2$ stimulated $Ca^{2+}-activated$ $K^+$ ($K_{Ca}$) currents but not delayed rectifier $K^+$ or transient outward $K^+$ currents, all of which are VDKCs. $H_2O_2-stimulated$ $K_{Ca}$ currents were blocked by iberiotoxin (IbTX, a large conductance $K_{Ca}$ blocker). The $H_2O_2-stimulating$ effect on large-conductance $K_{Ca}$ ($BK_{Ca}$) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated $BK_{Ca}$ currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the $H_2O_2-stimulating$ effect on $BK_{Ca}$ currents. Using RT-PCR and western blot analysis, three subtypes of $K_{Ca}$ channels were detected in HCFs: $BK_{Ca}$ channels, small-conductance $K_{Ca}$ ($SK_{Ca}$) channels, and intermediate-conductance $K_{Ca}$ ($IK_{Ca}$) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to $H_2O_2$, but IbTX decreased $H_2O_2$-induced apoptosis. These data suggest that among the VDKCs of HCFs, $H_2O_2$ only enhances $BK_{Ca}$ currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through $BK_{Ca}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• 제18권1호
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• pp.25-32
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• 2014

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

• 한국수산과학회지
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• 제26권6호
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• pp.549-556
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• 1993

코끼리조개를 fast foods의 원료로 이용하기 위한 기초자료를 얻기 위하여 물리적 성상, 유리 및 구성 아미노산의 함량, 핵산관련물질, 월별 성분변화 및 냉동기간중의 선도변화를 측정 하였다. 3년생과 5년생의 경우 내장을 제거한 가식부의 수율은 각각 29.92, $38.04\%$이었다. 7월에 채취한 코끼리조개의 유리아미노산중 taurine함량이 가장 높았으며 Gly, Ala 및 Glu 함량도 비교적 높게 나타났다. 구성아미노산에 있어서는 Glu 함량이 가장 높았고 그 다음으로는 Ala이었다. 필수아미노산으로는 Leu과 Lys의 함량이 비교적 높았다. 월별 성분변화에 있어서 수분, 단백질은 3월, 4월에 가장 높았으며 지방과 글리코겐은 7월에 가장 높았다. 핵산관련물질중 5'-GMP의 량은 4월에 채취된 시료보다 7월에 채취된 것에서 더 높게 나타났다. 냉동저장중 VBN, TMA, $NH_2-N$의 함량변화에 있어서는 $95^{\circ}$에서 2분간 열처리한 시료는 열처리하지 않은 시료에 비하여 증가율이 낮았기 때문에 열처리조작이 선도유지에 효과적이었으며 냉동저장중 총 creatine함량의 변화에 있어서는 열처리 조작의 영향은 나타나지 않았다.

• 약학회지
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• 제56권3호
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• pp.191-197
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• 2012

We previously reported that the novel isoflavone-free peptide mixture (black soybean peptide, BSP) had several beneficial effects like antiobesity and hypotriglyceridemic effect. However, there are no reports for BSP on anti-hypertensive activity. BSP induced heme oxygenase-1 (HO-1) in HUVECs, thus investigated the HO-1-induced activity in HUVECs and the anti-hypertensive effects in SHR animal model. BSP significantly induced HO-1 expression both at transcriptional and protein levels in a time- and dose-dependent manner as measured by RT-PCR and Western blot analysis, respectively. These inductions were abolished by pretreatment of N-acetyl-cystein (NAC, 1~10 mM), but not by employing Tempol, a superoxide dismutase (SOD) mimetic (1~5 mM). As expected, enzymatic activity (~2 fold) determined by bilirubin formation assay and cGMP concentration (~6 fold) were significantly increased in BSP-treated cells. Based on the numerous evidences on the beneficial effects of HO-1 and our results, we investigated in vivo effects of BSP on the antihypertensive activity. The administration of BSP (1% in drinking water) significantly decreased mean blood pressure (BP) (from $218.6{\pm}6.99$ to $190.0{\pm}3.42$ mm Hg, p<0.01). This result indicates that BSP is strong inducer of HO-1 expression, which may be triggered by oxidative stress, and has anti-hypertensive activity.

• The Korean Journal of Pain
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• 제22권1호
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• pp.16-20
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• 2009

Background: Zaprinast is an inhibitor of phosphodiesterase 5, 6 and 9. Phosphodiesterase inhibitors could produce anti-nociceptive effects by promoting the accumulation of cGMP. We hypothesized that intrathecal zaprinast could attenuate the allodynia induced by chronic constriction injury of the sciatic nerve in rat. Methods: Sprague-Dawley rats were prepared with four loose ligations of the left sciatic nerve just proximal to the trifurcation into the sural, peroneal and tibial nerve branches. Tactile allodynia was measured by applying von Frey filaments to the lesioned hindpaw. The thresholds for the withdrawal responses were assessed. Zaprinast ($3-100{\mu}g$) was administered intrathecally by the direct lumbar puncture method to obtain the dose-response curve and the 50% effective dose ($ED_{50}$). Measurements were taken before and 15, 30, 45, 60, 90, 120, and 180 min after the intrathecal doses of zaprinast. The side effects were also observed. Results: Intrathecal zaprinast resulted in a dose-dependent antiallodynic effect. The maximal effects occurred within 15-30 min and then they gradually decreased down to the baseline level over time in all the groups. There was a dose dependent increase in the magnitude and duration of the effect. The $ED_{50}$ value was $17.4{\mu}g$ (95% confidence intervals; $14.7-20.5{\mu}g$). No severe motor weakness or sedation was observed in any of the rats. Conclusions: Intrathecally administered zaprinast produced a dose-dependent antiallodynic effect in the chronic constriction injury neuropathic pain model. These findings suggest that spinal phosphodiesterase 5, 6 and 9 may play an important role in the modulation of neuropathic pain.

• The Korean Journal of Physiology and Pharmacology
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• 제4권1호
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• pp.33-40
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• 2000

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on contraction, cytosolic $Ca^{2+}$ level $([Ca^{2+}]_i)$ and ionic currents in guinea-pig ileum. SIN-1 $(0.01{\sim}100\;{\mu}M)$ inhibited 25 mM KCl- or histamine $(10\;{\mu}M)-induced$ contraction in a concentration-dependent manner. SIN-1 reduced both the 25 mM KCl- and the histamine-stimulated increases in muscle tension in parallel with decreased $[Ca^{2+}]_i.$ Using the patch clamp technique with a holding potential of -60 mV, SIN-1 $(10\;{\mu}M)$ decreased peak Ba currents $(I_{Ba})$ by $30.9{\pm}5.4%$ (n=6) when voltage was stepped from -60 mV to +10 mV and this effect was blocked by ODQ $(1\;{\mu}M),$ a soluble guanylyl cyclase inhibitor. Cu/Zn SOD (100 U/ml), the free radical scavenger, had little effect on basal $I_{Ba},$ and SIN-1 $(10\;{\mu}M)$ inhibited peak $I_{Ba}$ by $32.4{\pm}5.8%$ (n=5) in the presence of Cu/Zn SOD. In a cell clamped at a holding-potential of -40 mV, application of $10\;{\mu}M$ histamine induced an inward current. The histamine-induced inward current was markedly and reversibly inhibited by $10\;{\mu}M$ SIN-1, and this effect was abolished by ODQ $(1\;{\mu}M).$ In addition, SIN-1 markedly increased the depolarization-activated outward $K^+$ currents in the all potential ranges. We concluded that SIN-1 inhibits smooth muscle contraction mainly by decreasing $[Ca^{2+}]_i$ resulted from the inhibition of L-type $Ca^{2+}$ channels and the inhibition of nonselective cation currents and/or by the activation of $K^+$ currents via a cGMP-dependent pathway.