• The Korean Journal of Physiology and Pharmacology
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• 제2권6호
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• pp.733-742
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• 1998

Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

• Archives of Pharmacal Research
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• 제18권6호
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• pp.434-439
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• 1995

In pancreatic cells, NO formation is associated with increased levels of cGMP and endocrine/exocrine secretion. In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat pancreatic tissues. Treatment of rat pancreatic tissue with sholecystokinin-pancreozymin (CCK-PZ) resulted in an significant increase in arginine conversion to citruline, the amount of nitrite/nitrate, the release of amylase, and the level of cGMP. Furthermore, CCK-PZ stimulated increase of amylase release and conversion of arginine to citrulline transformation were counteracted by the inhibitor of NO synthase, $N^G-nitro-L-arginine$ methyl ester. The results on the time course of CCK-PZ-induced citrulline formation within the first seconds of simulation. The kinetics of citrulline accumulation correlate well with those of cGMP rise, which further confirms the conclusion that NO mediates the response to CCK-PZ by cGMP.

• 한국동물학회지
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• 제36권3호
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• pp.433-438
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• 1993

본 연구를 통해서 근세포 융합과정에서 신호 전달물질의 가능성이 제기되고 있는 세포내 cGMP peak가 guanylate cyclase activity의 변화와 관련이 있으며, guanylate cyclase는 L-arginine: NO synthase에 의해서 촉진될 것임을 입증할 수 있는 간접적 증거를 제시한다. 즉, SNP는 근세포의 융합과 guanylate cyclase activity를 아울러 증가시키며, L-arginine: NO synthase inhibitor인 L-NG-monomethyl arginine은 biochemical differentiation에는 영향을 주지 않고 근세포 융합만을 억제한다. 이러한 결과들과 muthylene blue가 근세포의 융합만을 억제하면서도 biochemical differentiation에는 영향을 주지 않으며 guanylate cyclase activity를 억제하는 사실들을 종합해서 생각할 때, 근세포 융합에서 cGMP peak가 guanylate cyclase activity의 활성화와 관련이 있으며, L-arginine: NO synthase가 $Ca^2$+ influx와 guanylate cyclase 사이를 매개할 가능성을 암시한다.

• Archives of Pharmacal Research
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• 제25권6호
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• pp.873-878
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• 2002

OA-8159, a new Phosphodiesterase (PDE) 5 inhibitor, has exhibited potent erectogenic potential in a penile erection test in rats and anesthetized dogs. In this study, we investigated the mechanism of its erectogenic activity by measuring the activity of OA-8159 against a various PDE isozymes and assessing cGMP and cAMP formation in a rabbit corpus cavernosum in vitro. DA-8159 inhibited the PDE 5 activity in rabbit and human platelets, which the $IC_{50}$ was 5.84$\pm$1.70 nM and 8.25$\pm$2.90 nM, respectively. The $IC_{50}$ of DA-8159 on PDE 1, PDE2, PDE 3 and PDE 6 were 870$\pm$57.4 nM, $101\pm$5 $\mu$M, 52.0$\pm$3.53 $\mu$M and 53.3$\pm$2.47 nM, respectively. This suggests that DA-8159 is a potent, highly selective, competitive inhibitor of PDE 5-catalyzed cGMP hydrolysis. The rates of cGMP hydrolysis catalyzed by human platelets-derived PDE 5 as a function of the cGMP concentration (5~100 nM) and two-fixed DA-8159 concentration (11.3 and 18.8 nM) were investigated in order to characterize the mode of PDE 5 inhibition by DA-8159. DA-8159 increased the apparent 4K_{m}$ value for cGMP hydrolysis but had no effect on the apparent $V_{max}$, indicating a competitive mode of inhibition. DA-8159 increased the cGMP concentrations in the rabbit corpus cavernosum dose dependently. In the presence of sodium nitroprusside (SNP), DA-8159 significantly sti\mulated the accu\mulation of cGMP when compared to the control level. This indicated that the enhancement of a penile erection by DA-8159 involved the relaxation of the cavernosal smooth \muscle by NO-sti\mulated cGMP accu\mulation. In conclusion, DA-8159 is a selective inhibitor of PDE 5-catalyzed cGMP hydrolysis and the enhancement of a penile erection by DA-8159 is mediated by the relaxation of the cavernosal smooth \muscle by the NO-sti\mulated cGMP accu\mulation.

• The Korean Journal of Pain
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• 제18권2호
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• pp.99-106
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• 2005

Background: Cyclic guanosine monophosphate (cGMP) and opioid receptors are involved in the modulation of nociception. Although the opioid receptors agonists are active in pain, the effect of an phospodiesterase inhibitor (zaprinast) for increasing the level of cGMP has not been thoroughly investigated at the spinal level. This study examined the effects of intrathecal zaprinast and morphine in a nociceptive test and we also examined the nature of the pharmacological interaction after the coadministration of zaprinast with morphine. The role of the nitric oxide (NO)-cGMP-potassium channel pathway on the effect of zaprinast was further clarified. Methods: Catheters were inserted into the intrathecal space of male SD rats. For the induction of pain, $50{\mu}l$ of 5% formalin solution was applied to the hindpaw. Isobolographic analysis was used for the evaluation of the drug interaction between zaprinast and morphine. Furthermore, NO synthase inhibitor ($_L-NMMA$), guanylyl cyclase inhibitor (ODQ) or a potassium channel blocker (glibenclamide) were intrathecally administered to verify the involvement of the NO-cGMP- potassium channel pathway on the antinociception effect of zaprinast. Results: Both zaprinast and morphine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed a synergistic interaction after the intrathecal administration of the zaprinast-morphine mixture in both phases. Intrathecal $_L-NMMA$, ODQ and glibenclamide did not reverse the antinociception of zaprinast in either phase. Conclusions: These results suggest that zaprinast, morphine and the mixture of the two drugs are effective against acute pain and they facilitated pain state at the spinal level. Thus, the spinal combination of zaprinast with morphine may be useful for the management of pain. However, the NO-sensitive cGMP-potassium channel pathway did not contribute to the antinocieptive mechanism of zaprinast in the spinal cord.

• 대한수의학회지
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• 제43권4호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.445-453
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• 2000

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of $Ca^{2+}-activated$ potassium channel $(K_{Ca}\;channel)$ and is involved in the activation of $K_{Ca}$ channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide $(SIN-1,\;50\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate $(8-pCPT-cGMP,\;100\;{\mu}M),$ a membrane-permeable analogue of cGMP, increased the $K_{Ca}$ channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP $(100\;{\mu}M),$ cGMP $(100\;{\mu}M),$ ATP+cGMP $(100\;{\mu}M\;each),$ PKG $(5\;U/{\mu}l),$ ATP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l),$ or cGMP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ did not increase the channel activity. ATP $(100\;{\mu}M)+cGMP\;(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer $(Rp-pCPT-cGMP,\;100\;{\mu}M),$ a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The $K_{Ca}$ channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of $K_{Ca}$ channels, or some associated proteins in the membrane patch increase the activity of the $K_{Ca}$ channel, and the activation may be associated with the vasodilating action.

• Biomolecules & Therapeutics
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• 제7권2호
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• pp.105-111
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• 1999

Nitric oxide (NO), a cellular messenger synthesized from L-arginine by NO synthase (NOS, EC.1.14.13.39), is considered to be a regulator of gastric secretion. In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat gastric chief cells. Treatment of chief cells with carba-chol resulted in an increase in the arginine conversion to citrulline, the amount of $NO_{x}$, the release of pepsine-gen, and the level of cGMP Especially, carbachol-stimulated increase of arginine to citrulline transformation, the amount of $NO_{x}$, cGMP level and the release of pepsinogen were partially reduced by the natural NOS inhibitor, $N^{G}$-monomethyl-L-arginine (MMA) and $N^{G}$, $N^{G}$-dimethyl-L-arginine (DMA). Furthermore, MMA- and DMA-induced decrease of pepsinogen secretion showed dose-dependent patters. Activation of NOS is one of the early events in receptor-mediated cascade of reactions in gastric chief cells and NO, not completely, but partially mediates gastric secretion. Agonist-stimulated pepsinogen secretion in chief cells has been considered to be mediated in adenosine 3',5'-cyclic monophosphate pathway and/or guanosine 3', 5'-cyclic monophosphate (cGMP) pathway. Taken together, the above results suggest that partial decrease of exocrine secretion following treatment of NOS inhibitor may result from the inactivation of NOS and subsequent guano- late cyclase, and NO/cGMP pathway may play a pivotal role in exocrine secretion.

• The Korean Journal of Physiology and Pharmacology
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• 제8권2호
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• pp.95-100
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• 2004

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium $(K_{ATP})$ channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that $K_{ATP}$ channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide $(100\;{\mu}M)$, SNAP $(300\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) $(100\;{\mu}M)$ followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide $(50\;{\mu}M)$, 5-hydroxydecanoate (5-HD) $(100\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) $(100\;{\mu}M)$. These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of $K_{ATP}$ channels in rat ventricular myocytes.

• 대한수의학회지
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• 제40권4호
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• pp.691-699
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• 2000

We investigated the effects of nitric oxide (NO) donors, S-nitroso-L-cysteine (Cys-NO) and 3-morpholinosydnonimine hydrochloride (SIN-1), on the contractile and electrical activity of the circular muscle of guinea pig gastric antrum by using intracellular microelectrode technique. The gastric antral circular muscle showed spontaneous phasic contraction and slow wave of membrane potential. Cys-NO ($0.001{\sim}10{\mu}M$) and SIN-1 ($0.001{\sim}100{\mu}M$) reduced not only the tonic and phasic contraction but also the amplitude of slow wave in a concentration dependent manner. NO donors were more potent to inhibit phasic contraction than to do slow wave. These inhibitory effects of NO donors were mimicked by the membrane permeable guanosine-3',5'-cyclic monophosphate (cGMP) analogue, 8-bromo-cyclic GMP (8-br-cGMP, $10{\sim}300{\mu}M$). The inhibitory effects of SIN-1 and Cys-NO were antagonized by the guanylate cyclase inhibitor, 1H[ [1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, $10{\mu}M$). These results suggest that the inhibitory effects of NO donors on the mechanical and electrical activity is mainly mediated by cGMP pathway.