Interaction of Divalent Metal Ions with Zn^(2+) -Glycerophosphocholine Cholinephosphodiesterase from Ox Brain

Lee, Kun Jong,Kim, Mee Ree,Kim, Yun-Bae,Myung, Pyung-Keun,Sok, Dai-Eun 충남대학교 약학대학 의약품개발연구소 1997 藥學論文集 Vol.13 No.-

The effect of divalent metal ions on the activity of glyccrophosphocholinc cholincphosphodiesterse from ox brain was examined. Zn^2+_ and Co^2+-glyccrophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn^2+ and Co^2+, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu^2+, they differed in the susccptibility to the inhibition by thiocholine or tellurte; while Co^2+-phosphodiesterase was more sensitive to tellurites, Zn^2+-phosphodiesterase was more susccptible to inhibition by thiocholine. In addition, Zn^2+-phosphodiesterase was more thermo-stable than Co^2+ enzyme. Separately, when propertics of native phosphodiesterase were compared to those of each metallo-phosphodiesterasc, native phosphodicsterase was found to be quite similar to Zn^2+-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn^2+-phosphodicsterase rather than Co^2+-enzyme. Consistent with this, the stability of natvie phosphodiesterase was maintained in the presence of Zn^2+, but not Co^2+, Mn^2+ was also as effective as Zn^2+ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu^2+ with a Ki value of 20 μM, and its inhibitory action was antagonized effectively by Zn^2+ or Co^2+. Also choline, another competitiive inhibitor of the enzymc, appeared to antagonize the inhibitory action of Cu^2+. Taken together, it is suggested that there may be multiple binding sites for divalent meta ions in the molccute of glyccrophosphocholine cholinephosphodicsterase.

Streptozotocin으로 당뇨병을 유발시킨 흰쥐의 지방세포에서 일어나는 Insulin-Sensitive Phosphodiesterase의 조절에 관한 연구

박경선(Kyung Sun Park),이명순(Myung Soon Lee),김경환(Kyung Hwan Kim) 대한약리학회 1993 대한약리학잡지 Vol.29 No.2

Streptozotocin으로 당뇨병을 유발시킨 흰쥐를 모델로 하여 당뇨병으로 인한 인슐린의 antilipolytic action을 매개하는 insulin-sensitive cyclic nucleotide phosphodiesterase의 역할의 변화 가능성을 연구하였다. 흰쥐의 epididymal adipose tissue로부터 분리한 지방세포를 여러 약물과 toxin으로 전처치한 다음, insulin을 처치 또는 처치하지 않고 37˚C에서 15분 동안 incubation하였다. 그리고 나서 differential centrifugation으로 3 fractions로 분리한 다음 cAMP phosphodiesterase activity를 assay하였다. Insulin에 의한 PDE activities의 증가는 당뇨병군과 대조군 모두 crude microsomal (P2) fraction에서만 볼 수 있었다. P2 fraction을 2 nM insulin, 100μM isoproterenol, 또는 두 약물을 함께 처치하여 나타난 maximal effect는 두 군 모두에서 유의한 차이가 없었다. 그러나 basal PDE activities는 당뇨병균이 대조군에 비해 증가한 것으로 나타났다. 당뇨병군의 P2 fraction의 insulin-sensitive PDE activities는 A₁ adenosine receptor agonist 인 PIA에 의해서 영향을 받지 않은 반면, 대조군의 경우 PIA에 의해 basal PDE activities와 같게 감소하였다. 그리고 지방세포의 pertussis toxin에 대한 sensitivity는 당뇨병군이 대조군보다 더욱 민감하였다. 그러나 cholera toxin에 대한 sensitivity는 당뇨병군과 대조군 사이에 큰 차이를 볼 수 없었다. 이러한 결과로 보아 streptozotocin으로 당뇨병을 유발시킨 흰쥐의 지방세포에서, adenosine receptor와 같은 inhibitory receptor를 경유한 signalling의 감소는 G_i proteins의 소실 또는 기능의 감소와 관련이 있으며, 또한 basal state에서 insulin-dependent PDE의 활성을 증가시키는 것으로 사료된다. Possible changes in the role of insulin-sensitive cyclic nucleotide phosphodiesterase(PDE) in mediating the antilipolytic action of insulin were investigated in adipocytes from streptozotocin-induced diabetic rats. Isolated adipocytes prepared from epididymal adipose tissue were incubated, with or without insulin, at 37˚C for 15 min following pretreatment with various drugs or toxins, and three (plasma membranes, microsomal membranes, and cytosol) fractions prepared by differential centrifugation were then assayed for cAMP phosphodiesterase activity. The PDE activities only in the crude microsomal (P2) fractions were activated by insulin both in diabetic and control rats. The basal PDE activities in P2 fractions of adipocytes from diabetic rats were higher than those from control rats, although the maximal effects observed at 2 nM of insulin, 100μM of isoproterenol or the combination of both were not significantly different from each other. The insulin-stimulated PDE activities in P2 fractions of adipocytes from diabetic rats were not changed by PIA, a A₁ adenosine receptor agonist, whereas they were decreased to the basal PDE activities in those from control rats. In addition, the adipocytes from diabetic rats showed an increased sensitivity to pertussis toxin compared to those from controls. There were no differences between diabetic and control rats in the sensitivity of adipocytes to cholera toxin. These data indicate that the impaired signalling through inhibitory receptors such as adenosine receptors in adipocytes from streptozotocin-induced diabetes relates to the loss or the decreased function of G_i proteins, and leads to the increased activity of the insulin-dependent PDE at the basal states.

Regulation of Insulin-Sensitive Cyclic Nucleotide Phosphodiesterase in Adipocytes of Streptozotocin-Induced Diabetic Rats

박경선,이명순,김경환,Park, Kyung-Sun,Lee, Myung-Soon,Kim, Kyung-Hwan The Korean Society of Pharmacology 1993 대한약리학잡지 Vol.29 No.2

Streptozotocin으로 당뇨병을 유발시킨 흰쥐를 모델로 하여 당뇨병으로 인한 인슐린의 antilipolytic action을 매개하는 insulin-sensitive cyclic nucleotide phosphodiesterase의 역할의 변화 가능성을 연구하였다. 흰쥐의 epididymal adipose tissue로부터 분리한 지방세포를 여러 약물과 toxin으로 전처치한 다음, insulin을 처치 또는 처치하지 않고 $37^{\circ}C$에서 15분 동안 incubation하였다. 그리고 나서 differential centrifugation으로 3 fractions로 분리한 다음 cAMP phosphodiesterase activity를 assay하였다. Insulin에 의한 PDE activities의 증가는 당뇨병군과 대조군 모두 crude microsomal (P2) fraction에서만 볼 수 있었다. P2 fraction을 2 nM insulin, $100\;{\mu}M$ isoproterenol, 또는 두 약물을 함께 처치하여 나타난 maximal effect는 두 군 모두에서 유의한 차이가 없었다. 그러나 basal PDE activities는 당뇨병균이 대조군에 비해 증가한 것으로 나타났다. 당뇨병군의 P2 fraction의 insulin-sensitive PDE activities는 $A_{1}$ adenosine receptor agonist 인 PIA에 의해서 영향을 받지 않은 반면, 대조군의 경우 PIA에 의해 basal PDE activities와 같게 감소하였다. 그리고 지방세포의 pertussis toxin에 대한 sensitivity는 당뇨병군이 대조군보다 더욱 민감하였다. 그러나 cholera toxin에 대한 sensitivity는 당뇨병군과 대조군 사이에 큰 차이를 볼 수 없었다. 이러한 결과로 보아 streptozotocin으로 당뇨병을 유발시킨 흰쥐의 지방세포에서, adenosine receptor와 같은 inhibitory receptor를 경유한 signalling의 감소는 $G_{i}$ proteins의 소실 또는 기능의 감소와 관련이 있으며, 또한 basal state에서 insulin-dependent PDE의 활성을 증가시키는 것으로 사료된다. Possible changes in the role of insulin-sensitive cyclic nucleotide phosphodiesterase(PDE) in mediating the antilipolytic action of insulin were investigated in adipocytes from streptozotocin-induced diabetic rats. Isolated adipocytes prepared from epididymal adipose tissue were incubated, with or without insulin, at $37^{\circ}C$ for 15 min following pretreatment with various drugs or toxins, and three (plasma membranes, microsomal membranes, and cytosol) fractions prepared by differential centrifugation were then assayed for cAMP phosphodiesterase activity. The PDE activities only in the crude microsomal (P2) fractions were activated by insulin both in diabetic and control rats. The basal PDE activities in P2 fractions of adipocytes from diabetic rats were higher than those from control rats, although the maximal effects observed at 2 nM of insulin, $100\;{\mu}M$ of isoproterenol or the combination of both were not significantly different from each other. The insulin-stimulated PDE activities in P2 fractions of adipocytes from diabetic rats were not changed by PIA, a $A_{1}$ adenosine receptor agonist, whereas they were decreased to the basal PDE activities in those from control rats. In addition, the adipocytes from diabetic rats showed an increased sensitivity to pertussis toxin compared to those from controls. There were no differences between diabetic and control rats in the sensitivity of adipocytes to cholera toxin. These data indicate that the impaired signalling through inhibitory receptors such as adenosine receptors in adipocytes from streptozotocin-induced diabetes relates to the loss or the decreased function of $G_i$ proteins, and leads to the increased activity of the insulin-dependent PDE at the basal states.

Active Site of Brain Zn^2+ - Glycerophosphocholine Cholinephosphodiesterase and Regulation of Enzyme Activity

Sok, Dai-Eun 충남대학교 약학대학 의약품개발연구소 1998 藥學論文集 Vol.14 No.-

Properties of active site of Zn^2+-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quatermary ammonium compounds such as phosphocholine, choline or carmitine inhibited the enzyme, divalent metal ions such as Co^2+, Mn^2+ or Zn^2+ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6-9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or dietylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tryrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tryrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen perxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.

Effect of Phosphodiesterase-5 Inhibitors on the Treatment of Male Infertility: A Systematic Review and Meta-Analysis

Dong Liang,Zhang Xiaojin,Yan Xuhong,Shen Yifeng,Li Yulin,Yu Xujun 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.4

Purpose: Male infertility is a worldwide problem with limitations in the treatment. Phosphodiesterase-5 inhibitors (PDE5is) is the first choice for the treatment of erectile dysfunction, more and more studies show that it has a certain effect on male infertility in recent years. But there was currently no high quality of systematic review to evaluate the effects of PDE5is on semen quality. Materials and Methods: We retrieved the electronic databases of MEDLINE, PubMed, Web of Science, EMBASE, etc. Related randomized controlled trials (RCTs) were collected and selected up to May 20, 2020. We have searched literature with terms “male infertility”, “phosphodiesterase-5 inhibitors”, “PDE5i”, “Tadalafil”, “Sildenafil”, “Vardenafil”, “Udenafil”, “Avanafil”, “semen”, and “sperm”. Mean value and its standard deviation were used to perform quantitative analysis. All statistical analyses were conducted by RevMan 5.3 and Stata software. Results: There were a total of 1,121 participants in the nine included studies. There was a statistically significant improvement treated with PDE5is compared with sham therapy, which including sperm concentration (mean difference [MD]=1.96, 95% confidence interval [CI]=1.70–2.21, p<0.001; MD=3.22, 95% CI=1.96–4.48, p<0.001), straight progressive motility (%) Grade A (MD=3.71, 95% CI=2.21–5.20, p<0.001), sperm motility (MD=8.09, 95% CI=7.83–8.36, p<0.001), morphologically normal spermatozoa (%) (MD=0.67, 95% CI=0.20–1.15, p=0.005; MD=1.27, 95% CI=0.02–2.52, p=0.05), sperm abnormalities (%) (MD=-0.64, 95% CI=-0.81–-0.47, p<0.001), and progressive motile sperm (MD=5.34, 95% CI=3.87–6.81, p<0.001). Conclusions: In this meta-analysis of nine RCTs, treatment with PDE5is could improve some indicators of male sperm.

GS 283의 평활근 억제 작용기전

김시환(Si Hwan Kim),이영수(Young Soo Lee),정원석(Won Seog Chong),장기철(Ki Churl Chang) 대한약리학회 1994 대한약리학잡지 Vol.30 No.1

Pharmacological characterization of GS 283, a tetrahydroisoquinoline derivative has been elucidated using rat thoracic aorta, guinea pig tenea coli and rabbit mesentery artery in vitro. GS 283 showed calcium antagonistic action in vascular smooth muscle, since high K⁺-induced contraction was concentration dependently inhibited. GS 283 also inhibited the contraction induced by α₁ receptor activation. Vasodilating action of GS 283 was not modified by the propranolol, indicating that GS 283 has no β receptor stimulatory action. Simultaneous measurement of intracellular calcium change and muscle tension indicated that the inhibitory effect of GS 283 was accompanied by the increase in tissue fluorescence. This increment was not due to fura 2 fluorescence but to endogenous pyridine nucleotide, suggesting that GS 283 has an effect to inhibit mitochondrial function. GS 283 had an inhibitory action on cyclic AMP and GMP-dependent phosphodiesterases from rat brain with Ki values of 2.5 and 6.7 mM. From these findings we concluded that GS 283 has multiple action such as the inhibition of cyclic nucleotide-dependent phosphodiesterases, blocking of calcium channel as well as inhibition of mitochondrial function which are responsible for vasodilatation. Tetrahydroisoquinoline유도체인 GS 283의 약리학적 특성을 흰쥐 흉부대동맥, 기니픽 결장띠 및 토끼 장간막 동맥 및 흰쥐 뇌를 사용하여 조사하였다. 혈관 평활근에서 GS283은 고 K⁺에 의한 수축을 농도 의존적으로 억제하여 Ca²⁺ 길항작용을 보였다. 또한α₁- 수용체 자극에 의한 수축도 억제하였다. GS 283의 혈관이완 작용은 propranolol영향을 받지 않으므로 β-수용체 자극작용에 의한 것이 아니었다. 세포내 칼슘이온과 근장력 변화를 동시에 측정하였을 때 GS 283의 억제효과는 조직내 형광의 증가를 수반했다. 이 증가는 fura 2형광에 의한것이 아니라 내인성 pyridine nucleotide에 의한 것이며 이는 GS 283이 미토콘드리아 기능을 억제하는 효과가 있음을 시사했다. 흰쥐 뇌의 cAMP와 cGMP 의존성 phosphodiesterase에 대한 GS 283의 K_i,값은 2.5와 6.7mM이었다. 이상의 결과에서 GS 283의 약리 작용은 Ca²⁺ 길항작용, α₁- 수용체억제 작용 및 cyclic nucleotide 의존성 phosphodiesterase 억제 등 다양한 작용이 있으며 평활근 수축 억제에 대한 GS283 작용에는 Ca²⁺ 길항이 가장 중요한 요인이 될 것으로 생각된다.

Simultaneous Determination of Synthetic Phosphodiesterase-5 Inhibitors in Dietary Supplements by Liquid Chromatography-High Resolution/Mass Spectrometry

Kim, So-Hee,Kim, Ho-Jun,Son, Jung-Hyun,Jeon, Byoung-Wook,Jeong, Eun-Sook,Cha, Eun-Ju,Lee, Jae-Ick Korean Society for Mass Spectrometry 2012 Mass spectrometry letters Vol.3 No.2

After success of sildenafil for the treatment of erectile dysfunction, a large number of its analogues have been approved from FDA. Recently, the illegal dietary supplements which include sildenafil, vardenafil, tadalafil, or analogues of these drugs as ingredient have been widely distributed. Therefore, the determination of the residue of synthetic phosphodiesterase- 5 (PDE-5) inhibitors in dietary supplements is highly required due to indiscriminate and unintentional overdose caused nausea, chest pains, fainting and irregular heartbeat. In this paper, we report a rapid and sensitive analytical method for the simultaneous determination of nine phosphodiesterase-5 inhibitors by liquid chromatography-high resolution mass spectrometry. The present method was found to be accurate and reproducible with 40 ${\mu}g$/g of the limit of quantification for the nine PDE-5 inhibitors. The developed method can be successfully applied to the analysis of the seven illegal dietary supplements.

DA-8159, a Potent cGMP Phosphodiesterase Inhibitor, Attenuates Monocrotaline-Induced Pulmonary Hypertension in Rats

KyungKooKang,GookJunAhn,YongSungSohn,ByoungOkAhn,WonBaeKim 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.8

In this study, we evaluated the effects of oral administration of DA-8159, a selective phosphodiesterase- 5 inhibitor, on the development of pulmonary hypertension (PH) induced by monocrotaline (MCT). Rats were administered either MCT (60 mg/kg) or saline. MCT-treated rats were divided into three groups and received orally administered vehicle, or 1 mg/kg or 5 mg/kg of DA-8159, twice a day for twenty-one days. The MCT group demonstrated increased right ventricular weights, medial wall thickening in the pulmonary arteries, myocardial fibrosis and the level of plasma cyclic guanosine monophosphate (cGMP), along with decreased body weight gains. However, DA-8159 markedly and dose-dependently reduced the development of right ventricular hypertrophy and medial wall thickening. DA-8159 also amplified the increase in plasma cGMP level and significantly increased the level of lung cGMP, compared with the MCT group. Although the body weight gain was still lower from the saline-treated control group, DA-8159 demonstrated a significant increase in body weight gains, in both 1 mg/kg and 5 mg/kg groups, when compared with the MCT group. In myocardial morphology, MCTinduced myocardial fibrosis was markedly prevented by DA-8159. These results suggest that DA-8159 may be a useful oral treatment option for PH.

Mechanisms Underlying the Inhibitory Effect of GS 283 in Various Smooth Muscles

김시환,이영수,정원석,장기철,Kim, Si-Hwan,Lee, Young-Soo,Chong, Won-Seog,Chang, Ki-Churl The Korean Society of Pharmacology 1994 대한약리학잡지 Vol.30 No.1

Tetrahydroisoquinoline유도체인 GS 283의 약리학적 특성을 흰쥐 흉부대동맥, 기니픽 결장띠 및 토끼 장간막 동맥 및 흰쥐 뇌를 사용하여 조사하였다. 혈관 평활근에서 GS283은 고 $K^+$에 의한 수축을 농도 의존적으로 억제하여 $Ca^{2+}$ 길항작용을 보였다. 또한 ${alpha}_1$- 수용체 자극에 의한 수축도 억제하였다. GS 283의 혈관이완 작용은 propranolol영향을 받지 않으므로 ${\beta}$-수용체 자극작용에 의한 것이 아니었다. 세포내 칼슘이온과 근장력 변화를 동시에 측정하였을 때 GS 283의 억제효과는 조직내 형광의 증가를 수반했다. 이 증가는 fura 2형광에 의한것이 아니라 내인성 pyridine nucleotide에 의한 것이며 이는 GS 283이 미토콘드리아 기능을 억제하는 효과가 있음을 시사했다. 흰쥐 뇌의 cAMP와 cGMP 의존성 phosphodiesterase에 대한 GS 283의 $K_i$,값은 2.5와 6.7mM이었다. 이상의 결과에서 GS 283의 약리 작용은 $Ca^{2+}$ 길항작용, ${\alpha}_1$- 수용체억제 작용 및 cyclic nucleotide 의존성 phosphodiesterase 억제 등 다양한 작용이 있으며 평활근 수축 억제에 대한 GS283 작용에는 $Ca^{2+}$ 길항이 가장 중요한 요인이 될 것으로 생각된다. Pharmacological characterization of GS 283, a tetrahydroisoquinoline derivative has been elucidated using rat thoracic aorta, guinea pig tenea coli and rabbit mesentery artery in vitro. GS 283 showed calcium antagonistic action in vascular smooth muscle, since high $K^+-induced$ contraction was concentration dependently inhibited. GS 283 also inhibited the contraction induced by ${\alpha}_1$ receptor activation. Vasodilating action of GS 283 was not modified by the propranolol, indicating that GS 283 has no ${\beta}$ receptor stimulatory action. Simultaneous measurement of intracellular calcium change and muscle tension indicated that the inhibitory effect of GS 283 was accompanied by the increase in tissue fluorescence. This increment was not due to fura 2 fluorescence but to endogenous pyridine nucleotide, suggesting that GS 283 has an effect to inhibit mitochondrial function. GS 283 had an inhibitory action on cyclic AMP and GMP-dependent phosphodiesterases from rat brain with Ki values of 2.5 and 6.7 mM. From these findings we concluded that GS 283 has multiple action such as the inhibition of cyclic nucleotide-dependent phosphodiesterases, blocking of calcium channel as well as inhibition of mitochondrial function which are responsible for vasodilatation.

Simultaneous Determination of Synthetic Phosphodiesterase-5 Inhibitors in Dietary Supplements by Liquid Chromatography-High Resolution/Mass Spectrometry

So-Hee Kim,Ho Jun Kim,Junghyun Son,Byoung Wook Jeon,Eun Sook Jeong,Eun Ju Cha,Jaeick Lee 사단법인 한국질량분석학회 2012 Mass spectrometry letters Vol.3 No.2

After success of sildenafil for the treatment of erectile dysfunction, a large number of its analogues have beenapproved from FDA. Recently, the illegal dietary supplements which include sildenafil, vardenafil, tadalafil, or analogues ofthese drugs as ingredient have been widely distributed. Therefore, the determination of the residue of synthetic phosphodiesterase-5 (PDE-5) inhibitors in dietary supplements is highly required due to indiscriminate and unintentional overdose causednausea, chest pains, fainting and irregular heartbeat. In this paper, we report a rapid and sensitive analytical method for thesimultaneous determination of nine phosphodiesterase-5 inhibitors by liquid chromatography-high resolution mass spectrometry. The present method was found to be accurate and reproducible with 40 μg/g of the limit of quantification for the nine PDE-5inhibitors. The developed method can be successfully applied to the analysis of the seven illegal dietary supplements.