[논문]Cytotoxicity by Lead-induced nNOS Phosphorylation in a Dopaminergic CATH.a Cells: Roles of Protein Kinase A

Kwon, Yong-Hyun; Choi, Ji-Young; Shin, Mi-Kyung; Lim, Woo-Sung; Lee, Sung-Keun; Kang, Ju-Hee; Park, Chang-Shin

Cytotoxicity by Lead-induced nNOS Phosphorylation in a Dopaminergic CATH.a Cells: Roles of Protein Kinase A 원문보기

Molecular & cellular toxicology, v.3 no.4, 2007년, pp.215 - 221

Kwon, Yong-Hyun (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project) , Choi, Ji-Young (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project) , Shin, Mi-Kyung (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project) , Lim, Woo-Sung (Department of Urology, Inha University College of Medicine) , Lee, Sung-Keun (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project) , Kang, Ju-Hee (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project) , Park, Chang-Shin (Department of Pharmacology, Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project)

Abstract ▼ AI-Helper

Neuronal cell toxicity induced by decreased nitric oxide (NO) production may be caused by modulation of constitutive neuronal NO synthase (nNOS). We used lead acetate ($Pb^{2+}$) to modulate physiological NO release and the related pathways of protein kinases like PKC, CaM-KII, and PKA in CATH.a cells, a dopaminergic cell line that has constitutive nNOS activity. In the cells treated with $Pb^{2+}$, cell viability and modulation (phosphorylation) levels of nNOS were determined by MTT assay and Western blot analysis, respectively. nNOS reductase activity (cytochrome c) was also assessed to compare the phosphorylation site-specific nNOS activity. nNOS activity was also determined by NADPH consumption rates. $Pb^{2+}$ treatment alone increased the phosphorylation of nNOS with decreased reductase activity. The phosphorylation levels increased markedly with decreased nNOS reductase activity, when $Pb^{2+}$ was combined with inhibitors for two (PKC and CaM-KII) or three (PKA, PKC and CaM-KII) protein kinases. Interestingly, when the cells were exposed to $Pb^{2+}$ plus PKC or CaM-KII inhibitor, the nNOS was phosphorylated strongly with the lowest activity. However, the levels of phosphorylated nNOS following $Pb^{2+}$ treatment decreased significantly after combined treatment with the PKA inhibitor, and $Pb^{2+}$-induced suppression of reductase activity did not occur. These results demonstrate that physiological NO release in the neuronal cells exposed to $Pb^{2+}$ can be decreased by PKA-mediated nNOS phosphorylation that may be caused by interactions with PKC and/or CaM-KII.

주제어

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가설 설정

plus the PKC (Ro-31-8220) and/or CaM-kinase II inhibitors (KN-93). In this study, it is important to determine if phosphorylation of nNOS alters the catalytic activity of nNOS. We have assessed catalytic activity using a NADPH oxidation assay (Figure 6).

대상 데이터

Lead acetate (Pb²⁺), MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), 2, 6-Dichloroindophenol (DCPI), Cytochrome c, and triethanolamine were purchased from Sigma (St. Louis, MO). PKC inhibitor (RO 31-8221), CaM-kinase II (KN-93), and PKA inhibitor (H-89) were purchased from Calbiochem (La Jolla, CA).
Louis, MO). PKC inhibitor (RO 31-8221), CaM-kinase II (KN-93), and PKA inhibitor (H-89) were purchased from Calbiochem (La Jolla, CA). RPMI 1640 medium, Fetal Bovine Serum (FBS), Horse Serum (HS) and streptomycin/penicillin were obtained from Gibco BRL (Gaithersburg, MD).

이론/모형

Cell viability was determined using a MTT assay. After 24 hrs of culture in 96-well culture plates, cells were pre-treated with a number of protein kinase inhibitors in 100 μL of serum-free medium for 10 min before treatment with 100 μL of Pb²⁺ (100 μM) in serum-free medium.
The crude extracts were centrifuged at 500×g for 5 minutes and the supernatants, containing the total cellular protein, were mixed with glycerol to a final concentration of 20% of total volume, and stored at -80℃. The concentration of protein in the extracts was quantified using the Bio-Rad protein assay.

성능/효과

In conclusion, these results demonstrate that physiological NO production in dopaminergic neuronal cells is reduced by nNOS phosphorylation, mediated by the PKA pathway, and with possible interactions with PKC and/or CaM-KII. Thus, Pb²⁺-induced cytotoxicity of neuronal cells may be induced by modulation of NO production.

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