Yoo Jung Yang

Mechanisms of L-DOPA-induced cytotoxicity in rat adrenal pheochromocytoma cells: implication of oxidative stress-related kinases and cyclic AMP

L-DOPA therapy for Parkinsons disease has a double-edge effect on nigrostriatal dopaminergic neurons: L-DOPA increases the intracellular level of dopamine, but it induces neuron cytotoxicity in a concentration-dependent manner. To investigate the molecular signaling mechanisms that underlie the concentration-dependent effects of L-DOPA on cell viability, the activities of mitogen-activated protein kinases (MAPKs) and apoptotic enzymes were measured in rat adrenal pheochromocytoma (PC12) cells in the presence of a low concentration (20 muM) and high concentrations (100-200 muM) of L-DOPA. At the low concentration, L-DOPA was not cytotoxic and its presence increased the activities of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, BadSer112, Bcl-2, and caspase-12. At the high concentrations, L-DOPA was cytotoxic and stimulated the activities of ERK1/2, p38 MAPK, c-Jun N-terminal kinase (JNK)1/2, BadSer155, caspase-12 and caspase-3. The increased levels of ERK1/2 and BadSer155 in the presence of high concentrations of L-DOPA did not protect against L-DOPA-mediated cytotoxicity. In addition, the levels of L-type Ca(2+) channel-sensitive intracellular cyclic AMP (cAMP) and Ca(2+) were elevated in the presence of L-DOPA, and the increase in the levels of intracellular cAMP may also play a role in cellular viability, since cAMP levels and cytotoxicity increased in parallel with L-DOPA concentrations and the addition of forskolin in the medium increased cytotoxicity in a concentration-dependent manner. These results suggest that, at a low and non-toxic concentration, L-DOPA may promote cell survival by increasing the activities of ERK1/2, BadSer112 and Bcl-2, while, at high concentrations, L-DOPA activates the caspase-3 cell death enzyme through the JNK1/2 and p38 MAPK signaling pathways as well as endoplasmic reticulum stress that activates caspase-12. Intracellular cAMP levels may also play a role here. The results may lead to an effective therapy for Parkinsons disease.

Effect of scoparone on neurite outgrowth in PC12 cells.

The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 microM, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 microM also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 microM scoparone. In addition, scoparone at 200 microM increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells.

Enantio-selective inhibition of (1R,9S)- and (1S,9R)-β-hydrastines on dopamine biosynthesis in PC12 cells

The inhibitory effects of (1R,9S)- and (1S,9R)-enantiomers of beta-hydrastine (BHS) on dopamine biosynthesis in PC12 cells were investigated. (1R,9S)-BHS decreased the intracellular dopamine content with the IC50 value of 14.3 microM at 24 h, but (1S,9R)-BHS did not. (1R,9S)-BHS was not cytotoxic at concentrations up to 250 microM towards PC12 cells. In these conditions, (1R,9S)-BHS inhibited tyrosine hydroxylase (TH) activity mainly in a concentration-dependent manner (33% inhibition at 20 microM) and decreased TH mRNA level in PC12 cells. The inhibitory patterns of dopamine content and TH activity by (1R,9S)-BHS showed similar behavioral curves. (1R,9S)-BHS at 10-50 microM also reduced the intracellular cyclic AMP level and Ca2+ concentration. In addition, treatment of L-DOPA at 20-50 microM for 24 h increased the intracellular dopamine content to 198-251% compared with the control in PC12 cells. However, the increase in dopamine levels induced by L-DOPA (20-50 microM) was reduced when L-DOPA was combined with (1R,9S)-BHS (10-50 microM). These results indicate that (1R,9S)-BHS, but not (1S,9R)-BHS, reduced dopamine content and L-DOPA-induced increase in dopamine content, in part, through the inhibition of TH activity and TH gene expression in PC12 cells: thus, (1R,9S)-BHS proved to have a function to regulate dopamine biosynthesis.

Induction of neurite outgrowth by (-)-(7R, 8S)-dihydrodehydrodiconiferyl alcohol from PC12 cells.

A lignan derivative, (-)-(7R, SSJ-dihydrodehydrodiconiferyl alcohol (DHDA), was isolated fromKalopanax septemlobus L. and was observed to have neuritogenic activity. DHDA at 50 μ? caused a marked induction of neurite outgrowth and an enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. However, it did not exhibit any neu-rotrophic action. At 50 μ?, DHDA enhanced NGF-induced neurite-bearing activity. This activity was partially blocked by the mitogen-activated protein kinase inhibitor PD98059 and by GF109203X, a protein kinase inhibitor. These results suggest that DHDA can induce neurite outgrowth and enhance NGF-induced neurite outgrowth from PC12 cells by amplifying up-stream steps such as and PKC.

Effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 microM and 100 microM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC50 values of harman and norharman were 21.2 microM and 103.3 microM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 microM harman and 100 microM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 microM and 150 microM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10-30 microM harman and 50-150 microM norharman inhibited L-DOPA (20-50 microM)-induced increases in dopamine content at 48 h. Harman at 20-150 microM and norharman at 100-300 microM also enhanced L-DOPA (20-100 microM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.

Induction of dopamine biosynthesis by l-DOPA in PC12 cells: Implications of l-DOPA influx and cyclic AMP

The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).

Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells.

The inhibitory effects of liriodenine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced dopamine content increases in PC12 cells were investigated. Treatment of PC12 cells with 5–10 μM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 μM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 μM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 μM liriodenine to 20–70% and 10–14% of control levels at 3–12 h, respectively; TH activity was more influenced than AADC activity. The levels of TH mRNA, intracellular cyclic AMP and basal Ca2+concentration were also decreased by 10 μM liriodenine. In addition, 10 μM liriodenine reduced L-DOPA (20–100 μM)-induced increases in dopamine content. However, 10 μM liriodenine resulted in a protective effect against L-DOPA (50–-100 μM)-induced cytotoxicity. These results suggest that liriodenine regulates dopamine biosynthesis by partially reducing TH activity and TH gene expression and has protective effects against L-DOPA-induced cytotoxicity in PC 12 cells.

Effects of scoparone on dopamine release in PC12 cells.

The effects of scoparone on dopamine release in PC12 cells were investigated. Scoparone at 50-200 microM increased dopamine release into the culture medium. However, the released levels of dopamine by scoparone were not altered in the absence of extracellular Ca(2+) and by adenylyl cyclase inhibitor MDL-12,330A. Scoparone increased phosphorylation of PKA, CaMK II and synapsin I. Scoparone also enhanced K(+)-induced levels of dopamine release by CaMK II phosphorylation. These results suggest that scoparone increases dopamine release by synapsin I phosphorylation via activation of PKA and CaMK II, which are mediated by cyclic AMP levels and Ca(2+) influx.

Effects of (+)-eudesmin from the stem bark ofMagnolia kobus DC. var.borealis Sarg. on neurite outgrowth in PC12 cells

Abstract(+)-Eudesmin [4,8-bis (3,4-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane] was isolated from the stem bark ofMagnolia kobus DC. var.borealis Sarg. and found to have neuritogenic activity. 50 μM (+)-eudesmin induced neurite outgrowth and enhanced nerve growth factor (NGF)-mediated mediated neurite outgrowth from PC12 cells. At this concentration, (+)-eudesmin also enhanced NGF-induced neurite-bearing activity and this activity was partially blocked by various protein kinase inhibitors. These included PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor. GF109203X, a protein kinase C (PKC) inhibitor and H89, a protein kinase A (PKA) inhibitor. These results suggest that (+)-eudesmin can induce neurite outgrowth from PC12 cells by stimulating up-stream MAPK, PKC and PKA pathways.

Inhibitory effects of (1R,9S ) -β -hydrastine on calcium transport in PC12 cells

Abstract(1R,9S)-β-Hydrastine (BHS), at 100 μM, has been shown to mainly reduce the K+-induced dopamine release and Ca2+ influx by blocking the L-type Ca2+ channel and inhibit the caffeine activated store-operated Ca2+ channels, but not those activated by thapsigargin, in PC12 cells. In this study, the effects of BHS on Ca2+ transport from Ca2+ stores in the absence of external Ca2+ were investigated in PC12 cells. BHS decreased the basal intracellular Ca2+ concentration ([Ca2+]i) in the absence of external Ca2+ in PC12 cells. In the absence of external Ca2+, pretreating PC12 cells with 100 μM BHS reduced the rapid increase in the [Ca2] elicited by 20 mM caffeine, but not that by 1 μM thapsigargin. In addition, BHS inhibited the increase in the [Ca2+]i elicited by restoration of 2 mM CaCI2 after the Ca2+ stores had been depleted by 20 mM caffeine, but not those depleted by 1 μM thapsigargin, in the absence of external Ca2+. These results suggested that BHS mainly inhibited Ca2+ leakage from the Ca2+ stores and the caffeine-stimulated release of Ca2+ from the caffeine-sensitive Ca2+ stores in PC12 cells.