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 microM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 microM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 microM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 microM 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 microM liriodenine. In addition, 10 microM liriodenine reduced L-DOPA (20-100 microM)-induced increases in dopamine content. However, 10 microM liriodenine resulted in a protective effect against L-DOPA (50-100 microM)-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 PC12 cells.     

Sodium-dependence and ouabain-sensitivity of the synthesis of dopamine in renal tissues of the rat.

1. The present study has examined the influence of sodium chloride (0-160 mM) and ouabain (100 and 500 microM), an inhibitor of the enzyme Na(+)-K+ ATPase, on the synthesis of dopamine in slices of rat renal cortex loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). The deamination of newly-formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also examined. The assay of L-DOPA, dopamine and DOPAC in kidney slices was performed by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. The accumulation of newly-formed dopamine and DOPAC in kidney slices loaded with L-DOPA (50 and 100 microM) was found to be dependent on the concentration of NaCl in the medium. A similar picture could be observed for DOPAC. The fractional rate of accumulation (k; mM NaCl-1) was at 50 and 100 microM L-DOPA, respectively, 0.00305 +/- 0.00036 and 0.00328 +/- 0.00029 for dopamine and 0.00672 +/- 0.00072 and 0.00641 +/- 0.00069 for DOPAC. The sodium-dependent formation of dopamine was completely abolished when the experiments were performed in the absence of oxygen. 3. In experiments performed in the presence of 120 mM NaCl, but not in conditions of low sodium (20 mM NaCl in the medium), ouabain (100 and 500 microM) was found to inhibit the accumulation of newly-formed dopamine and DOPAC (14-57% reduction; P less than 0.05); this effect was more marked at 50 and 100 microM L-DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 microM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 microM. Asimilobine at 0.15 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2+ concentrations. In addition, asimilobine at 0.05-5.0 microM, but not 10 microM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 microM) associated with l-DOPA (20, 50, and 100 microM) for 24 h inhibited L-DOPA-induced increases in dopamine levels and enhanced L-DOPA-induced cell death when compared with L-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.     

Effects of tributyltin chloride on L-DOPA-induced cytotoxicity in PC12 cells

Tributyltin chloride (TBTC) at concentrations of 0.5-1.0 microM inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of TBTC on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were investigated. TBTC at concentrations up to 1.0 microM neither affected cell viability, nor induced apoptosis after 24 or 48 h in PC12 cells. However, TBTC at concentrations higher than 2.0 microM caused cytotoxicity through an apoptotic process. In addition, exposure of PC12 cells to non-cytotoxic (0.5 and 1.0 microM) or cytotoxic (2.0 microM) concentrations of TBTC in combination with L-DOPA (20, 50 and 100 microM) resulted in a significant increase in cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTC or L-DOPA alone. The enhancing effects of TBTC on L-DOPA-induced cytotoxicity were concentration- and treatment time-dependent. These data demonstrate that TBTC enhances L-DOPA-induced cytotoxicity in PC 12 cells.     

Rasagiline enhances L-DOPA-induced contralateral turning in the unilateral 6-hydroxydopamine-lesioned guinea-pig

The modification of L-3,4-dihydrooxyphenylalanine- (L-DOPA-) induced turning response by the new selective monoamine oxidase type B (MAO-B) inhibitor rasagiline was studied in guinea-pigs bearing a unilateral 6-hydroxydopamine-induced lesion in the substantia nigra. In an initial experiment, it was established that contralateral turning is induced in lesioned guinea-pigs in response to apomorphine (18 mg/kg i.p.) and L-DOPA/carbidopa (15/3.5 mg/kg i.p.), while ipsilateral turning is induced by S(+)-methamphetamine (7 mg/kg i.p.). The effect of rasagiline was studied in a chronic treatment regimen, in which animals were treated with rasagiline (0.05 mg/kg s.c.) or saline s.c. daily commencing 2 weeks after lesioning, and L-DOPA/carbidopa (4:1 mg/kg) was administered once daily for 21 days. Only guinea-pigs with 95% or more depletion of striatal dopamine were included in this study. Guinea-pigs treated with rasagiline had a significantly increased intensity and duration of turning in response to L-DOPA (P <0.05 by repeated measures ANOVA) over the 21-day period. On day 21, turning averaged 806+/-105 (n=10) vs 442+/-123 (n=11) turns per 180 min for rasagiline and vehicle treated animals, respectively (P <0.05); turning duration half-time averaged 81+/-15.4 (n=10) as opposed to 33+/-7.6 (n=11) min for rasagiline and vehicle treatments (P <0.01). Concentration of dopamine in intact striatum was significantly increased (69.3+/-2.1 and 60.3+/-2.4 pmol/mg tissue for rasagiline and vehicle, P <0.05) and levels of dihydroxyphenylacetic acid and homovanillic acid were decreased by the rasagiline treatment. Activity of brain MAO-B was 8.6+/-2.9% and MAO-A was 71+/-1.5% that of control in rasagiline-treated animals. Chronic, selective inhibition of MAO-B by rasagiline potentiated L-DOPA-induced turning in this rodent model.     

Effects of tri butyl tin acetate on dopamine biosynthesis and l-dopa-lnduced cytotoxicity in pc12 cells

The effects of tributyltin acetate (TBTA) on dopamine biosynthesis and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were examined. TBTA at concentrations of 0.1-0.2 microM inhibited dopamine biosynthesis by reducing tyrosine hydroxylase (TH) activity and TH gene expression in PC12 cells. TBTA at 0.1-0.4 microM also reduced L-DOPA (20-50 microM)-induced increases in dopamine content for 24 h in PC12 cells. TBTA at concentrations up to 0.3 microM did not affect cell viability. However, TBTA at concentrations higher than 0.4 microM caused apoptotic cytotoxicity. Exposure of PC12 cells to non-cytotoxic (0.1 and 0.2 microM) or cytotoxic (0.4 microM) concentrations of TBTA with L-DOPA (20, 50 and 100 microM) significantly increased the cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTA or L-DOPA alone. These data suggest that TBTA inhibits dopamine biosynthesis and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

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.     

Effects of (1R,9S)-beta-hydrastine on l-DOPA-induced cytotoxicity in PC12 cells

(1R,9S)-beta-Hydrastine in lower concentrations of 10-50 microM inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of (1R,9S)-beta-hydrastine on L-DOPA (L-3,4-dihydroxyphenylalanine)-induced cytotoxicity in PC12 cells were investigated. (1R,9S)-Hydrastine at concentrations up to 250 microM did not reduce cell viability. However, at concentrations higher than 500 microM it caused cytotoxicity in PC12 cells, as determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) method and flow cytometry. Exposure of PC12 cells to cytotoxic concentrations of (1R,9S)-beta-hydrastine (500 and 750 microM) in combination with L-DOPA (20, 50 and 100 microM) after 24 or 48 h resulted in a significant decrease in cell viability compared with the effects of (1R,9S)-beta-hydrastine or L-DOPA alone, and apoptotic cell death was observed. However, the decrease in cell viability induced by (1R,9S)-beta-hydrastine was not prevented by the antioxidant N-acetyl-L-cysteine, indicating that it is not mediated by membrane-based oxygen free radical damage. These data suggest that (1R,9S)-beta-hydrastine has a mild cytotoxic effect and at higher concentration ranges aggravates L-DOPA-induced cytotoxicity in PC12 cells.     

Enantio-selective inhibition of (1R,9S)- and (1S,9R)-beta-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.     

Analysis of 3-morpholinosydnonimine and sodium nitroprusside effects on dopamine release in the striatum of freely moving rats: role of nitric oxide, iron and ascorbic acid

The effects of intrastriatal infusion of 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) on dopamine (DA), 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L-dihydroxyphenylalanine (L-DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis. SIN-1 (1 mM) infusion for 180 min increased microdialysate DA and 3-MT concentrations, while L-DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co-infusion with ascorbic acid (0.1 mM) inhibited SIN-1-induced increases in DA and 3-MT dialysate concentration. SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3-MT were negligible. In addition, SNP decreased ascorbic acid and L-DOPA but increased uric acid concentration in the dialysate. Co-infusion with deferoxamine (0.2 mM) inhibited the late SNP-induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations. SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3-MT, but decreased L-DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co-infusion with ascorbic acid (0.1 mM) inhibited the SNP-induced increase in DA and 3-MT, but did not affect the decrease in L-DOPA and increase in uric acid dialysate concentrations. These results suggest that NO released from SIN-1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.     

Assessment of renal dopaminergic system activity in the nitric oxide-deprived hypertensive rat model.

1. The present paper reports changes in the urinary excretion of dopamine, 5-hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long-term administration of NG-nitro-L-arginine methyl ester (L-NAME). Aromatic L-amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly-formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined. 2. Twenty four hours after exposure to L-NAME, both systolic (SBP) and diastolic (DBP) blood pressure were increased by 20 mmHg; heart rate was slightly decreased. During the next 13 days both SBP and DBP increased progressively reaching 170 +/- 3 and 116 +/- 3 mmHg, respectively. 3. Baseline urinary excretion of L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) during the 4 day period of stabilization averaged 4.4 +/- 0.5, 13.8 +/- 0.3, 37.4 +/- 0.8, 180.0 +/- 2.7 and 206.1 +/- 6.7 nmol day-1, respectively. The urinary excretion of L-DOPA, dopamine and DOPAC, but not that of 3-MT and HVA, were increased from day 6-8 of L-NAME administration onwards (L-DOPA, up to 13.4 +/- 2.1; dopamine, up to 23.0 +/- 1.6; DOPAC, up to 62.8 +/- 3.7 nmol day-1). Baseline daily urinary excretion of 5-hydroxytryptamine and 5-hydroxyindolacetic acid (5-HIAA) averaged 73.5 +/- 1.1 and 241.7 +/- 5.4 nmol day-1, respectively. During the first week of L-NAME administration, the urinary excretion of both 5-hydroxytryptamine and 5-HIAA did not change significantly; however, as was found with dopamine and DOPAC, changes in the urinary excretion of 5-hydroxytryptamine were evident during the second week of L-NAME administration. 4. In experiments performed on homogenates of isolated renal tubules, the decarboxylation of L-DOPA to dopamine was dependent on the concentration of L-DOPA used (10 to 5000 microM) and saturable at 1000 microM. AAAD activity as determined in homogenates (Vmax, in nmol mg-1 protein h-1; Km in microM) was significantly (P < 0.01) higher in rats given L-NAME for 14 days (Vmax = 25 +/- 2; Km = 72 +/- 10) than in control rats (Vmax = 14 +/- 1; Km = 63 +/- 7), rats given L-NAME for 7 days (Vmax = 15 +/- 1; Km = 69 +/- 5) and rats given L-NAME plus L-arginine (Vmax = 13 +/- 1; Km = 60 +/- 3) for 14 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nanomolar L-dopa facilitates release of dopamine via presynaptic beta-adrenoceptors: comparative studies on the actions in striatal slices from control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57 black mice, an animal model for Parkinson's disease.

Effects of L-DOPA (0.1-10,000 nM) on spontaneous release (Sp), evoked release (S) and tissue content (C) of dopamine (DA) were studied comparatively in superfused striatal slices from control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57 black mice to obtain evidence for L-DOPA-induced facilitation of S via presynaptic beta-adrenoceptors. In control slices, isoproterenol-induced concentration-dependent increases in S were propranolol-sensitive. L-DOPA at 0.1-3 nM tended to increase the S of DA with a concomitant tendency of increases in Sp. L-DOPA at 10-1 x 10(4) nM concentration-dependently increased Sp. L-DOPA at 1-10 microM tended to increase S and 10 microM increased C. In slices from MPTP-treated mice, the absolute amounts of Sp, S and C decreased by half compared to those in control slices. L-DOPA at 3 nM facilitated S without increasing Sp. This facilitation was antagonized by propranolol at 3 nM. L-DOPA at 30 nM decreased S from the peak facilitation, which contrasted with no effect in the control slices. However, 10-100 nM L-DOPA increased Sp more markedly than that in the control slices. L-DOPA at 100 nM increased S and C, which contrasted with no effect in the control slices. In conclusion, nanomolar L-DOPA facilitates the S of DA via presynaptic beta-adrenoceptors at concentrations lower than those required to induce conversion to DA even in striatal slices from the MPTP-treated mice model for Parkinson's disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.     

Actin cytoskeleton, tubular sodium and the renal synthesis of dopamine.

The present study has examined the effect of colchicine and cytochalasin B, two cytoskeleton disrupter compounds, on the formation of dopamine in slices of rat renal cortex loaded with exogenous L-3,4-dihydroxyphenylalanine (L-DOPA); the deamination of newly formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also examined. The accumulation of newly formed dopamine and DOPAC in kidney slices loaded with L-DOPA (10-100 microM) was found to be dependent on the concentration of L-DOPA, being similar in control conditions and in preparations treated with increasing concentrations of colchicine (5, 10 and 50 microM). By contrast, cytochalasin B (5, 10 and 50 microM) was found to produce a concentration-dependent reduction in the formation of dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with L-DOPA (10-100 microM). The inhibitory effect of cytochalasin B on the formation of dopamine was found to be completely abolished in kidney slices pretreated with ouabain (500 microM) or when sodium concentration in the incubation was reduced from 120 to 20 mM. On its own, ouabain (500 microM) was found to reduce the formation of dopamine by 55%; the effect of reducing sodium concentration in the incubation medium to 20 mM was also a significant reduction (53% decrease) in the formation of dopamine. The accumulation of DOPAC did always parallel that of its parent amine. It is concluded that the renal formation of dopamine is dependent on the concentration of sodium in the medium and the integrity of the tubular transport of sodium, namely on the association between actin cytoskeleton and Na+,K(+)-ATPase, appears to be determinant.     

L-dopa facilitates the release of endogenous norepinephrine and dopamine via presynaptic beta 1- and beta 2-adrenoceptors under essentially complete inhibition of L-aromatic amino acid decarboxylase in rat hypothalamic slices

In rat hypothalamic slices, L-aromatic amino acid decarboxylase (AADC) was assayed, and the actions of L-DOPA on impulse (2 Hz)-evoked norepinephrine (NE) and dopamine (DA) release were studied under inhibition of AADC. Slices were incubated with L-DOPA, and DA and NE produced by conversion of the precursor were analyzed by high performance liquid chromatography with electrochemical detection (HPLC-ECD). In the slices, the Km and Vmax of AADC were 131 microM and 122 pmol/min/mg protein, respectively. NSD-1015, an AADC inhibitor, caused a noncompetitive type of inhibition, and the K1 value was 0.086 microM. In the presence of 20 microM NSD-1015, which was expected to cause 99.6% inhibition of AADC, L-DOPA (0.01-100 nM) concentration-dependently facilitated the release of NE from the superfused slices, and the L-DOPA (10 nM)-induced facilitation was antagonized by 100 nM ICI 89,406 and 100 nM ICI 118,551, a selective beta 1- and beta 2-adrenoceptor antagonist, respectively. This action of L-DOPA was not modified by 30 microM tropolone, an inhibitor of catechol-O-methyl-transferase. L-DOPA at 0.01-1 nM similarly facilitated the release of DA. A quantitative analysis revealed that the L-DOPA-induced increase in NE and DA release was much higher by a factor of 3 to 4 orders than was the amount of DA and NE converted from L-DOPA. These results add further support to the hypothesis that L-DOPA itself acts as a neuroactive substance in the rat central nervous system.     

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

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5–30 μM decreased intracellular dopamine content with the IC₅₀ value of 22.1 μM. Catalpalactone at 5–20 μM, but not 30 μM, did not alter cell viability. Catalpalactone at 20 μM inhibited tyrosine hydroxylase (TH) and aromatic-l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20 μM reduced the increases in dopamine levels induced by L-DOPA (20–50 μM). Catalpalactone (5–30 μM) associated with L-DOPA (50–100 μM) enhanced L-DOPA-induced cytotoxicity at 48 h, which was prevented by N-acetyl-l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.     

Inhibition by L-3,4-dihydroxyphenylalanine of hippocampal CA1 neurons with facilitation of noradrenaline and gamma-aminobutyric acid release

Electrophysiological studies were performed to elucidate whether L-3,4-dihydroxyphenylalanine (L-DOPA) acted on hippocampal CA1 neurons, since this drug has been reported to act as a neurotransmitter in the hypothalamus and striatum. Hippocampal slices (450 microM thick) obtained from male Wistar rats (4-7 weeks of age) were placed in a bath (maintained at 30+/-1 degrees C) continuously perfused with artificial cerebrospinal fluid. The population spikes elicited by electrical stimuli applied to the Schaffer collateral/commissural fibers were recorded in the hippocampal CA1 region, using a glass micropipette filled with 3 M NaCl. Drugs were applied in the bath through a perfusion system. The population spikes were inhibited by L-DOPA (1 nM-10 microM) with a bell-shaped concentration-response curve (n=7-15). Maximum inhibitory effects were obtained at 100 nM. L-DOPA cyclohexyl ester, a putative L-DOPA recognition site antagonist, antagonized the L-DOPA-induced inhibition of population spike. However, the inhibition remained unaffected in the presence of 3-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor. Furthermore, bath application of either phentolamine, an alpha-adrenoceptor antagonist, or bicuculline, a GABA(A) receptor antagonist, antagonized the inhibitory effects of L-DOPA on population spikes. In addition, bicuculline (1 microM) antagonized the inhibition of population spike induced by 6-fluoronorepinephrine (10 microM), an alpha-adrenoceptor agonist, while phentolamine (10 microM) did not affect the muscimol (1 microM)-induced inhibition. These results suggested that L-DOPA itself acted on L-DOPA recognition sites to release noradrenaline, and that the latter facilitates gamma-aminobutyric acid (GABA) release via alpha-adrenoceptors located on the GABA-containing cells and/or their nerve terminals, thereby inhibiting the population spikes in the hippocampal CA1 field.     

Aggravation of L-DOPA-induced neurotoxicity by tetrahydropapaveroline in PC12 cells

Tetrahydropapaveroline (THP) is formed in Parkinsonian patients receiving L-DOPA therapy and is detected in the plasma and urine of these patients. In this study, we have investigated the effects of THP on L-DOPA-induced neurotoxicity in cultured rat adrenal pheochromocytoma, PC12 cells. Exposure of PC12 cells up to 10 microM THP or 20 microM L-DOPA after 24 or 48 hr, neither affected the cell viability determined by MTT assay, nor induced apoptosis by flow cytometry and TUNEL staining. However, at concentrations higher than 15 microM, THP showed cytotoxicity through an apoptotic process. In addition, THP at 5-15 microM for both incubation time points significantly enhanced L-DOPA-induced neurotoxicity (L-DOPA concentration, 50 microM). Exposure of PC12 cells to THP, L-DOPA and THP plus L-DOPA for 48 hr resulted in a marked increase in the cell loss and percentage of apoptotic cells compared with exposure for 24hr. The enhancing effects of THP on L-DOPA-induced neurotoxicity were concentration- and treated-time-dependent. THP, L-DOPA and THP plus L-DOPA produced a significant increase in intracellular reactive oxygen species generation and decrease in ATP levels, supporting the involvement of oxidative stress in THP- and L-DOPA-induced apoptosis. The antioxidant N-acetyl-L-cysteine strongly inhibited changes in apoptosis, decreases in cell viability and ROS generation induced by THP associated with L-DOPA. These results suggest that THP aggravates L-DOPA-induced oxidative neurotoxic and apoptotic effects in PC12 cells. Therefore, Parkinsonian patients treated with L-DOPA for long-term need to be monitored for the relationship between plasma concentration of THP and the symptoms of neurotoxicity.