Norsalsolinol Uptake Into Secretory Vesicles Via Vesicular Monoamine
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FULL PAPER Toxicology Norsalsolinol Uptake into Secretory Vesicles Via Vesicular Monoamine Transporter and Its Secretion by Membrane Depolarization or Purinoceptor Stimulation in PC12 Cells Yutaka MARUYAMA1), Yuko SUZUKI1), Akio KAZUSAKA1) and Shoichi FUJITA1)* 1)Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060–0818, Japan (Received 8 November 2000/Accepted 5 January 2001) ABSTRACT. The intracellular dynamics of norsalsolinol, a neurotoxin candidate causing parkinsonism-like symptoms, in PC12 cells was studied. We found that dopamine and norsalsolinol are co-localized to secretory granule layer by sucrose density gradient in norsalsoli- nol-treated PC12 cells. The norsalsolinol was actively taken up into isolated secretory vesicle fraction from PC12 cells with a Km value of 41.5 ± 6.8 µM. The uptake of 10 µM of norsalsolinol was sensitive to reserpine (1 µM), an inhibitor of vesicular dopamine transporter, and dopamine, an endogenous substrate, but insensitive to GBR-12909, an inhibitor of dopamine transporter on plasma membrane. In norsalsolinol-treated PC12 cells, exposure to high K+ or ATP resulted in simultaneous release of norsalsolinol and dopamine. Time course of a release of dopamine and that of norsalsolinol evoked by 50 mM KCl or 100 µM ATP corresponded to each other. These releases were dependent on the concentrations of secretagogues. These data suggest that norsalsolinol is taken up with dopamine into secretory vesicle via vesicular catecholamine transporter. KEY WORDS: exocytosis, norsalsolinol, PC12 cell, vesicular monoamine transporter. J. Vet. Med. Sci. 63(5): 493–497, 2001 Parkinson’s disease is one of the most common neurode- 30]. However, the intracellular localization of TIQs are not genarative diseases and its characteristic pathological fea- well understood. ture is selective cell death of dopamine-containing neurons A metabolite of MPTP, 1-methyl-4-phenylpyridinium in the substantia nigra [1]. The etiology of this disease has ion (MPP+), a compound structurally similar to TIQs accu- been intensively studied, but it has not been elucidated yet. mulates in chromaffin granules in adrenal grand [22] or The discovery of a potent dopaminergic neurotoxin, 1- serotonin granules in platelet [3]. These organelles contain methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) high concentration of monoamines taken up through the induced a hypothesis that endogenous or exogenous neuro- monoamine transporter utilizing an electrochemical gradi- toxins structurally similar to MPTP may be responsible for ent of protons established by vacuolar H+-ATPase [9, 10, the degeneration of dopamine neurons in Parkinson’s dis- 19], and this transporter also involves MPP+ uptake [16]. ease [28]. Isoquinoline alkaloids, which belong to a large Norsalsolinol, dihydroxy TIQ, has been shown to accumu- group of naturally occurring and synthetic compounds, have late in human brain [17]. This compound can be formed by been proposed as candidates for dopaminergic neurotoxins spontaneous condensation of dopamine and formaldehyde [6, 7, 23]. Elevated levels of tetrahydroisoquinoline (TIQ) by Pictet-Spenglar condensation. Our group reported that and its derivatives (TIQs) were detected in the human brain, dopamine secretion was inhibited by norsalsolinol in PC12 cerebrospinal fluid, and the urine of Parkinson’s disease cells, a well-established chromaffin cell line with abundant patients [17, 27]. It has been shown that TIQs cause selec- catecholamine secretory vesicles [14]. However, intracellu- tive dopaminergic neurotoxic effect, leading to artificial lar dynamics of norsalsolinol is not clear in dopaminergic parkinsonism in experimental animals [18]. Previous stud- neuron. Studies on the intracellular localization of the neu- ies have shown that TIQ alkaloids decrease cell viability in rotoxin may be helpful for understanding the mechanism of SH-SY5Y human neuroblastoma cells or mesencephalic- its toxicity. striatal primary culture [8, 30] and inhibit the activities of In this paper, we examined the intracellular dynamics of both tyrosine hydroxylase [L-tyrosine, tetrahydropteridine, norsalsolinol using PC12 cells. oxygen: oxidoreductase (3-hydroxylating), EC 1.14.16.2; TH] [15, 25], and monoamine oxidase [monoamine: oxygen MATERIALS AND METHODS oxidoreductase (deaminating), EC 1.4.3.4.] [2]. Further- more, it is reported that TIQs was taken up into SH-SY5Y Materials: Norsalsolinol was purchased from Dojin labo- cells or PC12 cells via dopamine transporter (DAT) [13, ratories (Kumamoto, Japan). Adenosine triphosphate diso- dium salt (ATP) was purchased from Sigma Chemical Co. *CORRESPONDENCE TO: FUJITA, S., Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate (St Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s School of Veterinary Medicine, Hokkaido University, Sapporo medium (DMEM), penicillin, streptomycin and amphoteri- 060–0818, Japan. cin B were purchased from Life Technologies (Rockville, 494 Y. MARUYAMA, Y. SUZUKI, A. KAZUSAKA AND S. FUJITA MD, U.S.A.). Nerve growth factor (NGF) was purchased The standard medium used was a modified Krebs solution from Upstate Biotechnology (Lake Placid, NY, U.S.A.). All with the following composition: NaCl, 118 mM; KCl, 4.7 other chemicals were purchased from Wako pure chemicals mM; KH2PO4, 1.2 mM; MOPS, (pH 7.4 adjusted with (Osaka, Japan). NaOH) 25 mM; CaCl2, 2.5 mM; MgSO4, 1.2 mM; and glu- Cell culture: PC12 cells were grown in DMEM supple- cose, 10 mM. In order to stimulate dopamine and norsal- mented with 5 % (v/v) fetal bovine serum, 10 % (v/v) horse solinol release, cells were incubated with secretagogues for serum, 50 U/ml of penicillin, 50 µg/ml streptomycin, and 10 min at 37°C. At the end of the incubation, the medium 0.125 µg/ml amphotericin B. The cells were incubated in a and cells adherent to the culture dish were quickly separated humidified atmosphere containing 5 % CO2 in air at 37°C using a pipette, and released into the medium was acidified for 5–7 days on 15 cm diameter or 24-well plastic culture with 0.4 M perchloric acid [13, 14, 29]. plates. PC12 cells were also differentiated with NGF (50 High performance liquid chromatography: Dopamine ng/ml). The medium was changed every other day [13, 14]. and norsalsolinol were measured by means of HPLC appa- Sucrose density gradient study: PC12 cells, grown on 15 ratus (Shimadzu, Kyoto, Japan) with electrochemical detec- cm diameter dish, were washed three times with modified tor (ECD-300, Eicom, Kyoto, Japan). A reverse-phased Krebs buffer and removed by gentle scraping into 5 ml column, Inertsil ODS-2 (particle size 5 µm, column size 150 buffer. The cells were centrifuged at 800 × g for 2 min and × 4.6 mm, GL Science, Tokyo, Japan) was used. The the pellet resuspended in 2 ml 10 mM Hepes, pH 7.2, con- mobile phase consisted of a degassed solution of 200 mg/l 1- taining 0.3 M sucrose and 0.1 M NaCl. The cell suspension sodium-octansulfonate, 5 mg/l EDTA-2Na, 150 ml/l metha- was then homogenized using a Polytron homogenizer nol and 0.1 M citrate buffer, pH 3.5. The detector cell was (Kinematica, Luzern, Switzerland) at volume 5 for 10 sec at operated at +700 mV [13, 14, 29]. 0°C. Homogenates were centrifuged at 800 × g for 10 min Protein content: The total protein contents were mea- twice in succession to rigorously remove broken cells, and sured according to the method of Lowry et al. [11]. the postnuclear supernatant was centrifuged at 5,000 × g for Statistics: The data is presented as arithmetic means ± 10 min to recover rapidly sedimenting secretory vesicles. standard error. Statistical significance was determined by The 5,000 × g pellet fractions were re-suspended in 2 M Student’s t-test, and p-values of less than 0.05 were consid- sucrose in 20 mM Tris, pH 7.8, 150 mM NaCl, 2 mM EGTA ered to be statistically significant. (TNE buffer), diluted 5-fold, and loaded on to discontinuous sucrose gradients. Discontinuous gradients consisting of 2 RESULTS ml each of 0.25, 0.5, 1.0, 1.5 and 2 M sucrose in TNE buffer were centrifuged at 20,000 × g for 30 min and collected Subcellular localization of norsalsolinol: To explore the from the bottom [12]. Each fraction was acidified by same subcellular localization of the norsalsolinol in cells, we first volume of 0.8 M perchloric acid, and protein was remove by performed sucrose gradient subcellular fractionation of centrifugation at 12,000 × g for 30 min. Norsalsolinol con- PC12 cell homogenates. PC12 cells were treated with 3 µM tent in supernatant was quantitated by HPLC-electrochemi- norsalsolinol for 4 hr, homogenized, and layered over a dis- cal detector (ECD). In norsalsolinol uptake study, fraction 7 continuous sucrose density gradient (0.3–2.5 M sucrose). was used. Following centrifugation, fractions were collected, and nor- Norsalsolinol uptake study in purified secretory vesicles: salsolinol, endogenous dopamine, and sucrose concentra- The secretory vesicle contained fraction (fraction 7) was tion were determined. Norsalsolinol and dopamine were diluted with 10 volume of 20 mM MOPS-Tris buffer (pH colocalized to the same subcellular fractions with a major 8.0) containing 100 mM KCl, 0.2 M sucrose, 2 mM MgCl2, peak at fractions 5–9 (1.5 M sucrose) (Fig. 1). The 1.5 M and was centrifuged at 12,000 × g for 15 min. The 12,000 × sucrose peak is consistent with the buoyant density which g pellet were re-suspended in the MOPS-Tris buffer supple- have demonstrated previously for chromaffin granules iso- mented with 5 mM ATP. The uptake of norsalsolinol by lated from PC12 cells [21]. In addition, some dopamine and secretory vesicles was assayed at 25°C by the addition of norsalsolinol were found at the top (fractions 17–18, 0.25 M norsalsolinol to purified secretory vesicles in the MOPS- sucrose) of the gradient. The additional peak at the top of Tris buffer supplemented with ATP.