Abbreviations

PD, Parkinson’s disease; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; SNpc, substantia nigra pars compacta; K_ATP, ATP-sensitive potassium channel; ACSF, artificial cerebrospinal fluid; Ipt, iptakalim; 6-OHDA, 6-hydroxydopamine; GABA, ϒ-aminobutyric acid; HPLC, high performance liquid chromatography

Introduction

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. The primary pathological change of PD is the loss of the dopaminergic neurons in substantia nigra pars compacta (SNpc). Its etiology remains unclear to date. It has been demonstrated that neurotoxity of excitatory glutamate is responsible for the onset of PD [₁]. The glutamate acts as a principal excitatory neurotransmitter in the mammalian central nervous system as well as a potent neurtoxin [₂]. According to current theories of functional anatomy of basal ganglia [₃], the neurotransmitters dopamine (DA) and glutamate interactions underlie brain functions, including regulation of normal movements [₄]. The information from cortex to neostriatium, the input nuclei of basal ganglia, is processed within neostriatium and transmitted to the output nuclei of basal ganglia by direct and indirect pathways. Under-activity of ϒ-aminobutyric acid (GABA)ergic neurons which activated by D₁ receptors within the direct pathway and over-activity of glutamatergic neurons which inhibited by D₂ receptors within the indirect pathway [₅] are thought to be a common mechanism for the dopaminergic neuron degeneration in PD [₆]. Therefore, enhancing the activity of DA in direct pathway and reducing the activity of glutamate in indirect pathway have been proposed as two therapeutic strategies for PD.

ATP-sensitive potassium (K_ATP) channels are distributed widely in the brain and directly couple the metabolic state of a cell to its electrical activity. K_ATP channels comprise heteromultimers of two pore-forming subunits of the inward rectifer (Kir6.1, Kir6.2) family and two regulatory sulfonylurea receptor subunits (SUR1, SUR2). These channels are not only relevant to acute metabolic challenges, but also to chronic genesis of neurodegenerative disorders like PD [₇]. Moreover, studies of mid-brain dopaminergic neurons in the weaver mouse, a genetic mouse model used to study dopaminergic degeneration similar to that in PD, support the idea of K_ATP channel activation as a neurprotective strategy [₈].

A new compound we identified in a previous study called iptakalim hydrochloride (Ipt) has been demonstrated to be a novel K_ATP channel opener through pharmacological, electrophysiological, biochemical studies and receptor binding test [₉]. Further studies indicated that Ipt can pass the blood-brain barrier. In vitro, Ipt can enhance the glutamate uptake activity of astrocytes, PC12 cells and synaptosomes [₁₀,₁₁,₁₂], suppress the currents of rat hippocampal neurons induced by the glutamate and NMDA in a dose-dependent manner [₉]. In vivo, we found that Ipt can significantly ameliorate hypolocomotion and catalepsy which are induced by haloperidol in rats (Wang et al, unpublished observation). It has been demonstrated that Ipt can significantly decrease rotational turns induced by apomorphine after unilateral 6-OHDA-lesioned rats were treated with oral gavage Ipt 3mg/kg /day for three weeks (Wang et al, unpublished observation). Therefore, we hypothesize that Ipt may affect the extracellular neurotransmitters of striatum in rats. This study was designed to investigate the effects of Ipt on the levels of extracellular neurotransmitters such as glutamate, DA in the striatum of unilateral 6-OHDA-lesioned rats by using the reverse microdialysis technique.

Materials And Methods

Male Sprague-Dawley rats (250-280g) were kept under room temperature ( 22±1°) and humidity with a 12h light/dark cycle. These rats were allowed free access to water and food throughout the experiment. Under pentobarbital (40mg/kg) anesthesia, the rats were fixed on a stereotaxic apparatus. 6-hydroxydopamine (8µg of 6-OHDA in 4 µl of 0.2% ascorbic acid saline solution, Sigma-Aldrich Corp. St. Louis, MO USA) was injected unilaterally into SNpc on the left (coordinates: A: -5.3mm from bregma, L: +1.8mm from midline and H:-7.8mm from dura) with a Hamilton syringe. The injection rate 1µl per min. After the injection, the needle was kept in place for 15min to allow the diffusion of the toxin away from the injection site to prevent back-flow. The control rats received sham vehicle (0.2% ascorbic acid saline solution) infusion. Rats’ rotation ability in response to apomorphine (0.05mg/kg subcutaneously, SC) was measured at 21-days after the injection. Thirty minutes before rotation test, each of the rats was placed in a rodent residence. Contralateral rotations induced by apomorphine were measured once a week for two weeks. Only those rats showing at least 210 turns during the 30 min test (unilateral 6-OHDA-lesioned rats) were selected for further study [₁₃].

Under pentobarbital (40mg/kg) anesthesia, both control and unilateral 6-OHDA-lesioned rats were placed in a stereotaxic apparatus and implanted with a stainless steel dialysis guide cannula to both sides of the striatum (coordinates: A: +1.0mm from bregma, L: ±2.9mm from midline and H: -3.5mm from dura). The guide cannula was cemented in place through affixing dental acrylic to three stainless steel screws tapped into skull of the rats.

After rats recovered for 7 days, the concentric microdialysis probes (MD-2200, 2mm membrane Bioanalytical system, Inc, Indiana, USA) were inserted through the guide cannula into the striatum. Artificial cerebrospinal fluid (ACSF, NaCl 140 mM, CaCl₂ 1.4 mM, MgCl₂ 1.2 mM, KCl 2.7 mM, and Glucose 5mM) was infused through the probe via a syringe pump Bioanalytical system, Inc, Indiana, USA). ACSF was adjusted to pH at 7.4 with phosphate-buffered saline and filtered by a 0.22µm vacuum filtration before use. Perfusion with ACSF started at 12 h after the probe insertion and lasted for 2h at a rate of 2µl per min for equilibrium. After that, five baseline samples were collected at a 20-min interval. Ipt was then administered into the striatum via perfusion through the microdialysis probe. Dose-response curves were determined by passing increasing concentration of Ipt through the microdialysis probe after the baseline samples were collected. Dose-response curves were used to measure the capacity of Ipt to alter the extracellular glutamate and DA levels. Typically, five different doses of Ipt (0.01, 0.1, 1, 10, and 100µM) were used in each experiment , each dose was passed through the probe for 60 min and three 40µl of dialysis samples were collected for each dosage in a 20-min interval. Each collected sample was aliquoted into two microcentrifuge tubes with 20 µl in each tube for analysis of the glutamate, dopamine and DOPAC levels. All samples were frozen at -80° before they were analyzed.

After both control and unilateral 6-OHDA-lesioned rats were decapitated, the striatum, cortex and hippocampi were homogenized in ice-cold HClO₄ solution (0.4mol/L) at a ratio of 1:9 (w/v) and centrifuged at 10 000×g for 15 min. KHCO₃ (2mol/L) was added to the supernatant at a ratio of 3:4 (v/v). After being centrifuged at 4000×g for 5 min, the supernatant was recovered and frozen at -80° before the glutamate was assayed.

Dialysate samples were assayed for DA and DOPAC by high-performance liquid chromatography (HPLC) with electrochemical detection (Waters, Milford, MA, USA). The samples were thawed and placed in the 717 Plus Autosampler (Waters, Milford, MA, USA) connected to the 2465ECD (Waters, Milford, MA, USA) equipped with a C18 reverse phase column (4.6×75mm, 3.5µm, Waters, Milford, MA, USA). The samples was eluted by a mobile phase made of 100mM Na-citrate, 0.1mM EDTA, 75mM Na₂HPO₄, 2mM NaCl, 1mM C-7 at pH 3.9, 10% methanol at a flow rate of 0.6ml/min. DA and DOPAC levels in the samples were calculated by extrapolating the peak area from a standard curve (ranging from 1nM to 100nM of mixed DA and DOPAC). The calculation was done through the Millennium³² Workstation (Waters, Milford, MA, USA). The threshold for DA and DOPAC detection was set at 1nM.

The concentration of the glutamate was determined by HPLC with fluorescent detection [₁₄]. The HPLC-fluorescent detector system (Shimadzu Corp, Tokyo, Japan) consisted of a reverse phase C18 column (4.6×250mm, 5µm, Waters, Milford, MA, USA). The emission and excitation wavelengths were set at 425nm and 338nm, respectively. The pre-column derivation solution contains 20mmol/L of o-phthaldialdehyde (OPA), 2mmol/L of β-mercaptoethanol, 25mmol/L of tetraborate, and 50% methanol (pH 9.6). Samples were mixed with equivalent volume of derivation solution, and incubated at the room temperature for 4 min. The derived reaction solutions were used to assay the glutamate concentration at 37° with a flow rate of 0.8ml/min. At the end of the experiments, the control and unilateral 6-OHDA-lesioned rats were given an overdose of pentobarbital (>50mg/kg i.p) and perfused intracardially with 10% formalin. Their brain was removed and stored in 10% formalin for at least 1 week. The coronal sections (100µm thick) of the brain were cut to verify the location of the probe. Samples obtained from the rats in which the probe was not correctly positioned (< 2%) were excluded from the analysis.

Results

All data were presented as the means±S.E.M. The second and third samples collected from each dosage of Ipt treatment were averaged and compared to the average over the third, fourth and fifth baseline samples. General linear model with random effects are used to analyze dosage effects. Post-hoc analysis was performed with Dunnet t-test. General linear model with repeated measurements are used for analysis of the difference between groups. When the results showed a significant difference, post-hoc analysis was performed by contrast method. All analyses are performed by SAS software package (Ver 8.12, by SAS Institute Inc., Cary, NC.USA). P values less than 0.05 were considered significant.

The glutamate level in homogenized tissue of the lesion-side striatum of unilateral 6-OHDA-lesioned rats increased 2 folds (P< 0.01) in comparison with that from intact-side striatum of unilateral 6-OHDA-lesioned and control rats, respectively. The glutamate concentration in homogenized tissue of the lesion-side hippocampi of unilateral 6-OHDA-lesioned rats increased by 20% (P< 0.05) compared to that of control rats. However, the glutamate concentrations were not significantly different between lesion- and intact-sides of cortex of unilateral 6-OHDA-lesioned rats or control rats (P>0.05) (Table 1).

Figure 1

Table 1: The glutamate contents in various areas of brain of unilateral 6-OHDA-lesioned rats

The effect of Ipt perfused through the dialysis probe at concentrations from 0.01 to 100µM on the extracellular glutamate in the striatum was evaluated in five rats (Fig. 1). Before Ipt was administrated on the dialysate (baseline), a significant higher level of extracellular glutamate was observed in the lesion-side striatum of unilateral 6-OHDA-lesioned rats than that in the intact-side striatum of unilateral 6-OHDA-lesioned rats (P<0.05), or that in the striatum of control rats (P<0.05). After different concentrations of Ipt was administered on the dialysate, the extracellular glutamate levels in both sides striatum of unilateral 6-OHDA-lesioned rats and control rats decreased in a concentration-dependent manner. These indicated that there was a high level of extracellular glutamate in striatum of unilateral 6-OHDA-lesioned rats, and Ipt lead to a significant decrease in the extracellular glutamate level in the striatum of rats.

Figure 2

Figure 1: Effects of iptakalim on the extracellular glutamate levels in the striatum of unilateral 6-OHDA-lesioned rats. B stands for baseline. * P<0.05 vs baseline; # P<0.05 vs intact-side of unilateral 6-OHDA-lesioned rats; + P<0.05 vs control rats(sham). (n=5,5,4).

The effects of Ipt on the extracellular DA level in the striatum of unilateral 6-OHDA-lesioned rats by microdialysis were indicated in Fig. 2. Before Ipt was administered (baseline), the extracellular DA levels in lesion-side and intact-side striatum of unilateral 6-OHDA-lesioned rats were significantly lower than those in the control rats (P < 0.05). Interestingly, in the striatum of control rats, Ipt significantly reduced the extracellular DA levels at high doses (10, 100µM), whereas it significantly increased the extracellular DA levels at low doses (0.01, 0.1, 1µM) in the lesion-side striatum of unilateral 6-OHDA-lesioned rats. In the intact-side striatum of unilateral 6-OHDA-lesioned rats, Ipt showed no effect on the extracellular DA levels. These data suggested that unilateral 6-OHDA-lesioned rats usually have a significant lower extracellular DA level in both sides of striatum and low doses of Ipt may increase the extracellular DA levels in the striatum of unilateral 6-OHDA-lesioned rats.

Figure 3

Figure 2: Effects of iptakalim on extracellular DA levels in the striatum of unilateral 6-OHDA-lesioned rats. B stands for baseline. * P<0.05 vs baseline; # P<0.05 vs intact-side of unilateral 6-OHDA-lesioned rats; + P<0.05 vs control rats(sham). (n=5,5,4).

The effects of Ipt on the extracellular DOPAC levels in the striatum of unilateral 6-OHDA-lesioned rats by microdialysis were showed in Fig. 3. Before Ipt was administered (baseline), the extracellular DOPAC levels in the lesion-side striatum of unilateral 6-OHDA-lesioned rats were significantly lower than those in the striatum of control rats (P<0.05). After Ipt was administered (from 0. 01µM to 100µM) on the dialysate, the extracellular DOPAC levels in the lesion-side striatum of unilateral 6-OHDA-lesioned rats significantly decreased in comparison with baseline samples (P<0.05). There were no significant changes in extracellular DOPAC levels for the intact-side striatum of unilateral 6-OHDA-lesioned and control rats (P>0.05). The extracellular DOPAC levels in the lesion-side striatum of unilateral 6-OHDA-lesioned rats were significantly lower than those in the intact-side of unilateral 6-OHDA-lesioned rats and in the striatum of control rats (P<0.05). Therefore, Ipt may also reduce the metabolite of DA in the lesion-side striatum of unilateral 6-OHDA-lesioned rats, but may have no effect on the intact-side striatum of unilateral 6-OHDA-lesioned rats and the striatum of control rats.

Figure 4

Figure 3: Effects of iptakalim on extracellular DOPAC levels in the striatum of unilateral 6-OHDA-lesioned rats. B stands for baseline. * P<0.05 vs baseline; # P<0.05 vs intact-side of unilateral 6-OHDA-lesioned rats; + P<0.05 vs control rats(sham). (n=5,5,4).

In a previous study, we have found that Ipt can significantly ameliorate the hypolocomotion and catalepsy induced haloperidol in rats and decrease the contralateral rotational turns induced by apomorphine in unilateral 6-OHDA-lesioned rats. These data demonstrated that Ipt could produce anti-PD like effects. However, little is known about the mechanisms of these effects. In the present study, we found that the glutamate levels in the homogenized striatum of lesion-side are higher than those in the intact-side of unilateral 6-OHDA-lesioned rats and those in the striatum of control rats. Meanwhile, the extracellular glutamate levels significantly increased and the extracellular DA and its metabolite DOPAC levels greatly reduced in the lesion-side striatum of unilateral 6-OHDA-lesioned rats. One of the mechanisms may be that 6-OHDA can inhibit the glutamate uptake activity of astrocytes, PC12 cells and synaptosomes [₁₀,₁₁], therefore lead to a decrease in the expression of glutamate transporters in unilateral 6-OHDA-lesioned rats (YL, Yang et al, unpublished observation). Recent evidence suggests that the neurotransmitters balance of DA, GABA and glutamate interactions is critical for maintaining the normal function of basal ganglia. Moreover, an imbalance between GABA ergic neurons activated by D₁ receptors and glutamate neurons inhibited by D₂ receptors is thought to play a major role in the pathogenesis of movement disorder [₆]. Ultimately, the increase in the glutamate activity of the subthalamic nucleus is responsible for the over-activity in basal ganglia output structures that is also directly under the control of the striatum [₁₅]. Therefore, one of the therapeutic strategies to control the PD is to reduce the glutamate in striatum.

Conclusions

Based on result from the present study, Ipt can reduce the extracellular glutamate level in the striatum of rats in a concentration-dependent manner. One of possible mechanisms could be that Ipt enhances the glutamate uptake activity of neuronal terminals and astrocytes [₁]. Surprisingly, Ipt can significantly increase the extracellular DA levels at low doses (0.01~1µM) in the lesion-side striatum of unilateral 6-OHDA-lesioned rats, while this effect disappears at high doses from 10µM to 100µM or in the intact-side striatum of unilateral 6-OHDA-lesioned rats. This result is coincident with the result from a previous study in our laboratory that Ipt could enhance the extracellular DA only in lesion-side striatum of unilateral 6-OHDA-lesioned rats via oral gavage Ipt 3mg/kg/day for 3 weeks (Wang et al, unpublished observation). There are many factors that may affect the release of DA in the striatum, such as glutamate spillover in the striatum [₁₆] and the condition of K_ATP channels in the dopaminergic neurons [₁₇]. Although some previous reports have suggested that the K_ATP channel openers can decrease the DA in striatum of the normal rats [₁₈], our experiments demonstrated that Ipt has different effects on the control and unilateral 6-OHDA-lesioned rats. Moreover, the extracellular DA levels in the striatum depend on many aspects such as the DA release, the function of DA transporter and the DA degradation etc. In the present study, Ipt can reduce the extracellular glutamate levels and decrease the excitotoxicity, which leads to the enhancement of the DA release in the lesion side striatum of unilateral 6-OHDA-lesioned model rats. These results are supported by Avshalumov et al. [₁₉]. On the other hand, Ipt can reduce the metabolite of DA—DOPAC in the lesion-side striatum of unilateral 6-OHDA-lesioned rats. These results imply that Ipt maybe inhibit the metabolic enzyme of DA, and reduce the DA degradation in the lesion-side striatum of unilateral 6-OHDA-lesioned rats. Based on these results, we infer that Ipt can ameliorate hypolocomotion, catalepsy and rotational behavior in PD model rats, by reducing the extracellular glutamate levels in the striatum of rats, and enhancing the extracellular DA levels in the lesion-side striatum of unilateral 6-OHDA-lesioned rats.

The present findings suggest that Ipt, a novel ATP-sensitive potassium channel opener, have potential to be used to develop a new therapeutic agent to treat Parkinson’s disease.

Acknowledgments

Correspondence to

Gang Hu. Department of Pharmacology & Neurobiology, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. CHINA. Tel: +86 25 86863169. Fax: +86 25 86863108. E-mail address: ghu@njmu.edu.cn