盐酸埃他卡林对PD模型大鼠脑内突触体谷氨酸摄取的影响

目的　研究谷氨酸转运体功能改变与帕金森病(Parkinson’sdisease ,PD)发病的相关性 ,探讨新型ATP敏感性钾通道 (ATPsensitivepotassiumchannel,KATP)开放剂盐酸埃他卡林 (Iptakalimhydrochloride ,Ipt)对PD模型大鼠脑内突触体摄取谷氨酸的影响及其机制。方法　采用 6 hydrox ydopamine(6 OHDA)建立PD大鼠模型 ,制备脑组织突触体 ;用同位素标记法测定L [3 H] glutamate摄取活性。 结果　PD模型大鼠纹状体和皮层的谷氨酸转运功能明显降低 ;Ipt(10、5 0和 10 0 μmol·L-1)具有恢复转运功能的作用 ,此作用可被KATP阻断剂Glibenclamide (2 0 μmol·L-1)逆转。结论　谷氨酸转运体功能下降与PD发病密切相关 ;Ipt通过激活KATP发挥促进谷氨酸摄取的作用 ,有望成为新一代PD治疗药物     

Nicergoline enhances glutamate re-uptake and protects against brain damage in rat global brain ischemia

Whereas a 2-3 degrees C decrease in intraischemic brain temperature can be neuroprotective, mild brain hyperthermia significantly worsens outcome. Our previous study suggested that an ischemic injury mechanism which is sensitive to temperature may not actually increase the extracellular glutamate concentration ([Glu](e)) during the intraischemic period, but rather impairs the Glu re-uptake system, which has been suggested to be involved in the reversed uptake of Glu. We speculated that enhancing Glu re-uptake, pharmacologically or hypothermically, may shorten exposure to high [Glu](e) in the postischemic period and thereby decrease its deleterious excitotoxic effect on neuronal cells. In the present study, rats treated with nicergoline (32 mg/kg, i.p.), an ergot alkaloid derivative, showed minimal inhibition of the [Glu](e) elevation which characteristically occurs during the 10-min intraischemic period, while Glu re-uptake was dramatically improved in the postischemic period, when severe transient global ischemia was caused by mild hyperthermia. Moreover, the nicergoline (32 mg/kg, i.p.) treated rats showed reduced cell death morphologically and clearly had a far lower mortality. The present study suggests that the development of therapeutic strategies aimed at inhibition or prevention of the reversed uptake of glutamate release during ischemia, i.e., activation of the glutamate uptake mechanism, is a promising approach to reduce neural damage occurring in response to brain ischemia.     

Methylmercury increases glutamate release from brain synaptosomes and glutamate uptake by cortical slices from suckling rat pups: modulatory effect of ebselen.

During the early postnatal period the brain is extremely sensitive to external agents. Here, we examined the effect of subcutaneous injections of methylmercury (MeHg; 2 mg/kg) during the suckling period (postnatal days [PND] 3-10, 3-17, or 3-24) on glutamate release from brain synaptosomal preparations and on glutamate uptake by brain cortical slices of rat pups. The possible antagonist effect of ebselen against MeHg effect was also examined at PND 24. MeHg increased the basal (but not K+-stimulated) glutamate release and glutamate uptake at PND 24. A strong tendency of increase in the basal glutamate release from synaptosomes (p= 0.088) was observed at PND 17. Ebselen, which did not affect glutamate release and uptake per se, prevented both effects of MeHg. This study indicates that (1) the effect of MeHg on glutamate release could be involved in its toxicity; (2) the increase in the glutamate uptake could represent a pathophysiological response to MeHg-induced glutamate release; (3) the inhibitory effect of ebselen on MeHg-induced glutamate release could be related to its reported neuroprotective effects.     

Measurement of glutamate uptake and reversed transport by rat synaptosome transporters

To establish an assay system for evaluation of the uptake and reversed transport of glutamate, we examined the effects of Na(+)-concentration and pharmacological agents on the extracellular glutamate concentration ([Glu](o)) in rat cortical synaptosomes in vitro. There was a decrease and increase of the [Glu](o) at high and low Na(+) concentrations, respectively, in a Ca(2+)-free medium. The changes in [Glu](o) in both directions were temperature-sensitive, and reversed at around 30 mM of Na(+). Dihydrokainate (DHK), a non-transportable inhibitor selective for glial glutamate transporter GLT-1, suppressed the decrease in [Glu](o), and the reversal of [Glu](o) change was shifted to about 60 mM Na(+). There was no change in the maximum [Glu](o) at total Na(+) substitution. Further pharmacological analysis revealed that D-aspartate and DL-threo-beta-hydroxy-aspartate (THA), transportable substrates of glutamate transporters, increased the [Glu](o) in standard media. In contrast, beta-phenylglutamic acid, a structural analogue of glutamate, suppressed both the decrease in [Glu](o) in standard medium and the increase in [Glu](o) in low Na(+) medium. It is, thus, concluded that both the direction and the amount of [Glu](o) changes are determined by a balance of the uptake and reversed transport of glutamate, and that this assay system is suitable for evaluation of the effect of this on glutamate transporters.     

Effects of calcium antagonists on KCl-evoked calcium uptake by rat cortical synaptosomes

A series of calcium antagonists were used to study their blocking effect on high potassium-induced calcium uptake into rat cortical synaptosomes; these antagonists were classified into five groups: dihydropyridine group (i.e. nifedipine and nitrendipine), benzothiazepine group (i.e. diltiazem), phenylalkylamine group (i.e. verapamil and D600), phenothiazine group (i.e. trifluoperazine) and diphenylpiperazine group (i.e. flunarizine and cinnarizine). Voltage-dependent 45Ca2+-uptake into this fraction was measured after 20 sec KCl-induced depolarization. The ID30 values of the above-mentioned antagonists affecting 45Ca2+-uptake were calculated to be nitrendipine (80 microM), nifedipine (100 microM), verapamil (50 microM), D600 (15 microM), diltiazem (70 microM), trifluoperazine (7 microM), cinnarizine (1.2 microM) and flunarizine (0.7 microM). Our results reveal that in rat brain synaptosomal fractions, calcium influx via the voltage-gated calcium channel appears to be more sensitive to diphenylpiperazine and phenothiazine groups; whereas, phenylalkylamine, benzothiazepine and dihydropyridine groups were relatively insensitive. This contrasts with the well known data obtained from vascular smooth muscle, in which the dihydropyridine group is the most sensitive of all the groups studied. Our results suggest that calcium channels in neuronal tissue are most likely different from those in non-neuronal tissue.     

埃他卡林增强星形胶质细胞摄取谷氨酸的作用

目的研究新型ATP敏感性钾通道开放剂(KATPCO)埃他卡林(iptakalim,Ipt)对星形胶质细胞摄取谷氨酸的影响。方法取新生大鼠脑星形胶质细胞作原代培养，将Ipt直接作用于细胞，观察它对正常和6-羟基多巴胺(6-OHDA)损伤模型细胞摄取谷氨酸的影响；分别加入阳性对照药吡那地尔和非特异性钾通道阻断药格列本脲分析Ipt的作用机制。根据［³H］标记的D,L-谷氨酸摄入量判断细胞谷氨酸摄取作用强度。结果Ipt和吡那地尔都能增强星形胶质细胞的谷氨酸摄取作用、逆转6-OHDA引起的谷氨酸摄取抑制效应，预先加入格列本脲后，上述作用均被取消。结论埃他卡林可能通过促进钾通道开放增强星形胶质细胞摄取谷氨酸的作用。     

Studies of [3H]nitrendipine binding and KCl-induced calcium uptake in rat cortical synaptosomes

Rat cortical synaptosomal fraction was used to study whether there is a direct link between [3H]nitrendipine binding and KCl-induced calcium uptake. [3H]Nitrendipine exhibited reversible and saturable binding to this preparation. The equilibrium dissociation constant Kd was 0.6 nM and the maximal binding capacity, Bmax, was 120 fmol/mg of protein. The binding could be displaced by certain calcium channel antagonists, the potency of which was in the order: nitrendipine greater than nifedipine greater than D600 greater than verapamil greater than flunarizine. Voltage-dependent 45Ca2+-uptake into this fraction was measured after 20 sec KCl-induced depolarization. Nitrendipine at high concentration (10 microM) had little effect on 45Ca2+-uptake into brain synaptosomes. The order of the above-mentioned calcium antagonists affecting 45Ca2+-uptake was flunarizine greater than D600 greater than verapamil greater than nifedipine greater than nitrendipine. Our results suggest that high-affinity binding of [3H]nitrendipine is not directly linked to voltage-dependent calcium uptake in brain.     

Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes

Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.     

Release of endogenous glutamate from rat cortical slices in presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid

Summary The effect of the new glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), on the electrically evoked release or, rather, overflow of endogenous glutamate in superfusates from rat cortical slices was compared with that of dihydrokainate. In the absence of these presumed uptake inhibitors, electrical stimulation for 4 min at 1 Hz did not elicit a measurable glutamate overflow over baseline at all. Basal overflow increased concentration-dependently in the presence of 10–100 M L-trans-PDC, about 5-fold at 100 M. Also, electrical stimulation caused increases of glutamate overflow over basal levels progressive with increasing concentrations of trans-PDC; a stimulated overflow corresponding to about 50% of basal overflow was obtained at 100 M. Basal as well as evoked release in the presence of dihydrokainate did not exceed ca. 60% of that obtained with 100 M L-trans-PDC. In synaptosomes, L-trans-PDC much more than dihydrokainate caused a transient increase of spontaneous glutamate release which was diminished in the absence of Na⁺, indicating that it is transported into the cytoplasm by the glutamate carrier and induces some efflux of the amino acid from this compartment. Moreover, trans-PDC caused a weak to moderate inhibition of K⁺-evoked glutamate release from synaptosomes at 10–300 M, without obvious concentration-dependence.Glutamate overflow elicited from rat cortical slices by electrical field stimulation at 1 Hz was Ca²⁺-dependent to about 80%. Tetrodotoxin (0.3 M) reduced it by about 90%. Lowering the temperature from 37°C to 22°C increased the ratio between evoked and basal overflow.As an application for L-trans-PDC as a glutamate uptake inhibitor in release studies, the regulation of glutamate release by GABA_B receptors was investigated. At 1 Hz, (–)-baclofen reduced evoked glutamate overflow at and above 3 M by maximally 40% at 30 M. This maximal effect was not increased when higher or lower stimulation frequencies were used nor when the Ca²⁺ concentration in the medium was increased or lowered, nor when the slices were prepared from other brain areas (hippocampus or striatum). The GABA uptake inhibitor, SK&F 89976, had no significant effect on evoked glutamate overflow, and the potent GABA_B antagonist, CGP 55845, induced only a small increase, indicating that tonic inhibition of glutamate by GABA via GABA_B receptors was not marked. On the other hand, the GABA_B antagonist was able to prevent the inhibitory effect of (–)-baclofen when applied before it and to abolish it when applied afterwards. The conclusion is that L-trans-PDC is a useful tool in glutamate release studies in brain slices for many purposes, with the reservation that its inhibitory effect on evoked glutamate release in synaptosomes is not yet understood. Correspondence to: P. C. Waldmeier at the above address     

Inhibition of sodium-dependent L-leucine uptake in rat brain synaptosomes

Synaptosomes isolated from adult rat cerebral cortices were used for studying the uptake of L-leucine by the Na(+)-dependent route. Three non-metabolizable amino acid analogues, which had been used previously to discriminate the Na(+)-dependent A-type uptake system of animal cells, were employed in this study. It was found that Na(+)-dependent uptake of leucine was insensitive to inhibition by 2-aminoisobutyric acid (AIB) and N-methylaminoisobutyric acid (MeAIB) whereas N-methylalanine (NMA) was markedly inhibitory. Inhibition by NMA was stereospecific--only the L-isomer had a pronounced effect. Na(+)-dependent uptake of leucine as well as its inhibition by L-NMA were rather insensitive to changes in pH from 6 to 9. Kinetic analysis of inhibition by L-NMA of Na(+)-dependent uptake revealed a non-competitive type of inhibition with a Ki value of approximately 0.5 mM.     

Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes

1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol.
2. [³H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [³H]-5-HT uptake with apparent K_is of 0.27±0.04 and 0.76±0.04 μM, respectively. Morphine essentially failed to inhibit [³H]-5-HT uptake (K_i 0.50±0.30 M).
3. Methadone, morphine and tramadol were active in the hot plate test with ED₅₀s of 3.5, 4.3 and 31 mg kg⁻¹, respectively. At the highest tested dose (80 mg kg⁻¹) tramadol produced only 77±5.3% of the maximal possible effect.
4. When [³H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg⁻¹, s.c., equal to the ED₅₀ in the hot plate test) and methadone (35 mg kg⁻¹, s.c., equal to the ED₉₀ in the hot plate test) decreased uptake.
5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg⁻¹ and 15 to 120 mg kg⁻¹, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg⁻¹, s.c.) or tramadol (31 mg kg⁻¹, s.c.) was administered. [³H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%.
6. Rats chronically-treated with methadone showed a significant increase in [³H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [³H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals.
7. These results further support the hypothesis that [³H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [³H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

     

Nontransportable inhibitors attenuate reversal of glutamate uptake in synaptosomes following a metabolic insult.

Na+-dependent, high-affinity glutamate transporters in the central nervous system are generally credited with regulating extracellular levels of L-glutamate and maintaining concentrations below those that would induce excitotoxic injury. Under pathological conditions, however, it has been suggested that these same transporters may contribute to excitotoxic injury by serving as sites of efflux for cellular L-glutamate. In this study, we examine the efflux of [3H]D-aspartate from synaptosomes in response to both alternative substrates (i.e., heteroexchange), such as L-glutamate, and a metabolic insult (5 mM potassium cyanide and 1 mM iodoacetate). Exposure of synaptosomes containing [3H]D-aspartate to either L-glutamate or metabolic inhibitors increased the efflux of the radiolabeled substrate to over 200% of control values. Two previously identified competitive transport inhibitors (L-trans-2, 3-pyrrolidine dicarboxylate and dihydrokainate) failed to stimulate [3H]D-aspartate efflux but did inhibit glutamate-mediated heteroexchange, consistent with the action of nontransportable inhibitors. These compounds also attenuated the efflux of [3H]D-aspartate from synaptosomes exposed to the metabolic inhibitors. These results add further strength to the model of central nervous system injury-induced efflux of L-glutamate through its high-affinity transporters and identify a novel strategy to attenuate this process.     

The effect of desipramine on the uptake of mescaline by cortical synaptosomes

The uptake of ¹⁴C-labelled noradrenaline and mescaline into a synaptosome-rich preparation from the rat cerebral cortex was studied. Noradrenaline was accumulated by the synaptosome-rich preparation by a temperature-dependent process which was competitively inhibited by desipramine. Mescaline was accumulated by a temperature- and sodium-dependent process. The uptake of mescaline was not affected by desipramine. The uptake of noradrenaline was inhibited by mescaline in a non-competitive manner; this suggests that the uptake of mescaline is not brought about by the noradrenaline uptake mechanism.     

Effects of lithium on adrenergic amine uptake in rat brain synaptosomes

The effects of lithium on the uptake and retention of (?)-erythro-metaraminol (MA) and (?)-m-octopamine (OA) by synaptosomes from rat whole brain were examined after (a) addition of lithium chloride (1 mmol/l.) to synaptosomal fractions in vitro and (b) administration of lithium chloride (1 mmol/kg) i.p. to rats twice daily for 10 days prior to isolation of synaptosomes. Under control conditions, apparent K_m concentrations (5 min incubation) for MA and OA were 33 and 3 μM respectively. As incubation times were increased to 15 min, retention of MA increased and OA decreased. Retention of both amines was reduced by low temperature. Short term application of lithium to synaptosomes in vitro did not alter the uptake and retention of either amine during 5 or 10 min incubation periods. Chronic pretreatment of rats with lithium resulted in greater uptake and retention of low concentrations of MA when compared with synaptosomes from control (saline-treated) animals. Chronic lithium pretreatment did not alter retention of OA. The data support the hypothesis that lithium acts to increase synaptosomal membrane uptake of aromatic amines and decrease binding of accumulated amine.     

Troglitazone enhances glucose uptake induced by alpha-adrenoceptor stimulation via phosphatidylinositol 3-kinase in rat heart

1. Thiazolidinedione-derived agents have been reported to act as insulin sensitizers by augmenting insulin-dependent stimulation of phosphatidylinositol 3-kinase (PI3K) activity in a specific manner. It has been suggested that alpha-adrenoceptor stimulation mediates glucose uptake through PI3K in the heart. 2. To elucidate whether the thiazolidinedione-derived agent troglitazone (TRO) affects glucose uptake induced by alpha-adrenoceptor stimulation through PI3K, the rate of glucose uptake was quantified from the rate of accumulation of sugar phosphate (d[SP]/dt) using [(31)P] nuclear magnetic resonance spectroscopy after substitution of glucose with 2-deoxyglucose in rat perfused heart. Hearts were stimulated with 100 micromol/L phenylephrine plus 10 micromol/L propranolol (alpha-adrenoceptor stimulation), or 1 micromol/L isoproterenol plus 10 micromol/L phentolamine (beta-adrenoceptor stimulation). 3. The d[SP]/dt in the alpha- and beta-adrenoceptor-stimulated groups (0.45 +/- 0.06 and 0.42 +/- 0.04 micromol/min per g, respectively) was higher than that of the control group (0.27 +/- 0.02 micromol/min per g; P < 0.01). The addition of 2 microg/mL troglitazone to alpha-adrenoceptor stimulation augmented d[SP]/dt (0.72 +/- 0.08 micromol/min per g; P < 0.05 vs the alpha-adrenoceptor-stimulated group), which was effectively blocked by 3 micromol/L wortmannin (0.35 +/- 0.06 micromol/min per g; P < 0.01 vs troglitazone + alpha-adrenoceptor stimulation group). However, addition of troglitazone to beta-adrenoceptor stimulation did not alter d[SP]/dt (0.33 +/- 0.02 micromol/min per g; P = NS vs the beta-adrenoceptor-stimulated group). 4. These results indicate that troglitazone acutely enhances alpha-adrenoceptor stimulation on glucose uptake through a PI3K-dependent pathway, thus possibly improving glucose utilization in a catecholamine-released state.     

Na(+) channel effects of remacemide and desglycinyl-remacemide in rat cortical synaptosomes

The effects of the novel anticonvulsant, remacemide hydrochloride and its active metabolite, desglycinyl-remacemide, on veratridine-induced Na(+) influx in rat cortical synaptosomes were investigated and compared to established Na(+) channel blocking antiepileptic drugs. Remacemide and desglycinyl-remacemide reduced veratridine-stimulated Na(+) influx to 30.7% (IC(50)=160.6 microM) and 13.2% (IC(50)=85.1 microM) of control, respectively. Carbamazepine, phenytoin and lamotrigine similarly reduced Na(+) influx to 20.1% (IC(50)=325.9 microM), 79.8% and 27.9% (IC(50)=23.0 microM) of control, respectively. Resting internal Na(+) concentrations were significantly increased by desglycinyl-remacemide (1 and 10 microM) and, conversely, decreased by desglycinyl-remacemide and carbamazepine (both 1000 microM). These studies support previous electrophysiological investigations, which suggest that remacemide and desglycinyl-remacemide exert their antiepileptic effects, at least in part, by an inhibitory action on voltage-gated Na(+) channels. Desglycinyl-remacemide may have an additional action on Na(+) homeostasis that merits further exploration.     

Effects of lipid peroxidation on neurotransmitters uptake by rat synaptosomes

This paper described the effect of in vitro peroxidation achieved by 60 s or 5 min exposure to 60 microM Fe2+ with 200 microM ascorbic acid, on selected properties of rat brain synaptosomes reflecting some steps of chemical neurotransmission. The studies have revealed dramatic differences between dopamine, GABA and choline high affinity uptake systems in response to peroxidation. The uptake of calcium by synaptosomes submitted to free radical oxidation, mostly its K+-depolarization-dependent portion, was significantly suppressed. In contrast, peroxidation appeared not to influence the transmembrane synaptosomal potential. It is concluded, that peroxidation of synaptic endings modifies the lipid content of synaptoplasmatic membranes and consequently leads to severe disturbances in the function of neurotransmitter uptake systems and depolarization-dependent calcium channels.     

Halothane inhibits 5-hydroxytryptamine uptake by synaptosomes from rat brain

The activity of 5-hydroxytryptamine (serotonin; 5-HT) in the central nervous system modulates sleep, the perception of pain and other functions of the body which might possibly relate to mechanisms of general anesthetic action. While administration of anesthetics has inconsistent effects on the content of 5-HT in brain, in vivo, accumulated data suggest that anesthetic drugs alter 5-HT homeostasis in the central nervous system. In an effort to identify one possible site of anesthetic action, the effect of halothane on the uptake of 5-HT was studied in synaptosomes isolated from the brain of rat. Established techniques were used to prepare the synaptosomal fractions and measure high affinity transport of radiolabelled 5-HT. Halothane inhibited synaptosomal accumulation of 5HT in a concentration-dependent manner, but had little effect on the passive or spontaneous release of the accumulated 5-HT. Rates of uptake of 5HT were inhibited by 43% in the presence of 1 mM halothane and by 75% of control in the presence of 5 mM halothane; the apparent I50 for halothane was 1.0 +/- 0.1 mM and Lineweaver-Burk analysis indicated the inhibition to be competitive at concentrations around the I50.