GAT-1 (IC50 = 0.28 μM); GAT-2 (IC50 = 137.34 μM); GAT-3 (IC50 = 202.8 μM)

SKF89976A 显示出较弱的抗异常疼痛效果。 SKF89976A 轻度抑制中国仓鼠卵巢 (CHO) 细胞中的去甲肾上腺素转运蛋白 (NET)、多巴胺转运蛋白 (DAT) 和血清素转运蛋白 (SERT)，根据底物摄取实验确定，这些细胞是每种转运蛋白的稳定转导物。 3514、202.13 和 728.8 分别是 IC50 值 [1]。 GABA 传输阻断剂是 SKF89976-A。 GABA (1 mM) 产生的内向电流不受 100 μM 印防己毒素的影响，但被 GABA 转运抑制剂噻加宾 (10 μM) 和 SKF89976A (100 μM) 完全抑制。众所周知，100 μM SKF 89976-A 通过阻断 GABA 转运到细胞中来完全、可逆地阻断 GABA 诱导的电流 [2]。 GAT-1 被 SKF89976A 不可转移地阻断。此外，SKF89976-A 还可抑制强直性 GAT 的背景 GABA 激活可能导致的基线内向电流。在所研究的每个细胞中，SKF89976A (100 μM) 可逆地将 GAT 电流降低 67.9±4.4% (n= 19)。 GABA 诱导的 GAT 电流逐渐降低并被细胞内输注 20 μM SKF89976-A 所抑制，而 GABAAR 介导的电流 (n=4) 并未被阻止 [3]。

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GAT抑制剂抑制效力的比较为了分析GAT抑制剂对[3H]GABA摄取的影响，我们之前使用CHO细胞建立了稳定表达GAT亚型的细胞系。使用这些细胞系和稳定表达大鼠单胺转运蛋白SERT、NET和DAT的CHO细胞来比较抑制GAT亚型的效力，我们分析了GAT抑制剂对GABA和单胺类摄取的影响。抑制剂SKF89976A和（S）-SNAP5114分别对GAT-1（IC50:0.28 mM）和GAT-3（IC50:5.31 mM）表现出亚型选择性（表1）。然而，NNC05-2090能有效抑制GAT-1（IC50:29.62 mM），BGT-1（10.60 mM）的IC50值低2.5倍以上。NNC05-2090对GAT-2（45.29 mM）和GAT-3（22.51 mM）的IC50值也明显较高。这些结果表明，在所有检查的GAT抑制剂中，NNC05-2090在GAT抑制剂之间抑制BGT-1的效力最高。NNC05-2090还抑制SERT、NET和DAT（IC50:5.29 mM、7.91 mM和4.08 mM），这些IC50值与BGT-1的IC50值相似[1]。

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尽管已经在体外研究了胶质细胞GABA的摄取和释放，但切片中胶质细胞中的GABA转运蛋白（GATs）尚未得到表征。从大鼠小脑切片中的Bergmann胶质细胞获得全细胞膜片钳记录，以表征载体介导的GABA流入和流出。GABA在-70 mV下诱导内向电流，在药理学上可分为GABA（A）受体和GAT电流。在GABA（A/B/C）受体阻断剂存在的情况下，-70 mV下测得的平均GABA诱导电流为-48 pA，在-70和+50 mV之间向内整流，受到外部Na（+）去除的抑制，并因外部Cl（-）的减少而减少。GAT-1的不可转运阻断剂（SKF89976A和NNC-711）和所有GAT亚型的可运输阻断剂（尼泊苷酸）分别可逆地将GABA诱导的转运电流降低了68%和100%。BGT-1阻断剂（甜菜碱）没有效果。SKF89976-A和NNC-711也抑制了基线内向电流，这可能是由背景GABA激活的紧张性GAT引起的。底物激动剂，尼泊苷酸和β-丙氨酸，而不是甜菜碱，诱导了电压和钠（+）依赖性电流。在记录过程中，当Na（+）和GABA进入贴片移液管或细胞内GABA灌注时，SKF89976A阻断了在-60 mV时激活的基线外向电流，并且随着去极化电位的增加而增加。这种载体介导的GABA外排诱导了记录细胞上GABA（a）受体激活检测到的细胞外GABA的局部积累。总体而言，这些结果表明Bergmann胶质细胞表达GAT-1，其被环境GABA激活。此外，胶质细胞中的GAT-1可以反向工作，释放足够的GABA来激活附近的GABA受体[3]。

静脉注射 SKF89976A (0.3 mg/kg) 可引起轻微的镇痛作用。在PSL模型小鼠中，注射SKF89976A可以以剂量依赖的方式改善戒断阈值的降低[1]。

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GAT抑制剂对PSL小鼠模型中机械性异常性疼痛的影响部分坐骨神经结扎（PSL）模型小鼠同侧的机械性戒断阈值明显低于假手术动物（图1）。对侧的机械性戒断阈值没有降低（图2B、3B、4和5）。PSL小鼠模型中的机械性异常性疼痛不受盐水治疗的影响（图1B）。腹腔注射0.1mg/kg NNC05-2090 3小时后，PSL模型小鼠的机械戒断阈值有所增加（图1B）。另一方面，0.3 mg/kg的SKF89976A腹腔注射对PSL模型小鼠的戒断阈值没有产生任何显著影响（图1B）。静脉注射BGT-1抑制剂NNC05-2090显著逆转了PSL模型小鼠的机械性异常性疼痛（图2）。静脉注射NNC05-2090在0.01-0.1 mg/kg范围内显示出剂量依赖性反应。i.t.注射BGT-1抑制剂NNC05-2090也显著逆转了PSL模型小鼠的机械性异常性疼痛，并且通过i.t.向PSL模型鼠注射NNC05-2009，在15-150pmole范围内也显示出剂量依赖性反应（图3）。除静脉注射0.01mg/kg外，NNC05-2090的抗痛觉过敏作用在注射后1小时内达到峰值（图2和图3）。NNC05-2090给药后，对侧的戒断阈值没有显著差异。静脉注射（0.3 mg/kg）时，SKF89976A产生微弱的抗痛觉过敏反应（图4A）。如图5A所示，腹腔注射SKF89976A剂量依赖性地改善了PSL模型小鼠戒断阈值的降低。我们还注射了GAT-3抑制剂（S）-SNAP5114，以检测其对PSL小鼠机械性异常性疼痛的抗异常性疼痛作用。静脉注射和腹腔注射（S）-SNAP5114均未对PSL模型小鼠戒断阈值的降低产生显著影响（图4B和5B）[1]。

记录过程中GABA转运蛋白阻断剂的细胞内灌注。[3]
GABA转运蛋白阻断剂的细胞内灌注如其他人之前报道的那样，用于单药或多药应用（Tang等人，1990）。我们使用了带有灌注口的直移液管支架（EH-U2，E.W.Wright）。通过灌注口，将聚乙烯管（内径0.86mm，外径1.27mm）引入足够远的地方，以很好地到达贴片移液管溶液中。在记录过程中，将装有LY填充的细胞内溶液的1ml注射器通过细长的塑料移液管尖端连接到聚乙烯管上。在添加贴片移液管之前，手动施加正压，将试管充满到底，去除气泡，并观察溶液的流出情况。然后，在施加负压以防止任何溶液泄漏但不在管端添加气泡后，将贴片移液管插入支架中。为了灌注含有GABA或SKF89976A的LY填充溶液，手动施加正压以添加足够的溶液，使贴片移液管中的体积加倍（约20μl）。GABA和SKF89976A的浓度加倍，以获得细胞中预期的最终浓度。

Drug preparation and administration [1]
Animals were used in experiments approximately 2 weeks (11 – 16 days) following nerve ligation. Animals were administered NNC05-2090, SKF89976A, (S)- SNAP5114, or amitriptyline. NNC05-2090, SKF89976A, and (S)-SNAP5114 were dissolved in dimethyl sulfoxide (DMSO) and diluted appropriately with ACSF or saline (the final concentration of DMSO was less than 0.5%). The composition of ACSF (in mM) was 142 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.25 mM NaH2PO4, 10 mM d-glucose, 10 mM HEPES, and 0.05% fatty acid-free bovine serum albumin (pH 7.4). The intraperitoneal (i.p.) injection of drugs was administered in a volume of 0.1 ml/10 g body weight. When given intravenously (i.v.), solutions were injected into the tail vein in a volume of 0.1 ml/10 g body weight. The head of a mouse was placed into a plastic cap and the body was held with one hand for an intrathecal (i.t.) injection. A 27-gauge needle attached to a Hamilton microsyringe was inserted into the subarachnoid space between the L5 and L6 vertebrae of the conscious mouse and 5 ml of the drug solution was slowly injected, as described by Hylden and Wilcox (31). Accurate placement of the needle was confirmed by a quick “flick” of the mouse’s tail, as described by Honda et al.

[1]. Antiallodynic action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor. J Pharmacol Sci. 2014;125(2):217-26.

[2]. Glutamate modulation of GABA transport in retinal horizontal cells of the skate. J Physiol. 2003 Feb 1;546(Pt 3):717-31.

[3]. GAT-1 and reversible GABA transport in Bergmann glia in slices. J Neurophysiol. 2002 Sep;88(3):1407-19.

1-(4,4-Diphenylbut-3-enyl)-3-piperidinic acid is a diarylmethane. The GABAergic system in the spinal cord has been shown to be involved in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In this study, we used a sciatic nerve ligation mouse model to compare betaine/GABA transporter (BGT-1) with other GAT isoforms to determine its role in neuropathic pain. 1-(3-(9H-carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol (NNC05-2090) is a moderately selective inhibitor of BGT-1 that showed anti-hyperalgesic effects in model mice via both intrathecal and intravenous administration. On the other hand, the selective GAT-1 inhibitor SKF89976A showed weak anti-hyperalgesic activity, while the selective GAT-3 inhibitor (S)-SNAP5114 had no anti-hyperalgesic activity. A systematic analysis of GABA uptake by these compounds in CHO cells stably expressing BGT-1 revealed that NNC05-2090 inhibited not only BGT-1 but also serotonin, norepinephrine, and dopamine transporters. Substrate uptake assays in CHO cells stably expressing each transporter showed IC50 values of 5.29, 7.91, and 4.08 μM for NNC05-2090, respectively. These values are similar to the IC50 value of BGT-1 (10.6 μM). These results indicate that the anti-hyperalgesic effect of NNC05-2090 is due to its inhibitory effect on BGT-1 and monoamine transporters. [1] The transport of the amino acid GABA to neurons and glial cells plays a key role in regulating the effects of GABA on the vertebrate retina. We investigated the regulatory effect of glutamate (a potential neurotransmitter in vertebrate photoreceptor cells) on GABA-induced transport currents in retinal level cells. We used whole-cell voltage clamp technology to detect the effects of enzymatically isolated ray lateral level cells. The inward current induced by GABA (1 mM) was completely inhibited by the GABA transport inhibitors tiagabin (10 μM) and SKF89976A (100 μM), but was not affected by 100 μM cucurbitacin. Pre-application of 100 μM glutamate significantly reduced the GABA-induced current. Glutamate inhibited the dose-response curve of GABA, but did not cause a transverse shift in the curve or change the voltage dependence of the current. The ionotropic glutamate receptor agonists fucoidan and AMPA also reduced GABA-induced currents, the effects of which could be blocked by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither induced nor altered GABA-induced currents, and metabolized glutamate analogs had no effect. The inhibitory effects of glutamate and fucoidan on GABA-induced currents were weakened when extracellular calcium ions were removed or when a high concentration of the calcium chelator BAPTA was added to the recording electrode. Caffeine (5 mM) and carotenoids (2 nM), which are known to alter intracellular calcium ion levels, also reduced GABA-induced currents, but simple depolarization-induced increases in calcium ion concentration did not have this effect. Our data suggest that glutamate regulates GABA transport in retinal cells through a calcium-dependent process, implying a close physical link between calcium-permeable glutamate receptors and GABA transporters in these cells. [2]

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Although the uptake and release of GABA by glial cells has been studied in vitro, GABA transporters (GATs) have not yet been characterized in glial cells in brain slices. We used whole-cell patch-clamp technique to record the electrophysiological activity of Bergmann glial cells in rat cerebellar slices to characterize carrier-mediated GABA influx and efflux. GABA induces an inward current at -70 mV, which can be pharmacologically separated into GABA(A) receptor current and GAT current. In the presence of GABA(A/B/C) receptor blockers, the mean GABA-induced current at -70 mV was -48 pA, exhibiting inward rectifying characteristics between -70 and +50 mV. This current was inhibited by extracellular Na⁺ removal and attenuated by extracellular Cl⁻ reduction. Nontransporter inhibitors of GAT-1 (SKF89976A and NNC-711) and transporter inhibitors of all GAT isoforms (nipoic acid) reversibly reduced GABA-induced transport currents by 68% and 100%, respectively. BGT-1 inhibitors (betaine) did not have this effect. SKF89976-A and NNC-711 also inhibited baseline inward currents that could be caused by persistent activation of GAT by background GABA. The substrate agonists nipoic acid and β-alanine (but not betaine) induced voltage-dependent and sodium-dependent currents. SKF89976A blocked baseline outward currents activated at -60 mV and enhanced with increasing depolarization potential when sodium ions and GABA were added to the patch-clamp electrode or when intracellular GABA was perfused during recording. This carrier-mediated GABA efflux leads to the local accumulation of extracellular GABA, which can be detected by recording the activation of GABA_A receptors on the cell. Overall, these results indicate that Bergman's glial cells express GAT-1 and that GAT-1 can be activated by surrounding GABA. Furthermore, GAT-1 in glial cells can also have a reverse effect, releasing enough GABA to activate nearby GABA receptors. [3]

C22H25NO2.HCL

371.90034

371.165

C, 71.05; H, 7.05; Cl, 9.53; N, 3.77; O, 8.60

85375-15-1

85375-15-1 ( free base);85375-85-5 (HCl);

92409

Light yellow to khaki solid powder

531.4ºC at 760 mmHg

275.2ºC

5.044

40.54

1

3

6

25

427

0

Cl.O=C(C1CCCN(CC/C=C(\C2C=CC=CC=2)/C2C=CC=CC=2)C1)O

TXQKSMSLZVKQBI-UHFFFAOYSA-N

InChI=1S/C22H25NO2/c24-22(25)20-13-7-15-23(17-20)16-8-14-21(18-9-3-1-4-10-18)19-11-5-2-6-12-19/h1-6,9-12,14,20H,7-8,13,15-17H2,(H,24,25)

1-(4,4-diphenylbut-3-enyl)piperidine-3-carboxylic acid

SKF89976A HCl; 85375-85-5; Skf 89,976A; SKF89,976A; 1-(4,4-diphenylbut-3-en-1-yl)piperidine-3-carboxylic acid; N-(4,4-Diphenyl-3-butenyl)nipecotic acid; N-(4,4-Diphenyl-3-butenyl)homo-beta-proline; SKF-89976; CHEMBL38686; SKF 89976-A; SKF-89976-A; SKF89976A hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~268.89 mM)
H2O : ~20 mg/mL (~53.78 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.72 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.72 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.72 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 25 mg/mL (67.22 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。