Kainate receptors (IC50: 35 nM)

To broaden the survey of pharmacological agents we used SYM 2081, which is a potent and highly selective kainate receptor agonist, with an IC50 for inhibition of [3H]-kainate binding of 35 nM and almost 3000- and 200-fold selectivity for kainate receptors over AMPA and NMDA receptors respectively. In addition, we examined a selective allosteric AMPA receptor antagonist GYKI 52466 (Lodge, 2009). But GYKI 52466 at even 100 μM would not dissolve in HL3 saline. This might be due the salt concentration and solubility in HL3 compared to water. So we could not further assess the actions of GYKI 52466 as it would not dissolve in the media. As far as we know, these compounds have not been tried before at the Drosophila NMJ. Surprisingly, SYM 2081 at 1 mM depolarized the muscle in 6 out of 6 preparations (Fig. 6A; n = 6; p < 0.05 Wilcoxon rank sum non-parametric). On average there was a 12% increase (less negative) in the resting membrane potential (a 100% change would be considered 0 mV). The SYM 2081 (1 mM) also presented a 40% decrease in the EPSP amplitude which was more of a decrease than expected given and slight depolarization in the muscle (Fig. 6B; n = 6; p < 0.05 Wilcoxon rank sum non-parametric). However, SYM 2081 at 0.1 mM had no significant effect on membrane depolarization or on the amplitude of the EPSP. [1]

Glutamate acts at central synapses via ionotropic (iGluR--NMDA, AMPA and kainate) and metabotropic glutamate receptors (mGluRs). Group I mGluRs are excitatory whilst group II and III are inhibitory. Inhibitory mGluRs also modulate peripherally the mechanosensitivity of gastro-oesophageal vagal afferents. Here we determined the potential of excitatory GluRs to play an opposing role in modulating vagal afferent mechanosensitivity, and investigated expression of receptor subunit mRNA within the nodose ganglion. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of selective GluR ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors, which respond only to mucosal stroking. The selective iGluR agonists NMDA and AMPA concentration-dependently potentiated afferent responses. Their corresponding antagonists AP-5 and NBQX alone attenuated mechanosensory responses as did the non-selective antagonist kynurenate. The kainate selective agonist SYM 2081 had minor effects on mechanosensitivity, and the antagonist UBP 302 was ineffective. The mGluR5 antagonist MTEP concentration-dependently inhibited mechanosensitivity. Efficacy of agonists and antagonists differed on mucosal and tension receptors. We conclude that excitatory modulation of afferent mechanosensitivity occurs mainly via NMDA, AMPA and mGlu5 receptors, and the role of each differs according to afferent subtypes. PCR data indicated that all NMDA, kainate and AMPA receptor subunits plus mGluR5 are expressed, and are therefore candidates for the neuromodulation we observed.[2]

Effects of GluR agonists and antagonists on mechanical sensitivity of vagal afferents [2]
After mechanical sensitivity of gastro-oesophageal vagal afferents had been established, the effects of various agonists and antagonists on mechanical sensitivity were determined. Kynurenate (10−7–10−5m) was added to the superfusing Krebs' solution, and was allowed to equilibrate for 20 min, after which the stimulus–response curves were re-established. The agonists N-methyl d-aspartate (NMDA) (10−7–10−5m), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA; 10−5–10−4m), and (25, 4R)-4-methylglutomic acid (SYM 2081; 10−6–10−5m) were added to the superfusion solution. The antagonists d(−)-2-amino-5-phosphono-pentanoic acid (AP-5) (10−6–10−5m), 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) (10−9–10−8m) and (s)-1-(2-amino-2-carboxyethyl)-2-(2-carboxybenzyl)pyrimidine-2,4-dione (UBP 302; 10−7–10−5m) were added to a ring placed on the preparation over the receptive field.

[1]. Furthering pharmacological and physiological assessment of the glutamatergic receptors at the Drosophila neuromuscular junction. Comp Biochem Physiol C Toxicol Pharmacol. 2009 Nov;150(4):546-57.

[2]. Potentiation of mouse vagal afferent mechanosensitivity by ionotropic and metabotropic glutamate receptors. J Physiol. 2006 Nov 15;577(Pt 1):295-306.

(4R)-4-methyl-L-glutamic acid is a 4-methyl-L-glutamic acid in which the methyl group at position 4 adopts R-configuration. It has a role as an excitatory amino acid agonist. It is a conjugate acid of a (4R)-4-methyl-L-glutamate(1-).
2S,4R-4-Methylglutamate has been reported in Lathyrus japonicus with data available.

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In this study we showed, in a concentration-dependent manner, that kainate rapidly reduced the amplitude of evoked EPSPs as well as the spontaneous quantal events. The lack of any response to t-ACPD and ATPA as well as the inability of these compounds to mimic kainate's action suggest that there is not a presynaptic mechanism of kainate-like or metabotropic autoreceptors on these motor nerve terminals. However, the kainate receptor agonist, domoic acid, did follow the actions of kainate in reducing the evoked EPSPs. We suggest this action is due to domoic acid and kainate acting as an antagonist on the postsynaptic quisqualate receptors present on muscle and that no presynaptic contribution can be proposed for the rapid reduced amplitude of evoked EPSP and quantal responses. The agonist action of SYM 2081 at high concentration suggests that this compound may have some action on quisqualate receptors, perhaps even in other invertebrates. However, SYM 2081 does behave like kainate in this preparation. The reduced frequency in the spontaneous quantal responses in the presence of kainate or domoic acid appears to be due to the responses decreasing in size so they are not detectable from the noise or that the postsynaptic receptors are fully blocked. [1]

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In conclusion, this study in conjunction with a previous one from our group, has completed a comprehensive characterization of the influence of all major GluR subtypes on vagal afferent mechanosensitivity. There is clearly scope for both excitatory and inhibitory modulation of afferent sensitivity by glutamate from both exogenous and endogenous sources, providing a balance to achieve normal vagal afferent function. This balance is clearly possible to manipulate pharmacologically. How it may be altered in disease states we hope will be the subject of our continuing investigations.[2]

C6H10NO4-

160.1479

161.069

C, 44.72; H, 6.88; N, 8.69; O, 39.71

31137-74-3

95883

White to off-white solid powder

329.4ºC at 760mmHg

153ºC

Lathyrus japonicus

0.209

3

5

4

11

168

2

C[C@H](C[C@@H](C(=O)O)N)C(=O)O

KRKRAOXTGDJWNI-DMTCNVIQSA-N

InChI=1S/C6H11NO4/c1-3(5(8)9)2-4(7)6(10)11/h3-4H,2,7H2,1H3,(H,8,9)(H,10,11)/t3-,4+/m1/s1

(2S,4R)-2-amino-4-methylpentanedioic acid

(2S,4R)-2-amino-4-methylpentanedioic acid; (2R,4S)-4-amino-4-carboxy-2-methylbutanoate; 31137-74-3; sym 2081; (2S,4R)-4-Methylglutamic acid; 2S,4R-4-Methylglutamate; SYM-2081; 77842-39-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

H2O : ~50 mg/mL (~310.25 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)