GABAB receptor

3-Aminopropylphosphinic acid (10 μM) functions as an anti-aging drug for skin[4]. It also induces a concentration-dependent suppression of the cholinergic twitch contraction in the electrically stimulated ileum (IC50=1.84-0.23 μM)[2].

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The effects of phosphonic analogues of GABA, beta-alanine and glycine on guinea-pig ileum longitudinal muscle were measured. 3-Aminopropylphosphinic acid (AMPh) and 2-aminoethylphosphonic acid (2-AEPh) were devoid of any effect both in non-stimulated preparations and in electrically-stimulated preparations. The phosphonic analogue of GABA, 3-aminopropylphosphonic acid (3-APPh) possessed a GABAB agonistic effect (relaxation and inhibition of twitch response) at doses of 10(-3)M. No agonistic effect on GABAA receptors was observed. 3-APPh at doses tested (2 X 10(-4)M and 10(-3)M) also displayed antagonistic action on the effects of GABAB agonists producing a parallel shift of the log dose-effect curves of GABA- and (-)-baclofen-inhibition of twitch responses. In contrast 3-APPh did not antagonize the inhibitory effect of morphine and noradrenaline. The contractile effect of GABA, mediated via GABAA receptors, was unaffected by 3-APPh(10(-3)M). It is concluded that 3-APPh is a partial agonist at the GABAB site in guinea-pig ileum. [1]

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1. 3-Aminopropylphosphinic acid, a gamma-aminobutyric acid (GABA) analogue, was tested for activity on guinea-pig isolated ileum and rat isolated anococcygeus muscle preparations. The effects of 3-aminopropylphosphinic acid were compared with those of GABA and baclofen. 2. In the electrically stimulated ileum, 3-aminopropylphosphinic acid, like GABA and baclofen, caused a concentration-dependent inhibition of the cholinergic twitch contraction, the IC50 value being 1.84 +/- 0.23 microM (n = 12). Unlike GABA, but like baclofen, 3-aminopropylphosphinic acid did not produce an initial contraction. 3. The inhibitory effects of 3-aminopropylphosphinic acid and baclofen in the guinea-pig ileum were not significantly antagonized by bicuculline (10 microM), phentolamine plus propranolol (both 1 microM), yohimbine (1 microM), naloxone (1 microM), impromidine (1 microM) or 8-phenyltheophylline (10 microM). The inhibitory effects of 3-aminopropylphosphinic acid, but not of baclofen, were however antagonized by phaclofen (500 microM). In addition the effects of 3-aminopropylphosphinic acid were abolished by baclofen desensitization in the guinea-pig ileum. 4. 3-Aminopropylphosphinic acid, GABA and baclofen reduced the twitch contraction evoked by electrical field stimulation in the rat anococcygeus muscle. The IC50 for 3-aminopropylphosphinic acid inhibition of the anococcygeus contraction was 0.89 +/- 0.15 microM (n = 8). 5. It is concluded that 3-aminopropylphosphinic acid is a potent, selective GABAB agonist, being seven times more potent than baclofen in the guinea-pig ileum and five times more potent than baclofen in the rat anococcygues muscle preparations. [2]

3-Aminopropylphosphinic acid (5 mg/kg; iv) inhibits the guinea pigs' vagal bronchospasm by blocking the actions of GABA[3].

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GABA is a known inhibitory neurotransmitter in the CNS. Recent studies have also demonstrated the presence of GABA in peripheral tissue, including lung. To delineate a role for GABA in lung, the effect of GABA and selective GABA agonists and antagonists on neuronally-induced airway contractions in guinea pigs were studied. In vitro, tracheal contractions induced by electrical field stimulation (EFS) were inhibited by tetrodotoxin and atropine indicating that the contractions were mediated by neuronal release of acetylcholine. The contractions caused by EFS, but not those by exogenous acetylcholine, were inhibited by GABA (EC50 = 4.5 microM) and the selective GABA-B agonist baclofen (EC50 = 9 microM), but not by the GABA-A agonist, muscimol. The inhibitory effect of baclofen was not affected by the GABA-A antagonist, bicuculline, but was significantly reversed with the GABA-B antagonists, 3-Aminopropylphosphinic acid (3-APPA) (pA2 = 4.5) and 2-hydroxysaclofen (pA2 = 4.1). In vivo, vagal nerve stimulation (5 V, 20 Hz, 0.5 ms, 5 s) in anesthetized, mechanically ventilated guinea-pigs caused cholinergic-dependent bronchospasms that were inhibited by intravenous GABA (3 and 10 mg/kg) and baclofen (1-10 mg/kg), but not by muscimol. The inhibitory effects of GABA and baclofen against vagal bronchospasm were blocked by 3-APPA (5 mg/kg, i.v.), but not by bicuculline. Responses to the GABA-B agonists were unaltered after the treatment of animals with phentolamine or propranolol to block alpha-adrenergic and beta-adrenergic receptors, respectively. Bronchospasm due to intravenous methacholine was also unchanged by GABA and baclofen [3].

‘In vitro’ preparations [1]
Experiments were performed in male guinea-pigs (weight range 300-500 g); animals weregUed by a blow on the head; segments ofterminal ileum were quickly removed and placedin a modified Krebs solution of the followingcomposition (mM): KHzP041.3, KCI 3.4, NaCl134.7, CaC12 2.8, MgS04 0.6, NaHC03 16.3,glucose 7.7. Strips of ileal longitudinal musclewith myenteric plexus attached were obtained bythe method of Paton & Zar (1968). Segmentswere mounted in an organ bath, bubbled with amixture of 5% COZ and 95% 02and maintained at37°C. When necessary, electrical stimulation wasperformed following the method described byPaton (1963) and Paton & VLzi (1969). Thestimuli ( 1.5 times maximal rectangular pulses of 1msec duration, at a frequency of 6 per min) were applied using two coaxial platinum electrodesfrom a MARB stimulator. The ileum wasconnected to an isometric transducer under aresting tension of 0.5 g and responses wererecorded on a MARB polygraph. Preparationswere allowed to equilibrate for 60 min beforedrug administration. Drugs were administered ina volume that never exceeded 1% of the total bathvolume (4 ml). The occurrence of desensitization to GABA was prevented by allowing 20 min toelapse between drug administrations. In fact, aswe previously observed (Giotti et al., 1983a;b),both in non-stimulated and in electrically-stimulated preparations, submaximal doses of GABA, repeated at intervals ranging between 15-30 min, evoked the same effects.

Animal/Disease Models: guinea pigs[3]
Doses: 5 mg/kg
Route of Administration: IV
Experimental Results: Blocked the inhibitory effects of GABA against vagal broncho-spasm.

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Guinea-pig ileum [2]
Male guinea-pigs (300-450g) were killed by a blow to the head and bled out. Segments of ileum of approximately 3 cm in length were removed from an area 10-15cm proximal to the ileo-caecal junction. Preparations were immediately placed in a modified Krebs solution (BUlbring, 1953) which was continually bubbled with 95% 02 and 5% CO2. Segments were freed of their mesenteric attachment and suspended in 10 ml organ baths containing Krebs solution under an isometric tension of 1 g. Isometric contractions to transmural stimulation (Paton, 1954) were recorded with strain gauge transducers and displayed on an Ormed Multitrace pen recorder. Electrical stimulation of preparations was achieved by passing rectangular pulses (duration 0.5 ms; frequency 0.1 Hz; supramaximal voltage (25-35 V)), from a Grass SD11 stimulator via platinum electrodes. Preparations were allowed to equilibrate for 1 h prior to addition of compounds to the organ bath.

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Rat anococcygeus [2]
Male Wistar rats (200-300 g) were killed by a blow to the head and bled out and their anococcygeus muscles removed as previously described (Gillespie, 1972). The muscles were mounted in organ baths (10 ml) containing modified Krebs solution which was continually gassed with 95% 02 and 5% CO2. A resting tension of 0.5 g was applied and the preparations were field stimulated from Grass SD11 stimulators via platinum ring electrodes with the following stimulus parameters: pulse duration 1 ms; frequency 10 Hz; for 1 s. Isometric muscle responses were measured with a strain gauge transducer and displayed on an Ormed Multitrace pen recorder. Typical tension responses generated in either preparation as a result of electrical stimulation were between 2 and 4 g force. Preparations generating less tension were rejected. In both preparations, sequential agonist concentration-response curves were constructed, allowing 30 min between additions of agonist to minimize tachyphylaxis. When antagonists were used, concentration-response curves to 3-Aminopropylphosphinic acid in the presence of the antagonist were constructed after an initial equiliThe following drugs were used: y-amino-n-butyric acid, (±+baclofen, (±)-propranolol (ICI), phentolamine mesylate, yohimbine hydrochloride, naloxone hydrochloride, 8-phenyltheophylline, impromidine oxylate, bicuculline methiodide, phaclofen and 3-Aminopropylphosphinic acid (prepared by the method of Dingwall et al., 1987a, b). With the exception of 8-phenyltheophylline, all compounds were dissolved in distilled water, subsequent dilutions being made in distilled water and compounds were added to the organ bath in volumes no greater than 1% total volume. 8- Phenyltheophylline was made up in 80% methanol/ 2 M NaOH, subsequent dilutions being made in distilled water and all vehicle controls were negative. Propranolol and phentolamine were added directly to the Krebs solution.

[1]. GABA-related activities of amino phosphonic acids on guinea-pig ileum longitudinal muscle. J Auton Pharmacol. 1986 Sep;6(3):163-9.
[2]. 3-Aminopropylphosphinic acid--a potent, selective GABAB receptor agonist in the guinea-pig ileum and rat anococcygeus muscle. Br J Pharmacol. 1989 Aug;97(4):1292-6.
[3]. Prejunctional GABA-B inhibition of cholinergic, neurally-mediated airway contractions in guinea-pigs. Pulm Pharmacol. 1991;4(4):218-24.
[4]. 3-Aminopropyl dihydrogen phosphate (3-APPA; 3-aminopropane phosphoric acid); a novel anti-aging substance. Journal of Investigative Dermatology, vol. 4, no. 106, 2015, p. 895.

Bioulac, De Tinguy-Moreaud, Vincent, and Neuzil (1979) described the inhibitory effect of 3-APPh on central neuronal firing, an effect insensitive to dicyclopentene (possibly a GABAB receptor); furthermore, Cates, Li, Yakashe, et al. (1984) recently confirmed that this compound has a certain affinity for the GABA binding site. Regarding GABAA receptors, none of the tested phosphonates showed agonistic activity against this subtype: only 3-APPh caused ileal contraction, but this contraction was insensitive to dicyclopentene and bitter substances, and GABA could not desensitize this contraction. The ineffectiveness of 3-APPh against GABAE receptors is also consistent with previous binding studies at the central level (Galli, Zilletti, Scotton, Adembri, and Giotti, 1980; Cates et al., 1984). However, our primary focus was on investigating the antagonistic effects of these drugs on GABA receptors: none showed interference with GABAA receptor-mediated contraction; conversely, high doses of 3-APPh exhibited significant antagonism against GABAB-mediated inhibition; the antagonism of 3-APPh was reversible and specific to GABAergic drugs. 2-AEPh and 3-APPh were ineffective. One question arising from this is whether the agonistic effect of 3-APPh on GABAB receptors interferes with its antagonistic effect on the same receptor through desensitization. The fact that 3-APPh rapidly reverses the effect of GABAA on muscle twitching (Figure 6) supports the idea of a direct antagonistic effect. Furthermore, at lower test doses (2 × 10⁻⁵ M), the antagonistic effect of 3-APPh on the GABAB effect appeared competitive: the dose-response curves shifted parallel, with little change in the maximum effect. On the other hand, desensitization may occur at the highest dose (10⁻³ M); in fact, at this concentration, the dose-response curve tends to flatten, which may be due to desensitization. In summary, the GABA phosphonate analog 3-aminopropylphosphonic acid (3-APPh) exhibits characteristics of both a weak GABA_B receptor antagonist and a weak GABA_B receptor agonist, while having no effect on GABA_A receptors (agonist or antagonist). This characteristic may be interpreted as partial agonism, unlike other drugs considered GABA_A receptor antagonists (such as 3-APS, which has characteristics of both a strong GABA_A receptor agonist and a weak GABA_B receptor antagonist) (Table 2). Therefore, it appears that no effective GABA_B receptor antagonist has yet been discovered. However, a precise understanding of the differences between drugs acting on GABAB receptors can help in using them as experimental tools and in developing more selective GABAB antagonists. [1] Several studies have explored the requirements for GABAB receptor activity, showing that even small changes to the baclofen molecule can lead to a complete loss of its activity (Olpe et al., 1980; Krogsgard-Larsen, 1988). To date, no GABAB agonist has been found to be more effective than baclofen in the in vitro systems described herein. 3-aminopropylphosphonic acid is 7 times more potent than racemic baclofen in the guinea pig ileum and 5 times more potent than racemic baclofen in the rat anal and caudal muscles. Studies reported by Dingwall et al. (1987a, b) have shown that 3-aminopropylphosphonic acid has a 20-fold greater affinity for GABAB receptors than baclofen. Interestingly, a comparison is made between GABA phosphonate analogs described in this paper as potent agonists and those described as weak partial agonists/antagonists (Luzzi et al., 1986), despite the only structural difference being the acidic moiety. 3-Aminopropylphosphonic acid has a distorted tetrahedral arrangement around its phosphorus atom, contains only one acidic proton, and has a negative charge distributed across the two oxygen atoms, thus making it similar to GABA in many respects. 3-Aminopropylphosphonic acid has a nearly tetrahedral arrangement around its phosphorus atom, contains two acidic protons (pH-dependent), and has a negative charge distributed across the three oxygen atoms. In guinea pig ileum, 3-aminopropylphosphonic acid appears to interact with presynaptic GABA receptors located at cholinergic nerve endings. Since the Sid value of 3-aminopropylphosphonic acid is not significant and it does not induce initial contraction, it may not possess GABAA receptor agonist activity. Although a more specific O value is needed for final statistical validation of this hypothesis, the results using the known specific receptor antagonist Iic acidm(l) indicate that 3-aminopropylphosphonic acid does not interact with any other type of receptor. Furthermore, tissues desensitized to baclofen no longer respond to 3-aminopropylphosphonic acid. Clonidine (with a mean above average, p < 0.05) was used to test the specificity of GABAB receptors. Clonidine showed comparable potency to baclofen during GABAB receptor desensitization, with significant differences before and after desensitization. Faclofen is considered a weak but selective GABAB antagonist (Kerr et al., 1987; Dutar and Nicholl, 1988). We were able to demonstrate that the response to 3-aminopropylphosphonic acid was inhibited in guinea pig ileum, but the response to baclofen was not inhibited. The reason is currently unclear, although recent studies have claimed that faclofen exhibits GABA antagonistic activity in multiple testing systems (Karlsson et al., 1988; Soltesz et al., 1988), but its structure does support our hypothesis that the 3-aminopropylphosphonic acid (MPA) interacts with the GABA receptor. [2]
In guinea pig ileum and rat anal-coccygeal muscle, the response time course of MPA was similar to that of GABA and baclofen. In guinea pig ileum, the response to the GABAB receptor agonist was generally transient, while in rat anal-coccygeal muscle, the response was more persistent (Bowery et al., 1981; Muhyaddin et al., 1982). Our pharmacological studies support the view of Dingwall et al. (1987a, b) that MPA is a potent and selective GABAB receptor agonist. Further research on this compound may help elucidate the physiological role of the GABAB receptor in the mammalian gut. [2]

C3H10NO2P

122.08286

122.037

103680-47-3

6335948

Typically exists as solid at room temperature

0.001mmHg at 25°C

-1.4

2

3

3

7

66

0

NCCCP(=O)O

MQIWYGZSHIXQIU-UHFFFAOYSA-O

InChI=1S/C3H8NO2P/c4-2-1-3-7(5)6/h1-4H2/p+1

3-aminopropyl-hydroxy-oxophosphanium

3-aminopropylphosphinic acid; 103680-47-3; 3-aminopropyl-hydroxy-oxophosphanium; 3-Aminopropanephosphinic acid; (3-aminopropyl)phosphinic acid; CGP-27492; 3-APPA; Cgp 27492;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)