Moreover we revealed that alanine and

Moreover, we revealed that β-alanine and taurine, a structural analog of β-alanine, have different receptor affinity in the SG neurons of the spinal dorsal horn. Additionally, previous studies indicate that taurine regulates nociceptive information at the spinal cord level, and we have previously demonstrated that taurine activates glycine and GABAA receptors in SG neurons at high concentrations [22]. However, the present study demonstrated that β-alanine activates glycine receptors selectively at a high concentration (3 mM). Both β-alanine and taurine activate glycine and/or GABAA receptors; however, previous studies have demonstrated that the agonist potency of β-alanine on glycine receptors is stronger than taurine [[32], [33], [34], [35]]. Additionally, Stephan et al. reported that β-alanine can behave as a full agonist for glycine receptors, whereas taurine cannot [36]. Considering the above, β-alanine may be an amino AZD7687 sale that acts on glycine receptors more selectively than taurine. We used a concentration of 0.3 mM in the pharmacological study, which has been used in previous studies [37,38] and is lower than the EC50, as the present study we demonstrated. It was reported that β-alanine in the CNS is widely distributed in region-specific concentrations [39]. The mean concentration of β-alanine in the cerebrospinal fluid is between 0.02 and 0.1 mM [2,40]. Massive concentration increases of β-alanine occur following electrical stimuli [[4], [5], [6],11]. These data suggest that the concentration of β-alanine used in our study is physiologically relevant. In conclusion, β-alanine hyperpolarizes membranes in SG neurons by activating glycine receptors and increasing Cl− conductance. Taken together, these results show that together with the classical neurotransmitters, glycine and GABA, β-alanine is an important neurotransmitter and/or modulator in SG neurons, and may be involved in antinociception.
Funding This work was supported by a Grant-In-Aid for Scientific Research (No. 17K16725) by the Japan Society for the Promotion of Science, Tokyo, Japan.
Introduction Neurosteroids and neuroactive steroids act in various ways in many physiological and pathological processes of the central nervous system. This becomes possible because of their ability to modulate the functions of excitatory receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA), or inhibitory γ-aminobutyric acid (GABAA) and glycine (Gly) receptors (for a review see King, 2013). In addition, it has been shown that some neurosteroids are able to act on multiple targets (Korinek et al., 2011; O'Dell et al., 2005). For example, endogenous neurosteroids allopregnanolone or pregnanolone sulfate (Fig. 1B and D), can potentiate GABAA receptors (GABAARs) as well as inhibit the NMDA receptors (NMDARs). In contrast, pregnenolone sulfate (Fig. 1C) was found to potentiate NMDARs while it inhibits AMPA, kainate, and GABAA receptors (Wu et al., 1991). The compounds' configuration and substitution at the C-3 carbon of the steroidal skeleton and configuration at C-5 play a critical role in their activity at both NMDA and GABAA receptors (Fig. 1). NMDARs are glutamate-gated ionotropic receptors that are involved in excitatory neurotransmission and plasticity (Traynelis et al., 2010). There have been several reports showing that neurosteroids acting as negative modulators of NMDARs exert neuroprotective activity in both in vitro and in vivo models of neurodegeneration (Lapchak, 2004; Rambousek et al., 2011; Vyklicky et al., 2016). The inhibitory GABAA receptors are the major type of receptors that underlie the physiologically and clinically relevant effects of neurosteroids, and they are the most extensively studied type (Belelli and Lambert, 2005). In view of the above, it seems reasonable to continue the evaluation and development of neurosteroids because of their important regulatory activities, neuroprotective properties and multiple mechanisms of action. Therefore, this paper describes the effects of selected neuroactive steroids 1–6 (Fig. 2), which are potent negative modulators of excitatory NMDARs (Borovska et al., 2012; Adla et al., 2017; Weaver et al., 1997; Vyklicky et al., 2016; Chodounska et al., 2016), on inhibitory GABAA and Gly receptors of rat hippocampal neurons. The primary goal of this study was to obtain a structure-activity relationship for neurosteroid actions on GABAARs and GlyRs, and then to compare it with their effect on NMDARs. Such a complex structure-activity relationship study (SAR) has not been reported previously and could be used for the further development of neuroprotective therapeutics, which is a topic of great interest. Pregnanolone glutamate (PA-Glu, compound 1, Fig. 2), pregnanolone hemisuccinate (PA-hSuc, compound 2, Fig. 2), and pregnanolone hemipimelate (PA-hPim, compound 3, Fig. 2) were chosen as the main candidates for further screening, as all three compounds were potent inhibitors of NMDA-induced currents, with IC50 values varying from 17 to 51 μM (Borovska et al., 2012; Adla et al., 2017; Weaver et al., 1997; Vyklicky et al., 2016; Chodounska et al., 2016). Moreover, PA-Glu, PA-hSuc, and PA-hPim were shown to exert neuroprotective effects in vivo in several biological models (Rambousek et al., 2011; Holubova et al., 2014; Kleteckova et al., 2014; Lapchak, 2004; Vyklicky et al., 2016). Our previous SAR study showed that non-polar modification of the steroid D-ring in combination with isosteric amide-based C-3 substitution of various lengths and types leads to an increase in the ability of steroids to inhibit the NMDARs current (Kudova et al., 2015, 2016; Adla et al., 2017). In this paper, androstane glutamate (AND-Glu, compound 4, Fig. 2), androstane aspartylamide (N-2′-L-Asp-AND, compound 5, Fig. 2) and 17β-methyl-androstane hemimalonylamide (17β-Me-AND-3β-hMalAmide, compound 6, Fig. 2) were selected for the screening.