Amino Acids as Neurotransmitters. The Balance between Excitation and Inhibition as a Background for Future Clinical Applications

Abstract

For more than 30 years, amino acids have been well-known (and essential) participants in neurotransmission. They act as both neuromediators and metabolites in nervous tissue. Glycine and glutamic acid (glutamate) are prominent examples. These amino acids are agonists of inhibitory and excitatory membrane receptors, respectively. Moreover, they play essential roles in metabolic pathways and energy transformation in neurons and astrocytes. Despite their obvious effects on the brain, their potential role in therapeutic methods remains uncertain in clinical practice. In the current chapter, a comparison of the crosstalk between these two systems, which are responsible for excitation and inhibition in neurons, is presented. The interactions are discussed at the metabolic, receptor, and transport levels. Reaction-diffusion and a convectional flow into the interstitial fluid create a balanced distribution of glycine and glutamate. Indeed, the neurons’ final physiological state is a result of a balance between the excitatory and inhibitory influences. However, changes to the glycine and/or glutamate pools under pathological conditions can alter the state of nervous tissue. Thus, new therapies for various diseases may be developed on the basis of amino acid medication.

1. Introduction

Even for students just beginning to study biochemistry and physiology, it is immediately apparent that amino acids (AAs) are among the most important molecules in nature. Their functions are broad and varied. Indeed, protein synthesis relies on the well-known polymerization of AAs to form a peptide bond. This property is the most famous aspect of AAs. However, many AAs have specific individual functions, such as neurotransmission [1], cellular energy metabolism [2], and detoxification [3, 4]. Accumulating evidence in recent years has demonstrated that AAs also regulate both the expression of genes and the protein phosphorylation cascade. Moreover, hormones and different low-molecular-weight biologically important chemical compounds can be synthesized from AAs [5]. AAs can be divided into essential and nonessential categories. If the body cannot synthesize the carbon skeleton of an amino acid, then it is considered nutritionally essential. Indeed, the diet must contain such AAs. The dietary essentiality of other AAs (e.g., arginine, glycine, proline, and taurine) is determined by the developmental stage and species [6]. In contrast, if AAs can be synthesized de novo in a species-dependent manner, they are considered nonessential. Accumulating evidence has led to the concept of functional AAs (FAAs), which are defined as AAs that regulate key metabolic pathways to improve the health, survival, growth, development, lactation, and reproduction of organisms [7]. Since the late 1970s, researchers have generally agreed that amino acids can also function as inhibitory or excitatory neurotransmitters [8]. It should be noted that in neurochemistry, the term “neurotransmitter” is usually used synonymously with “neuromediator,” another term for a chemical participant in connections between neurons and neuroglia cells. Because these terms are exchangeable, they will both be used in the text. Based on their effects on vertebrate nerve cells, γ-aminobutyric acid (GABA), glycine, and taurine fall into the class of inhibitory amino acids, whereas glutamate and aspartate fall into the class of excitatory compounds [9]. Indeed, GABA is considered the main inhibitory neurotransmitter in the central nervous system (CNS) [10], but it is not truly a member of the AA family. Although taurine also plays a role in inhibitory neuromediation [11] and serves as an osmoeffector to regulate volume in astrocytes [12], this compound is considered a derivative of cysteine, and, similar to GABA, not a true amino acid. Thus, the remaining excitatory/inhibitory amino acid neurotransmitters are glutamate, aspartate, and glycine. The first and third are the most prominent members of the AA family. The processes that regulate glutamate and glycine in the CNS are (i) transportation, (ii) biochemical transformations in metabolic pathways, and (iii) interactions with membrane receptors. In the current chapter, the crosstalk between the processes mentioned above for both glutamate and glycine is presented because the final state of neurons seems to be a result of the balance between these excitatory and inhibitory influences.

2. The membrane transport system of amino acids

Glutamate and glycine are nonessential amino acids; their levels differ depending on the location. The extracellular glutamate concentration around quiescent neurons is less than 1 μM, while its concentration in the cytoplasm is much higher, at approximately 2 mM [13]. The brain sequesters glycine in concentrations of 600 μM [14], with a basal concentration in the cerebrospinal fluid (CSF) of ~6 μM [15], compared to a plasma concentration of ~250 μM [16]. Because no extracellular enzymes degrade glutamate and glycine, maintaining these low extracellular concentrations requires cellular uptake of both compounds. Thus, the activity of the carriers directly regulates receptor response to neuron activation. Indeed, glutamate and glycine serve as neuromediators in the extracellular fluid because the binding site of AA receptors is exposed to the outer surface of cells. Consequently, the release of AA into the extracellular fluid controls receptor activation and active states are controlled by the removal of AAs from the extracellular fluid [17]. This uptake is catalyzed by a family of transporter proteins located on the cell surface of both astrocytes and neurons [17]. A high-affinity glutamatergic uptake system was observed in the mammalian brain in the 1970s. Subsequently, excitatory amino acid transporters (EAATs) were experimentally identified. They transport glutamate and aspartate across the plasma membrane. Notably, EAATs are part of the well-known solute carrier 1 (SLC1) family of transmembrane amino acid transporters [18]. Thus, released glutamate molecules can be removed from the synaptic cleft by the brain transporters; this process will initiate the glutamate-glutamine cycle, eventually restoring the pool of the neuromediator in synaptic vesicles [19]. Five EAAT isoforms, human EAAT1-5, have been identified; they correspond to GLAST1/GLT-1/EAAC1/EAAT4/EAAT5 in rodents, respectively [20]. In addition, the EAAT4 and EAAT5 subtypes were identified, with EAAT5 predominantly expressed in the retina. Notably, the transport cycle times of EAATs are relatively slow and their high affinity for glutamate makes it possible to sequester low glutamate concentrations from the extracellular space, preventing excitotoxicity. The slow transportation rate may in part be overcome by rapid surface diffusion and transporter tracking of EAATs upon glutamate stimulation [21]. The SLC1 family also contains two neutral amino acid transporters, alanine serine cysteine transporters 1 and 2 (ASCT1 and 2), which share high sequence homology with the EAATs [22]. EAAT1 and EAAT2 are glutamate transporters that are mostly expressed in astrocytes. These two glutamate transporters are responsible for most of the glutamate clearance in the brain. EAAT2 is widely expressed in the cerebral cortex and the hippocampus [13]. Moreover, GLT-1/EAAT2 accounts for approximately 90% of the total glutamate uptake in the brain, and thus, it is considered the most important glutamate transporter subtype in the CNS. This transporter is predominantly but not exclusively expressed in astrocytes [22]. Glutamate transporters couple glutamate uptake to the transport of inorganic ions. It is now generally accepted that 3 Na⁺ ions and 1 H⁺ ion are cotransported and 1 K⁺ ion is counter-transported with the uptake of each glutamate molecule. Based on this stoichiometry, glutamate transporters were calculated to concentrate glutamate up to 5 × 10⁶-fold inside cells under physiological conditions. This glutamate transport is electrogenic [23].

The extracellular levels of glycine in inhibitory and excitatory synapses are controlled by glycine transporters (GlyTs). Both subtypes, GlyT1 and GlyT2, belong to the sodium-dependent solute carrier 6 (SLC6) family of transporters, but they have different regional and cellular expression patterns in the CNS, different stoichiometries (that is, different numbers of sodium ions that are co-transported with every glycine molecule) and varying abilities to reverse-transport glycine into the extracellular space. To date, five variants of GlyT1 (GlyT1a, GlyT1b, GlyT1c, GlyT1d, and GlyT1e) and three variants of GlyT2 (GlyT2a, GlyT2b, and GlyT2c) have been identified and occur as a result of alternative promoter usage and/or splicing, but the relative distributions of these within the CNS have not been fully characterized [21].

The essential function of membrane transporters is to accumulate neuromediators in vesicles. At presynaptic terminals, vesicular glutamate transporters (vGluTs; SLC17A7, -6, and -8) load glutamate into synaptic vesicles. The two subtypes of vGluTs, vGluT1, and vGluT2, are expressed in excitatory neurons in a complementary manner in the brain, composing two subsets of excitatory neurons [13]. Glycine also actively accumulates in synaptic vesicles through vesicular inhibitory amino acid transporter (VIAAT); currently, only one type of transporter (SLC32A1) is known to be responsible for this process [18]. The scheme of balanced neuromediator transport is represented in Figure 1.

Figure 1.

Membrane carriers are responsible for clearance of glutamate/glycine from interstitial fluid (ISF) in the CNS. The scheme indicates two types of neurons. Some are excitatory and glutamatergic (the upper part of the scheme). Other neurons are inhibitory and glycinergic (the lower part of the scheme). Both types of neurons are interconnected with astrocytes. Moreover, glycine and glutamate are accessible for both types of cells. AA transporters (EAAT, GlyT, etc.) are found in all cell membranes but have differing isoenzyme compositions.

Remarkably, both glutamate and glycine transporters have mechanisms that include sodium ion transport. This means that neuromediator uptake is accompanied by changes in membrane potential. Moreover, the intake of both glutamate and glycine initiates several metabolic reactions in neurons and astrocytes. However, these reactions are spatially distributed, and the fate of the neuromediators is functionally determined by different cells. Interestingly, the metabolic transformations of AAs are closely related to ATP production by mitochondria and the oxidation of glucose.

3. Transformations of amino acids in the cell metabolic network

As mentioned above, any example of metabolic transformation in brain tissue is tightly connected with glycolysis Therefore, glutamate/glycine participation in metabolic pathways seems to be considered correctly including the main neighbor reactions of glucose oxidation. The primary source of energy for the brain is glucose. This sugar is almost entirely oxidized under basal physiological conditions, providing nearly all the energy necessary to support brain function. However, when supplemental energy is needed, necessary energy demands may be provided by other metabolites, such as ketones, fatty acids, acetate, lactate, and certain amino acids [19]. Pyruvate, the end product of aerobic glycolysis, can enter the tricarboxylic acid (TCA) cycle by two different routes: (1) via acetyl-CoA formation, catalyzed by the pyruvate dehydrogenase complex, and (2) by the formation of oxaloacetate, catalyzed by PC [24]. However, the end metabolite of anaerobic glycolysis, lactate, also participates in the energy supply of neurons (Figure 2). Pellerin and Magistretti originally proposed the astrocyte-neuron lactate shuttle (ANLS) model, wherein lactate released from astrocytes serves as a buffer compound in response to a glutamate-induced glycolysis stimulus [25]. Then, lactate is exported to neurons, where it is converted to pyruvate to fuel oxidative phosphorylation.

Figure 2.

A scheme of the metabolic pathways involved in general glutamate/glycine transformations. The reactions occur in various intracellular localizations and can be duplicated in different compartments. The main metabolic pathways (glycolysis and the tricarboxylic acid (TCA) cycle) are labeled. The enzyme abbreviations are as follows: GM: glutaminase; GS: glutamine synthetase; GDH: glutamate dehydrogenase; GL: glutamylcysteine ligase; GTS: glutathione synthetase; AG: asparaginase; AT: aminotransferase; PPC: phosphoenolpyruvate carboxykinase; PC: pyruvate carboxylase; PDC: pyruvate dehydrogenase complex; PK: pyruvate kinase; LDH: lactate dehydrogenase; SDH: serine dehydrogenase; STM: serine transhydroxymethylase; and GCS: the glycine cleavage system. Other abbreviations are as follows: NAD+: Nicotinamide adenine dinucleotide (oxidized); NADH: Nicotinamide adenine dinucleotide (reduced); ATP: Adenosine triphosphate; ADP: Adenosine diphosphate; and THF:Tetrahydrofolate.

Thus, the ANLS model suggests that lactate, not glucose, provides energetic support for firing neurons [26]. Glutamate and glycine are active participants in these metabolic processes. Exclusion of most blood-borne glutamate at the blood-brain barrier (BBB) and a net removal of glutamine from the brain indicate that the cerebral pools of glutamate are largely produced within the brain [27]. The stability of glutamate concentration is maintained by two main reactions. Glutamine synthetase (GS), which is found in astrocytes, is the only known enzyme to date that is capable of a reversible conversion between glutamine and glutamate and ammonia in the mammalian brain [28]. Furthermore, cells can convert glutamate to glutamine in an ATP-dependent process catalyzed by glutamine synthetase. Astrocytic uptake of glutamate and release of glutamine, together with neuronal uptake of glutamine and release of glutamate, constitute the glutamate-glutamine cycle [29]. However, much of the glutamate taken up by astrocytes is destined for oxidative degradation, which first requires conversion to the TCA cycle intermediate 2-oxoglutarate. This can take place via transamination by aminotransferase (AT) or via oxidative deamination by glutamate dehydrogenase (GDH) [30].

Once glycine passes into a cell by uptake by GlyTs, the intracellular glycine concentration can be regulated via synthesis from L-serine within the cell, which itself can be synthesized from glycolysis intermediates and L-glutamate [24]. The major pathway for the glycine catabolism involves the oxidative cleavage of glycine to CO2, NH⁴⁺, and a methylene group (–CH2–), which is accepted by tetrahydrofolate (H⁴folate) in a reversible reaction catalyzed by the glycine cleavage system (also called glycine synthase) [31]. The glycine cleavage system is essentially reversible but catalyzes glycine synthesis significantly only under anaerobic conditions, such as in anaerobic bacteria or anaerobic systems in vitro supplemented with NADH+H⁺ [32].

Taken together, all known information about the metabolic pathways suggests that glutamate and glycine self-regulate the processes of their concentration restoration and mutual transformation. Additionally, oxidative phosphorylation in the mitochondria also plays a key role in the balance of these AAs.

4. CNS receptors of amino acids

The neuromediator function of AAs in the CNS is performed through the activation of membrane receptors. After being released from the presynaptic membrane into a synaptic cleft, glutamate and glycine rapidly diffuse to a postsynaptic membrane, where appropriate receptors are further activated.

Glutamate receptors are divided into two groups: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Excitatory neurotransmission throughout the CNS is mediated by ligand-gated ion channels, including ionotropic glutamate receptors (iGluRs) [33]. Abnormalities in iGluRs lead to a wide range of neurological diseases. Glutamate, the primary neurotransmitter in almost all synapses in the CNS, is released from presynaptic terminals and diffuses to the postsynaptic membrane, where it binds to iGluRs. This process leads to the opening of ion channels, allowing cations to flow in. Thus, the transmembrane channel rapidly depolarizes the postsynaptic membrane. The decrease in membrane potential initiates signal transduction in the postsynaptic neuron. In the iGluR family, four subtypes of integral membrane proteins have been identified in vertebrates based on their pharmacological properties and sequence homologies: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), N-methyl-D-aspartate (NMDA), and δ-receptors [34]. Subsequent cloning studies have revealed that NMDARs are assembled as heteromers that differ in subunit composition. To date, seven different subunits have been identified and categorized into three subfamilies according to sequence homology [35]. Each iGluR family member exhibits specific kinetic and pharmacological properties in addition to playing a unique role in neurotransmission [36]. The iGluRs are ligand-gated ion channels that are permeable to Na⁺ and K⁺ (and Ca²⁺ in some instances), whereas the mGluRs are G protein-coupled receptors that trigger second messenger cascades. The early component and the late component of neurotransmission are assumed to be mediated by AMPARs and NMDARs/KARs, respectively. This assumption is based on receptor kinetics, as AMPARs are faster and NMDARs/KARs are slower. Nevertheless, acoustic signals are transferred by all of these iGluRs in a precise and reliable manner. Moreover, some auditory processing neurons have a fourth type of iGluR, the delta receptor [34]. The open, or conducting, conformation of the iGluR ion channel is nonselective for monovalent cations. Membrane excitation is often driven by channel permeability to Ca²⁺. This Ca²⁺ influx and its physiological and pathological consequences depend strongly on the specific iGluR subtype and the specific subunits in its oligomeric complex [37].

mGluRs are G protein-coupled receptors (GPCRs) that, following activation, regulate both G protein-dependent and G protein-independent signalling pathways. According to sequence homology, cell signalling activation, and agonist selectivity, the mGluRs have been divided into eight subtypes (from mGlu1 to mGlu8). These subtypes comprise three different subgroups (from I to III) [38]. Group I mGluRs (mGlu1 and mGlu5) are functionally linked to polyphosphoinositide (PI) hydrolysis and are negatively coupled with K⁺ channels. Both group II (mGlu2 and mGlu3) and group III (mGlu4, mGlu6, mGlu7, and mGlu8) mGluRs negatively regulate adenylate cyclase and activate mitogen-activated protein kinase (MAPK) and PI-3-kinase pathways [39]. mGluRs are usually localized on synaptic and extrasynaptic membranes in both glia and neurons. Group I mGluRs are generally postsynaptic, surrounding ionotropic receptors, and modulate depolarization and synaptic excitability. Groups II and III are mostly expressed at the presynaptic level and control the release of neurotransmitters [39, 40]. mGluRs are heavily expressed throughout the basal ganglia (BG), where they modulate neuronal excitability, transmitter release, and long-term synaptic plasticity [41]. These receptors are coupled to different G proteins and modulate slow postsynaptic neuronal responses, either through presynaptic or postsynaptic machinery or through modulation of astrocyte function [42]. mGluRs are highly and diffusely expressed in glial cells. On the one hand, this increases the options for therapeutic interventions, but on the other hand, it makes it even more difficult to selectively target single receptors to yield neuroprotection (Figure 3) [43].

Figure 3.

A reconstruction of possible AA ionotropic receptors in the CNS. The images were created using the data collected in the Protein Data Bank (PDB) (https://www.rcsb.org/). The scaled images show GlyR (6UBS, Danio rerio, [44]), AMPAR (5IDE, Rattus norvegicus, [45]), KAR (6KZM, Rattus norvegicus, [46]) and NMDAR (7EOQ , Homo sapiens, [47]).

Glycine receptors (GlyRs), along with certain γ-aminobutyric acid receptors (GABAARs), are the principal determinants of fast inhibitory synaptic neurotransmission in the central nervous system (CNS). GlyR and GABAAR belong to the superfamily of pentameric ligand-gated ion channels (pLGICs) [33]. The two neurotransmitters (glycine and GABA) may be functionally interchangeable, and the multiple receptor subtypes with inhibitory influences provide diverse mechanisms for maintaining inhibitory homeostasis [35]. Inhibitory glycine receptors (GlyRs) are anion-selective ligand-gated ion channels (LGICs), which, together with GABAA receptors (GABAARs), nicotinic acetylcholine receptors (nAChRs), and serotonin type 3 receptors (5HT-3), form the eukaryotic Cys-loop family [36]. Several endogenous molecules, including neurotransmitters and neuromodulators (such as glutamate, Zn, and Ni), and exogenous substances, such as anaesthetics and alcohols, modulate GlyR function [40].

5. Participation in development of pathological processes

Despite their obvious physiological roles in protein synthesis, the cellular effects of glycine and glutamate in the CNS seem to be quite different. If glycine has been contemplated an “angel” compound, due to its generally positive effects, then glutamate has usually been considered a “demon” compound, owing to its generally negative effects. Although the last claim is far from accurate, the first is supported by many experimental findings. Indeed, the effect of glycine has always been reported as positive. It protects against oxidative stress caused by a wide variety of chemicals, drugs, and toxicants at the cellular or organ level in the liver, kidneys, intestines, and vascular system [34, 37]. Glycine is a major component of collagen molecules that is vital to stabilizing them to form a triple helix [48]. Administration of glycine attenuates diabetic complications in a streptozotocin-induced diabetic rat model [49]. Supplemental glycine effectively protects muscles in a variety of wasting models, including cancer cachexia, sepsis, and dieting [50]. Glycine may prevent ischaemia–reperfusion injury by direct cytoprotection, presumably by inhibition of the formation of plasma membrane pores and of the inflammatory response [38]. The cytoprotective and modulatory effects of glycine have been observed in many nonneuronal cell types. The action of glycine is mediated by classic or unconventional GlyRs, both inside and outside of the nervous system [51]. Glycine cytoprotection substantially overlaps with the number of agents that act on neuronal receptors with glycine as an agonist or coagonist. This observation has been confirmed by molecular pharmacology studies from multiple laboratories. The studies indicate highly constrained steric and conformational requirements for the interaction, which, along with the rapid on-off timing of the effects, is consistent with the involvement of reversible ligand-binding site interactions [52].

In contrast, glutamate is considered a toxic agent that yields excitotoxicity at overload concentrations. Indeed, the neurotoxic potential of glutamate has been recognized since the 1950s [53]. For example, a major driver of white matter demise is excitotoxicity, a consequence of the excessive glutamate released by vesicular and nonvesicular mechanisms from axons and glial cells. This excessive glutamate concentration results in overactivation of iGluRs profusely expressed by all cell compartments in white matter [54]. Generally, excitotoxicity involves a large inflow of Ca²⁺ and Na⁺ into neurons up to the conditions when Ca²⁺ concentrations reach critical levels, leading to cell injury or death [55]. Moreover, ambient extracellular glutamate is lower than the concentration known to trigger excitotoxicity and subsequent neurodegeneration; excitotoxicity is known to occur at extracellular glutamate concentrations as low as 2 to 5 μM, with swelling and apoptosis predominating at <20 μM glutamate and fast necrosis at >100 μM glutamate [56]. Excitotoxic neuronal death is involved in neurodegenerative diseases of the CNS, such as multiple sclerosis [57], Alzheimer’s disease [58], Parkinson’s disease [59], Huntington’s disease [60], stroke, epilepsy, alcohol withdrawal, and amyotrophic lateral sclerosis [61]. However, the role of glutamate is not only excitotoxic. The assumption that neurodegenerative disease treatments should “fight against” glutamate is incorrect given the wrong function of glutamate in the CNS. As a part of normal physiological excitation, this AA must be properly regulated, but battling with glutamate receptors or the transport system will cause serious negative consequences. Instead, the level and functional activity of glutamate may be adjusted by metabolic processes, including glycine and oxidative phosphorylation, in mitochondria.

6. Balance is achieved through mutual interactions of the excitatory and inhibitory effects of amino acids

Because glutamate is the major mediator of excitatory signals as well as of nervous system plasticity, including cell elimination, it follows that glutamate needs to be present at the right concentrations in the right places at the right time [17]. These conditions are regulated by GS, GM, and EAATs and convectional diffusion in ISF. There is evidence that extracellular glutamate is not compartmentalized by EAATs under some conditions [62]. The most obvious shift in glutamate levels is observed under high GDH and AT activity. The general activation of bioenergetics decreases the excessive glutamate concentration by stimulating the TCA cycle. Moreover, glycine can participate in this shift in a variety of ways. GlyT-1 controls glycine release and reuptake, determines glycine availability at glycine binding sites on NMDA receptors [36] and coordinates neuronal-glial interactions at glutamatergic synapses [19]. Thus, glycine assists glutamate in the activation of astrocytes and further stimulates the mitochondria according to the ANLS hypothesis. Glycine can conjugate with glutamate in the GSH synthesis pathway (Figure 1). This mechanism is essential to maintain the redox status of neurons and to prevent oxidative stress and high levels of reactive oxygen species (ROS) synthesis. Neuronal mitochondria are the target of glutamate, which attenuates succinate dehydrogenase (a key enzyme of the TCA cycle) inhibition by oxaloacetate [63], with further induction of ROS production [64]. However, glycine can prevent excessive hydrogen peroxide production induced by glutamate in brain mitochondria [65], thereby reducing the prooxidant effects of the excessive glutamate concentrations.

Interestingly, the effects of amino acids can vary depending on the species. For example, in a chick model, injections of L-glutamate, NMDA, and AMPA attenuated total distress vocalizations and induced sedation [66]. The association between glutamate and inhibition/sedation is even stronger because the brain contains a considerable level of glutamate decarboxylase, which directly catalyzes the decarboxylation of glutamate to GABA [27]. Additionally, glycine is not always associated with direct inhibition in the CNS. Indeed, in mature neurons, where there is a low intracellular Cl⁻ concentration maintained by K⁺- Cl⁻ cotransporter 2 (KCC2), activation of GlyRs elicits an influx of Cl⁻, leading to rapid hyperpolarization and postsynaptic inhibition [67]. In contrast, in immature neurons, activation of GlyRs results in efflux of Cl⁻, leading to neuronal depolarization; this opens voltage-dependent Ca²⁺ channels, elicits action potentials, and establishes early network activity and excitation in the developing nervous system [68].

Thus, the balance between excitation and inhibition is the result of continuous interactions among different processes involving both glutamate and glycine. It is essential that the main reactions and regulatory sites are nonhomogenously distributed in neuronal space and are time-regulated. Convective flow does not restore the homogeneity of mediator and metabolite concentrations because of the tortuosity of the system [63]. A scheme of the balanced interactions between glycinergic and glutamatergic synapses is shown in Figure 4.

Figure 4.

The transport and activation of receptors in glycinergic and glutamatergic synapses. The transport system is tightly linked with glucose consumption. This transport system occurs in both astrocytes and neurons, but according to the ANLS model, the majority of glucose is consumed in astrocytes, with further diffusion of lactate to neurons. Lactate transport is facilitated by monocarboxylate transporters (MCTs), which have two different isoenzymes. MCT1 is expressed in astrocytes, and MCT2 is found in neurons [69]. Glutamate-glutamine cycling occurs between central astrocytes and neurons, mediated by sodium-coupled neutral amino acid transporters (SNATs). Transport is mediated by two isoforms, SNAT3 and SNAT1 [70]. ISF: interstitial fluid.

7. Clinical applications and perspectives

The first (and obvious) clinical application of AAs is as a reference level to indicate different pathologies. This suggestion covers more AAs than those mentioned above. For decades, the biochemical analysis of AAs in body fluids has been an important diagnostic tool in the detection of congenital errors of metabolism. Significant elevations of amino acids in plasma, urine, or CSF have been the backbone of many diagnostic procedures [71]. This is because defects in amino acid catabolic pathways can be detected by the characteristic accumulation of their metabolites. Well-known examples of this are elevated plasma concentrations of phenylalanine in phenylketonuria (PKU) and increased concentrations of homocysteine in homocystinuria [71].

In addition, the properties of glutamate/glycine discussed above indicate a wide range of potential medical applications for compounds that govern transport, receptors, and metabolic systems in the CNS. A classic pharmacological approach may be based on the search for chemicals that affect the indicated processes; interactions with the target protein site or reaction must be local and precisely unidirectional and wide metabolic participation of the candidate should be avoided. There are several examples to date. Each of the three mGlu subgroups can be considered a novel target for the treatment of schizophrenia. All three symptom domains could be effectively treated by mGlu5 positive allosteric modulators, which are devoid of toxicity and seizure liability according to preclinical data. Furthermore, the potential antipsychotic and cognitive-enhancing effects of drugs targeting mGlu1 and mGlu3 were supported by recent genetic investigations of schizophrenia patients [72]. Preclinical studies have revealed that specific mGluR subtypes mediate significant neuroprotective effects that reduce toxin-induced midbrain dopaminergic neuronal death in animal models of Parkinson’s disease [41]. Additionally, mGluRs have emerged as research targets in treating Alzheimer’s disease. In particular, mGluR-based compounds producing both symptomatic and disease-modifying effects in preclinical models of the disease are of special interest [73]. G protein-coupled mGluRs expressed by tumor cells, particularly cancer stem cells, might represent new candidate drug targets for the treatment of malignant brain tumors [74]. Group III mGluR agonists have been recently identified as promising tools for managing affective symptoms, such as the pathological anxiety observed in neuropathic pain. However, the use of mGluR ligands as anxiolytics was disappointing in clinical trials. Nevertheless, there is ground for a certain amount of optimism [75].

Pharmacological modulation of glycinergic inhibition could represent a novel therapeutic strategy for a variety of diseases involving altered synaptic inhibition, primarily in the spinal cord and brain stem but possibly also at supraspinal sites [74]. Among the inhibitors of GlyT-1, two candidates have attracted the most attention. Sarcosine, a known intermediate of glycine metabolism, had positive results as a short-term treatment of major depression and for acutely ill and chronically stable schizophrenia patients. Another GlyT-1 inhibitor, bitopertin, was expected to be effective in treating negative or positive schizophrenia symptoms. However, the phase III clinical trials fell short of the primary endpoint, and the investigation was halted due to its lack of efficacy in improving negative symptoms [76]. Gelsemium, a small genus of flowering plants from the family Loganiaceae, may be used as a pain treatment and for its mechanism of action. Gelsemium and its active alkaloids may produce antinociception by activating the spinal α3 glycine/allopregnanolone pathway in inflammatory, neuropathic, and bone cancer pain without inducing antinociceptive tolerance, in contrast to morphine [75].

Another strategy is to directly use AAs for medical treatment. In this scenario, glycine is the most appropriate candidate. Glycine has a wide spectrum of protective properties against different diseases and injuries. As such, it represents a novel anti-inflammatory, immunomodulatory and cytoprotective agent [77]. Oral supplementation of glycine at a proper dose is very successful in treating several metabolic disorders in individuals with cardiovascular diseases, various inflammatory diseases, cancers, diabetes, and obesity [34]. Glycine was well tolerated at a dose of 0.8 g/kg body weight a day, resulting in significantly increased serum glycine levels and a 7% reduction in negative symptoms in patients with treatment-resistant schizophrenia [78]. An acute high dosage of glycine attenuates the neurophysiological representation of the brain’s preattentive acoustic change detection system (mismatch negativity) in healthy controls, raising the possibility that the optimal effects of glycine and other glycine agonists may depend on the integrity of the NMDA receptor system [79]. The glycine was effective in the treatment of ischaemic stroke patients. In a randomized, double-blind, placebo-controlled study on 200 patients with acute (<6 h) ischaemic stroke in the carotid artery area, 1.0–2.0 g/day of glycine was accompanied by a tendency towards decreased 30-day mortality (5.9% in the 1.0 g/day glycine and 10% in the 2.0 g/day glycine groups vs. 14% in the placebo and 14.3% in the 0.5 g/day glycine groups), an improved clinical outcome on the Orgogozo Stroke Scale (p < 0.01) and the Scandinavian Stroke Scale (p < 0.01) and a favorable functional outcome on the Barthel Index for Activities of Daily Living (p < 0.01) in the 1.0 g/day glycine group compared to those in the placebo group in patients with no or mild disability [80]. The molecular mechanism of such an effect is based on the ability of glycine to initiate stable vasodilatation of arterioles, which has been demonstrated in rat pial vessels and in mesenteric arterioles [81, 82].

8. Conclusions

According to experimental and clinical evidence, AAs are especially useful nutrients for the treatment of patients with different diseases. These nutrients not only supply a background pool for biochemical reactions, but the functions of the metabolites cover a wide range of neurochemical processes, and they are always mutually dependent. Even though some processes are decreased or increased in illnesses, it does not mean that the treatment strategy must be targeted to only correct the single altered process. A prominent example is glutamate-induced excitotoxicity in neurons. The best strategy to prevent increased glutamate concentrations is to maintain bioenergetic processes in neurons and astrocytes at high activity levels and to activate glycine-dependent processes. Moreover, it helps to assign the exceeded content of the neuromediator to a physiological range and to form stable conditions for further health development, avoiding excitotoxicity (Figure 5). Searching for exogenous antagonists of metabolic receptors seems to be an incorrect therapeutic strategy because the function of the AA-dependent system depends on the basic metabolic regulatory core of metabolic processes. Indeed, to find appropriate therapeutic methods, further fundamental and clinical investigations are necessary.

Figure 5.

Scheme of the mutual influence of inhibition and excitation mediated by glycine and glutamate.

Conflict of interest

The author has no conflict of interest to declare.