2.   NEUROCHEMISTRY

 

2.1.     Criteria to identify neurotransmitters

A multitude of chemicals called neurotransmitters mediate intercellular communication in the nervous system. Although they exhibit great diversity in many of their properties, all are stored in vesicles in nerve terminals and are released to the extracellular space via process requiring calcium ions. Their action is terminated by reuptake into presynaptic terminal or glial cells by specific transporter proteins or by catabolism in synaptic cleft or in presynaptic terminal. Criteria to identify neurotransmitters are shown in the Table 2.1.

 

Table 2.1. Criteria to identify neurotransmitter

presence in presynaptic nerve terminal

synthesis by presynaptic neuron

releasing on stimulation (membrane depolarisation)

producing rapid-onset and rapidly reversible responses in the target cell

existence of specific receptor

 

From the chemical point of view are neurotransmitters monoamines, amino acids and peptides. There are two main groups of neurotransmitters:

1. classical neurotransmitters
2. neuropeptides

 

2.2.     Classical neurotransmitters

All classical neurotransmitters (Table 2.2) are synthesized in nerve terminals.

 

The first molecule to be implicated as neurotransmitter was acetylcholine (Ach). It was demonstrated that acetylcholine is the transmitter at neuromuscular synapses, as well as at a variety of neuron-neuron synapses. Ach is synthesized from choline and acetyl‑coenzyme A in the nerve endings; reaction is catalyzed by the enzyme choline acetyltransferase (Figure 2.1). ACh is rapidly degraded in synaptic cleft by the enzyme acetylcholinesterase to choline and acetate.

 

Catecholamines (dopamine, norepinephrine and epinephrine) are synthesized from tyrosine (Figure 2.2). Indolamines (serotonin and tryptamine) are synthesized from tryptophan (Figure 2.3.). Action of catecholamines and indolamines on target cells is terminated much more slowly than those of acetylcholine; they are removed from synaptic cleft by reuptake. The major enzymes involved in the catabolism of catecholamines are monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT). Serotonin is metabolized by MAO.

There are evidence that dysfunction in brain catecholamine or indolamine pathways contribute to affective disorders and schizophrenia. Some antidepressant prolongs monoamine neurotransmitters action by inhibiting their high affinity reuptake system. Catecholamine theory of psychotic illness focuses on dysfunction at dopaminergic synapses.

 

There are three major amino acid neurotransmitters in the nervous system: g-amino butyric acid (GABA), glycine and glutamic acid. GABA and glycine are inhibitory neurotransmitters; glutamate and aspartate are excitatory neurotransmitters.

 

Specific properties have nitric oxide, which does not interact with membrane receptors but diffuse to target intracellular receptor.

 

Table 2.2. Selected classical neurotransmitters

System	Transmitter	Precursor
cholinergic	acetylcholine	choline+acetylcoenzym A
aminoacidergic	GABA	glucose ® glutamate
	aspartic acid	glucose+glutamine; glutamate
	glutamic acid	glucose+glutamine; aspartate
	glycine	serine
	homocysteine	cysteine®cystine
monoaminergic
catecholamines	dopamine	tyrosine®DOPA®dopamine
	norepinephrine	®norepinephrine®epinephrine
	epinephrine
indolamines	tryptamine
	serotonin	tryptophan®5-hydroxytryptophan
others, related to aa	histamine	histidine
	taurine	cysteine®cysteamine
purinergic	adenosine
	ADP
	AMP
	ATP

 

 

Figure 2.1. Synthesis and release of acetylcholine

 

 

 

Figure 2.2. Catecholamine biosynthesis

 

 

Figure 2.3. Serotonin biosynthesis

 

 

2.3.     Neuropeptides

It was identified a great number of neuropeptides (Table 2.3) and new neuropeptides are discovered continually. b-endorphin is important in psychiatry, since it has a role in pain and in stress. All neuropeptides are synthesized in the cell body in following steps: 1. transcription of pre-propeptide gene and creation of pre-propeptide RNA; 2. translation into pre-propeptide, which enters the endoplasmic reticulum; 3. forming of the propeptide, which is direct precursor of neuropeptide; 4. propeptide enters vesicles, where it is converted into the neuropeptide. The action of neuropeptides in the synaptic cleft is terminated by peptidases; there is no reuptake for neuropeptides. Neuropeptides are co-transmitters usually and their transduction mechanism is coupled with G proteins.

 

Table 2.3. Selected bioactive peptides

Peptide	Group
substance P, substance K (tachykinins)	brain and gastrointestinal peptides
neurotensin
cholecystokinin (CCK)
gastrin
bombesin
galanin	neuronal
neuromedin K
neuropeptideY (NPY)
peptide YY (PYY)
cortikotropin releasing hormone (CRH)	hypothalamic releasing factors
growth hormone releasing hormone (GHRH)
gonadotropin releasing hormone (GnRH)
somatostatin
thyrotropin releasing hormone (TRH)
adrenocorticotropic hormone (ACTH)	pituitary hormones
growth hormone (GH)
prolactin (PRL)
lutenizing hormone (LH)
thyrotropin (TSH)
oxytocin	neurohypophyseal peptides
vasopressin
atrial natriuretic peptide (ANF)	neuronal and endocrine
vasoactive intestinal peptide (VIP)
enkephalines (met-, leu-)	opiate peptides
dynorphin
b-endorphin

 

 

2.4.     Membrane transporters

Neurotransmitters are removed from synaptic cleft by enzymatic degradation or by active transport to presynaptic button or to surrounding glia cells. The main way of acetylcholine removing from synaptic cleft is metabolism by acetylcholinesterase. But for majority of neurotransmitters speculated in biochemical hypothesis of mental disorders there are specific membrane transporters (Figure 2.4).

 

The main classes of membrane transporters:

1. Na⁺/Cl^- dependent transporters are embedded in the presynaptic membrane mainly; they transport serotonin, norepinephrine or dopamine
2. Vesicular transporters carries neurotransmitters into synaptic vesicles (it is important to prevent neurotransmitter degradation)
3. Na⁺ dependent transporters are localized in the membrane of glia cells; they transport GABA, glutamate or aspartate

 

Figure 2.4. Membrane transporters

 

 

2.5.     Growth factors

Growth factors are proteins that stimulate cellular proliferation and promote cellular survival. They are essential for nervous system development and function. Different cells including neurons and glia produce growth factors. They are released from cell and after interaction with membrane receptors changes in activity of intracellular enzymes occur. So, mechanism of their action is similar to action of neurotransmitters, but they are not released in response to membrane depolarization and changes intracellular calcium levels.

 

There are 6 major classes of growth factors, which act within nervous system (Table 2.4.). From the psychiatry point of view neurotrophins are most important. Neurotrophins support the survival and phenotypic specificity of subsets of neurons. Brain-derived neurotrophic factor (BDNF) has a role in response on stress and in action of antidepressants.

 

Table 2.4. Growth factors in the nervous system

Neurotrophins	Nerve growth factor (NGF) Brain-derived neurotrophic factor (BDNF) Neurotrophin 3 (NT3) Neurotrophin 4/5 (NT4/5)
Neurokines	Ciliary neurotrophic factor (CNTF) Leukemia inhibitory factor (LIF) Interleukin 6 (IL-6) Cardiotrophin 1 (CT-1)
Fibroblast growth factors	FGF-1 FGF-2
Transforming growth factor b superfamily	Transforming growth factors b (TGFb) Bone morphogenetic factors (BMPs) Glial-derived neurotrophic factor (GDNF) Neurturin
Epidermal growth factor superfamily	Epidermal growth factor (EGF) Transforming growth factor a (TGFa) Neuregilins
Other growth factors	Platelet-derived growth factor (PDGF) Insulin-like growth factor I (IGF-I)

 

 

2.6.     Membrane receptors

Receptor is macromolecule specialized on transmission of information. It is defined as binding site with functional relationships. A binding site is not necessarily a physiological receptor, since neurotransmitters must bind to uptake systems and metabolic enzymes also. To determine whether a binding site is real receptor pharmacology of binding site and biological response must coincide.

Receptor complex includes:

1. Specific binding site
2. Transduction element
3. Effector system (2^nd messengers)

Effector system includes enzymes adenylyl cyclase or phospholipase, which generate 2^nd messengers, such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate (IP₃), diacylglycerol (DG), calcium (Ca²⁺); second messengers activate protein kinases A, G, C or CaM‑II.

 

Receptors are able to adapt their properties to increased or decreased activation (Table 2.5, Figure 2.5). Changes in the number of receptors are known mechanism of their adaptation. But response to receptor activation can be altered at unchanged density of receptors too. Regulation of properties of receptors consists of decreased or increased activity of post receptor events, which results in decreased or increased final physiological response to receptor stimulation (Table 2.6).

 

Table 2.5. Regulation of receptors

Number of receptors (down-regulation, up-regulation)

Properties of receptors (desensitisation, hypersensitivity)

 

Table 2.6. Mechanisms of receptor desensitisation

Interactions of subunits of active molecules (by phosphorylation, ribosylation, changes in membrane lipid composition, etc.)

Production of messengers - inhibitors of receptors

Production of receptor clusters (by cytoskeleton)

Internalisation of receptors (by endocytosis)

 

Figure 2.5. Adaptation of receptors

 

 

 

2.7.     Receptor classification

There are many types of receptors and they can be classified by different criteria, for example according to their pharmacological properties (it is according to activating neurotransmitter or other agonist) or according to effectors system, which is connected with their function (Table 2.7).

 

Table 2.7. Classification of receptors by their effector system

Receptor coupled directly to the ion channel

Receptor associated with G proteins

Receptor with intrinsic guanylyl cyclase activity

Receptor with intrinsic tyrosine kinase activity

 

 

2.8.     Receptors coupled to ion channels

Direct coupling of the neurotransmitter receptor to the ion channel whose activity it regulates is the simplest and the most rapid way of signal transduction. An example of receptor with internal ion channel is GABA_A receptor, nicotinic acetylcholine receptors, or ionotropic glutamate receptors.

 

Nicotinic acetylcholine receptor complex is one of ligand-gated ion channels; it contains both the acetylcholine binding site and the ion channel that is activated by acetylcholine binding. Following receptor activation, nicotinic acetylcholine receptors as well as ionotropic glutamate receptors increase membrane permeability for cations Na⁺, K⁺ or Ca²⁺; they are excitatory receptors.

 

Receptor for g-amino butyric acid includes chlorine (Cl^-) channel, which is opened in response to binding of GABA (Figure 2.6). Chlorine inputs into cell and hyperpolarization of membrane occurs; so it is inhibitory receptor. There are many modulation sites on GABA_A receptor, for example benzodiazepines positively modulate activity of this receptor.

 

Figure 2.6. GABA_A receptor

 

 

 

2.9.     Receptors associated with G proteins

It is assumed that signal transduction mediated by receptors associated with guanyl nucleotide-binding proteins (or G proteins) and 2^nd messenger systems are altered at mental disorders and during treatment with psychotropic drugs. Scheme for hypothesis of second messengers in signal transduction (Figure 2.7) show that after activation of receptor by first messenger G proteins are activated and activated G proteins activate effectors enzymes, such as adenylyl cyclase or phospholipase C, and second messengers are produced. Second messenger activate protein kinase of type A, C or CaM-II, which catalyses phosphorylation of cell proteins and physiological response to receptor activation arises. Transcription factors can be phosphorylated too, and phosphorylated transcription factors serve as third messengers, which activate gene expression.

 

According to type of 2^nd messenger used there are two main pathways of signal transduction: 1. adenylyl cyclase system; 2. phosphoinositide system.

 

In adenylyl cyclase system (Figure 2.8) activated receptor activates G protein, which activates adenylyl cyclase and cyclic adenosine monophosphate (cAMP) is produced as 2^nd messenger. cAMP activates protein kinase A, which phosphorylates cellular proteins (receptors, ion channels, enzymes, transcription factors, etc.).

 

In phosphoinositide system (Figure 2.9) activated receptor activates G proteins, which activate phospholipase C, and inositoltriphosphate (IP₃) and diacylglycerol (DG) are produced as 2^nd messengers. DG activates protein kinase C, IP₃ activate releasing of Ca²⁺ from intracellular pool and Ca²⁺ activate many enzymes including calmoduline and protein kinase dependent on Ca²⁺ and calmoduline.

 

 

2.10. Receptors with guanylyl cyclase activity

Cyclic guanosine monophosphate (cGMP) is produced as second messenger after activation of membrane bound or soluble form of guanylyl cyclase system (Figure 2.10). cGMP can activate protein kinase, activate or inhibit several forms of phosphodiesterase, open up cation channels (in retina), etc.

 

Figure 2.7. Scheme for hypothesis of second messengers

 

 

 

Figure 2.8. Signal transduction: Adenylyl cyclase system

 

G_s/i/o/x – G proteins, CaM – calmodulin, GTP – guanosine triphosphate, ATP – adenosine triphosphate, ADP – adenosine diphosphate, cAMP – cyclic adenosine monophosphate, 5´-AMP - 5´-adenosine monophosphate, PKA – protein kinase A, PKC – protein kinase C

 

 

Figure 2.9. Signal transduction: Phosphoinositide system

 

PI-PLC - phospholipase C specific for phosphoinositides, PIP2 - phosphatidylinositol-4,5-biphosphate, IP3 ‑ inositol-1,4,5-triphosphate; DG - diacylglycerol; PKC – protein kinase C

 

 

Figure 2.10. Signal transduction: Guanylyl cyclase system

 

GC – guanylyl cyclase, CaM – calmodulin, NO – nitric oxide, nNOS – nitric oxide synthase, ATP – adenosine triphosphate, cGMP – cyclic guanosine monophosphate

 

 

2.11. Receptor types

Basic characteristics of receptors which can be used to their classification are shown in Table 2.8. Primary types and selected subtypes of receptors (according to the international agreement) are summarized in Tables 2.9, 2.10, 2.11, 2.12 and 2.13.

 

Table 2.8. Basic characteristics of receptors

Name
Endogenous ligands and their efficiency
Selective agonists
Selective antagonists
Blockers of ion channels
Selective radioligands
Effectors	cGMP (internal guanylyl cyclase)
	Gi/o (K+­, Ca2+¯, cAMP¯)
	Gq/11 (IP3, DG)
	Gs (cAMP­)
	internal ion channel (Na+/K+/Ca2+/Cl-)
Gen
Structure

 

Table 2.9. Type of receptors

System	Type
acetylcholinergic	acetylcholine nicotinic receptors
	acetylcholine muscarinic receptors
monoaminergic	a1-adrenoceptors
	a2-adrenoceptors
	b-adrenoceptors
	dopamine receptors
	serotonin receptor
aminoacidergic	GABA receptors
	glutamate ionotropic receptors
	glutamate metabotropic receptors
	glycine receptors
	histamine receptors
peptidergic	opioid receptors
	other peptide receptors
purinergic	adenosine receptors (P1 purinoceptors)
	P2 purinoceptors

 

Table 2.10. Subtypes of monoamine receptors

RECEPTORS	Subtype	Transducer		Structure (aa/TM)
a1-adrenoceptors	a1A	Gq/11	­IP3/DAG	466/7
	a1B	Gq/11	­IP3/DAG	519/7
	a1D	Gq/11	­IP3/DAG	572/7
a2-adrenoceptors	a2A	Gi/o	cAMP	450/7
	a2B	Gi/o	cAMP	450/7
	a2C	Gi/o	cAMP	461/7
	a2D	Gi/o	cAMP	450/7
b-adrenoceptors	b1	Gs	­cAMP	477/7
	b2	Gs	­cAMP	413/7
	b3	Gs, Gi/o	­cAMP	408/7
dopamine	D1	Gs	­cAMP	446/7
	D2	Gi Gq/11	cAMP ­IP3/DAG, ­K+, ¯Ca2+	443/7
	D3	Gi	cAMP	400/7
	D4	Gi	cAMP, ­K+	386/7
	D5	Gs	­cAMP	477/7
5-hydroxytryptamine	5-HT1A	Gi/o	cAMP	421/7
	5-HT1B	Gi/o	cAMP	390/7
	5-HT1D	Gi/o	cAMP	377/7
	5-ht1E	Gi/o	cAMP	365/7
	5-ht1F	Gi/o	cAMP	366/7
	5-HT2A	Gq/11	­IP3/DAG	471/7
	5-HT2B	Gq/11	­IP3/DAG	481/7
	5-HT2C	Gq/11	­IP3/DAG	458/7
	5-HT3	internal cationic channel		478
	5-HT4	Gs	­cAMP	387/7
	5-ht5A	?		357/7
	5-ht5B	?		370/7
	5-ht6	Gs	­cAMP	440/7
	5-HT7	Gs	­cAMP	445/7

 

Table 2.11. Subtypes of aminoacidergic receptors

RECEPTORS	Subtypes	Transducer		Structure (aa/TM)
GABA	GABAA	int. Cl-		more subunits
	GABAB	Gi/o, Gs	cAMP, ­cAMP, ­K+(G), ­Ca2+(G)	961,941/7
excitatory aa (ionotropic)	NMDA	int. Na+/K+/Ca2+		more subunits
	AMPA	int. Na+/K+/Ca2+		more subunits
	kainate	int. Na+/K+/Ca2+		more subunits
excitatory aa (metabotropic)	mglu1	Gq/11	­IP3/DAG	1194/7
	mglu2	Gi/o	cAMP	872/7
	mglu3	Gi/o	cAMP	877/7
	mglu4	Gi/o	cAMP	912/7
	mglu5	Gq/11	­IP3/DAG	1212/7
	mglu6	Gi/o	cAMP	877/7
	mglu7	Gi/o	cAMP	915/7
	mglu8	Gi/o	cAMP	908/7
glycine	glycine	int. Cl-		pentamer
histamine	H1	Gq/11	­IP3/DAG	487/7
	H2	Gs	­cAMP	359/7
	H3	Gi/o	cAMP	445/7

 

Table 2.12. Subtypes of acetylcholine receptors

RECEPTORS	Subtypes	Transducer		Structure (aa/TM)
muscarinic	M1	Gq/11, NO	­IP3/DAG	460/7
	M2	Gi/o	cAMP,­K+(G)	466/7
	M3	Gq/11, NO	­IP3/DAG	590/7
	M4	Gi/o	cAMP,­K+(G)	479/7
	M5	Gq/11, NO	­IP3/DAG	532/7
nicotinic	a1	int. Na+/K+/Ca2+		pentamer
	a2	int. Na+/K+/Ca2+		pentamer
	a3	int. Na+/K+/Ca2+		pentamer
	a4	int. Na+/K+/Ca2+		pentamer
	a6	int. Na+/K+/Ca2+		pentamer
	a7	int. Na+/K+/Ca2+		pentamer
	a8	int. Na+/K+/Ca2+		pentamer
	a9	int. Na+/K+/Ca2+		pentamer

 

Table 2.13. Subtypes of opioid and related peptidergic receptors

RECEPTORS	Subtypes	Transducer		Structure (aa/TM)
opioid and similar receptors	mu opioid peptidergic (m)	Gi/o	cAMP, ­K+(G),¯Ca2+(G)	400/7
	delta opioid peptidergic (d )	Gi/o	cAMP, ­K+(G),¯Ca2+(G)	372/7
	kappa opioid peptidergic (k)	Gi/o	cAMP, ­K+(G),¯Ca2+(G)	380/7
	N/OFQ receptor	Gi/o	cAMP, ­K+(G),¯Ca2+(G)	370/7

 

 

2.12. Feedback and crossconnection

There are many neurotransmitters, many receptors, less G proteins and a few effector systems. But there are many feedbacks at the cellular level and many cross-reactions at the intracellular level. That results in divergence or convergence of signals.

 

Example of negative feedback in synapse (Figure 2.11):

Norepinephrine released from noradrenergic synapse can diffuse to adjacent acetylcholinergic synapse and can activate presynaptic a₂ receptors. Because a₂ receptors are inhibitory receptors, activation of noradrenergic synapse can lead to inhibition of acetylcholinergic synapse. There is great number of similar feedbacks.

 

Example of intracellular cross connection (Figure 2.12):

Activation of serotonin 2 receptor leads to production of diacylglycerol (DG) as 2^nd messenger and DG activates protein kinase C (PKC). PKC can phosphorylate adenylyl cyclase and positive regulation of transduction system connected with adrenoreceptors occurs. PKC can affect noradrenergic signal transduction through direct phosphorylation of receptors or G proteins also.

 

General scheme of signal transduction implying feedbacks is shown on Figure 2.13.

 

Figure 2.11. Feedback to transmitter-releasing

 

(See: Atwood H.L., MacKay W.A.: Essentials of Neurochemistry. B.C. Decker Inc., Toronto, Philadelphia, 1989.)

 

 

Figure 2.12. Crossconnection of transducing systems on postreceptor level

 

AR – adrenoceptor, G – G protein, PI-PLC – phosphoinositide specific phospholipase C, IP3 – inositoltriphosphate, DG – diacylglycerol, CaM – calmodulin, AC – adenylyl cyclase, PKC – protein kinase C

 

 

Figure 2.13. Scheme of signal transduction (implying feedback)

 

G – G protein, GTP – guanosine triphosphate, PLC - phospholipase C, PLD - phospholipase D, PLA2 - phospholipase A2, AC – adenylyl cyclase, IP3 – inositoltriphosphate, DG – diacylglycerol, cAMP – cyclic adenosine monophosphate, FFA – free fatty acids, PKC – protein kinase C, PKA – protein kinase A, PKCaM – Ca2+ and calmodulin dependent protein kinase, PP – phosphoprotein phosphatase