Neurotransmitters 1. Types of Neurotransmitters Two general types: 1) small, nitrogen-containing molecules: Amino acids glutamate, aspartate, GABA, glycine Monoamines dopamine, norepinephrine, epinephrine, serotonin, histamine Others: acetylcholine, purines (ATP, adenosine) 2) neuroactive peptides (small polypeptides); more than 40!: Gut-Brain peptides eg substance P, neuropeptide Y, neurotensin, vasoactive intestinal peptide, insulin, CCK, POMC, others Pituitary hormones oxytocin, vasopressin, LH, GH, FSH, ACTH, MSH, TSH Hypothalamic releasing factors CRH, GnRH, GHRH, TRH, somatostatin Opioids b-endorphin, dynorphin, enkephalins Others angiotensin, bradykinin,. 2. Overview of Synaptic Transmission Knowledge of the general features of synaptic transmission is crucial to understanding how communication in the nervous system is regulated. The whole process involving transmitter synthesis, storage, release, receptor binding, degradation, and reuptake involves many molecules that are targets for mutations/disease as well as pharmacotherapy. Synthesis of Small Molecule Neurotransmitters The molecules and enzymes necessary for synthesizing small molecule neurotransmitters are contained in the presynaptic terminal. This ensures that the supply of neurotransmitter can keep up with electrical activity. NT synthesis is regulated by neuronal activity levels. Synthesis occurs in the cytosol, but then neurotransmitters are packaged in vesicles to protect them from degradation and to prepare them for release. Cofactors in NT synthesis include folic acid, SAM (S-adenosylmethionine), O 2, Cu 2, vitamins C, B6, and B12. Storage in Vesicles Neurotransmitters are concentrated into presynaptic vesicles. Vesicles are assembled in the terminal through a process of endocytosis that provides a mechanism for recycling material. Neurotransmitters enter vesicles using transporter proteins in the vesicular membrane. Transport depends on a vesicular ATPase that pumps protons into the lumen. The transporter exchanges H + in the lumen with transmitter in the cytoplasm. This efficient mechanism allows vesicles to concentrate neurotransmitters to 50-100 mm levels. Drugs like reserpine block the vesicular transporter. They prevent refilling of vesicles and inhibit synaptic transmission. Neurotransmitter Release Depends on Ca influx The fate of neurotransmitter released at a synapse includes: 1) binding to presynaptic receptors, 2) binding to postsynaptic receptors, 3) diffusion out of synaptic cleft, 4) enzymatic degradation, and 5) reuptake across the plasma membrane. Transmitter Receptors Ionotropic receptors: 1
Composed of 4-5 subunits that form a pore in the membrane for passage of ions. Diversity: different types of subunits are mixed to make receptors that vary in physiological, pharmacological and functional properties and these are distributed to different CNS locations. Subunit composition can change developmentally, causing receptors to have agedependent properties. Metabotropic- G protein coupled ionotropic Metabotropic receptors: Transmitter binding is coupled to G protein activation and second messenger pathways. Diversity come from the different types of G proteins (G s, G i/o, G q ) that are coupled to receptors and the specific subunits (a, b, g) associated with them. Reuptake by Plasma Membrane (pm)transporters Plasma membrane (pm) transporters efficiently allow neurotransmitters and other molecules to cross the cell membrane. pmtransporters depend on cotransport of Na + and other ions to move transmitters into the terminal against their concentration gradients. They can produce a 10,000x increase in presynaptic neurotransmitter concentration compared to extracellular space. This is a diverse group of molecules that is expressed within and outside the nervous system. Some transmitters have several transporter subtypes, which vary in NT location, specificity, and pharmacology. Implication: the CNS changes molecular structure to match specific needs at specific locations. Note: Most drugs cannot take advantage of these differences and consequently affect the entire class of molecules. Functions of reuptake: 1) terminate action of transmitter at receptor, 2) prevent transmitter diffusion to other synapses, 3) recycle supply of transmitter in presyn. terminal. pmtransporters can run in reverse when neurotransmitter levels are high intracellularly. Some molecules called false neurotransmitters (tyramine, guanethidine, ephedrine, amphetamine) mimic neurotransmitters and bind to pm transporters to enter terminals and then bind to the vesicular transporter to enter vesicles. They displace the real neurotransmitters, which accumulate in the terminal cytosol. This can result in a large, nonvesicular leak of real transmitter out of the terminal and massive stimulation of Na + 2
receptors. In addition, the false neurotransmitters may be released from synaptic vesicles to have reduced effects on postsynaptic receptors (inhibitor or partial agonist). 3. Criteria for Neurotransmitters: 1) synthesized in neuron, 2) stored in nerve terminal, 3) released in quantities sufficient to affect postsynaptic cell, 4) exogenous application mimics action, 5) mechanism for removal. 3
Overview of Synaptic Transmission 9. Neuromodulation by presynaptic receptors 1. Neurotransmitter synthesis 2. Storage in Vesicles 8. Recycling of vesicles 7. Reuptake transporter in neurons/glia 6. Degradation in cleft, metabolism, or diffusion from synapse 5. Binding to Receptors G Postsynaptic membrane 3. Ca ++ entry 4. Neurotransmitter Release 4
4. Amino Acid Neurotransmitters (glutamate, aspartate, GABA, glycine) these AAs are common to all cells/neurons!! To be transmitter, must be taken up into synaptic vesicles. Essential AAs cross the blood brain barrier (BBB) via transporters to enter brain. However, AA neurotransmitters DO NOT CROSS BBB. They are restricted from entry WHY? NTs must be synthesized by neurons and glia from TCA intermediates and other AAs. GLUTAMATE Synthesis: 1. 70% synthesized from glutamine by glutaminase 2. glucose a-ketoglutarate glutamate O NH 2 O C CHCH 2 CH 2 C OH Glutamate THE major excitatory neurotransmitter in CNS Vesicular transporter highly specific for glutamate; concentrates into synaptic vesicle. Receptors: ionotropic 14 possible subunits arranged in groups of 4 to form 3 types: AMPA, Kainate, NMDA metabotropic 8 types: mglur1, mglur2, mglur3, mglur4, mglur5, mglur6, mglur7, mglur8 SIGNIFICANCE: Potentially, many types of tetra/penta-meric ionotropic receptors can be made from differing combinations of subunits, all responding to the same neurotransmitter. However, each type may differ in its geographic distribution, physiological properties, pharmacological sensitivity, and functional role. Reuptake by plasma membrane Glu transporter The primary mechanism for inactivation of Glu in the synapse. pmglu transporter is found primarily on astrocytes (few on neurons). Thus, glia play a big role in Glu inactivation and recycling. Astrocytes take-up glutamate, convert it to glutamine via glutamine synthetase and transport it out to extracellular environment. Neurons take-up glutamine via a glutamine transporter and convert it to glutamate. 5 subtypes differing in affinity, specificity, location. highly effective at lowering extracellular Glu concentration. Elevated Glu levels are neurotoxic!! Glu transporter is important in buffering Glu especially if released in excessive amounts by neurons in pathological conditions. This reuptake system also plays a big role in neuronal metabolism. Glutamate is cotransported into astrocytes along with Na +, using its concentration gradient. The increase in intracellular Na that occurs, stimulates the Na + -K + ATPase, which then stimulates ATP synthesis. The consequent increase in astrocyte metabolism results in increased levels of lactate that is transported out of astrocytes and into surrounding neurons to be used in oxidative metabolism. 5
Nerve terminal astrocyte Nature Reviews Neuroscience 6
GABA (g-amino butyric acid) Synthesis: Glutamate GABA by glutamic acid decarboxylase (and from glutamine) THE major inhibitory neurotransmitter in CNS; major importance in controlling potential for seizures, anxiety, sedation. Drugs facilitate GABA binding. Vesicular transporter concentrates GABA in vesicles Receptors: ionotropic GABA-A, GABA-C; metabotropic GABA-B GABA A Ionotropic GABA receptor has specific modulatory sites where agonist and antagonist molecules bind to alter the inhibitory efficacy of the receptor. Agonists like benzodiazepines act allosterically to facilitate GABA s ability to activate the receptor and also prolong the time the channel stays open Flumazenil blocks Molecular Neuropharmacology by Nestler, Hyman, Malenka, McGraw Hill, 2001 Alcohol also binds to and facilitates the GABA A receptor. Because they bind at different sites to the same receptor, benzos, barbiturates, and alcohol can act synergistically to depress neuronal activity to lethal levels. In contrast, alcohol detoxification, which lowers GABA A receptor activity by removing its facilitory effect can be treated with benzos to lessen the effects of withdrawal, which can be life-threatening. Inactivation: reuptake by neurons and glia. 4 different pmgaba transporters identified that differ in structure, type of cell found (neuron/glia/other), pharmacology NH 2 GABA O CH 2 CH 2 CH 2 C (Anatagonist) OH Receptor (Anatagonist) 7
5. Monoamine Neurotransmitters (dopamine, norepinephrine, epinephrine, serotonin) Catecholamines (dopamine, norepinephrine, epinephrine) General characteristics of Catecholamines: Synthesized by a small percentage of neurons but terminals have wide distribution to large areas of brain. Act as excitatory and inhibitory neurotransmitters, but they also have powerful, modulatory effects (influence release of other transmitters) that influence motor activity, emotion, mood, attention, and arousal. eg impaired dopamine release causes movement disorders, Parkinson s disease, schizophrenia. All based on structure of catechol Dopamine system All synthesized from tyrosine or indirectly from phenylalanine. Remember: phenylalanine (essentialaa) tyrosine (nonessentialaa) via PAH phe and tyr are actively transported across BBB and then transported into neurons The disorder PKU (PAH defect) results in low catecholamine levels! One type of vesicular transporter in brain for all monoamines. Second type of vesicular monoamine transporter in adrenal medulla. Reserpine inhibits vesicular transporter. ALL receptors are metabotropic (G-protein coupled) affect ion channels directly or indirectly via second messenger pathways. Receptor activation is complex can cause excitation in some neurons, but inhibition in others based on how G-protein (phosphorylation) affects particular type of channel subunits expressed. Major mechanism for stopping synaptic action of monoamines is reuptake into cell. Two types of pmcatecholamine transporters: dopamine transporter and NE/E transporter. Importance: 1) terminates synaptic action, 2) limits diffusion to other synapses, 3) recycles unmetabolized transmitter for packaging in vesicles and its reuse. Catecholamines are degraded by 2 enzymes: monoamine oxidase (MAO) and catechol-omethyltransferase (COMT) found intra- and extra-cellularly in neurons and other cells. DOPAMINE Synthesized from tyrosine (see diagram). Also from phenylalanine. Tyrosine hydroxylase is rate limiting enzyme in synthesis. It s activity is saturated at normal levels of tyrosine in neuron. Tyrosine and phenylalanine cross the BBB via a single transporter, which is also saturated at normal blood AA levels. Thus, catecholamine synthesis cannot be increased by raising tyrosine levels. L-aromatic amino acid decarboxylase (AADC) has broad specificity for amino acid substrates; also present in many cell types outside nervous system. Carbidopa, which doesn t cross BBB, inhibits peripheral AADC to prevent conversion of dopa to dopamine peripherally. Peripheral dopamine affects gut and causes nausea/vomiting. Source: Neurons that release dopamine have cell bodies in only 2 locations: Midbrain and hypothalamus. Dopamine Receptors metabotropic D 1, D 2, D 3, D 4, D 5 ; excitatory or inhibitory depending on receptor subtype (eg D 1 receptors are excitatory, D 2 receptors are inhibitory). 8
Inactivated by reuptake via pmdopamine Transporter (DAT) Inhibited by cocaine Amphetamines interact with dopamine and NE transporters Neurotoxin MPTP is a substrate for pmdopamine transporter. Selectively kills dopaminergic neurons when internalized. NOREPINEPHRINE Dopamine-b-hydroxylase is unique bound to inner surface of synaptic vesicle; NE is synthesized inside vesicle from dopamine. Thus, uses vesicular monoamine transporter. Source: Pons (locus ceruleus) is the major source of NE cell bodies for CNS. The locus ceruleus influences arousal. Postganglionic sympathetic neurons also contain NE. NE Receptors: multiple types of a/b adrenergic receptors Inactivation by reuptake via pmne transporter (NET) Inhibited by several classes of antidepressants: tricyclics imipramine, amytriptyline; selective NE reuptake Inhibitors (SNRIs) venlafaxine, reboxetine; and cocaine. These drugs inhibit NE, DA, and SERT transporters to varying degrees. EPINEPHRINE (derivation: epi-nephron) Phenylalanine-N-methyltransferase (cytoplasmic) converts NE to E. Requires NE to exit vesicle, undergo conversion, and then be transported back into vesicle. Source: few E-neurons in CNS; E is primarily synthesized in adrenal medulla E Receptors: a/b adrenergic receptors Degradation of Monoamines by MAO and COMT MAO present in neurons and most mammalian cells; intracellular and extracellular location intracellularly localized to outer mitochondrial membrane; degrades monoamines not protected inside vesicles by deamination to aldehyde. Functions: degrades monoamines in neurons/regulates general neurotransmitter level. Dietary monoamines act as false neurotransmitters. MAO also 1) decreases availability of dietary monoamines in peripheral tissues (gut) and 2) prevents their entry across BBB. MAO A and MAO B forms: differ in CNS location, substrate specificity, pharmacology MAO A Distribution: CNS and gut Present in gut and liver to breakdown dietary monoamines (eg tyramine in cheese and wine). Tyramine gains entry into sympathetic neurons via the pmmonoamine transporter and concentrates in vesicles via the vesicular monoamine transporter where it displaces NE. Irreversibly inhibited by clorgyline MAO B Distribution: CNS (astrocytes, serotonergic neurons, histaminergic neurons) Irreversibly inhibited by selegiline MAO inhibitors (MAOIs) Nonspecific irreversible inhibitors: tranylcypromine, phenelzine, isocarboxazid 9
Newer MAOIs are more selective and reversible Increase presynaptic concentration of neurotransmitters and prolong availability of released neurotransmitter Caution on dangerous interactions When combined with foods containing tyramine (beer, red wine, cheese, salami, soy sauce, fava beans, liver), may result in release of large amounts of NE, inducing hypertensive crisis. WHY: MAO normally metabolizes tyramine in gut. Excess tyramine displaces NE in sympathetic vesicles and NE is released at synapses by reversal of the pmne transporter. COMT present in nervous system and peripheral tissues; present extracellularly in synaptic cleft and degrades neurotransmitter after release. broad catechol substrate specificity methylates (SAM cofactor) one of the catechol hydroxyl groups Inhibitors include: entacapone, tolcapone Indolamines: SEROTONIN (5-hydroxytryptamine/5-HT) Synthesized from tryptophan, an essential AA transported across BBB. synthesis similar to dopamine: tryptophan hydroxylase is rate-limiting enzyme Vesicular monoamine transporter concentrates serotonin into synaptic vesicles. Source: serotonergic cell bodies are located in midline (raphe) nuclei of the pons and medulla. Axons distribute widely to the cortex and spinal cord. Receptors: 14 different receptors identified so far (5-HT 1A-F, 5-HT 2, 5-HT 3..); All are metabotropic except 5-HT 3 (ionotropic). Have excitatory and inhibitory effects. All hallucinogenic drugs are 5-HT 2A partial agonists; Many antipsychotics are 5-HT 2A and D 2 dopamine receptor antagonists. The large number of serotonin receptor subtypes is linked to their unique roles in brain function. Development of drugs that are subtype-specific agonists or antagonists may be more selective for specific clinical disorders. Inactivation: Synaptic action stopped primarily by reuptake via specific pmserotonin transporter (SERT) Inhibitors: Many antidepressants (SSRIs Selective Serotonin Reuptake Inhibitors, tricyclics) bind with high affinity; also cocaine. These drugs inhibit NE, DA, and SERT transporters to varying degrees. Serotonin is metabolized by MAO Serotonergic Syndrome can occur from drug interactions that cause excessive serotonin activity, eg MAO combined with SSRIs or tricyclic antidepressants; SSRIs/SNRIs combined with serotonin agonists (triptans). Symptoms include mental status changes (agitation, anxiety, confusion, hallucinations), myoclonus, ataxia, fever, autonomic hyperactivity (shivering, diarrhea, life-threatening cardiovascular changes). 10
Catecholamine Synthesis and Degradation Tyrosine COOH CH 2 CH NH 2 terahydrobiopterin catechol Dopa (Dihydroxyphenylalanine) Tyrosine Hydroxylase COOH OH CH 2 CH NH 2 OH pyridoxal phosphate L-Aromatic Amino Acid Decarboxylase Dopamine H CH 2 CH NH 2 OH OH H Norepinephrine ascobate Dopamine-b-hydroxylase 1. MAO: Deamination to aldehyde + 2. COMT: Methylation of catechol hydroxyl HVA VMA MHPG OH CH CH NH 2 homovanillic acid vanillylmandelic acid 3-methoxy-4-hydroxy-phenylglycol Phenolethanolamine-N-methyl-transferase SAM OH H Epinephrine CH 3 CH CH N H OH 11
Serotonin Synthesis and Degradation Tryptophan COOH N CH 2 CH NH 2 terahydrobiopterin Tryptophan Hydroxylase 5-Hydroxytryptophan COOH CH 2 CH NH 2 N pyridoxal phosphate Aromatic amino acid decarboxylase 5-Hydroxytryptamine N CH 2 CH 2 NH 2 MAO Deamination to aldehyde 5-HIAA (5-Hydroxyindole acetic acid) Acetylcholine Synthesis and Degradation O CH 3 C SCoA Acetyl CoA + CH3 CH 2 CH 2 N CH3 Choline CH3 choline acetyltransferase SCoA O CH3 O + CH 3 C O CH 2 CH 2 N CH3 CH 3 C OH + Acetylcholine CH3 Acetate Acetylcholine esterase CH3 CH 2 CH 2 N CH3 Choline CH3 12
7. Other types of Small Molecule Neurotransmitters PURINES: ATP, adenosine Important neurotransmitter in pain system. Peripheral pain fibers have purinergic receptors; damaged tissues releases ATP causing excitation. Adenosine receptors are metabotropic and caffeine is an antagonist. Membrane-soluble molecules: nitric oxide, arachadonic acid. 8. Neuroactive Peptides These are small polypeptides consisting of 5-41 amino acids. They act as neurotransmitters to adjacent neurons, they can enter the circulation to act as hormones on distant target organs in the body, and they act as neuromodulators of activity and behavior by influencing release of other transmitters over long periods of time. Neuromodulators have slow onset, long duration effects on other neurons influencing excitability and transmitter release rather than acting as a fast on/off synaptic signal. For example, nicotine from 1 cigarette can improve mood, decrease anxiety, and enhance attention by influencing dopamine release at synapses for long periods. Their synthesis, packaging into vesicles, processing in presynaptic terminals, and function are different from that of the small molecule neurotransmitters. Neuropeptide synthesis requires DNA transcription and mrna translation to produce a protein. Generally, neuropeptides are synthesized as large precursor polypeptides (prepropeptides) that are subsequently cleaved into smaller molecules in a multistep process. Thus, each precursor may give rise to many different smaller peptides that each have bioactivity (eg opioids). Neuropeptides are synthesized in the cell body on ribosomes, they subsequently are processed through the endoplasmic reticulum, and then are transferred to the Golgi Apparatus where they are packaged into vesicles. Vesicles containing neuropeptides travel by axoplasmic transport down the axon to presynaptic terminals. Presynaptic vesicles undergo calcium-dependent release. They may act on postsynaptic cells or they may be transported by the circulation to distant targets where they act as hormones. Inactivation of neuropeptides is slow and depends on extracellular proteases, which results in long lasting effects. There are NO reuptake transporters for neuropeptides, so they cannot reenter the presynaptic terminal. Synaptic transmission depends on a continuous supply from the cell body and inactivation is slow. They can be co-released with small molecule neurotransmitters from the same terminal. They utilize a large variety of receptors, which are metabotropic, G-protein coupled. REFERENCES: **1. Molecular Neuropharmacology, E.J.Nesler, S.E.Hyman, and R.C.Malenka, McGraw-Hill, 2001; an excellent resource on neurotransmitters 2. I.B.Levitan and L.K.Kaczmarek, The Neuron, 3 rd edition, Oxford University Press, NY, NY, 2002 3. Biochemical Basis of Neuropharmacology, J.R.Cooper, F.E.Bloom, and R.H.Roth, 8 th edition, Oxford University Press, NY 2003 13
Mechanism of Action of Some Drugs 9. Modulation by presynaptic receptors a2 agonist/antagonist 8. Recycling vesicles 1. Neurotransmitter synthesis levodopa MAO-I selegiline 2. Storage in Vesicles Reserpine ß 7. Reuptake transporter Cocaine, antidepressants, cyclobenzaprine 6. Degradation in cleft, metabolism, or diffusion MAO-I - selegiline COMT-I - entacapone AChE-I - pyridostigmine Postsynaptic membrane 5. Binding to Receptors Agonists - benzos, baclofen, opioids, succinylcholine Antagonists antipsychotics, naloxone, atropine G 3. Ca entry 4. Neurotransmitter Release amphetamine, amantadine, botox 14