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  1. 1. Neurochemistry 1 2009 Tim Murphy Objective: To understand the metabolic processes underlying the synthesis and metabolism of amino acid and peptide neurotransmitters. Major points to be covered: -regulation of metabolism by enzymes -metabolic processes neurons share with other cells and organs -properties and functions of enzymes and pumps (transporters). -metabolic contingencies imposed by the existence of a blood-brain- barrier, i.e. the central role of glucose -synthesis and metabolism of amino acid transmitters and GABA. -glutamate -aspartate -glycine -neuropeptide synthesis and the pathway to regulated release
  2. 2. Neuronal metabolism. • Neurons share with other cells the need and ability to synthesize nucleic acids, proteins, carbohydrates and lipids. • Likewise they share the metabolic processes required to generate chemical energy for these processes: glycolysis, pentose-phosphate shunt, TCA cycle, oxidative phosphorylation. • Neurons must be able to synthesize and metabolize neurotransmitters. • Neurons must also synthesize second messenger molecules needed to mediate signal transduction.
  3. 3. The brain makes use of general metabolism to find precursors and in some cases the finished products for synaptic physiology. glycine
  4. 4. Enzymes • Help processes within neurons overcome activation energy, and provide a site of regulation. • Essentially all chemical reactions in cells are mediated by enzyme, protein catalysts. • A catalyst acts by bringing together the reactants, and thereby increasing the rate of a chemical reaction, without being permanently changed in the reaction. • Enzymes also allow the coupling of energetically unfavourable reactions with reactions that release free energy. If together the two reactions result in a negative ∆G, the coupled reaction can occur.
  5. 5. Enzymes lower activation energy for reactions.
  6. 6. Enzymes permit coupled reactions, for example falling rocks turn wheel to raise water for a different type of work.
  7. 7. ATP is a useful energy currency since it can form high-energy intermediates permitting the coupling of energetically unfavorable reactions to favorable ones, shown is the amination of glutamate.
  8. 8. General Properties of Enzymes • Enzymes are highly specific due to the specific structure of the active site • Substrate specificity • Reaction specificity • Enzymes bind substrates in specific ways that stabilize a reactive conformation, known as the TRANSITION STATE • Some enzymes require cofactors for complete activity (vitamin B6, pyridoxyl deficiency can impact GABA synthesis).
  9. 9. Velocity (V) versus substrate (S) plot. Km Saturation pseudo 1st order
  10. 10. V=Vmax * [S]/([S]+Km ) With a competitive inhibitor, the Km is increased but the Vmax is not effected. Km ’=Km *(1+[I]/Ki ), note when I= Ki the Km doubles With a noncompetitive inhibitor only the Vmax is reduced. Vmax’=Vmax*(1-[I]/([I]+Ki)), note when I= Ki the Vmax halves Michaelis-Menton Equation, describes saturable enzyme kinetics, also applicable to binding of ligands to receptors. know this, it describes many Interactions enzymes, receptors, protein-protein interactions
  11. 11. Km and Vmax • The activity of enzymes can be discussed in terms of their Km, a measure of the affinity of the enzyme for its substrate, and the Vmax, which is the maximal velocity of the enzymatic reaction. • Km has two meanings: 1) the concentration of substrate at which 1/2 the active sites on an enzyme are filled. 2) the ratio of dissociation to association rates for enzyme substrate interactions. Km=kdissoc/kassoc. Since the association rates of many reactions at going the speed of diffusion, the strength of binding and rates of reaction are often determined by the dissociation rate. • Although these terms are associated with enzymes they are related to other saturable systems such as transporters and receptors.
  12. 12. Competitive inhibitors. • Action: at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate. • Effect: Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.
  13. 13. Noncompetitive inhibitors. • Action:Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate. . • Effect: Vmax is reduced; Km, as defined by [S] required for 1/2 maximal activity, is unchanged. • Knowing if something is competitive or non- competitive is important since it determines how much inhibitor you need relative to substrate (practical implication!!)
  14. 14. 17 0.772727 0.361702128 0.128787879 21 0.807692 0.411764706 0.134615385 25 0.833333 0.454545455 0.138888889 29 0.852941 0.491525424 0.142156863 33 0.868421 0.523809524 0.144736842 37 0.880952 0.552238806 0.146825397 41 0.891304 0.577464789 0.148550725 45 0.9 0.6 0.15 49 0.907407 0.620253165 0.151234568 53 0.913793 0.638554217 0.152298851 57 0.919355 0.655172414 0.153225806 61 0.924242 0.67032967 0.154040404 65 0.928571 0.684210526 0.154761905 69 0.932432 0.696969697 0.155405405 73 0.935897 0.708737864 0.155982906 77 0.939024 0.719626168 0.156504065 81 0.94186 0.72972973 0.156976744 Receptor binding or enzyme Vel. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 [S] 21 45 69 93 117 141 165 189 substrate or ligand [ ] velocityorbinding V control V comp. Inh. V noncomp. Inh. Substrate or ligand concentration
  15. 15. Transport
  16. 16. Relative scales, simple diffusion rates will be low for polar substances.
  17. 17. Channels and carriers.
  18. 18. Since many transported compounds are charged their movement is governed by electrical and chemical gradients just like small ions such as K+, Na+, Cl-, and Ca2+.
  19. 19. Uniports-facilitative or uncoupled transport • Molecules or ions move down their concentration gradient via a specific carrier. • In contrast to a channel which will allow movement of thousands of ions per millisecond and whose specificity is primarily mediated by pore size, a facilitative carrier requires binding of a specific substrate which induces conformational changes in the carrier through which the substrate is moved, and then released, restoring the carrier to its original conformation.
  20. 20. Carrier-Mediated Transport, Uniporters. • Carrier types at the blood brain barrier: hexose, monocarboxylic acid, large neutral amino acid, basic amino acid, acidic amino acid, choline, purine, and nucleoside carriers. • These substances serve as building blocks for all brain macromolecules and neurochemicals.
  21. 21. Symports and antiports • Couple movement of one molecule with that of one or more other substrates. Energy is derived from concentration gradients no ATP needed (directly) although indirectly to establish gradient. • The high-affinity pumps for amino acids, and neurotransmitters are principally Na+-symporters, i.e. the movement of Na+ down its electrochemical gradient provides the free-energy required to move another substrate (neurotransmitter) up its concentration gradient • Na+/Ca++ antiporters, and Na+/H+ antiporters move these ions out of cells as Na+ enters.
  22. 22. Na+, Ca2+ exchangeGlutamate protons
  23. 23. The Na+ gradient can be used to pump glucose uphill.
  24. 24. Primary active transport • Systems utilize the free-energy obtained by ATP hydrolysis to move ions against concentration gradients (uphill), i.e. Na+-, K+-ATPase or the Ca2+ ATPase. • Estimated to require up to half the brain ATP, while other biochemical processes including protein, lipid and neurotransmitter synthesis together use perhaps 10%. • Other primary pumps, such as Ca2+-ATPases and proton pumps probably account for the rest. The brain uses 20% of total body oxygen consumption, thus 10% of total is used primarily to maintain neuronal ionic gradients via this pump.
  25. 25. Na+, K+ ATPase
  26. 26. For reference.
  27. 27. Na+, K+ ATPase • Energy is directed into the pumping process by the 3Na+-dependent phosphorylation, followed by the 2K+-dependent dephosphorylation. Phosphorylation induces a conformational change that moves 3Na+ to the outside of the cell. • Pump stoichiometry is 3/2 making it electrogenic.
  28. 28. Fundamental Neurosci. 2002 Zigmond et al.
  29. 29. Role of the pump in resting membrane potential. • If pump is blocked with ouabain (blocks binding of K+) an immediate small depolarization occurs (only a few mV), however membrane will remain relatively constant as it is largely determined by K+ permeability, however the membrane is also slightly permeable to Na+ and over time the membrane potential will depolarize if Na+ diffuses in unchecked by the pump.
  30. 30. Glucose • Is the major fuel of the brain because it is the only fuel which enters in sufficient amounts to support the energy requirements. • Glucose gains access to brain and into cells by specific carriers - blood levels much higher than brain levels, thus glucose moves down its concentration gradient via facilitative transport. • Glucose utilization of tied to neuronal activity and increased blood flow, basis of PET functional imaging with 2-deoxyglucose. • Isolated neurons can use other fuels such as pyruvate and lactate, but they normally are not BBB permeable.
  31. 31. Blood (~6 mM glucose). 4X Glut-1 expressed on the ab-lumenal side Farrell and Pardridge 1991 CSF (~4 mM glucose). Fundamental Neurosci. 2002 Zigmond et al.
  32. 32. Glucose transport • The Km of the BBB glucose transporter is about 7 mM, which is about the level of plasma glucose, thus brain glucose varies directly with changes in blood levels. The blood brain barrier transporter is Glut-1. • Neurons possess a carrier of higher affinity, Glut3 Km = 200 µM, allowing them to extract glucose from the extracellular space. Within neurons, glucose is immediately phosphorylated to a charged, impermeant metabolite, glucose-6- phosphate, thus the intracellular glucose concentration is effectively zero. Why is it advantageous to reduce the apparent free concentration of glucose.
  33. 33. Used in PET scanning. Fundamental Neurosci. 2002 Zigmond et al.
  34. 34. Glycolysis and TCA cycle • Within the cell, glucose enters the glycolysis pathway in the cytoplasm, and via pyruvate and acetyl-CoA, in the mitochondrial TCA cycle. In these systems, reducing equivalents are generated and via oxidative phosphorylation they generate ATP, the chemical fuel for the brain. • Glycolysis and the TCA cycle are also the source of non-essential amino acid precursors used to synthesize the neurotransmitters glutamate, aspartate, GABA, and glycine.
  35. 35. Blood brain barrier. • What is the blood brain barrier (BBB)? • The existence of a blood-brain-barrier prevents molecules in the circulation from freely entering the brain. • Prevents constant fluctuations in circulating metabolites, ions, and hormones from directly influencing neuronal activity. • Diffusion allows passage of gases, i.e. (O2 and CO2) and lipid soluble compounds, i.e. psychoactive drugs.
  36. 36. The blood brain barrier largely occurs at capillaries through astrocyte endfeet and endothelium tight junctions. Transport across it is selective. Carrier types at the blood brain barrier: hexose,monocarboxylic acid, large neutral amino acid, basic amino acid, acidic amino acid, choline, purine, and nucleoside carriers. Drewes LR. Adv Exp Med Biol. 1999;474:111-22. . Endothelium
  37. 37. Iadecola and Nedergaard 2007 Nat. Neurosci.
  38. 38. Astrocytes labeled and imaged in a live mouse with sulforhodamine; maximal intensity projection through 200 µm of adult C57 bl6 cortex. Blood vessels apparently enwrapped by astrocyte processes are indicated by arrows. The dark shadows are due to large superficial vessels that absorb the IR light preventing excitation below them. 30 µm
  39. 39. Perivascular glia contain high levels of the antioxidant tripeptide glutathione Sun et al. 2006.
  40. 40. Paulson, European Neuropsychopharmacology 12, 2002, Pg. 495 Fig. 1. Characteristics of the endothelium. In the muscle capillary (upper) there are pores or slits between the endothelial cells allowing bulk flow of water and smaller solutes between the blood and the extracellular space in the tissue. In contrast, the brain endothelial cells (lower) are connected by tight junctions. No pores or slits are present preventing bulk flow. Water therefore has to cross the blood–brain barrier by the mechanism of diffusion.
  41. 41. Brain activity and blood supply are tightly linked. • It has been known for over 100 years increased neuronal activity is associated with increases in blood flow. Roy CS, Sherrington CS (January 1890). "On the Regulation of the Blood-supply of the Brain". J. of Physiol. 11 (1-2): 85–158.17. • Changes in blood flow or oxygenation are used a surrogate measure of neuronal activity.
  42. 42. Imaging brain metabolism. • 2-deoxygluocose method radioactive detection or positron emission tomography (PET) scanning, need isotopes poor time resolution (Sokoloff 1977 J. of Neurochem.). • Functional magnetic resonance imaging (fMRI), second level time resolution, signals related to changes in oxy/deoxyhemoglobin potentially complicated (Ogawa et al. 1990 PNAS). • Intrinsic signal imaging more direct spectroscopy of brain signals related to changes in oxy/deoxyhemoglobin, can be performed with a video camera (Grinvald et al. 1986 Nature).
  43. 43. 10 µm Synapses are on average 13 µm from capillaries. RBC supply rates are normally ~100 cells/sec. Acute reduction in supply rate by >90% leads to damage within 10 min, which can reverse if reperfusion occurs early. Zhang et al. 2005
  44. 44. Scale bar=10 um region1 ctr at 49_5410 µm Control 10 min 30 min 1 hr 2 hr 3 hr Irreversible ischemia; red vessels, green dendrites. clot
  45. 45. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 99.90 99.95 100.00 100.05 100.10 100.15 100.20 reflected635nmlight Time (sec) 635 nm light 1) Reduced reflection, increased absorbance with elevated deoxyhemoglobin in active areas. 2) General increase in blood volume and oxyhemoglobin in surrounding areas leads to large late positive global signal. Intrinsic optical signals, light scattering provides a reflection of neuronal activity. Stim 1 secReflected light 2) General blood volume. 1) Local deoxyhemo- globin signal.
  47. 47. Change in light scattering in response to forelimb stimulation.
  48. 48. Neurotransmitters: small molecule and neuropeptide.
  49. 49. Small molecule Neurotransmitters (MW<300) are synthesized in the terminal. precursor uptake Transporters/carriers are required to transit the vesicle and plasma membrane. Fundamental Neurosci. 2002 Zigmond et al.
  50. 50. Neurotransmitter transporters: • Plasma membrane forms terminate neurotransmission, replenish neurotransmitter pool, and may have a signaling function. • Vesicular transporters use both concentration gradient and protons to concentrate transmitter in vesicles: these transporters make neurons transmitter specific.
  51. 51. Molecular structure of plasma memb. neurotransmitter transporters: • Norepinephrine, GABA, serotonin, dopamine, glycine, choline, and proline transporters • Homologous in their 12 transmembrane spanning domains. • In the case of GABA, transport results from the co-transport of Na+ and Cl- ions
  52. 52. Glutamate plasma memb. transporters form a distinct family. • Structure different from other transmitter transporters-8 transmembrane domains not related to other existing mammalian transporter clones. • Homologous to each other (~50%) and to a bacterial proton-coupled glutamate transporter. • Glast1 (EAAT1), Storck et al. PNAS 89, 10955- 10959 (1992). Expressed in glial cells. • GLT-1 (EAAT2), Pines et al. Nature 360, 464-467 (1992). Localized to glial cells • EAAC1 (EAAT3), Kanai and Hediger Nature 360, 467-471 (1992). Relatively neuron specific in brain, also expressed in intestinal tissues, and kidney.
  53. 53. Transporter is electrogenic allowing its current to be measured and studied with the patch clamp method.
  54. 54. Vesicle glutamate transporters. • Several members including VGLUT1 and VGLUT2, and VGLUT3 isolated by R. Edwards lab (Science;289:957-60, 2000). • Defines glutamatergic neuron classes, although all neurons contain glutamate only those expressing VGLUT’s can package it at high concentrations. • Transport mediated by a combination of H+ and ionic gradients.
  55. 55. Chaudhry et al. 2002 JCB. Vesicular accumulation of amino acids results from both a gradient of membrane potential and pH.
  56. 56. Neurotransmitter glycine. • Non-essential amino acid derived from glycolysis and TCA cycle intermediates. • Glycine made from glucose via amino acid serine. • High-affinity uptake system removes glycine from synapse. Shares a vesicular pump with GABA, VGAT • Glycine and its pump found in high levels in spinal cord, in neurons presynaptic to strychnine-sensitive glycine receptor-chloride channel. • Receptors mainly found in the spinal cord.
  57. 57. Neurotransmitter glutamate • Na+ -dependent, high-affinity uptake system has been well characterized, and occurs principally in glutamate nerve terminals (EAAC-1/EAAT3). • Glutamate uptake into glial cells allows metabolism via glutamine synthetase. Glutamine formed in glia then enters neurons to provide a precursor for glutamate synthesis via glutaminase activity. • Since glutamate transport is determined by ion concentration gradients it is described by the Nernst potential. At positive voltages the transporter can reverse (may occur during a stroke).
  58. 58. Astro Gln efflux through system N. Neuronal Gln uptake by system A. see Chaudhry et al. 2002 JCB. Fundamental Neurosci. 2002 Zigmond et al.
  59. 59. Fundamental Neurosci. 2002 Zigmond et al.
  60. 60. Fundamental Neurosci. 2002 Zigmond et al.
  61. 61. Reference, Glutamate metabolism, 4 possible synthetic pathways 1) From α-ketoglutarate (2-oxoglutarate) and ammonia via glutamate dehydrogenase. This pathway is of fundamental importance in the synthesis of all amino acids, since it is the key mechanism for the formation of α-amino groups directly from ammonia. Transamination of α-keto acids with glutamate as amino group donor then allows the introduction of α-amino groups into the synthesis of other amino acids. 2) From α-ketoglutarate and aspartate by aspartate aminotransferase; antibodies to this enzyme stain many presumed glutamate neurons 3) From glutamine by glutaminase; antibodies to this enzyme also stain some presumed glutamate neurons. Glutaminase removes the NH2 from the glutamine. 4) From α-ketoglutarate by ornithine-aminotransferase or via proline oxidase. Both these pathways form P5C (pyroline 5-carboxylic acid), which via P5C dehydrogenase can yield glutamate. There is no evidence yet that these are neuronal enzymes. However, a high-affinity proline uptake system has recently been found that appears to be associated with glutamate pathways.
  62. 62. (astrocyte) Fundamental Neurosci. 2002 Zigmond et al.
  63. 63. Neuropeptide neurotransmitters. • History i.e. regulated release of enzymes from exocrine cells, and hormones such as insulin from endocrine cells • The discovery of vasopressin release from posterior pituitary in the 1940s by du Vigneaud demonstrated that neurons could secrete peptides for intercellular communication • This was followed by the discovery of hypothalamic factors regulating the anterior pituitary by Guillemin and Schally • The discovery, in the mid-seventies, of enkephalins as endogenous ligands for discovered opiate receptors.
  64. 64. Fundamental Neurosci. 2002 Zigmond et al.
  65. 65. Synthesis and processing of neuropeptides, RNA. • mRNA splicing to generate different bioactive peptides, selective usage of some exons. A mechansim by which a single gene encodes polypeptides of varied function. Splicing occurs in the nucleus. Substance P and substance K are encoded by the same gene but are only found together in mature mRNA in some tissues. Calcitonin and CGRP are formed in different neurons by alternative splicing of introns. • mRNA moves through nuclear pores and into cytoplasm.
  66. 66. Peptide synthesis. • Proteolytic maturation then occurs in acidic, clathrin-coated secretory vesicles. Involves endopeptidases, which often cleave C-terminal to the paired dibasic amino acids, i.e. Lys-Arg, Arg- Arg. POMC can be processed into at least 6 different peptide hormones through proteolytic cleavage (ACTH, bendorphin, Clip, aMSH, gMSH, bLPH, etc). Processing can be specific to different brain or pituitary regions. • The dibasic residues are then removed by carboxypeptidase.
  67. 67. Peptide synthesis. • Some prohormones, i.e. somatostatin, are cleaved by other endopeptidases, N-terminal to dibasic pairs, which are then removed by aminopeptidases. • Many peptides end in a modified C-terminal amide. This is formed by the action of peptidyl- glycine-α-amidating monooxygenase (PAM) which converts the C terminal Gly to a amide group. Amidation is critical for the function of some peptides (such as substance P). • Vesicles containing peptides are moved via fast axonal transport to release sites
  68. 68. Degradation • specific uptake systems have not been identified • presumably, diffusion from synapses, and proteases of various sorts on the surface of neurons and glia cleave the peptides to their constitutive amino acids, which can then be reutilized
  69. 69. Methods of study • Purification via bioassay, chemical assay, molecular cloning • Synthesis allows antibody production, RIA, immunohistochemistry, radioligand binding, electrophysiology • Most peptides act via G protein-coupled receptors modulate K+ channels and Ca++ channels and can be studied electrophysiologically.
  70. 70. Anatomy, localization • Found in most, if not all neurons, can coexist with other peptides or with amine and amino acid transmitters, present in dense core large vesicles. • Made in the cell body on ribosomes and transported to terminals. • If a prohormone is cleaved prior to packaging in vesicles, it is possible to sort the mature peptides to different vesicles. In fact, recent work in Aplysia indicates that peptides in distinct vesicles can be sorted to distinct neuronal processes. This would appear to contravene Dales Law: 1) a neuron has only one transmitter and 2) a neuron is only excitatory or inhibitory.
  71. 71. Readings • Fundamental Neuroscience Fundamental Neuroscience 1st Ed., Chapter 8, p. 193-234 Chapter 14, p.389-392. Or 2nd Ed. Chapter 7 p. 167-196 and Chapter 13 339-360. In 3rd Edition Chap. 7 starting pg.133 and Chapter 12 starting pg. 271. • Cooper, Bloom & Roth, The Biochemical Basis of Neuropharmacology, Chaps. 7-13, 6th Ed or Chaps 6-11 7th Ed. • Molecular Biology of the Cell 4th ed. Chapter 11 or Molecular Biology of the Cell 3rd ed. Chapter 11 p 507-523.