CHAPTER OUTLINE
Nitric Oxide Synthesis and Function
Tyrosine-Derived Neurotransmitters
Tryptophan-Derived Neurotransmitters
GABA: Inhibitory Neurotransmitter
Creatine and Creatinine Biosynthesis
High-Yield Terms
Endothelium-derived relaxing factor (EDRF): produced and released by vascular endothelium to promote smooth muscle relaxation; was found to be nitric oxide, NO
Catecholamines: neurotransmitter family derived from tyrosine, a catechol is an organic compound composed of a benzene ring and at least 2 hydroxyl groups attached, hence the derivation of the name of these compounds
Norepinephrine (noradrenaline): principal catecholamine neurotransmitter of sympathetic postganglionic nerves, its actions are exerted by binding to receptors of the adrenergic family
Epinephrine (adrenaline): a catecholamine neurotransmitter and hormone that modulates numerous function in the body by binding to receptors of the adrenergic family
Dopamine: a catecholamine neurotransmitter that plays a major role in the reward-driven learning processes in the CNS, functions by binding dopaminergic receptors
Glutathione: a tripeptide composed of glutamate, cysteine, and glycine that serves as a critical biological reductant; it is also conjugated to drugs to make them more water soluble and is involved in amino acid transport across cell membranes
Polyamine: any of a family of organic compounds having 2 or more primary amino groups (–NH2), biologically important polyamines are involved in DNA replication, ion channel modulation, and blood-brain barrier permeability
Several amino acids possess distinct biochemical functions unrelated to their roles in protein synthesis and as sources of oxidizable carbon. Numerous biologically active compounds are derived from the amino acids such as signaling molecules and neurotransmitters.
Nitric Oxide Synthesis and Function
Vasodilators, such bradykinin, do not exert their effects upon the vascular smooth muscle cell in the absence of the overlying endothelium. For example, when bradykinin binds to bradykinin B2 receptors on the surface of endothelial cells, a signal cascade, coupled to the activation phospholipase Cβ (PLCβ), is initiated. The PLCβ-mediated release of inositol trisphosphate, IP3 (from membrane-associated phosphatidylinositol-4,5-bisphosphate, PIP2), leads to the release of intracellular stores of Ca2+. In turn, the elevation in Ca2+ leads to the liberation of endothelium-derived relaxing factor (EDRF) which then diffuses into the adjacent smooth muscle. EDRF was found to be the free radical diatomic gas, NO. NO is formed by the action of NO synthase (NOS) on the amino acid arginine (Figure 31-1).
FIGURE 31-1: The reaction catalyzed by nitric oxide synthase. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York: McGraw-Hill; 2012.
NO can also be formed from nitrite, derived from vasodilator drugs such as glycerin trinitrate (nitroglycerin) during their metabolism. The half-life of NO is extremely short, lasting only 2-4 seconds. This is because it is a highly reactive free radical and interacts with oxygen and superoxide. NO is inhibited by hemoglobin and other heme proteins which bind it tightly.
Within smooth muscle cells, NO reacts with the heme moiety of a soluble guanylyl cyclase, resulting in activation of the latter and a consequent elevation of intracellular levels of cGMP. The net effect is the activation of cGMP-dependent protein kinase (PKG) and the phosphorylation of substrates leading to smooth muscle cell relaxation.
Both eNOS and nNOS are constitutively expressed and regulated by Ca2+. The calcium regulation is imparted by the associated calmodulin subunits, thus explaining how vasodilators such as acetylcholine effect smooth muscle relaxation as a consequence of increasing intracellular endothelial cell calcium levels. Although iNOS contains calmodulin subunits, its activity is unaffected by changes in Ca2+ concentration. iNOS is transcriptionally activated in macrophages, neutrophils, and smooth muscle cells.
The major functions of NO production through activation of iNOS are associated with the bactericidal and tumoricidal actions of macrophages. Overproduction of NO via iNOS is associated with cytokine-induced septic shock such as occurs postoperatively in patients with bacterial infections. Bacteria produce endotoxins such as lipopolysaccharide (LPS) that activate iNOS in macrophages.
NO is also generated by cells of the immune system and as such is involved in nonspecific host defense mechanisms and macrophage-mediated killing. NO also inhibits the proliferation of tumor cells and microorganisms. Additional cellular responses to NO include induction of apoptosis (programmed cell death), DNA breakage, and mutation.
Chemical inhibitors of NOS are available and can markedly decrease production of NO. The effect is a dramatic increase in blood pressure due to vasoconstriction. Another important cardiovascular effect of NO is exerted through the production of cGMP, which acts to inhibit platelet aggregation.
Tyrosine-Derived Neurotransmitters
The majority of tyrosine that does not get incorporated into proteins is catabolized for energy production. One other significant fate of tyrosine is conversion to the catecholamines. The catecholamine neurotransmitters are dopamine, norepinephrine, and epinephrine (Figure 31-2).
FIGURE 31-2: Synthesis of the catecholamines from tyrosine. Reproduced with permission of themedicalbiochemistrypage, LLC.
High-Yield Concept
There are 3 isozymes of NOS in mammalian cells: neuronal NOS (nNOS), also called NOS-1; inducible or macrophage NOS (iNOS), also called NOS-2; endothelial NOS (eNOS), also called NOS-3. Nitric oxide synthases are very complex enzymes, employing 5 redox cofactors: NADPH, FAD, FMN, heme, and tetrahydrobiopterin (BH4 is also sometimes written as H4B).
Nitric oxide is involved in a number of other important cellular processes in addition to its impact on vascular smooth muscle cells. Events initiated by NO that are important for blood coagulation include inhibition of platelet aggregation and adhesion and inhibition of neutrophil adhesion to platelets and to the vascular endothelium.
Norepinephrine is the principal neurotransmitter of sympathetic postganglionic nerves. Norepinephrine functions as a stress hormone and also regulates vascular tone resulting in increases in blood pressure. Epinephrine exerts numerous functions in the body that include regulation of heart rate, vascular tone, and alterations in metabolic processes.
High-Yield Concept
Both epinephrine and norepinephrine actions underlie the fight-or-flight response which encompasses increased heart rate, increased blood flow to skeletal muscle, and increased glucose mobilization from glycogen.
Tyrosine is transported into catecholamine-secreting neurons and adrenal medullary cells where catecholamine synthesis takes place. The site of synthesis is why these neurotransmitters are also referred to as adrenergic neurotransmitters and their receptors are called adrenergic receptors.
The first step in the process requires tyrosine hydroxylase, which like phenylalanine hydroxylase requires tetrahydrobiopterin (BH4) as cofactor. The dependence of tyrosine hydroxylase on BH4 necessitates the coupling to the action of dihydropteridine reductase (DHPR) as is the situation for phenylalanine hydroxylase (Chapter 30) and tryptophan hydroxylase (see below).
The hydroxylation reaction generates DOPA (3,4- dihydrophenylalanine, also called L-DOPA). DOPA decarboxylase converts DOPA to dopamine, dopamine β-hydroxylase converts dopamine to norepinephrine, and phenylethanolamine N-methyltransferase converts norepinephrine to epinephrine. This latter reaction is one of several in the body that uses S-adenosylmethionine (SAM) as a methyl donor generating S-adenosylhomocysteine. Within the substantia nigra and some other regions of the brain, synthesis proceeds only to dopamine. Within the adrenal medulla dopamine is converted to norepinephrine and epinephrine. Once synthesized, dopamine, norepinephrine, and epinephrine are packaged in granulated vesicles. Within these vesicles, norepinephrine and epinephrine are bound to ATP and a protein called chromogranin A.
The actions of norepinephrine and epinephrine are exerted via receptor-mediated signal transduction events. There are 3 distinct types of adrenergic receptors: α1, α2, β. Within each class of adrenergic receptor there are several subclasses. The α1 class contains the α1A, α1B, and α1D receptors. The α1 receptor class is coupled to Gq-type G-proteins that activate PLCβ resulting in increases in IP3 and DAG release from membrane PIP2. The α2 class contains the α2A, α2B, and α2C receptors. The α2 class of adrenergic receptor is coupled to Gi-type G-proteins that inhibit the activation of adenylate cyclase, and therefore activation results in reductions in cAMP levels. The β class of receptors is composed of three subtypes: β1, β2, and β3 each of which couple to Gs-type G-proteins resulting in activation of adenylate cyclase and increases in cAMP with concomitant activation of PKA.
Dopamine binds to dopaminergic receptors identified as D-type receptors and there are 4 subclasses identified as D1, D2, D4, and D5. Activation of the dopaminergic receptors results in activation of adenylate cyclase (D1 and D5) or inhibition of adenylate cyclase (D2 and D4).
Epinephrine and norepinephrine are catabolized (Figure 31-3) to inactive compounds through the sequential actions of catecholamine-O-methyltransferase (COMT) and monoamine oxidase (MAO). Compounds that inhibit the action of MAO have been shown to have beneficial effects in the treatment of clinical depression, even when tricyclic antidepressants are ineffective. The utility of MAO inhibitors was discovered serendipitously when patients treated for tuberculosis with isoniazid showed signs of an improvement in mood; isoniazid was subsequently found to work by inhibiting MAO.
FIGURE 31-3: Metabolism of the catecholamine neurotransmitters. Only clinically important enzymes are included in this diagram. The catabolic by-products of the catecholamines, whose levels in the cerebrospinal fluid are indicative of defects in catabolism, are in blue underlined text. TH, tyrosine hydroxylase; DHPR, dihydropteridine reductase; BH2, dihydrobiopterin; BH4, tetrahydrobiopterin; MAO, monoamine oxidase; COMT, catecholamine-O-methyltransferase; MHPG, 3-methoxy-4-hydroxyphenylglycol; DOPAC, dihydroxyphenylacetic acid. Reproduced with permission of themedicalbiochemistrypage, LLC.
Tryptophan-Derived Neurotransmitters
Tryptophan serves as the precursor for the synthesis of serotonin (5-hydroxytryptamine, 5-HT), and melatonin (N-acetyl-5-methoxytryptamine). Serotonin is synthesized through 2-step process involving a tetrahydrobiopterin-dependent hydroxylation reaction (catalyzed by tryptophan 5-monooxygenase, also called tryptophan hydroxylase) and then a decarboxylation catalyzed by aromatic L-amino acid decarboxylase (Figure 31-4). The hydroxylase is normally not saturated and as a result an increased uptake of tryptophan in the diet will lead to increased brain serotonin content.
FIGURE 31-4: Biosynthesis and metabolism of serotonin and melatonin. ([NH4+], by transamination; MAO, monoamine oxidase; ~CH3, from S-adenosylmethionine.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York: McGraw-Hill; 2012.
Serotonin is present at highest concentrations in platelets and in the gastrointestinal tract. Lesser amounts are found in the brain and the retina. Serotonin containing neurons have their cell bodies in the midline raphe nuclei of the brain stem and project to portions of the hypothalamus, the limbic system, the neocortex, and the spinal cord. After release from serotonergic neurons, most of the released serotonin is recaptured by an active reuptake mechanism. The function of the antidepressant, fluoxetine, and related drugs called selective serotonin reuptake inhibitors (SSRI) is to inhibit this reuptake process, thereby resulting in prolonged serotonin presence in the synaptic cleft.
The function of serotonin is exerted upon its interaction with specific receptors. Several serotonin receptors have been cloned and are identified as 5HT1, 5HT2, 5HT3, 5HT4, 5HT5, 5HT6, and 5HT7. Within the 5HT1 group there are subtypes 5HT1A, 5HT1B, 5HT1D, 5HT1E, and 5HT1F. There are 3 5HT2 subtypes, 5HT2A, 5HT2B, and 5HT2C as well as 2 5HT5 subtypes, 5HT5A and 5HT5B. Most of these receptors are coupled to G-proteins that affect the activities of either adenylate cyclase or phospholipase Cβ (PLCβ). The 5HT3 receptors are ion channels.
Some serotonin receptors are presynaptic and others postsynaptic. The 5HT2A receptors mediate platelet aggregation and smooth muscle contraction. The 5HT2C receptors are suspected in control of food intake as mice lacking this gene become obese from increased food intake and are also subject to fatal seizures. The 5HT3 receptors are present in the gastrointestinal tract and are related to vomiting. Also present in the gastrointestinal tract are 5HT4receptors where they function in secretion and peristalsis. The 5HT6 and 5HT7 receptors are distributed throughout the limbic system of the brain and the 5HT6 receptors have high affinity for antidepressant drugs.
Melatonin is derived from serotonin within the pineal gland and the retina, where the necessary N-acetyltransferase enzyme is expressed. The pineal parenchymal cells secrete melatonin into the blood and cerebrospinal fluid. Synthesis and secretion of melatonin increases during the dark period of the day and is maintained at a low level during daylight hours. This diurnal variation in melatonin synthesis is brought about by norepinephrine secreted by the postganglionic sympathetic nerves that innervate the pineal gland. The effects of norepinephrine are exerted through interaction with β-adrenergic receptors. This leads to increased levels of cAMP, which in turn activate the N-acetyltransferase required for melatonin synthesis. Melatonin functions by inhibiting the synthesis and secretion of other neurotransmitters such as dopamine and GABA.
GABA: Inhibitory Neurotransmitter
Several amino acids and amino acid derivatives have distinct excitatory or inhibitory effects upon the nervous system. The glutamate derivative, γ-aminobutyrate (GABA; also called 4-aminobutyrate) is a major inhibitor of presynaptic transmission in the CNS, and also in the retina. Neurons that secrete GABA are termed GABAergic neurons.
The formation of GABA occurs via a metabolic pathway referred to as the GABA shunt (Figure 31-5). Glucose is the principal precursor for GABA production via its conversion to α-ketoglutarate in the TCA cycle. Within the context of the GABA shunt the α-ketoglutarate is transaminated to glutamate by GABA α-oxoglutarate transaminase (GABA-T). Glutamic acid decarboxylase (GAD) catalyzes the decarboxylation of glutamic acid to form GABA. There are 2 GAD genes in humans identified as GAD1 and GAD2. The GAD isoforms produced by these 2 genes are identified as GAD67 (GAD1 gene: GAD67) and GAD65 (GAD2 gene: GAD65), which is reflective of their molecular weights. Both the GAD1 and GAD2 genes are expressed in the brain and GAD2 expression also occurs in the pancreas.
FIGURE 31-5: Metabolism of γ-aminobutyrate. (α-KA, α-keto acids; α-AA, α-amino acids; PLP, pyridoxal phosphate.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York: McGraw-Hill; 2012.