Much of the tyrosine that does not get incorporated into proteins is catabolized for energy production. Another significant fate of tyrosine is conversion to the catecholamines. The catecholamines are dopamine, norepinephrine, and epinephrine. All three catecholamines exert effects in numerous locations in the body as either a neurotransmitter or as a hormone. Within the central and peripheral nervous systems (CNS and PNS, respectively) the catecholamines exert their effects as neurotransmitters, in the periphery they do so as hormones. The details of catecholamine effects exerted via activation of specific receptors are only covered briefly in this section. For greater detail go to the Biochemistry of Nerve Transmission page.
Tyrosine is transported into catecholamine-secreting neurons and adrenal medullary cells where catecholamine synthesis takes place. The first step in the process requires tyrosine hydroxylase which, like phenylalanine hydroxylase (of tyrosine synthesis), requires tetrahydrobiopterin (H4B, or written as BH4) as cofactor. The tyrosine hydroxylase reaction represents the rate-limiting reaction of catecholamine biosynthesis. The dependence of tyrosine hydroxylase on H4B necessitates the coupling to the action of dihydropteridine reductase (DHPR) as is the situation for phenylalanine hydroxylase and tryptophan hydroxylase (see below). The product of the tyrosine hydroxylase reaction is 3,4-dihydrophenylalanine (L-DOPA; more commonly just DOPA). The enzyme DOPA decarboxylase then converts DOPA to dopamine. The enzyme dopamine β-hydroxylase then converts dopamine to norepinephrine. Dopamine β-hydroxylase is a major vitamin C and copper (Cu2+)-dependent enzyme whose activity is negatively affected in Menkes disease. The last step of catecholamine biosynthesis is the conversion of norepinephrine to epinephrine which involves a methylation reaction. The enzyme phenylethanolamine N-methyltransferase catalyzes this methylation reaction utilizing SAM as a methyl donor. In addition to epinephrine synthesis, the last reaction generates S-adenosylhomocysteine. Within the substantia nigra locus of the brain, and some other regions of the brain, synthesis proceeds only to dopamine. Within the locus coeruleus region of the brain the end product of the pathway is norepinephrine. Within adrenal medulla chromaffin cells, tyrosine is converted to norepinephrine and epinephrine.
The tyrosine hydroxylase gene (symbol: TH) is located on chromosome 11p15.5 and is composed of 14 exons that generate three alternatively spliced mRNAs that encode tyrosine hydroxylase isoforms a, b, and c, Mutations in the TH gene are associated with the development of Segawa syndrome. Segawa syndrome is an autosomal recessive disorder that manifests in early infancy as a DOPA-responsive dystonia. Two distinct phenotypes are associated with Segawa syndrome, one that manifests very early and presents with symptoms of greater severity, and a later onset less severe type that responds better to L-DOPA therapy. DOPA decarboxylase (also known as aromatic L-amino acid decarboxylase) is encoded by the DDC gene which is located on chromosome7p12.2 and is composed of 18 exons that generate multiple alternatively spliced mRNAs. The DDC encoded enzyme is also responsible for the conversion of 5-hydroxytryptophan to serotonin (see next section) and tryptophan to tryptamine. The dopamine β-hydroxylase gene (symbol: DBH) is located on chromosome 9q34 and is composed of 12 exons that encode a protein of 617 amino acids. Dopamine β-hydroxylase is found as both a soluble and a membrane-bound enzyme dependent upon whether or not the signal peptide from the precursor protein is present. The phenylethanolamine N-methyltransferase gene (symbol: PNMT) is located on chromosome 17q12 and is composed of 5 exons that generate two alternatively spliced mRNAs, one of which is non-coding, the other encodes a protein of 282 amino acids.
Synthesis of the catecholamines from tyrosine. Tyrosine is converted to each of the three catecholamines through a series of four reactions. The tissue from which the neurotransmitter/hormone is derived expresses a specific set, or all, of these enzymes such that only dopamine (substantia nigra) is the result, or only norepinephrine (locus coeruleus), or both norepinephrine and epinephrine (adrenal medulla). DOPA decarboxylase (also known as aromatic L-amino acid decarboxylase) is encoded by the DDC gene. Dopamine β-hydroxylase is a critical vitamin C (ascorbate) and copper (Cu2+)-dependent enzyme.
Once synthesized, dopamine, norepinephrine and epinephrine are packaged in granulated vesicles for secretion in response to the appropriate nerve impulse. Within these vesicles, norepinephrine and epinephrine are bound to ATP and a protein called chromogranin A. Norepinephrine is the principal neurotransmitter of sympathetic postganglionic nerves. Both norepinephrine and epinephrine are stored in synaptic knobs of neurons that secrete it, however, epinephrine is not a mediator at postganglionic sympathetic nerve impulses. The major location, within the brain, for norepinephrine synthesis is the locus coeruleus of the brainstem. The major brain region for the synthesis of dopamine is the substantia nigra which is located below the posterior hypothalamus and next to the ventral tegmetal area. The presence of high concentrations of tyrosine in the locus coeruleus and the substantia nigra leads to increased melanin synthesis which confers on these brain regions a dark bluish coloration observable in brain sections. Indeed, these brain regions are so-called due to the dark bluish-black pigmentation. The Latin term, substantia nigra, means "black substance". The Latin word coeruleus means "dark blue, blue, or blue-green".
Outside the brain, the major site of norepinephrine and epinephrine synthesis is in adrenal medullary chromaffin cells. Outside the brain, dopamine is synthesized in several tissues including the gastrointestinal system where its actions reduce gastrointestinal motility, the pancreas where its actions inhibit insulin synthesis, and in the kidneys where its actions increase sodium excretion and urinary output.
The actions of norepinephrine and epinephrine are exerted via receptor-mediated signal transduction events. The receptors to which epinephrine and norepinephrine bind are referred to as adrenergic receptors. The adrenergic receptors are members of the G-protein coupled receptor (GPCR) family. There are two distinct classes of adrenergic receptor identified as the α (alpha) and β (beta) receptors. There are two distinct subclasses of α adrenergic receptor identified as the α1 and α2 sub-classes. For greater detail on the adrenergic receptors go to the Biochemistry of Nerve Transmission page. Within each class of adrenergic receptor there are several sub-classes. The α1 class contains the α1A, α1B, and α1D receptors. The α1 receptor class are 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 receptors are coupled to Gi-type G-proteins that inhibit the activation of adenylate cyclase and therefore, receptor activation results in reduced levels of cAMP and consequently reduced levels of active PKA. 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. However, the β2 class of receptor can switch from Gs to Gi/o signaling following phosphorylation of the receptor by PKA.
Dopamine binds to dopaminergic receptors identified as D-type receptors and there are five subclasses identified as D1, D2, D3, D4, and D5. All five dopamine receptors belong the the G-protein coupled receptor (GPCR) family. The D1 and D5 dopamine receptors are coupled to the activation of Gs-type G-proteins and, therefore, receptor activation results in activation of adenylate cyclase. The D2, D3, and D4 dopamine receptors are coupled to Gi-type G-proteins and, therefore, receptor activation results in the inhibition of adenylate cyclase. For more details on the dopamine receptors go to the Biochemistry of Nerve Transmission page.
Epinephrine and norepinephrine are catabolized 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.
Catabolism of the catecholamine neurotransmitters. Only clinically important enzymes are included in this diagram. The catabolic byproducts of the catecholamines, whose levels in the cerebrospinal fluid are indicative of defects in catabolism, are in blue underlined text. Abbreviations: TH: tyrosine hydroxylase, DHPR: dihydropteridine reductase, H2B: dihydrobiopterin, H4B: tetrahydrobiopterin, MAO: monoamine oxidase, COMT: catecholamine-O-methyltransferase, MHPG: 3-methoxy-4-hydroxyphenylglycol, DOPAC: dihydroxyphenylacetic acid.
Tryptophan serves as the precursor for the synthesis of serotonin (5-hydroxytryptamine, 5-HT, see also Biochemistry of Nerve Transmission) and melatonin (N-acetyl-5-methoxytryptamine).
Pathway for serotonin and melatonin synthesis from tryptophan. Abbreviations: THP: tryptophan hydroxylase; DHPR: dihydropteridine reductase; H2B: dihydrobiopterin; H4B: tetrahydrobiopterin, 5-HTP: 5-hydroxytryptophan; DDC: DOPA decarboxylase; AANAT: aralkylamine N-acetyltransferase; ASMT: acetylserotonin-O-methyltransferase.
Serotonin is synthesized through a two-step process involving a tetrahydrobiopterin-dependent hydroxylation reaction (catalyzed by tryptophan-5-monooxygenase, also called tryptophan hydroxylase) and then a decarboxylation catalyzed by DOPA decarboxylase (also known as aromatic L-amino acid decarboxylase). Tryptophan hydroxylase is normally not saturated and as a result, an increase in dietary uptake of tryptophan will lead to increased brain serotonin content. Tryptophan hydroxylase represents the rate-limiting step in serotonin and melatonin synthesis. Humans possess two tryptophan hydroxylase genes identified as TPH1 and TPH2. The TPH1 gene is located on chromosome 11p15.3–p14 and is composed of 10 exons that encode a protein (tryptophan 5-hydroxylase 1) of 444 amino acids. The TPH2 gene is located on chromosome 12q21.1 and is composed of 14 exons that encode a protein (tryptophan 5-hydroxylase 2) of 490 amino acids.
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 (serotonergic 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 re-uptake of serotonin is catalyzed by the SLC family transporter, SCL6A4. The function of the antidepressant, Prozac®, and related drugs called selective serotonin re-uptake inhibitors (SSRIs), is to inhibit this reuptake process, thereby, resulting in prolonged serotonin presence in the synaptic cleft. Mutations in the SLC6A4 gene affect the rate of serotonin re-uptake and have been shown to be correlated with obsessive-compulsive disorder, anxiety-related traits and are suspected to be involved in sudden infant death syndrome, aggressive behavior in Alzheimer disease patients, and depression-susceptibility in persons experiencing emotional trauma.
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 three 5HT2 subtypes, 5HT2A, 5HT2B, and 5HT2C as well as two 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 class of receptors are ion channels, referred to as ionotropic receptors.
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 involved in the regulation of emesis (vomiting). Also present in the gastrointestinal tract are 5HT4 receptors 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 rate-limiting enzyme for melatonin synthesis 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 activates PKA. PKA phosphorylates aralkylamine N-acetyltransferase activating this enzyme required for melatonin synthesis. The terminal reaction in melatonin synthesis is catalyzed by acetylserotonin O-methyltransferase which is synthesized from one of two genes. One gene is located on the X chromosome (Xp22.3) and the other on the Y chromosome (Yp11.3). Melatonin functions by inhibiting the synthesis and secretion of other neurotransmitters such as dopamine and GABA.back to the top
The amino acid derivative, γ-aminobutyrate (GABA; also called 4-aminobutyrate) is a major inhibitory neurotransmitter responsible for the regulation of presynaptic transmission in the CNS, and also in the retina. Neurons that secrete GABA are termed GABAergic. GABA cannot cross the blood-brain-barrier and as such must be synthesized within neurons in the CNS. The synthesis of GABA in the brain occurs via a metabolic pathway referred to as the GABA shunt. 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 the amino acid glutamate by GABA α-oxoglutarate transaminase (GABA-T). Glutamic acid decarboxylase (GAD) catalyzes the decarboxylation of glutamic acid to form GABA. There are two GAD genes in humans identified as GAD1 and GAD2. The GAD isoforms produced by these two 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. The activity of GAD requires pyridoxal phosphate (PLP) as a cofactor. PLP is generated from the B6 vitamins (pyridoxine, pyridoxal, and pyridoxamine) through the action of pyridoxal kinase. Pyridoxal kinase itself requires zinc for activation. A deficiency in zinc or defects in pyridoxal kinase can lead to seizure disorders, particularly in seizure-prone pre-eclamptic patients (hypertensive condition in late pregnancy). The presence of anti-GAD antibodies (both anti-GAD65 and anti-GAD67) is a strong predictor of the future development of type 1 diabetes in high-risk populations.
GABA exerts its effects by binding to two distinct receptors, GABA-A (GABAA) and GABA-B (GABAB) that are members of the ion channel (ionotropic) receptors. GABA-A receptors are chloride channels that in response to GABA binding increases chloride influx into the neuron. The GABA-B receptors are potassium channels that when activated by GABA leads to potassium efflux from the cell. For more details on the synthesis and function of GABA go the the Biochemistry of Nerve Transmission page.back to the top
Histamine is a potent neurotransmitter that binds to specific histamine receptors. Histamine is synthesized by the enzymatic decarboxylation of the amino acid histidine by the enzyme L-histidine decarboxylase (HDC). Within the gastrointestinal tract bacteria also produce histamine via a similar decarboxylation reaction. The principal cells that synthesize and release histamine are mast cells and basophils of the immune system, enterochromaffin-like cells of the gastrointestinal system, and neurons. The synthesis and storage of histamine by mast cells and basophils represents the greatest store (>90%) of the neurotransmitter. Within the brain the neurons that synthesize histamine are within the tuberomammillary nucleus of the hypothalamus.
The histidine decarboxylase gene (symbol: HDC) is located on chromosome 15q21.2 and is composed of 14 exons that generate two alternatively spliced mRNAs encoding two distinct isoforms of the enzyme. The isoform 1 protein is composed of 662 amino acids and the isoform 2 protein is composed of 629 amino acids.
Histamine functions by binding to specific histamine receptors. Humans express four distinct histamine receptors identified as H1R, H2R, H3R, and H4R. All four histamine receptors are members of the G-protein coupled receptor superfamily. The H1R protein is encoded by the HRH1 gene, the H2R protein is encoded by the HRH2 gene, the H3R protein is encoded by the HRH3 gene, and the H4R protein is encoded by the HRH4. The HRH1 gene is located on chromosome 3p25 and is composed of 8 exons that generate four alternatively spliced mRNAs all of which encode the same 487 amino acid protein. The HRH2 gene is located on chromosome 5q35.2 and is composed of 8 exons that generate two alternatively spliced mRNAs. HRH2 isoform 1 is 397 amino acids and HRH2 isoform 2 is 359 amino acids. The HRH3 gene is located on chromosome 20q13.33 and is composed of 4 exons that encode a 445 amino acid protein. The HRH4 gene is located on chromosome 18q11.2 and is composed of 5 exons that generate three alternatively spliced mRNAs. Isoform 1 is 390 amino acids. Isoform 2 is 302 amino acids. Isoform 3 is 67 amino acids. For more details on histamine function go to the Biochemistry of Nerve Transmission page.back to the top
Vasodilators, such as acetylcholine and bradykinin, do not exert their effects upon the vascular smooth muscle cell in the absence of the overlying endothelium. When acetylcholine (or bradykinin) binds its receptor on the surface of endothelial cells, a signal cascade, coupled to the activation phospholipase C-β (PLCβ), is initiated. The activation of PLCβ, via acetylcholine binding to vascular smooth muscle, results from the activation of the M3 (muscarinic) receptor. The activation of PLCβ via bradykinin results from its binding to the B2 bradykinin receptor. Both the M3 and B2 receptors are coupled to Gq-type G-proteins. The activation of PLCβ results in the release of inositol trisphosphate, IP3 (from membrane associated phosphatidylinositol-4,5-bisphosphate, PIP2), which in turn leads to the release of intracellular stores of Ca2+. The elevation in Ca2+ leads to the production of endothelium-derived relaxing factor (EDRF) which then diffuses into the adjacent smooth muscle. Within smooth muscle cells, EDRF reacts with the heme moiety of a soluble guanylate cyclase (sGC), 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; specifically the PKGI isoforms) and the phosphorylation of substrates leading to smooth muscle cell relaxation.
Quite unexpectedly, EDRF was found to be the free radical diatomic gas, nitric oxide, NO. So stunning was the elucidation of the pathway to, and actions of, NO that Drs. Murad, Ignarro and Furchgott were awarded the Nobel Prize in 1998 for their work on this system. Endogenous NO is formed by the action of NO synthase, (NOS) on the amino acid arginine. 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 anion. NO is also inhibited by hemoglobin and other heme proteins which bind it tightly. NO can also be formed from nitrite, derived from vasodilators such as glycerin trinitrate (nitroglycerin, NTG) and nitroprusside during their metabolism.
Neuronal NOS (nNOS), also called NOS-1. The NOS1 gene is located on chromosome 12q24.22 and is composed of 35 exons the generate multiple alternatively spliced mRNAs that encode three characterized isoforms.
Inducible or macrophage NOS (iNOS), also called NOS-2. The NOS2 gene is located on chromosome 17q11.2 and is composed of 29 exons that encode a protein of 1153 amino acids.
Endothelial NOS (eNOS), also called NOS-3. The NOS3 gene is located on chromosome 7q36 and is composed of 29 exons that generate multiple alternatively spliced mRNAs that encode four characterized isoforms.
Nitric oxide synthases are very complex enzymes, employing five redox cofactors: NADPH, FAD, FMN, heme, and tetrahydrobiopterin (H4B; also written BH4). The role of Ca2+ ions in the function of NOS is related to the calmodulin subunits of NOS enzymes (see below). All NOS enzymes also require Zn2+ ions for function. The role of zinc in NOS function is a structural one not a catalytic one. All three NOS enzymes function as homodimers and the interactions of the subunits is required for the process of electron flow from NADPH (bound in the reductase domain of the enzyme), through the various co-factors to the heme moiety which is bound to the N-terminal oxygenase domain of the enzyme. It is the oxygenase domain that binds the required oxygen and arginine substrates. The oxygenase domain also binds the H4B co-factor. The actual process of NO production by NOS enzymes involves two steps. In the first step the enzyme hydroxylates arginine to Nω-hydroxy-L-arginine. During the second step the enzyme oxidizes the Nω-hydroxy-L-arginine to L-citrulline and NO.
Nitric oxide synthase (NOS) activities in the synthesis of nitric oxide (NO). NOS enzymes function as homodimers when heme is bound. The heme moiety of one monomer is indeed essential for the process of interdomain electron transfer between NADPH and the flavins (FAD and FMN) to the heme of the opposite monomer. When intracellular Ca2+ rises it facilitates the binding of the calmodulin subunits to eNOS and nNOS. In the presence of the substrate, arginine, and the tetrahydrobiopterin cofactor (H4B; also can be written BH4) the intact NOS homodimers couple the reduction of ferric (Fe3+) heme to ferrous (Fe2+) heme while simultaneously coupling O2 reduction to the synthesis of NO. The other product of the reaction is citrulline. Electron transfer from the reductase domain in the C-terminus enables NOS ferric (Fe3+) heme to bind the required O2 and form a ferrous (Fe2+) heme dioxy species. This heme species can receive a second electron from H4B (preferentially) or from the reductase domain. Within the active NOS homodimer the resulting oxidized H4B has been shown to exist as the trihydrobiopterin radical (H3B•) or the trihydrobiopterin radical cation protonated at N5 (H3B•H+). The H3B• radical (or the radical cation) can be reduced back to H4B by electrons supplied by the flavins associated with functional NOS. Additional evidence indicates that ascorbic acid (AscH) can serve as the reducing agent which generates the ascorbate radical (Asc•) during the reduction process.
Both eNOS and nNOS are constitutively expressed and regulated by Ca2+. The calcium regulation is imparted be 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 interacts with calmodulin, its activity is unaffected by changes in Ca2+ concentration. iNOS is transcriptionally activated in macrophages, neutrophils, and smooth muscle cells. When intracellular Ca2+ levels rise the calmodulin subunits have an increased affinity for eNOS and nNOS. The calmodulin subunits facilitate the flow of electrons from NADPH to the heme moiety in the oxygenase domain of the enzyme.
Endothelial NOS (eNOS) is most highly expressed within endothelial cells. However, expression of eNOS is also detected in platelets, cardiac myocytes, specific renal tubular epithelial cells, certain neurons within the brain, and in placental syncytiotrophoblast cells. As indicated, the major regulator of eNOS (and nNOS) activity is Ca2+ ion. However, other factors regulate the activity of eNOS that are not related to changes in calcium concentrations. Several proteins, such as caveolin-1 and hsp90, interact with and affect the overall activity of eNOS. In addition, eNOS activity can be modulated by phosphorylation. The predominant trigger for eNOS phosphorylation is fluid shear stress in the vasculature. Additional triggers for eNOS phosphorylation include vascular endothelial growth factor (VEGF)-induced signal transduction, insulin-mediated activation of PKB/AKT, and bradykinin-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Phosphorylation of eNOS enhances the flow of electrons through the reductase domain of the enzyme. The predominant effects of NO produced in response to eNOS activation are vasodilation and inhibition of platelet adhesion and aggregation. Endothelial NOS-derived NO induces the dilation of all types of blood vessels by stimulating soluble guanylate cyclase (sGC) and increasing cGMP in smooth muscle cells. The cGMP, in turn, activates PKGI isoforms resulting in the phosphorylation of smooth muscle proteins that lead to relaxation. The ability of eNOS produced NO to prevent platelet adhesion and aggregation reduces the propensity for the development of abnormal fibrin clots (thrombi) and, therefore, will contribute to a reduced potential for the development of atherosclerosis. Endothelial cell produced NO can also interfere with leukocyte adhesion to the endothelium thus, further contributing to a reduction in the potential for atherosclerosis.
Neuronal NOS (nNOS) is constitutively expressed in specific neurons within the brain and spinal cord. Neurons that express nNOS and thus produce NO, are called nitrergic nerves. Neuronal NOS is also constitutively expressed within the adrenal glands, pancreatic islet cells, epithelial cells of numerous tissues, vascular smooth muscle, skeletal muscle, and renal macula densa cells. Within the brain the principal functions of nNOS produced NO are in the mediation of long-term synaptic transmission. Production of NO within the brain, via nNOS, is also important for the central regulation of blood pressure. Peripheral production of NO via the action of nNOS, like that produced by the action of eNOS, can effect changes in vascular tone. These vascular effects of nNOS are independent of the effects of nNOS within the CNS. Neuronal NOS is expressed within smooth muscle cells of the penile corpus cavernosum and thus, NO produced by this enzyme is involved in penile erection. Smooth muscle relaxation induced by NO in the corpus cavernosum is mediated by cyclic GMP (cGMP). The cGMP that is produced via NO activation of soluble guanylate cyclase (sGC) in this tissue is degraded by a specific cyclic nucleotide phosphodiesterase (PDE) identified as PDE5A. Residual corpus cavernosum nNOS activity is essential for the erectile promoting actions of selective PDE5 inhibitors such as sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis).
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 post-operatively in patients with bacterial infections. Bacteria produce endotoxins such as lipopolysaccharide (LPS) that activate iNOS in macrophages. Although predominately induced in macrophages, under appropriate stimulation conditions many other cell types will express the iNOS gene. Once expressed, the iNOS enzyme is catalytically active and does not respond with changes in enzyme activity to changes in intracellular calcium levels as is the case for nNOS and eNOS. The production of large amounts of NO by activation of iNOS expression in macrophages represents the major cytotoxic activity of these cells. The NO produced binds to the iron in numerous enzymes that contain the metal in their active sites. Examples of NO-inhibited enzymes include the Fe-S cluster-dependent activities of mitochondrial oxidative phosphorylation and ribonucleotide reductase. Macrophage generated NO can also cause DNA strand breaks and fragmentation. These effects of macrophage produced NO explain, to a large degree, the cytotoxic and cytostatic effects of NO on invading parasites as well as on certain tumor cells.
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.back to the top
Creatine synthesis occurs predominantly in the kidneys and the liver. The synthesis of creatine begins in the kidneys using the amino acids arginine and glycine. The formation of guanidinoacetate (GAA) from these two amino acids is catalyzed by the enzyme glycine amidinotransferase, also called L-arginine:glycine amidinotransferase (AGAT). Glycine amidinotransfersae is encoded by the GATM gene located on chromosome 15q21.1 and is composed of 12 exons that encode a 423 amino acid protein that localizes to the mitochondria. Guanidinoacetate it transported to the blood and picked up by heptocytes where it is methylated forming creatine. The methyl donor for this reaction is S-adenosylmethionine (SAM or AdoMet) and the reaction is catalyzed by the enzyme guanidinoacetate N-methyltransferase. This latter enzyme is encoded by the GAMT gene located on chromosome 19p13.3 and is composed of 6 exons that generate two alternatively spliced mRNAs that encode isoform a (236 amino acids) and isoform b (269 amino acids).
Following synthesis creatine is released to the blood where is is picked up, predominantly, by the brain and skeletal muscle cells through the action of the SLC family member transporter, SLC6A8. The SLC6A8 gene is located on the X chromosome (Xq28) and is composed of 14 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform. Within cells creatine is phosphorylated by creatine kinases (CK; also called creatine phosphokinase, CPK) that generate the high-energy storage compound, creatine phosphate. There are two creatine kinase genes in humans identified as the muscle creatine kinase gene (symbol: CKM) and the brain creatine kinase gene (symbol: CKB). The CKM gene is located on chromosome 19q13.32 and is composed of 9 exons that encode a protein of 381 amino acids. The CKB gene is located on chromosome 14q32 and is composed of 8 exons that encode a protein of 381 amino acids. Different combinations of the proteins encoded by these two genes generate tissue-specific isoforms of the functional enzymes. For details on the different forms of CK go to the Enzyme Kinetics page.
Synthesis of creatine and creatine phosphate: The synthesis of creatine begins from arginine and glycine via the renal enzyme glycine amidinotransferase (GATM). The product, guanidinoacetate, is transported to the liver where it is methylated via the action of guanidinoacetate N-methyltransferase (GAMT) forming creatine. The methyl group is donated from S-adenosylmethione (SAM). Creatine is released to the blood and picked up by the brain and skeletal muscle cells via the action of the SLC6A8 transporter. Creatine kinase (creatine phosphokinase) transfers a phosphate from ATP generating the high-energy intermediate, creatine phosphate.
Although creatine synthesized by the kidneys and liver can be transported from the blood into the brain, during both embryonic development and during postnatal life the brain can directly synthesize its own creatine. The GATM and GAMT genes are expressed in neural tissues. However, the level of creatine synthesis in the embryonic nervous system is not sufficient for needs and so the developling brain requires matrenally and peripherally synthesized creatine. In the adult brain glial cells and neurons continue to express the GATM and GAMT genes, however, cells express either only the GATM gene while others epress only the GAMT gene. Therefore, similar to the transport of GAA from the kidneys to the liver for creatine synthesis, the GAA produced in certain neural cells must be transported into GAMT expressing cells in order for brain creatine synthesis. This latter transport is likely catalyzed by SLC6A8. This explains why, in patients with SLC6A8 mutation, there is a lack of sufficient brain creatine. Like in skeletal muscle, brain creatine phosphate plays an important role in the regulation of a high energy balance in the brain. Creatine also functions as a neurotransmitter in the brain by acting as a partial agonist at postsynaptic GABAA receptors.
Creatine is used as a storage form of high energy phosphate. The phosphate of ATP is transferred to creatine, generating creatine phosphate, through the action of creatine phosphokinase. The reaction is reversible such that when energy demand is high (e.g. during muscle exertion) creatine phosphate donates its phosphate to ADP to yield ATP.
Both creatine and creatine phosphate are found in muscle, brain and blood. Creatinine is formed in muscle from creatine phosphate by a nonenzymatic dehydration and loss of phosphate. The amount of creatinine produced is related to muscle mass and remains remarkably constant from day to day. Creatinine is excreted by the kidneys and the level of excretion (creatinine clearance rate) is a measure of renal function.back to the top
Creatine deficiency syndromes (CDS) represent a group of disorders that result from defects in the synthesis and transport of creatine. These disorders are also referred to as primary metabolism disorders of creatine. As discussed in the previous section, there are two enzymes of creatine synthesis, glycine amidinotransferase (encoded by the GATM gene ) and guanidinoacetate N-methyltransferase (encoded by the GAMT gene), and one major creatine transporter encoded by the SLC6A8 gene. The first disorder in creatine metabolism to be characterized involved mutations in the GAMT gene. The initial GAMT deficient patient experinced severe psychomotor retardation, refractory epilepsy, and movement disorder. The patient exhibited no cerebral creatine along with high GAA concentrations in the brain and body fluids. Treatment of this patient was effected by oral administration of creatine monohydrate. Following this initial patient characterization, mutations in the GATM gene and the SLC6A8 gene were found in patient exhibiting similar pathology. The GATM and GAMT mutation-associated disorders are inherited as autosomal recessive diseases. The SLC6A8 gene is located on the X chromosome and, therefore, creatine transporter deficiencies are inherited in an X-linked recessive manner. Few GATM deficient patients have been identifed, 40 GAMT deficient patients have been identified, and around 150 SLC6A8 deficient patients have been identified to date.
Although skeletal muscle creatine synthesis will be impaired in individuals with GATM and GAMT mutations, impaired muscle function is not the major symptom in creatine deficiency syndrome patients. All of these disorders exert dramatic effects on central nervous system function and, therefore, neurological symptoms are the main clinical complications in these patients. The clinical features in creatine defficiency syndromes include intellectual disability, speech and language delay, epilepsy and autism spectrum disorder. Due to the different levels of enzyme defect with various mutations and the different genes there is wide variability in the severity of the clinical symptoms. Patients with GATM deficiencies have milder clinical manifestations which are generally limited to moderate intellectual disability and language disorder. Patients with GAMT deficiencies have high concentrations of GAA in body fluids and manifest with refractory epilepsy and severe movement disorders. Patients with SLC6A8 deficiencies exhibit mental retardation, epilepsy, autism spectrum disorder, and severe language delay.
Treatment of both GATM and GAMT deficient patients can be carried out with oral creatine monohydrate. However, this therapy does not lower the levels of the intermediate metabolite, GAA, seen with GAMT deficiency. In these latter patients the addition of ornithine supplementation and dietary restriction in arginine help to reduce the production of GAA. The reductions in GAA provide better control of the refractory epilepsy that is common in GAMT deficiency. Currently there is no effective treatment for patients with SLC6A8 deficiencyback to the top
Glutathione (abbreviated GSH) is a tripeptide composed of glutamate, cysteine and glycine that has numerous important functions within cells. Glutathione serves as a potent reductant eliminating hydroxy radicals, peroxynitrites, and hydroperoxides; it is conjugated to drugs to make them more water soluble; it is involved in amino acid transport across cell membranes (the γ-glutamyl cycle); it is a substrate for the peptidoleukotrienes; it serves as a cofactor for some enzymatic reactions; and it serves as an aid in the rearrangement of protein disulfide bonds.
GSH is synthesized in the cytosol of all mammalian cells via the two-step reaction shown in the Figure. The rate of GSH synthesis is dependent upon the availability of cysteine and the activity of the rate-limiting enzyme, γ-glutamylcysteine synthetase (also called glutamate-cysteine ligase, GCL). The second reaction of GSH synthesis involves the enzyme, glutathione synthetase, which condenses γ-glutamylcysteine with glycine. Both reactions of GSH synthesis require ATP. Functional GCL is a heterodimer composed of the catalytic subunit and a regulatory subunit called the modifier subunit. The GCL catalytic subunit is encoded by the GCLC gene and the modifier subunit is encoded by the GCLM gene. The GCLC gene is located on chromosome 6p12 and is composed of 16 exons that generate two alterntively spliced mRNA that encode GCL isoform a (637 amino acids) and GCL isoform b (599 amino acids). The GCLM gene is located on chromosome 1p22.1 and is composed of 8 exons that encode a protein of 274 amino acids. The glutathione synthetase gene (symbol: GSS) is located on chromosome 20q11.2 and is composed of 15 exons that encode a protein of 474 amino acids.
In the liver, major factors that determine the availability of cysteine are diet, membrane transport activities of the three sulfur amino acids cysteine, cystine and methionine, and the conversion of methionine to cysteine (see the Amino Acid Metabolism page). Numerous conditions can alter the level of GSH synthesis via changes in glutamate-cysteine ligase activity and expression of the GCLC gene such as oxidative stress, antioxidant levels, hormones, cell proliferation, and diabetes mellitus.
Synthesis of Glutathione. The synthesis of glutathione, which occurs in the cytosol of all cells, proceeds via a two-step reaction involving the ATP-requiring enzymes glutamate-cysteine ligase (GCL) and glutathione synthetase (GSS). The common designation for reduced glutathione (monomeric) is GSH. When glutathione reduces reactive oxygen or nitrogen species, or upon its involvement in many other reduction reactions, it becomes oxidized to the dimeric form which is abbreviated GSSG.
The role of GSH as a reductant is extremely important particularly in the highly oxidizing environment of the erythrocyte. The sulfhydryl of GSH can be used to reduce peroxides formed during oxygen transport. Endogenously produced hydrogen peroxide (H2O2) is reduced by GSH in the presence of selenium-dependent GSH peroxidase. Hydrogen peroxide can also be reduced by catalase, which is present only in the peroxisomes. In the mitochondria, GSH is particularly important because mitochondria lack catalase. The resulting oxidized form of GSH consists of two molecules disulfide bonded together (abbreviated GSSG). The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce GSSG back to two moles of GSH. Hence, the pentose phosphate pathway is an extremely important pathway of erythrocytes for the continuing production of the NADPH needed by glutathione reductase. In fact as much as 10% of glucose consumption, by erythrocytes, may be mediated by the pentose phosphate pathway.
Detoxification of xenobiotics or their metabolites is another major function of GSH. These compounds form conjugates with GSH either spontaneously or enzymatically in reactions catalyzed by members of the glutathione S-transferase (GST) family. The conjugates formed are usually excreted from the cell and, in the case of the liver they are excreted in the bile. There are several GST enzymes that are divided into three major families that comprise cytsolic, mitochondrial, and microsomal family members. The cytosolic GST enzymes comprise the largest family and based upon amino acid sequences and substrate specificities this family has been divided into seven subclasses identified as alpha (α, A), mu (μ, M), omega (ω, O), pi (π, P), sigma (σ, S), theta (θ, T), and zeta (ζ, Z). The human genome contains five GST alpha genes, five mu genes, two omega genes, one pi gene, one sigma gene, two theta genes, and one zeta gene. The cytosolic enzymes are functional as dimers and since the alpha and mu proteins can form functional heterodimers, the complexity of functional enzymes is greater than the number of individual subunit genes. Another soluble GST enzyme that is not found in the cytosol is the human GST kappa (κ, K) enzyme (encoded by the GSTK1 gene) that is localized to the peroxisomes. The GST enzymes function not only in detoxification and antioxidant reactions but also in the biosynthesis and metabolism of eicosanoids and steroids as well as in the modulation of signal transduction processes.
|Class||Enzyme Subunits||Encoding Genes||Comments|
|Alpha||1, 2, 3, 4, 5||GSTA1, GSTA2, GSTA3, GSTA4, GSTA5||the GSTA genes are clustered on chromosome 6; most abundantly expressed class of GSTs in the liver; also expressed in kidney and testis|
|Mu||1, 2, 3, 4, 5||GSTM1, GSTM2, GSTM3, GSTM4, GSTM5||the GSTM genes are clustered on chromosome 1; the mu genes are highly polymorphic accounting for a high degree of variability in a given individuals ability to detoxify certain toxins and carcinogens; GSTM2 expression highest in muscle; GSTM3 expression highest in brain; polymorphisms in GSTM1 gene associated with hypertension and increased risk for schizophrenia|
|Omega||1, 2||GSTO1, GSTO2||both genes found on chromosome 10|
|Pi||1||GSTP1||expression is highest in the brain, lung, and heart; polymorphisms in GSTP1 gene associated with increased risk for schizophrenia|
|Sigma||1||HPGDS||the sigma GST enzyme is a prostaglandin-D synthase encoded by the hematopoietic prostaglandin D synthase gene; catalyzes the conversion of PGH2 to PGD2|
|Theta||1, 2||GSTT1, GSTT2, GSTT2B||the GSTT genes are clustered on chromosome 22; the GSTT1 gene is absent in 38% of the population; in some populations the GSTT2B gene is a pseudogene; polymorphisms in GSTT1 associated with hypertension and increased risk for schizophrenia|
|Zeta||1||GSTZ1||this GST catalyzes the conversion of maleylacetoacetate to fumarylacetoacetate in the catabolism of phenylalanine and tyrosine; originally identified as 4-maleylacetoacetate isomerase or maleylacetoacetate cis-trans-isomerase|
Several mechanisms exist for the transport of amino acids across cell membranes. Many are symport or antiport mechanisms that couple amino acid transport to sodium transport. The γ-glutamyl cycle is an example of a group transfer mechanism of amino acid transport. Although this mechanism requires more energy input, it is rapid and has a high capacity. The cycle functions primarily in the kidney, particularly renal epithelial cells. The enzyme γ-glutamyl transpeptidase is located in the cell membrane and shuttles GSH to the cell surface to interact with an amino acid. Reaction with an amino acid liberates cysteinylglycine and generates a γ-glutamyl-amino acid which is transported into the cell and hydrolyzed to release the amino acid. Glutamate is released as 5-oxoproline and the cysteinylglycine is cleaved to its component amino acids. Regeneration of GSH requires an ATP-dependent conversion of 5-oxoproline to glutamate and then the two additional moles of ATP that are required during the normal generation of GSH.back to the top
The polyamines are highly cationic molecules that tend to bind nucleic acids with high affinity. Because of this interaction activity, it is believed that the polyamines are important participants in DNA synthesis, or in the regulation of that process. In addition to their function in DNA replication, polyamines and polyamine derivatives are involved in the processes of protein synthesis, ion channel function, regulation of gene expression, cell proliferation, and apoptosis (programmed cell death).
One of the earliest signals that cells have entered their replication cycle is the appearance of elevated levels of mRNA for ornithine decarboxylase 1 (ODC1), and then increased levels of the enzyme. Ornithine decarboxylase is the first enzyme in the pathway to synthesis of the polyamines. The human genome contains two ODC sequences with only the ODC1 gene being functional. The other locus, identified as ODCP, is a pseudogene. The ODC1 gene is located on chromosome 2p25 and is composed of 13 exons that generate four alternatively spliced mRNAs. Three of the ODC1 splice variants encode the same enzyme identified as isoform 1 (461 amino acids). ODC isoform 2 is composed of 332 amino acids. Functional ODC is a homodimer and requires pyridoxal phosphate (PLP), derived from vitamin B6, as a cofactor. The catalytic activity of ODC produces the 4-carbon saturated diamine, putrescine.
Principal pathway of polyamine synthesis in humans. The key features of the pathway are that it involves putrescine, an ornithine catabolite, and S-adenosylmethionine (SAM; AdoMet) as a donor of two propylamine residues. The first propylamine conjugation yields spermidine and addition of another to spermidine yields spermine. The form of SAM utilized in the polyamine biosynthesis pathway is decarboxylated SAM (abbreviated dcSAM or dcAdoMet).
An important intermediate required for the synthesis of spermidine and spermine from putrescine is derived from S-adenosylmethionine (SAM). The enzyme, adenosylmethionine decarboxylase 1 (AMD1; also known as S-adenosylmethionine decarboxylase or SAM-decarboxylase) cleaves the SAM carboxyl residue, producing decarboxylated SAM (S-adenosymethylthiopropylamine; also known as decarboxylated S-adenosylmethionine, dcAdoMet or dcSAM), which retains the methyl group usually involved in SAM methyltransferase activity. Adenosylmethionine decarboxylase 1 activity is regulated by product inhibition and allosterically stimulated by putrescine. The AMD1 gene is located on chromosome 6p21 and is composed of 10 exons that generate four alternatively spliced mRNAs that encode four distinct isoforms of the enzyme. Spermidine synthase catalyzes a condensation reaction between dcAdoMet and putrescine, producing spermidine and 5'-methylthioadenosine. Spermidine synthase is encoded by the SRM gene (also known as SPDSY) located on chromosome 1p36–p22. The SRM gene is composed of 8 exons that encode a protein of 302 amino acids. A second propylamine residue from dcAdoMet is added to spermidine producing spermine by the enzyme spermine synthase encoded by the SMS gene (also known as SPMSY). The SMS gene is located on the X chromosome (Xp22.1) and is composed of 13 exons that generate two alternatively spliced mRNAs encoding SMS isoform 1 (366 amino acids) and SMS isoform 2 (313 amino acids).
The signals for regulating the production of ODC are complex. The promoter of the ODC1 gene contains elements that are regulated by growth factors, hormones, and tumor promoters. Production of ODC protein is also regulated at the level of translation by the product of the reaction, putrescine. The ODC1 mRNA contains a long 5'-untranslated region (5'-UTR) that folds into a complex secondary structure. Within the 5'-UTR there are two domains that reduce translational efficiency. One domain is a GC-rich sequence, the other is a small functional upstream open reading frame (uORF). Another significant mechanism for regulating ODC1 mRNA translation is particularly important during mitosis. Cap-dependent translation is frequently repressed during mitosis and the ODC1 mRNA contains an internal ribosome entry site (IRES) that allows the mRNA to be translated under these conditions.
The stability of the ODC1 protein also contributes to the level of enzyme activity in cells. The half-life of ODC protein is short (< 1hr) and this half-life is controlled by proteosomal degradation. However, entry into the proteosome, in the case of ODC, does not require prior ubiquitination. The ability of the proteosome to recognize, and degrade, non-ubiquitinated ODC involves a family of proteins called antizymes. The antizyme proteins interact with ODC monomers preventing formation of functional homodimers while simultaneously altering the conformation of the monomers so that they are recognized by the proteosome. Humans express three distinct ODC antizymes identified as antizyme 1, 2, and 3. Antizyme 1 is encoded by the OAZ1 (ornithine decarboxylase antizyme 1) gene which is located on chromosome 19p13.3 and is composed of 5 exons that generate two alternatively spliced mRNAs. These two mRNAs encode antizyme 1 isoform 1 (228 amino acids) and antizyme 1 isoform 2 (226 amino acids). The translation of the functional antizyme 1 proteins is regulated by a polyamine-regulated ribosome frameshifting mechanism. The antizyme 1 mRNAs contain two overlapping open reading frames (ORFs). These ORFs are identified as ORF1 which is the shorter of the two, and ORF2. The reading frame of ORF2 is in the +1 frame relative to reading frame of ORF1. The ORF2 reading frame does not contain an AUG translation initiation codon and as such translation through ORF2 requires the failure of the ribosome to terminate at the end of ORF1 and instead shift to the +1 reading frame. This ribosomal frameshifting event is stimulated by polyamines. In the presence of high concentrations of polyamines the ribosome frameshifting occurs resulting in the synthesis of functional antizyme 1 which comprises amino acids from both ORF1 and ORF2. In addition to the two different isoforms, the antizyme 1 mRNA contains two in-frame AUG codons that have been shown, in rodents, to be alternatively utilized resulting in differentially localized antizyme 1 isoforms.
Antizyme 2 is encoded by the OAZ2 gene which is located on chromosome 15q22.31 and is composed of 5 exons that generate two alternatively spliced mRNAs that encode two distinct protein isoforms. Although widely expressed like the OAZ1 gene, the level of OAZ2 expression is much lower. Antizyme 3 is encoded by the OAZ3 gene which is located on chromosome 1q21.3 and is composed of 7 exons that generate three alternatively spliced mRNAs encoding three distinct protein isoforms. Antizyme 3 isoform 1 is the longest protein encoded by the OAZ3 gene and the translation of this form of the enzyme begins from a CUG codon and not an AUG codon. Expression of the OAZ3 gene is restricted to haploid germ cells in the testis.
As discussed, the synthesis of the polyamines is regulated by several independent mechanisms. In addition to these mechanism of control, intracellular polyamine levels are also regulated via their catabolism. Putrescine is catabolized by amine oxidase, copper containing 1 encoded by the AOC1 gene, while spermidine and spermine are catabolized by spermidine/spermine N1-acetyltransferase 1 (also known as diamine acetyltansferase 1) encoded by the SAT1 gene (also known as SSAT). The SAT1 gene is located on the X chromosome (Xp22.1) and is composed of 7 exons that generate two alternatively spliced mRNAs. Only one of the mRNAs encodes a functional SAT1 enzyme as the other mRNA is subjected to nonsense-mediated mRNA decay. The functional SAT1 enzyme is a 171 amino acid protein. In addition to putrescine, the AOC1 encoded enzyme is responsible for the catabolism of histamine and other related biogenic amines.
The butylamino group of spermidine is used in a posttranslational modification reaction important to the process of translation. A specific lysine residue in the translational initiation factor eIF-5A is modified. Following the modification the residue is hydroxylated yielding a residue in the protein termed hypusine. The term hypusine is derived from hydroxyputrescine and lysine. The hypusination of eIF-5A is necessary for the promotion of translational elongation. The hypusination of eIF-5A involves a two-step reaction process. The first reaction involves the enzyme deoxyhypusine synthase which transfers the butylamine moiety from spermidine to the specific eIF-5A lysine residue. The second reaction involves the enzyme deoxyhypusine hydroxylase which hydroxylates the deoxyhypusine to hypusine. Deoxyhypusine synthase is encoded by the DHPS gene located on chromosome 19p13.2 which is composed of 13 exons that generate three alternatively spliced mRNAs, each of which encode a distinct isoform of the enzyme. Deoxyhypusine hydroxylase is encoded by the DOHH (deoxyhypusine hydroxylkase/monooxygenase) gene located on chromosome 19p13.3 which is composed of 7 exons that generate two alternatively spliced mRNAs that both encode the same 302 amino acid protein.back to the top