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).

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).

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.

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 α_1A, α_1B, and α_1D receptors. The α₁ receptor class is coupled to G_q-type G-proteins that activate PLCβ resulting in increases in IP₃ and DAG release from membrane PIP₂. The α₂ class contains the α_2A, α_2B, and α_2C receptors. The α₂ class of adrenergic receptor is coupled to G_i-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 G_s-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.

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.

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 5HT_1A, 5HT_1B, 5HT_1D, 5HT_1E, and 5HT_1F. There are 3 5HT₂ subtypes, 5HT_2A, 5HT_2B, and 5HT_2C as well as 2 5HT₅ subtypes, 5HT_5A and 5HT_5B. 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 5HT_2A receptors mediate platelet aggregation and smooth muscle contraction. The 5HT_2C 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.

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