Fundamental Aspects of Catecholamine Biology



Fundamental Aspects of Catecholamine Biology





Introduction: Overview of the Sympathoadrenal System

Epinephrine (E) was discovered in extracts of adrenal glands at the end of the 19th century and was reasonably well characterized shortly thereafter. It was thus the first hormone with a known chemical structure (Fig. 1.1). Over the ensuing century, catecholamine research resulted in many important discoveries that elucidated many fundamental biologic processes with wide applicability (Tables 1.1 and 1.2). These processes included neurochemical transmission, cell surface receptors, transmitter inactivation by reuptake, storage in subcellular granules, release by exocytosis, signal transduction and second messengers, G-proteins, and reversible protein phosphorylation in the activation and deactivation of enzymes and receptors. Nobel prizes have been awarded to 10 scientists for research involving catecholamines (Tables 1.1 and 1.2).

The overview provided in this chapter gives a general outline of the autonomic nervous system (ANS) and prepares the groundwork for appreciating the physiology and pharmacology detailed in subsequent sections.


The naturally occurring catecholamines

The naturally occurring, biologically important catecholamines are epinephrine (E), norepinephrine (NE), and dopamine (DA) (Table 1.3; Fig. 1.1). Present throughout the animal kingdom, catecholamines are found in protozoa and invertebrates, as well as vertebrates. In invertebrates, the predominant catecholamine is DA, which appears to serve as a neurotransmitter. In vertebrates, all three major
catecholamines are found with variation in the relative proportions in the peripheral nerves and adrenal medulla. For example, in amphibians, E is the predominant adrenergic neurotransmitter in distinction to mammals that utilize NE at sympathetic neuroeffector junctions. In mammals, for practical purposes, E is limited to the central nervous system (CNS) and to chromaffin cells of the adrenal medulla. (The term “chromaffin” is a portmanteau word derived from chromium and affinity; chromaffin cells darken markedly on exposure to dilute solutions of chromium salts.) Catecholamines, therefore, are widely distributed in nature, phylogenetically ancient, and it may thus be inferred that they act as signaling molecules throughout the animal kingdom.






FIGURE 1.1. Structures of naturally occurring catecholamines and related compounds. The conventional numbering system for ring and side chain substituents is shown for phenylethylamine, which may be considered the parent compound of many sympathomimetic amines. Catecholamines are hydroxylated at positions 3 and 4 on the ring. (From Landsberg L, Young JB. Catecholamines and the adrenal medulla. In: Bondy PK, Rosenberg LE, eds. Metabolic Control and Disease. 8th ed. Philadelphia, PA: WB Saunders; 1980:1621-1693.)


Embryology of the sympathoadrenal system

The sympathoadrenal (SA) system is derived from the neural crest. Early in embryonic development SA precursor cells migrate ventrally from the neuraxis and establish the sympathetic ganglia and the chromaffin cells of the adrenal medulla. The differentiation into sympathetic neurons and adrenal medullary chromaffin cells occurs after migration out of the neural crest but before the precursors
destined to become chromaffin cells invade the anlagen of the developing adrenal cortex. Bone morphogenetic proteins derived from the dorsal aorta play a role in the differentiation of the neural crest cells into SA precursors. Nerve growth factor(s) derived from innervated tissues are involved in the development and maintenance of the sympathetic nervous system (SNS). Although the adrenal cortex is not required for the differentiation of adrenal medullary chromaffin cells, it is necessary for the synthesis of E, accomplished by the induction of the E-forming enzyme in the cytoplasm of chromaffin cells. In the sympathetic nerves innervating sweat glands, a phenotypic change in neurotransmitter from NE to acetylcholine (Ach) occurs during development, an example of the plasticity that exists in the developing nervous system.








TABLE 1.1 Landmark Discoveries in Catecholamine Research: Structure of First Circulating Hormone (Epinephrine); First Evidence for Both Neurochemical Transmission and for Specific Cell Surface Receptors; and Termination of Action by Uptake into the Sympathetic Nerve Endings









  • Oliver and Shafer (1894)


    – Adrenal extracts raise blood pressure



  • Abel (1897) and Takamine (1900)


    – Isolated and purified active principle


    – Called epinephrine in U.S., adrenaline in U.K.



  • Stolz (1904) and Dakin (1905)


    – Synthesized racemic adrenaline


    – First hormone with identified chemical structure



  • Lewandowsky (1899), Langley (1904), and Elliott (1904)


    – Adrenal extracts mimic the effects of sympathetic nerve stimulation


    – The concept of neurochemical transmission is born



  • Barger and Dalea (1910)


    – Structure activity relationships of “sympathomimetic amines”


    – Primary amines (like NE) more closely resemble sympathetic stimulation than secondary amines (like E)



  • Cannon (1921)


    – Stimulation of sympathetic nerves releases an adrenaline-like substance



  • Von Eulera (1946)


    – NE identified as the adrenergic neurotransmitter



  • Ahlquist (1948)


    – Based on differential potencies for stimulatory and excitatory actions of sympathomimetic agonists the concept of distinct alpha and beta receptors is proposed



  • Axelroda (1961)


    – Identified NE uptake (and reuptake) into sympathetic nerve endings


a Denotes Nobel prize.


NE, norepinephrine; E, epinephrine.










TABLE 1.2 Landmark Discoveries in Catecholamine Research: Signal Transduction; Subcellular Storage and Release by Exocytosis; Second Messengers; G-proteins; Reversible Protein Phosphorylation in Receptor Desensitization and Enzyme Activation











  • Blaschko and Welch (1953), Hillarp (1953)


    – Storage in subcellular organelles



  • Krebsa and Fischer (1955)


    – Protein phosphorylation activates hepatic phosphorylase; inactivated by dephosphorylation



  • De Robertis and Vaz Ferreira (1957), Coupland (1965)


    – Release by exocytosis



  • Sutherlanda (1962)


    – Catecholamine action through stimulation of adenylyl cyclase; cyclic AMP as second messenger; phosphodiesterase system metabolizes cyclic AMP


    – Eventually established (several groups) that cyclic AMP activated phosphorylation



  • Rodbella (1972), Gilmana (1981)


    – G-protein transducers between receptor activation and cellular effect



  • Murada (1978)


    – Inhibitory G proteins; cyclic GMP; nitrous oxide



  • Lefkowitza and Kobilkaa (1983)


    – Beta adrenergic receptor coupled to G proteins; desensitization associated with beta receptor phosphorylation


a Denotes Nobel prize.


AMP, adenosine monophosphate.









TABLE 1.3 Biologically Important Catecholamines in Humans: Epinephrine, Norepinephrine, Dopamine









  • Epinephrine


    – Circulating hormone of the adrenal medulla


    – Neurotransmitter within the CNS (brainstem)



  • Norepinephrine


    – Neurotransmitter at peripheral sympathetic nerve endings


    – Neurotransmitter within the CNS



  • Dopamine


    – Neurotransmitter within the CNS


    – Peripheral neurotransmitter in selected areas (small intensely fluorescent cells in sympathetic ganglia and in carotid body)


    – Autocrine or paracrine function (kidney, gut) after synthesis from circulating DOPA


CNS, central nervous system; DOPA, 3,4-dihydroxyphenylalanine.




The extra-adrenal chromaffin cells

As the SA precursor cells migrate ventrally from the neuraxis, some of these precursors destined to become chromaffin cells remain associated with the sympathetic neuronal precursors and form the extra-adrenal chromaffin cells. Aggregates of these cells are located in and around the preaortic plexuses, the largest of which is known as the organ of Zuckerkandl. These cells are prominent in fetal and neonatal life and tend to regress with aging. They store NE (not E) and are not innervated; their function is unknown, although they serve as a nidus for the subsequent development of extra-adrenal pheochromocytomas.


Central nervous system control of the sympathoadrenal system

The SNS and the adrenal medulla form a distinct unit that operates under direction of the CNS. Although the activity of these two components is always coordinated centrally to defend the constancy of the internal environment, the activity of the two systems varies widely in different physiologic circumstances; the adrenal medulla, for example, is frequently stimulated when the SNS is suppressed. The major factor responsible for the generation of catecholamine-mediated effects is the stimulatory output from the CNS centers that regulate the SA activity. Although the full physiologic expression of SA stimulation is modified by a variety of factors operating at the neuroeffector junctions, the level of central sympathetic outflow is the major determinant of the physiologic actions regulated by the SA system. Central SA outflow sets the gain; other factors fine-tune the responses. Autonomic reflexes do exist independent of descending central regulation but these are clinically important only after central connections are interrupted as in spinal cord injury.

CNS regulation of SA outflow has a number of important implications for the maintenance of homeostasis, but none more interesting or important than the integration of SA outflow with voluntary activity. CNS regulation permits anticipation of an event to initiate SNS-mediated changes in the circulation and metabolism before the actual event (“fight or flight” for example) takes place, thus limiting the impact on the internal milieu. In other words, the SA response can be anticipatory rather than merely reactive.


Structural organization of the autonomic nervous system

The ANS is comprised of two major divisions: the sympathetic and the parasympathetic. In distinction to the somatic nervous system, which controls the movement of the voluntary striated musculature, the ANS regulates involuntary or vegetative functions. Although useful for heuristic purposes, the distinction between somatic and autonomic is not complete as somatic activation is frequently accompanied by autonomic discharge. Some distinctions between the somatic nervous system and ANS are shown in Table 1.4.

In Figure 1.2, the overall anatomic organization of the SA system is shown; Figure 1.3 and Table 1.5 present the basic anatomy of the ANS in schematic form.
Preganglionic neurons in the intermediolateral cell column of the spinal cord synapse with postganglionic sympathetic neurons in the sympathetic ganglia. Note that the adrenal medulla is analogous to the postganglionic sympathetic nerves; it receives a preganglionic cholinergic innervation from the splanchnic nerves and releases E into the circulation. Nerves that utilize Ach as their neurotransmitter are referred to as cholinergic; those utilizing NE are called adrenergic, per the suggestion of Sir Henry Dale in the 1930s (Table 1.6).








TABLE 1.4 Somatic and Autonomic Nervous Systems





























Autonomic


Somatic


Unconscious


Conscious


Innervates smooth muscle and glands


Innervates striated muscle


Synapse in ganglia outside the CNS


Direct innervation from the CNS


Preganglionic fibers are myelinated; postganglionic are unmyelinated


Somatic nerves are myelinated


Ground plexus of terminal fibers in innervated tissues


Discrete motor endplates


Dispersion of central outflow at level of the ganglia


Discrete innervation of motor units


Representative functions: cardiac stimulation; vasomotor tone; glandular secretion; heat conservation and dissipation; visceral smooth muscle contraction


Function: voluntary movement


CNS, central nervous system.



Intracellular storage of catecholamines in sympathetic nerve endings and chromaffin cells

After biosynthesis from tyrosine in sympathetic nerve endings and in adrenal medullary chromaffin cells, catecholamines are stored in discrete subcellular organelles referred to as “chromaffin granules” in the adrenal medulla and dense core vesicles in the sympathetic nerve endings. These organelles have many similarities and a few differences notably the larger size of the chromaffin granules (Figs. 1.4 and 1.5). They play a role in catecholamine biosynthesis and represent a large storage pool in which the catecholamines are protected from enzymatic degradation by monoamine oxidase (MAO).


General functions of the autonomic nervous system: steady state and “fight or flight”

Maintaining homeostasis is the overriding function of the ANS, a concept pioneered by Harvard physiologist Walter B. Cannon. Functioning below the conscious level, the ANS regulates bodily processes that maintain the constancy of the internal environment. The circulation, digestion, and metabolism, for
example, are all controlled from the CNS by the ANS. The sympathetic branch of the ANS is also structured to promptly address external threats to the integrity of the organism, by supporting the organism for “fight or flight,” as described by Cannon early in the 20th century. Each preganglionic neuron of the SNS synapses with many postganglionic neurons in the sympathetic ganglia (average 1:20 pre to postganglionic cells), including neurons in ganglia above or below the level at which the preganglionic neuron exits the neuraxis. The adrenal medulla, furthermore, secretes E into the circulation thereby supporting the function of the SNS. That said, it is important to recognize that the SNS functions continuously
in the regulation of normal physiology and that this regulation is characterized by discriminating rather than generalized responses.






FIGURE 1.2. Organization of the sympathoadrenal system. Descending tracts from the medulla, pons, and hypothalamus synapse with preganglionic sympathetic neurons in the spinal cord, which in turn innervate the adrenal medulla directly or synapse in paravertebral ganglia with postganglionic sympathetic neurons. The latter give rise to sympathetic nerves, which are distributed widely to viscera and blood vessels. Release of E or NE at the adrenal medulla or at sympathetic nerve endings occurs in response to a downward flow of nerve impulses from regulatory centers in the brain. E, epinephrine; NE, norepinephrine. (From Landsberg L, Young JB. Catecholamines and the adrenal medulla. In: Bondy PK, Rosenberg LE, eds. Metabolic Control and Disease. 8th ed. Philadelphia, PA: WB Saunders; 1980:1621-1693).






FIGURE 1.3. Organization of the peripheral autonomic nervous system. (From Moskowitz MS. Diseases of the autonomic nervous system. Clin Endocrinol Metab. 1977;6:745-768.)


Relationship between the sympathetic nervous system and the parasympathetic nervous system

The SNS and the parasympathetic nervous system (PSNS) are reciprocally related in terms of both activation and physiologic function. An increase in SNS
activity is associated with a decrease in PSNS outflow and vice versa. The actions stimulated by SNS and the PSNS are also antagonistic. Pulse rate, for example, is increased by the SNS and decreased by the PSNS. Gut motility and secretion, similarly, are suppressed by the SNS and stimulated by the PSNS. This dual control of autonomic functions permits more precise regulation in the maintenance of homeostasis than would be possible with a unidirectional system.








TABLE 1.5 Anatomic Organization of the Sympathetic and the Parasympathetic Nervous Systems


















Sympathetic


Parasympathetic


Short preganglionic nerves


Long preganglionic nerves


Ganglia in paravertebral chains and preaortic area


Ganglia in innervated organs


Thoracolumbar outflow: preganglionic fibers originate in the intermediolateral cell column of the spinal cord, exit the spinal cord from T1-L2 and synapse in paravertebral and preaortic ganglia or the adrenal medulla


Craniosacral outflow: preganglionic fibers originate in the midbrain, the medulla oblongata exiting the neuraxis in cranial nerves III, VII, IX, X, and in the pelvic nerves from S2 to S4 regions of the spinal cord


Preganglionic dispersion: each preganglionic neuron synapses with many postganglionic sympathetic nerves


Much less preganglionic dispersion except for the vagal innervation of the enteric plexuses









TABLE 1.6 Chemical Neurotransmission in the Autonomic Nervous System: Adrenergic and Cholinergic Nerves




















Sympathetic nervous system


Parasympathetic nervous system


Preganglionic nerves


Acetylcholine


Acetylcholine


Most postganglionic sympathetic nerves


Norepinephrine


Acetylcholine


Termination of action at neuroeffector junction


Reuptake into sympathetic nerve terminals


Hydrolysis by cholinesterase



Biosynthesis of Catecholamines



Biosynthetic pathway from L-tyrosine

Tyrosine is sequentially hydroxylated to 3,4-dihydroxyphenylalanine (DOPA), decarboxylated to DA, and hydroxylated at the β position to NE. The biosynthesis

of NE is carried out in peripheral adrenergic nerves and in central neurons that utilize NE as a neurotransmitter. In the chromaffin cells of the adrenal medulla, and certain neurons of the CNS, NE is N-methylated to E.






FIGURE 1.4. Electron photomicrograph of a sympathetic nerve ending in rat pineal gland. Note vesicles with electron-dense cores containing norepinephrine. Magnification ×45,000. (Courtesy of Dr. Floyd Bloom)






FIGURE 1.5. Electron photomicrograph of human adrenal medulla. Cells at the lower left containing small, electron-dense particles are adrenomedullary chromaffin cells with chromaffin granules; those above are adrenocortical cells. Magnification ×7,250. Inset shows chromaffin granules with clearly defined limiting membranes under higher magnification (×50,000). (Courtesy of Dr. James Connolly)






FIGURE 1.6. Biosynthetic pathway for catecholamines. TH, AADC, and DBH catalyze formation of NE from tyrosine. Subsequent formation of E, catalyzed by PNMT, takes place in the adrenal medulla and in neurons of the CNS and peripheral ganglia that use epinephrine as a neurotransmitter. TH, tyrosine hydroxylase; AADC, aromatic-L-amino acid decarboxylase; DBH, dopamine β-hydroxylase; NE, norepinephrine; E, epinephrine; PNMT, phenylethanolamine-N-methyltransferase. (Modified from Levine RJ, Landsberg L. Catecholamines and the Adrenal Medulla. In: Bondy PK, Rosenberg LE, eds. Duncan’s Disease of Metabolism. Philadelphia, PA: WB Saunders; 1974.)


Catecholamine biosynthetic enzymes

Tyrosine hydroxylase (TH) is the rate-limiting step in catecholamine biosynthesis. The enzyme is localized to those peripheral tissues that synthesize and store catecholamines and those central neurons that utilize catecholamines as neurotransmitters. Tetrahydrobiopterin and Fe2+ are essential cofactors. The biosynthesis of NE and E is linked to release by changes in the activity of TH and, after prolonged stimulation, by the induction of TH synthesis. The coupling of synthesis and release assures a constant pool of stored NE or E despite wide variations in SA activity.

The DOPA formed by the action of TH on tyrosine is decarboxylated by aromatic-L-amino acid decarboxylase (AADC), also known as DOPA decarboxylase, to form DA. Unlike the other enzymes involved in catecholamine biosynthesis AADC has a widespread distribution in non-neural tissues. The decarboxylation of circulating DOPA in the kidney to form DA, which then acts in autocrine or paracrine fashion to influence renal function, is an example of how a dopaminergic system might be involved in physiologic regulation.

In adrenergic neurons and the adrenal medulla, DA formed by the action of AADC in the cytoplasm is β-hydroxylated by dopamine β-hydroxylase (DBH) to form NE. This reaction, unique among the biosynthetic steps, occurs in the storage granules (dense core vesicles) of the SNS or chromaffin granules of the adrenal medulla. DBH, an enzyme that is similar to TH in many respects, uses
ascorbate as a hydrogen donor; it is not substrate specific for DA, so it may hydroxylate a variety of phenylethylamines. The subcellular localization of DBH to the storage granules means that the final step in NE synthesis occurs within the storage site. DBH is both a structural component of the granule wall as well as a soluble component of the granule contents. The latter is released along with NE or E during SA activation, a fact accounting for the short-lived, and long gone, interest in plasma DBH as a marker of SA activity. Granular localization of DBH protects newly formed DA from degradation by cytoplasmic MAO.

In the adrenal medulla, NE is N-methylated to E by phenylethanolamine N-methyltransferase (PNMT). S-adenosyl methionine is the methyl donor. In humans, about 80% of chromaffin cells synthesize and store E while the remainder store NE. The unique adrenal circulation that features a portal blood supply from the cortex to the medulla induces PNMT in the E-producing cells by exposing the chromaffin cells to very high levels of glucocorticoids. Interestingly, PNMT-positive chromaffin cells contain glucocorticoid receptors, while those lacking this enzyme do not. Although not required for differentiation of precursor cells into chromaffin cells, the capacity to produce E (at least in the adrenal medulla) does depend on the adjacent cortex and the steroid exposure that the portal system affords.

Note that PNMT is a cytosolic enzyme, so NE synthesized in the granules must diffuse out into the cytosol for conversion to E which is then taken up in the granule and stored. Although cumbersome, there is no feasible alternative to this sequence.

Another important point: although those chromaffin cells that produce E are phenotypically distinguishable from NE chromaffin cells, prolonged and intense adrenal medullary stimulation results in progressive decrease in E and increase in NE secretion, presumably due to a lack of time for regeneration of E stores in PNMT-positive cells. This means that in situations of strong adrenal medullary activation, the provenance of NE is uncertain; it cannot be assumed to derive from the SNS.


Regulation of catecholamine biosynthesis

In the sympathetic nerve endings and the adrenal medulla, the levels of NE and E respectively remain relatively constant despite wide variation in the degree of SA activity. This is accounted for in large measure by the coupling of catecholamine synthesis to catecholamine release, which is accomplished by increases in TH activity in the short term and by induction of TH synthesis in response to prolonged stimulation of SA activity. The increase in enzyme activity involves TH phosphorylation which alters the binding of catecholamines and tetrahydrobiopterin to the enzyme. The increase in enzyme biosynthesis with prolonged stimulation (termed “trans-synaptic induction”) is related to increased Ach which reflects increased preganglionic impulse traffic. The importance of TH in maintaining NE stores in peripheral sympathetic nerve endings in the
face of increased SNS activity is demonstrated by the depletion in tissue NE when TH is inhibited.

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