Tyrosine hydroxylase is the rate-limiting enzyme in the overall biosynthesis of epinephrine and as such is subject to several regulatory inputs that operate over both short (minutes) and long (hours) time frames. The enzyme is allosterically inhibited by the penultimate product of the pathway in the adrenal medulla, norepinephrine. Also, since tyrosine hydroxylase rarely functions at maximal velocity, its activity is controlled, to a certain extent, by the level of substrate (tyrosine) and cofactor (tetrahydrobiopterin). A number of external signals stimulate tyrosine hydroxylase activity through signaling pathways that result in the phosphorylation of specific serine residues near the N-terminal domain of the enzyme. This occurs primarily, but not exclusively, at serine-40 and counters the feedback inhibition by norepinephrine. In the longer term, there is regulation of tyrosine hydroxylase gene transcription, a major way in which the adrenal medulla responds to sustained stress. Increased tyrosine hydroxylase gene transcription is a major part of adaptation to various types of stress in animal studies, including physical stressors, such as immobilization, exercise, or hypo glycemia, and social stress such as crowding. Agents that increase cyclic AMP levels as well as glucocorticoids are mediators of the effects of these stressors on tyrosine hydroxylase gene transcription.
The expression of the final enzyme in the path way of epinephrine synthesis phenyl ethanolamine-N- methyltransferase, PNMT in the adrenal medulla is dependent on glucocorticoids. Glucocorticoids also mediate the increase in PNMT transcription in response to stress, which requires the presence of an intact hypothalamic-pituitary-adrenal (HPA) axis. This enzyme, along with tyrosine hydroxylase, is also transcriptionally increased by the 27 amino acid neuropeptide pituitary adenyl cyclase activating peptide (PACAP) which is co-secreted with acetylcholine and stimulates catecholamine secretion from the chromaffin cell.
The stimulus for release of epinephrine from the secretory vesicles into the blood stream is acetylcholine, secreted by the preganglionic splanchnic nerve terminus. As shown in Figure 1, when acetylcholine binds to the α subunits of the nicotinic acetylcholine receptor in the membrane of the chromaffin cell, the ligand gated ion channel opens, allowing the influx of sodium. This causes the depolarization of the membrane, which brings about the activation of one or more voltage- dependent calcium channels. The influx of calcium is the signal for exocytosis of the contents of the chromaffin vesicles into the nearby capillaries.
Fig1. Epinephrine secretion by the chromaffin cell. A. Acetylcholine-stimulated secretion of epinephrine by the chromaffin cell. The axon of the preganglionic splanchnic nerve terminates on the chromaffin cell of the adrenal medulla where it releases acetylcholine (triangles) which binds to the α subunits of the nicotinic cholinergic receptor leading to an influx of Na+. The resulting membrane depolarization causes the opening of voltage-dependent calcium channels (blue); the rise in intracellular calcium in the vicinity of the mature vesicles brings about their exocytosis. The contents of the vesicle, epinephrine (red circles), peptides and proteins (blue circles), and other small molecules such as ATP (not shown) are released. B. Proteins involved in the fusion and exocytosis of chromaffin granules. The SNARE proteins, named as receptors for SNAP proteins (soluble N-ethylmaleimde-sensitive attachment protein) shown are: synaptobrevin-2 (green; also known as VAMP2) in the vesicular membrane; SNAP-25 (red); and syntaxin-1 (blue) in the plasma membrane. In the unprimed vesicle, syntaxin-1 forms a “closed” complex with Munc18-1 (purple), a member of a family of proteins first described in secretion deficient yeast mutants (Munc=mammalian uncoordinated). Munc13 (light purple) opens the closed complex, allowing Munc18-1 to bind to the SNAP25 and synaptobrevin, leading to the formation of a four-helical bundle, tethering the vesicle to the plasma membrane. The increase in intracellular calcium (see panel A), which interacts with synaptotagmin-1 (tan), leads to the opening of the vesicle with the extrusion of its contents.
Although the precise mechanism of calcium-mediated exocytosis in these cells is not yet clear, some features are beginning to emerge from studies with chromaffin cells and neurons. The two exocytotic systems share the properties of Ca2+ dependency and secretion from a readily releasable pool of vesicles which have undergone several steps of maturation prior to and following docking at the membrane. Neuronal secretion occurs from active zones in which there is tight coupling between Ca2+ entry and the secretion release sites, whereas chromaffin cells do not have such active areas. Nevertheless the features of the docking process, depicted in Figure 1B, appear to be similar in both of these cell types as well as in platelets. The membrane SNARE proteins (see legend, Figure 1) syntaxin-1 and SNAP-25 are present in the docking site along with Munc18, a member of an ancient highly con served family of secretory proteins also found in yeast, plants, and nematodes. Upon stimulation of secretion, Munc18 participates in the formation of a four-helical bundle made of up the vesicular SNARE protein syn aptobrevin and the two membrane SNARE proteins, syntaxin-1 and SNAP-25. This complex, along with Munc13, tethers the vesicle to the plasma membrane. Synaptotagmin-1, in the vesicle membrane, participates as the calcium sensor and, along with other proteins such as CAPS (calcium-activated proteins of secretion), mediates the stimulatory effect of Ca2+ on exocytosis.
