The first step in the synthesis of 5-HT is hydroxylation of the essential amino acid, tryptophan, by the enzyme tryptophan hydroxylase (Fig. 9.4). This enzyme has several features in common with tyrosine hydroxylase, which converts tyrosine to /-DOPA in
Figure 9.4 The synthesis and metabolism of 5-HT. The primary substrate for the pathway is the essential amino acid, tryptophan and its hydroxylation to 5-hydroxytryptophan is the rate-limiting step in the synthesis of 5-HT. The cytoplasmic enzyme, monoamine oxidase (MAOa), is ultimately responsible for the catabolism of 5-HT to 5-hydroxyindoleacetic acid
Figure 9.4 The synthesis and metabolism of 5-HT. The primary substrate for the pathway is the essential amino acid, tryptophan and its hydroxylation to 5-hydroxytryptophan is the rate-limiting step in the synthesis of 5-HT. The cytoplasmic enzyme, monoamine oxidase (MAOa), is ultimately responsible for the catabolism of 5-HT to 5-hydroxyindoleacetic acid the noradrenaline synthetic pathway. First, it has an absolute requirement for O2 and the reduced pterin co-factor, tetrahydrobiopterin. Second, hydroxylation of tryptophan, like that of tyrosine, is the rate-limiting step for the whole pathway (reviewed by Boadle-Biber 1993) (see Chapter 8). However, unlike the synthesis of noradrenaline, the availability of the substrate, tryptophan, is a limiting factor in the synthesis of 5-HT. Indeed, the activated form of tryptophan hydroxylase has an extremely high Km for tryptophan (50 ^M), which is much greater than the concentration of tryptophan in the brain (10-30 ^M). This means that not only is it unlikely that this enzyme ever becomes saturated with its substrate but also that 5-HT synthesis can be driven by giving extra tryptophan.
This influence of tryptophan availability on the rate of synthesis of 5-HT has some interesting implications. First, it predicts that a dietary deficiency of tryptophan could lead to depletion of the neuronal supply of releasable 5-HT. Indeed, this has been confirmed in humans to the extent that a tryptophan-free diet can cause a resurgence of depression in patients who were otherwise in remission (see Chapter 20). In contrast, a tryptophan-high diet increases synthesis and release of 5-HT. In fact, when given in combination with other drugs that augment 5-HT transmission (e.g. an MAO inhibitor or a 5-HT reuptake inhibitor), tryptophan can cause a life-threatening delirium known as the 'serotonin syndrome' (Gillman 1999).
Transport of tryptophan across the blood-brain barrier and neuronal membranes relies on a specific carrier for large neutral amino acids (LNAAs). Thus, although an increase in the relative concentration of plasma tryptophan, either through dietary intake or its reduced metabolism in a diseased liver, increases its transport into the brain, other LNAAs (such as leucine, isoleucine or valine) can compete for the carrier. This process forms the basis of an intriguing theory linking the intake of carbohydrates in the diet with an individual's mood. It is known that consumption of carbohydrates increases secretion of insulin which, in addition to its well-known glucostatic role, promotes uptake of LNAAs by peripheral tissues. However, it seems that tryptophan is less affected by insulin than the other LNAAs in this respect and so its relative concentration in the plasma increases, thereby increasing its transport into the brain (see Rouch, Nicolaidis and Orosco 1999). The resulting increase in synthesis and release of 5-HT is claimed to enhance mood. Although this scheme is rather controversial, it has been suggested as an explanation for the clinical improvement in some patients, suffering from depression or premenstrual tension, when they eat carbohydrates. It has also been suggested to underlie the carbohydrate-craving experienced by patients suffering from Seasonal Affective Disorder (Wurtman and Wurtman 1995).
Not a great deal is known about factors that actually activate tryptophan hydroxylase. In particular, the relative contribution of tryptophan supply versus factors that specifically modify enzyme activity under normal dietary conditions is unknown. However, removal of end-product inhibition of tryptophan hydroxylase has been firmly ruled out. Also, it has been established that this enzyme is activated by electrical stimulation of brain slices, even in the absence of any change in tryptophan concentration, and so other mechanisms are clearly involved.
So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca2+ and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca2+ in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the Km of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca2+/calmodulin protein kinase inhibitor. However, whereas depolarisation alone increases enzyme Fmax, it does not appear to affect the enzyme Km and so a firm link between neuronal depolarisation and enzyme phosphorylation has yet to be established.
The apparent reliance of enzyme activation on phosphorylation and intracellular Ca2+ gives a clue as to how the rate of 5-HT synthesis might be coupled to its impulse-evoked release. Certainly, the impulse-induced increase in intracellular Ca2+, and/or activation of the G protein-coupled receptors that govern synthesis of cAMP, could modify the activity of tryptophan hydroxylase. Indeed, this could explain why activation of either somal 5-HT1A autoreceptors in the Raphe nuclei (which depress the firing rate of 5-HT neurons) or terminal 5-HT1B autoreceptors (which depress 5-HT release) can reduce the production of cAMP and attenuate 5-HT synthesis.
The product of the hydroxylation of tryptophan, 5-hydroxytryptophan, is rapidly decarboxylated to 5-HT by a specific decarboxylase enzyme. This is generally thought to be a soluble enzyme which suggests that 5-HT is synthesised in the cytoplasm, before it is taken up into the storage vesicles. If this is the case, then considerable losses might be incurred from its metabolism by monoamine oxidase before it reaches the storage vesicles. Indeed, this could explain why 5-HT turnover seems to greatly exceed its rate of release.
The high affinity of the decarboxylase enzyme for its substrate (10 ^M in the brain) makes it unlikely that this stage could ever become rate-limiting for the pathway as a whole. Nevertheless, the Km for this enzyme is considerably higher than tissue concentrations of 5-hydroxytryptophan and so, again, supply of this substrate is likely to be a crucial factor.
Finally, as with the noradrenergic system, there is evidence for long-term changes in the rate of synthesis of 5-HT that are triggered by prolonged changes in its rate of release. These can be traced to the rate of gene transcription and the ensuing synthesis of enzyme protein ('enzyme induction'). It has even been shown that mRNA for tryptophan hydroxylase shows a daily rhythm in cultured eye-cups maintained in the dark. Again, not a great deal is known about the underlying control mechanisms but the synthesis of tryptophan hydroxylase, at least, is increased by exposure of 5-HT neurons in vivo to the growth factor, brain-derived neurotrophic factor ('BDNF; Siuciak et al. 1998). Steroid hormones also seem to modulate tryptophan hydroxylase gene transcription but research in this area is confounded by the variation in this effect across different tissues and different hormones, with both increases and decreases being reported.
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