Tyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme of catecholamine synthetic pathway, and its regulation is critical for controlling catecholamine synthesis. The well recognized regulatory mechanisms are inhibition by catecholamine binding and re-activation upon Ser40 phosphorylation. Catecholamines bind to TyrH tightly, while phosphorylation of TyrH at Ser40 decreases the binding affinity by several hundred-fold. Regulation of TyrH is accompanied by conformational changes of the protein. This study focuses on the identification of the conformational changes of TyrH upon dopamine binding and Ser40 phosphorylation, using hydrogen deuterium exchange mass spectrometry (HDMS) and fluorescence spectroscopy. HDMS identifies three peptides undergoing conformational changes upon dopamine binding, peptide 35-41, 42-71 and 295-299. Peptides 35-41 and 42-71 are on the regulatory domain, while peptide 295-299 is at the active site entrance. Upon dopamine binding, all three peptides are protected from exchange; phosphorylation of TyrH at Ser40 has opposite effects on the exchange kinetics of peptide 295-299, but peptides 35-41 and 42-71 could not be detected by MS after phosphorylation. This suggests that the structural effects of dopamine binding and Ser40 phosphorylation are opposite. The fluorescence spectroscopy of mutant enzymes containing a single tryptophan at position 14, 34 or 74 was performed before and after phosphorylation. F34W/F3W TyrH has a significant decrease in steady-state fluorescence anisotropy, an increase in the bimolecular quenching rate constant kq and dynamic anisotropy upon phosphorylation at Ser40, while F14W/F3W TyrH and F74W/F3W TyrH exhibit much smaller differences. This suggests that phosphorylation of TyrH at Ser40 increases the flexibility of the regulatory domain. The results are consistent with TyrH existing in two conformations, a closed conformation stabilized by dopamine in which the N-terminal regulator domain of TyrH covers the active site entrance and an open conformation stabilized by phosphorylation in which the regulatory domain has moved away from the active site entrance.

Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Serotonin is a very important neuromodulatory agent that affects many physiological and behavioral responses of both vertebrates and invertebrates. In helminths, especially parasitic ones, not much is known about the biosynthesis and mode of action of serotonin or any of the related biogenic amine neurotransmitters, such as catecholamines (dopamine and noradrenaline). In this study, we cloned two full length cDNAs from Schistosoma mansoni encoding tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). TPH and TH catalyze the rate limiting steps in the biosynthesis of serotonin and catecholamines, respectively. Both enzymes were expressed in Escherichia coli and the purified proteins were shown to have TPH and TH activities. This indicates that S. mansoni, and possibly other parasitic helminths, may be capable of synthesizing serotonin and catecholamines endogenously. In the second part of our studies, we looked at the mode of action of serotonin in helminths, in particular the molecular properties of serotonergic G protein-coupled receptors (GPCR). We cloned two helminth GPCRs, one from the free living nematode Caenorhabditis elegans and the second from S. mansoni. The C. elegans receptor (5-HT2Ce) was shown to encode a functional serotonin receptor with structural and signaling properties similar to those of mammalian 5-HT2 receptors. However, its agonist I antagonist binding profile differed from previously characterized serotonin receptors. The cloned S. mansoni receptor (SmGPCRx) was found to represent a new structural class of receptor, which shared about the same level of amino acid sequence homology with various biogenic amines receptors, such as serotonin, catecholamines, and octopamine receptors. Additional sequence analysis and immunolocalization studies confirmed that SmGPCRx possesses structural characteristics of a GPCR. SmGPCRx is the first GPCR ever cloned from a parasitic flatworm. Taken together, these studies mark an important first step to

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Tyrosine hydroxylase belongs to the group of hydroxylases of aromatic acids and catalyzes a key step in the biosynthesis of catecholamine neurotransmitters. The tyrosine hydroxylase possesses the homotetrameric structure and contains three structural domains: the N-terminal regulatory domain, the catalytic domain and the C-terminal tetramerization domain. The activity of tyrosine hydroxylase is regulated by phosphorylation and through the regulation of its expression. Phosphorylation at Ser-19 induces binding of the 14-3-3 protein, which affects the structure of the regulatory domain and protects it against both dephosphorylation and degradation. Since the structure of the regulatory domain is still unknown, we decided to perform its structural characterization using NMR techniques. First, the expression and purification protocol of the regulatory domain of tyrosine hydroxylase was optimized. The protein was expressed as a His-tag fusion protein and its purification is composed from two steps: the chelating chromatography and the size-exclusion chromatography. The dynamic light scattering and the 1 H nuclear magnetic resonance were used to verify its monodispersity, and hence its suitability for further experiments.

The lateral and basal nuclei of the amygdala receive dopaminergic projections from the ventral tegmental area and substantia nigra, and noradrenergic projections from the locus coeruleus (LC). Previously, we demonstrated postmortem indices of altered dopaminergic (amygdala) and noradrenergic (LC) neurochemistry in subjects with major depressive disorder (MDD). For example, decreased levels of dopamine transporter were observed in the amygdala in MDD, while concentrations of tyrosine hydroxylase (TH) were elevated in the LC in MDD. The present study investigated the quantitative distribution of TH in nuclei of the human amygdala from 5 control subjects, and measured amounts of TH in specific amygdaloid nuclei and the LC from 8-10 matched pairs of MDD and psychiatrically normal control subjects. Matched pairs included 3 females and 7 males (controls and MDD), average ages of 50±5 y (controls) and 51±5 y (MDD), average postmortem intervals of 16±2 h (controls) and 21±1 h (MDD), and average pH values of 6.58±0.08 (controls) and 6.59±0.09 (MDD). The lateral, basal, accessory basal, and central nuclei of the amygdala and the LC were punched from frozen sections of postmortem brain. TH-immunoreactivity was measured by quantitative Western blotting. In normal control subjects, TH levels in the LC were between 3000- and 4000-fold higher than TH levels in the nuclei of the amygdala. Within the amygdala, amounts of TH were highest in the basal and central nuclei, and lowest in the lateral nucleus. TH levels in the basal nucleus were highly variable across subjects. TH levels were significantly lower (-50%) in the lateral amygdaloid nucleus in MDD subjects as compared to control subjects. In contrast, TH levels in the LC were significantly higher (+75 %) in MDD subjects. This report is the first demonstration of altered TH levels in the human amygdala. The direction of change associated with MDD of TH in the lateral nucleus of the amygdala was opposite to that found in the LC. Whether abnormal amounts of TH in the amygdala are a result of altered dopaminergic or noradrenergic input to the amygdala requires further study.

The postnatal development of rat retinal phenylethanolamine-N-methyltransferase (PNMT) activity was measured by radiometric assay. Activity was detected on day 1 of life. Retinal PNMT activity of day 1 neonates approximated 10% that of the adult. There is an increase in enzyme activity before eye opening. By day 30, enzyme activity has peaked. The enzyme during this early period possesses the same substrate specificity and inhibitor sensitivity as that of the adult enzyme. PNMT activity is detected before tyrosine hydroxylase activity.

KOSTRZEWA, R. M., J. W. KLARA, J. ROBERTSON AND L. C. WALKER. Studies on the mechanism of sprouting of noradrenergic terminals in rat and mouse cerebellum after neonatal 6-hydroxydopa. BRAIN RES. BULL. 3(5) 525-531, 1978.-The effect of various pharmacologic agents on the noradrenergic innervation of rat cerebellum was observed. It was found that the neurotoxin 6-hydroxydopa (6-OHDOPA), when given to rats at birth, caused a 46% reduction at 5 weeks of age in tyrosine hydroxylase activity in the locus coeruleus, the nucleus of origin for noradrenergic fibers innervating the cerebellum. At the same time, however, both tyrosine hydroxylase activity and NE content were elevated by 50% in the cerebellum. By treating gravid mice with the 6-OHDOPA, which crosses the placental barrier to affect the brains of developing pups, a dissociation has been shown between the elevated cerebellar NE levels and reduced telencephalic NE content. None of the other assorted pharmacological agents-namely amphetamine, metaraminol, apomorphine, α-methyl-ρ-tyrosine, L-dihydroxyphenylalanine and tyramine-when given at birth, caused a permanent elevation in cerebellar NE content. This series of studies suggests that a reduced number of noradrenergic perikarya are providing a greater innervation of the cerebellum than in control rats. Also, alteration of the telencephalic noradrenergic fibers, which are also derived from the locus coeruleus, does not appear to be a necessary event for the initiation of sprouting of noradrenergic fibers in the cerebellum. Because none of the acute-acting pharmacological agents caused a permanent elevation of NE in the cerebellum, it appears that damage, and not mere stimulation or blockade, is a necessary event for initiation of sprouting.

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid-stimulating hormone. Prolactin therapy initiated before 21 days maintains normal-sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 μg of ovine prolactin (oPRL), 50 μg of oPRL, or 50 μg of recombinant mouse prolactin (rmPRL) beginning at 3days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P ≤ 0.01) TIDA neurones than dw/dw, regardless of treatment. TIDA neurones in 50 μg oPRL-treated dw/dw mice were greater (P ≤ 0.05) than those in 5 μg oPRL- and rmPRL-treated dw/dw mice, which were greater (P ≤ 0.01) than those in saline-treated dw/dw mice. Fifty microgram oPRL-treated dw/dw mice also had greater (P < 0.01) TIDA neurone numbers than the maximum numbers observed in untreated dw/dw mice development. Among saline, 5 μg oPRL and 50 μg oPRL treatments, but not rmPRL, A14 neurone numbers were higher (P ≤ 0.01) in normal compared to in dw/dw mice. The mechanism of TIDA neurone recruitment was investigated using bromodeoxyuridine (BrdU) treatment at intervals after 21 days. Mice treated with rmPRL, but not oPRL, had increased BrdU incorporation in the periventricular area surrounding the third ventricle and median eminence and in the arcuate nucleus. The data obtained in the present study indicate that oPRL, but not rmPRL, when given at a high enough dose, induces TIDA neurone differentiation in dw/dw mice. This supports neurotrophic effects of prolactin on TIDA neurones in early postnatal development that extends beyond maintenance of the cell population.