J.Y. Lew

Localization and characterization of phenylethanolamine N-methyl transferase in the brain of various mammalian species

The distribution of PNMT activity in various regions and nuclei of the rat, monkey and human brain was investigated. The distribution of PNMT activity in the rat brain correlates with the distribution of PNMT immunofluorescence. The PNMT activity in the primate brain is more widely distributed than in the rat brain. High and intermediate enzyme activity values were dound in the same regions of the primate brain as in the rat brain. Intermediate or low levels were also found in various other regions of the primate brain, e.g., basal ganglia, amygdala, septum, habenula. The brain PNMT has the same substrate specificity and similar kinetic properties as the adrenal enzyme. Immunotitration studies revealed cross-reactivity between the homologous adrenal and brain PNMT.

Dopaminergic transmission in the rat retina: evidence for volume transmission

The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site-and phosphospecific antibodies and site-directed mutagenesis

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT‐20 cells expressing wild‐type or [Leu19]‐recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l‐DOPA biosynthesis rates in intact AtT‐20 cells. Treatment of either PC12 cells or AtT‐20 cells expressing wild‐type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT‐20 cells expressing wild‐type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT‐20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization‐induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.

On the distribution patterns of D1, D2, tyrosine hydroxylase and dopamine transporter immunoreactivities in the ventral striatum of the rat

The distribution of dopamine D1 and D2 receptor immunoreactivities in the nucleus accumbens and the olfactory tubercle of adult and postnatal male rats were compared with the distribution of tyrosine hydroxylase and dopamine transporter immunoreactivities. An overall co-distribution of D1 and D2 receptor immunoreactivities with tyrosine hydroxylase immunoreactivity was found in the nucleus accumbens and the olfactory tubercle. However, the major finding in this study was, following a more detailed analysis in coronal sections of the shell part of the nucleus accumbens, the existence of nerve cell patches of strong D1 receptor immunoreactivity associated with low D2 receptor, dopamine transporter and tyrosine hydroxylase immunoreactivities. These patches were mainly surrounded by areas of strong D2 receptor, tyrosine hydroxylase and dopamine transporter immunoreactivities and could be found also in the olfactory tubercle. Similar observations were made in postnatal rats. Serial reconstructions of the patches of strong D1 receptor immunoreactivity in the rostrocaudal direction were made. The patches formed a continuous tubular nerve cell system in the shell part of the nucleus accumbens. Since this nerve cell system was found to be surrounded by a high density of dopamine terminals, it may represent a compartment where dopamine transmission mainly acts on D1 receptors via local diffusion (i.e. via volume transmission). However, it must be noted that the D1 receptor rich patches constitute only a small fraction of the nucleus accumbens and the overall density of tyrosine hydroxylase immunoreactive terminals correlates with the density of both D1 and D2 receptors in the nucleus accumbens. In conclusion, the present paper gives new aspects on the chemical microarchitecture of the nucleus accumbens.

Binding Interactions of Ergot Alkaloids with Monoaminergic Receptors in the Brain

The interactions of ergot alkaloids and of other drugs with dopamine (DA) and alpha-adrenergic receptors were investigated. The tested ergot alkaloids inhibit synaptosomal tyrosine hydroxylase activity and reverse the apomorphine-elicited inhibition of synaptosomal tyrosine hydroxylase activity. Thus, ergot alkaloids interact as both agonists and antagonists with the presynaptic DA receptors. Ergot alkaloids also compete effectively for the binding of 3H-DA and 3H-haloperidol to bovine striatal membranes. These results show that these drugs are mixed agonist-antagonists with respect to the postsynaptic DA receptors. To determine the effects of ergot alkaloids and of neuroleptics on the alpha-adrenergic receptors in the CNS, we have measured their effects on the binding of 3H-dihydroergocryptine and 3H-WB-4101 to cerebral cortical membranes. The displacing potencies of the tested ergot alkaloids and of the neuroleptics indicated that they have a high affinity for the alpha-adrenoreceptors in the CNS. The mechanisms underlying the therapeutic efficacy of mixed agonist-antigonists of DA and alpha-adrenergic receptors in Parkinsons disease and in geriatric disorders were considered.

Solubilization and characterization of striatal dopamine receptors

Abstract: Dopamine receptor binding proteins were sol‐ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio ‐ 2 ‐ hydroxy ‐ 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [3H]spiroperidol ([3H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO‐solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga‐tion at 100,000 ×g for 2 h. The CHAPSO‐solubilized dopamine receptor proteins, as well as the prelabeled [3H]Spi‐receptor protein complex, bind specifically to wheat germ agglutinin (WGA)‐agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO‐solubilized, prelabeled [3H]Spi‐receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA‐agarose‐bound [3H]Spi‐receptor protein complex with digitonin (CHAPSO‐digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [3H]Spi‐receptor complex in the CHAPSO‐digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO‐solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [3H]Spi to CHAPSO‐solubilized dopamine receptor proteins are similar to those for membrane‐bound proteins. Chronic treatment with haloperidol increases the Bmax, and does not change the KD for [3H]Spi in the CHAPSO‐solubilized and in the membrane‐bound preparations. Thus, the CHAPSO‐solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane‐bound dopamine receptors.

The blockade of α2-adrenoceptors by the PNMT inhibitor SK&F 64139

Three tetrahydroisoquinolines were tested for activity on central α2-adrenoceptors by determining their ability to inhibit [3H]clonidine binding to rat cerebral cortical membrane homogenates. The compounds included the PNMT inhibitors SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline) and SK&F 29661 (1,2,3,4-tetrahydroisoquinoline 7-sulfonamide) as well as SK&F 72223 (5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline) a structural analogue inactive as a PNMT inhibitor. SK&F 64139 and SK&F 72223 but not SK&F 29661 inhibited [3H]clonidine binding. Studies in the isolated guinea pig atrium confirmed that SK&F 64139 and SK&F 72223 are α2-adrenoceptor antagonists.

Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using antibodies raised to phosphorylated forms of the enzyme

Abstract Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) to the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX–forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the Serine 40-phosphorylated form of tyrosine hydroxylase. Treated slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre lexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggest that antibodies directed against phosphorylated forms of tyrosine hydroxylase can be used to evaluate the state of tyrosine hydroxylase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo .

Catecholamine-stimulated cyclic AMP formation in phenylethanolamine n-methyltransferase containing brain stem nuclei of normal rats and of rats with spontaneous genetic hypertension

Stimultaion of cyclic AMP formation by epinephrine and norepinephrine has been studied in discrete areas of rat brain that include the epinephrine-containing brain stem nuclei C-1 and C-2. In the C-1 area, epinephrine-stimulated cyclic AMP formation was partially reversed by 100 microM phentolamine and by 10--100 microM propranolol or alprenolol and hence appeared to involve activation of a mixture of both alpha- and beta-adrenergic receptors as has been reported for other rat brain areas such as the cerebral cortex. However, in the C-2-area, the epinephrine and norepinephrine stimulated cyclic AMP formation involved the activation of a single receptor type which was alpha-like in character. Stimulation of cyclic AMP formation by epinephrine in the C-2 area was antagonized by nanomolar concentrations of both phentolamine and yohimbine. The epinephrine-stimulated formation of cyclic AMP in the C-2 but not in the C-1 area was augmented in a strains of rats which exhibit spontaneous genetic hypertension (SHR) vs. Wistar-Kyoto controls. It is suggested that the enhanced epinephrine-stimulated cyclic AMP formation in the C-2 area of SHR rats could be a physiological compensatory response to some other hypertension-causing lesion which, for example, results in chronically reduced epinephrine release or in ruduced availability of epinephrine at its postsynaptic receptor thereby leading to receptor supersensitivity. Supporting this possibility was the finding that treatment of SHRs and control animals and reserpine resulted in enhancement of epinephrine-stimulated cyclic AMP formation in the C-2 area of control rats, essentially obliterating the difference between control and SHR. The findings are also interepreted as supporting the involvement of epinephrine neurons in central vaso-depressor mechanisms.

Antibodies to a segment of tyrosine hydroxylase phosphorylated at serine 40.

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti‐pTH32–47 antibodies with phospho‐ and dephospho‐Ser40 forms of TH protein and peptide TH32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody‐capture ELISAs, anti‐pTH32–47 was more reactive with the phospho‐TH than with the dephospho‐TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho‐TH forms. In western blots, labeling of the ∼60‐kDa TH band by anti‐pTH32–47 was readily detectable in lanes containing protein kinase A‐phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti‐pTH32–47. Similarly, anti‐pTH32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8‐bromoadenosine 3′,5′‐cyclic monophosphate or forskolin enabled removal of TH activity by anti‐pTH32–47. Lastly, in immunohistochemical studies, anti‐pTH32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion‐fixed rat brain.