High Levels Of Tyrosine Hydroxylase Research Articles

Treatment of PC12 cells by nerve growth factor, dexamethasone, and forskolin. Effects on cell morphology and expression of neurotensin and tyrosine hydroxylase.

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.

Individual Differences in Locomotor Activity Are Associated with Levels of Tyrosine Hydroxylase and Neurofilament Proteins in the Ventral Tegmental Area of Sprague-Dawley Rats

We have demonstrated previously that chronic morphine and cocaine treatments increase levels of tyrosine hydroxylase (TH), and decrease levels of neurofilament (NF) proteins, in the ventral tegmental area (VTA), a major dopaminergic brain reward region, of outbred Sprague-Dawley rats. We have also found inherent differences in levels of these proteins in the VTA of inbred rat strains that differ in their behavioral responses to opiates, cocaine, and other drugs of abuse, with the Lewis rat showing higher levels of TH and lower levels of NFs in the VTA compared to the Fischer 344 rat. Based on recent reports that individual differences in drug responses among outbred Sprague-Dawley rats are highly correlated with the animals' locomotor response to novelty, we determined in the present study whether such within-strain differences in locomotor behavior are also associated with differences in levels of TH and NFs in the VTA. Groups of 42 Sprague-Dawley rats were assessed for locomotor activity in a novel environment. The four animals from each group with the lowest locomotor responses (designated L rats), and the four with the highest locomotor responses (designated H rats), were analyzed for TH and NF immunoreactivity by immunoblotting procedures. It was found that the VTA of L rats exhibited higher levels of TH and lower levels of three major NF proteins, NF-200, NF-160, and NF-68, compared to the VTA of H rats. A tendency for similar L versus H differences in TH and NF levels were observed when groups of rats with the second lowest and second highest locomotor responses were compared; no differences were seen in groups whose locomotor responses were closer to the median. These biochemical differences between H and L rats showed regional specificity, with no significant differences seen in several other regions of brain or spinal cord studied. Differences were also observed between L and H rats in their locomotor responses to acute and repeated cocaine exposure. The possible relationship between the individual differences in TH and NFs and individual differences in locomotor activity and other drug-related behaviors is discussed.

The generation of monoclonal antibodies that bind preferentially to adrenal chromaffin cells and the cells of embryonic sympathetic ganglia

Adrenal chromaffin cells, sympathetic neurons, and small intensely fluorescent (SIF) cells are each derived from the neural crest, produce catecholamines, and share certain morphological features. These cell types are also partially interconvertible in cell culture (Doupe et al., 1985a,b; Anderson and Axel, 1986). Thus, these cells are said to be members of the sympathoadrenal (SA) lineage and could share a common progenitor. To investigate the origins of this lineage further, we used the cyclophosphamide immuno-suppression method (Matthew and Patterson, 1983) to generate five monoclonal antibodies (SA1-5) that bind strongly to chromaffin cells, with little or no labeling of sympathetic neurons or SIF cells in frozen sections from adult rats. Competition experiments indicate that these antibodies bind to at least three distinct epitopes in tissue sections. The SA antibodies also label most of the cells of embryonic sympathetic ganglia and adrenal primordia. Labeling of sympathetic ganglia appears as the cells initially coalesce and express high levels of tyrosine hydroxylase (TH). Not all TH+ cells in the embryo are SA 1-5+, however; carotid body SIF cells, nodose ganglion TH+ cells, and the transiently TH+ cells in the dorsal root ganglia do not display detectable SA 1-5 labeling. Thus, the expression of these markers for the SA 1-5 lineage is selective. SA antigen expression is hormonally controlled; removal of glucocorticoid and addition of NGF to cultured adrenal chromaffin cells result in the loss of SA 1-5 labeling. These results suggest that the presumed precursors for sympathetic neurons and SIF cells initially express chromaffin cell markers.

Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system

Immunohistochemical distributions of tyrosine hydroxylase and calmodulin in the rat forebrain were analyzed quantitatively to confirm our previous results that the activities of central catecholamine-synthesizing enzymes are regulated by a calcium-calmodulin-dependent system. The adjacent slices of adult rat brain were stained immunohistochemically for tyrosine hydroxylase and for calmodulin, and the distributions and amounts of these proteins were measured by a fluorescence microphotometry system that was developed in our laboratory. Immunohistochemical fluorescence intensity was measured stepwise at 40 μm intervals through a 6 μmφ (on the slice) pin hole. Each stained brain slice was divided into approximately 100,000 areas, and measured for fluorescence intensity and displayed two- and three-dimensionally. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all areas of the brain, but its intensity varied. The relatively high levels of calmodulin could be observed in brain regions with high levels of tyrosine hydroxylase distribution, though high levels of tyrosine hydroxylase could not always be observed in brain regions where high levels of calmodulin were distributed. In the present study, high levels of tyrosine hydroxylase and calmodulin were distributed in the nucleus accumbens septi and the lateral part of the neostriatum regions in which the amount of dopamine was increased by the intraventricular administration of calcium. These findings suggest that the synthesis of central catecholamines is regulated by a calcium-calmodulin-dependent system.

L-3,4-Dihydroxyphenylalanine Synthesis by Genetically Modified Schwann Cells

We have investigated whether Schwann cells can be modified by gene transfer to synthesize L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L-DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L-DOPA while maintaining their capacity to myelinate neurons in vitro. This study indicate that it may be feasible to utilize autotransplantation of genetically modified Schwann cells to alleviate the movement disorders in Parkinson's disease.

Ionic excitation of a clone of mouse neuroblastoma

The electrical properties and the possible regulation of these properties were studied by means of intracellular microelectrode recordings in cells of mouse neuroblastoma clone N1E-115. This clone has high levels of tyrosine hydroxylase and regulates this enzyme. Cells treated for 24 h with 4 μM aminopterin followed by at least 5 days in culture developed rhythmic discharge of action potentials when superfused with phosphate-buffered saline containing ⩽ 0.2mMcalcium or⩽ 0.2mM calcium and zero potassium. This ionic excitation occurred in no cells at less than 5 days after treatment with aminopterin but at 5 days or more after treatment, 20% of cells responded to low calcium while 52% responded to low calcium and zero potassium. Concomitant with the development of a susceptibility to ionic excitation was an increase in the average resting membrane potential and morphologic maturation. This ionic excitation of cultured mouse neuroblastoma cells may be useful for studying biochemical events associated with repetitive discharge of action potentials.

Studies on the Transport of l-Tyrosine into an Adrenergic Clone of Mouse Neuroblastoma

Abstract Studies on the transport of l-[U-14C]tyrosine into cultured mouse neuroblastoma clone N1E-115, which has high levels of tyrosine hydroxylase (EC 1.14.16.2), are presented. This transport was by a saturable process, exhibited marked exchanging properties, and was inhibited by a reagent which attacks sulfhydryl groups but not by metabolic inhibitors. No major ion requirements were found for this transport, which was sensitive to changes in temperature but not to changes in pH in the range of 6 to 8. Molecules with close structural analogy to l-tyrosine such as l-3,4-dihydroxyphenylalanine, l-3-iodotyrosine, and l-phenylalanine were potent competitive inhibitors of this transport with inhibitor constants (Ki) in the range of 2 to 6 x 10-5 m. In addition, compounds such as l-isoleucine, l-leucine, l-methionine, and l-valine caused inhibition of l-tyrosine transport. These results are consistent with the conclusion that l-tyrosine transport into this clone is by facilitated diffusion and by a mechanism similar to the leucine-preferring system which has been described by others.

Stimulation of Tyrosine Hydroxylase Activity in an Adrenergic Clone of Mouse Neuroblastoma by Dibutyryl Cyclic AMP

Regulation of catecholamine biosynthesis can be studied in mouse neuroblastoma cells growing in tissue culture. We have isolated adrenergic clones1 and have studied in particular clone N1E-115 which has very high levels of tyrosine hydroxylase (EC 1.14.3a), the first enzyme within the adrenergic cell in the biosynthetic pathway to catecholamines. We found that tyrosine hydroxylase activity in N1E-115 is related inversely to the rate of cell division and/or cell density in culture (ref. 2 and E. R. and M. Nirenberg, manuscript in preparation) so that from early log to late stationary phase of growth, its activity increases more than 30-fold. The catecholamine precursors, phenylalanine and tyrosine, enter these cells by high affinity transport systems3 and are converted into catechols (E. R., unpublished data).