Regulation of tyrosine hydroxylase from human pheochromocytoma, bovine adrenal and rat striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum can be activated by Mg²⁺, ATP and cyclic AMP. In pheochromocytoma, this activation is due to a decreased Km for the pterin cofactor, whereas in adrenal medulla, it is a result of an increase in the Vmax. Norepinephrine increases the Km for pterin cofactor for tyrosine hydroxylase from both of these tissues. The Ki for norepinephrine is not altered by the presence of Mg²⁺, ATP and cyclic AMP with enzyme from pheochromocytoma or adrenal medulla. On the other hand, striatal tyrosine hydroxylase shows a two-fold increase in the Ki for dopamine after exposure to Mg²⁺, ATP and cyclic AMP.     

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [³²P]P_i and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. ³²P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of ³²P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of ³²P into tyrosine hydroxylase, with 50 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca²⁺. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of ³²P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus,hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of ³H₂O from L-4-³H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of ³H₂O from L-4-³H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank ³H₂O produced from L-4-³H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank ³H₂O produced from L-3,5-³H-tyrosine. The K_m values of tyrosine hydroxylase for phenylalanine determined by the production of ³H₂O from L-4-³H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of ³H₂O formed from L-4-³H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.     

Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of tyrosine hydroxylase

Studies are presented which demonstrate that rat pheochromocytoma tumors are a convenient material for the preparation of tyrosine hydroxylase mRNA. Total pheochromocytoma poly(A)⁺mRNA has been extracted from tumors, then translated in a reticulocyte lysate cell-free system. Neo-synthesized tyrosine hydroxylase has been identified by direct immunoprecipitation followed by sodium dodecyl sulfate acrylamide gel electrophoresis. The proportion of this specific mRNA has been calculated; it represents 0.15 per cent of the total poly(A)⁺mRNA. The molecular weight of the $\text{in}\text{vitro}$ synthesized tyrosine hydroxylase is 62,000.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Tyrosine hydroxylase: mechanisms of oxygen radical formation

Summary

A new mechanism of oxygen radical formation in dopaminergic neurons is proposed, based on the oxidative mechanism of tyrosine hydroxylase. The cofactor (6R,6S)-5,6,7,8-tetrahydrobiopterin can rearrange in solution which allows an autoxidation reaction producing O₂^.-, H₂O₂ and HO^.. The combination of tyrosine hydroxylase and the cofactor produces more oxygen radicals than does the autoxidation of the cofactor. This production of oxygen radicals could be damaging to dopaminergic neurons. In the presence of tyrosine, the enzyme produces less radicals than it does in the absence of tyrosine. Mechanisms are proposed for the generation of reactive oxygen species during the autoxidation of the cofactor and during enzymatic catalysis. The generation, by tyrosine hydroxylase, of very small amounts of oxygen radicals over the period of 65 years could contribute to the oxidative stress that causes Parkinson's disease.     

A point mutation in the tyrosine hydroxylase gene associated with Segawa's syndrome

We have examined the molecular basis of Segawa's syndrome in six families with seven affected children. In one family two siblings with this disease carried a point mutation in exon 11 of the tyrosine hydroxylase gene, resulting in an amino acid exchange of Gln³⁸¹ to Lys³⁸¹. These results suggest that a change in tyrosine hydroxylase causes this form of Segawa's syndrome.     

Evidence for a functionally important histidine residue in human tyrosine hydroxylase

Summary Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M^–1 min^–1 at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per enzyme subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoenzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.Abbreviations BH₄ (6R)-l-erythro-tetrahydrobiopterin - DEP diethylpyrocarbonate - DOPA 3,4-dihydroxyphenylalanine - hTH1 human tyrosine hydroxylase isoenzyme 1 - apo-hTH1 apoenzyme of hTH1 - Fe(II)-hTH1 holoenzyme (iron reconstituted) of hTH1 - dopamine-Fe(III)-hTH1 holoenzyme of hTH1 with dopamine bound - TH tyrosine hydroxylase     

Formation of Catechol Compounds from Phenylalanine and Tyrosine with Isolated Nerve Endings

THERE is much evidence that catecholamines may act as synaptic transmitters in the mammalian brain¹. Enzymatic activities necessary for the synthesis of catecholamines have been located in central neurones¹ and it is generally believed that tyrosine hydroxylase² is the rate limiting enzyme in brain as well as peripheral tissues containing catecholamines³. While it is clear that tyrosine can serve as a precursor of catecholamine synthesis in the brain^{1, 3, 4}, the significance of phenylalanine is problematic. It was believed that the mammalian brain is devoid of enzymatic activity necessary to convert phenylalanine to tyrosine^{6, 7}, while liver is known to be rich in the enzyme phenylalanine hydroxylase⁸. The earlier attempts to demonstrate hydroxylation of phenylalanine in brain tissue may have been unsuccessful due to methodological problems⁹. Recent evidence suggests that tyrosine hydroxylase prepared from peripheral sympathetically innervated tissues or from brain can hydroxylate either phenylalanine or tyrosine⁹. Initially, the rate of hydroxylation of phenylalanine by tyrosine hydroxylase was thought to be as little as 5% that of tyrosine⁹. It has been found recently, however, that structural variations in the pteridine cofactor present in the incubation mixture lead to striking changes in the ability of partially purified tyrosine hydroxylase from bovine adrenal medulla to hydroxylate phenylalanine¹⁰. Thus, tetrahydrobiopterin allowed the hydroxylation of phenylalanine to proceed at least as rapidly as that of tyrosine or faster¹⁰. As the structure of the endogenous pteridine cofactor of tyrosine hydroxylase is not known, it is possible that synthesis of catecholamines from phenylalanine as well as tyrosine could occur in intact neuronal tissues. Evidence has been presented that after the injection of large quantities of ¹⁴C-phenylalanine into the lateral ventricle of the rat brain, small amounts of labelled tyrosine and traces of newly synthesized catecholamines were detected in brain tissues, giving qualitative evidence that catecholamines may be synthesized in brain from phenylalanine in vivo¹¹.     

Evidence for the lack of direct phosphorylation of bovine caudate tyrosine hydroxylase following activation by exposure to enzymatic phosphorylating conditions.

Highly purified bovine caudate tyrosine hydroxylase can be activated by exposure to enzymatic phosphorylating conditions. This activation is due to both a decrease in the K_m for the pterin cofactor and to some increase in V_max. The K_m for the enzyme's substrate, tyrosine, is unchanged by activation. After tyrosine hydroxylase was activated in the presence of [γ-³²P]-ATP, no incorporation of ³²P into the enzyme was observed by either immunoprecipitation studies or by sucrose gradient studies.     

A denaturant-insoluble form of tyrosine hydroxylase in PC12 pheochromocytoma cells

A 6M urea-insoluble form of tyrosine hydroxylase (TH_i) was detected in PC12 pheochromocytoma cells by western blotting immunodetection methods, and the characteristics and mechanisms of formation of this insoluble species were investigated. TH_i accounts for about 4% of the immunodetectable tyrosine hydroxylase in exponentially dividing pheochromocytoma cells. It is unlikely that a subpopulation of dead or dying cells is the source of TH_i since essentially no changes in TH_i levels were detected when cell death was intentionally increased. To measure the kinetics of formation of cellular TH_i, exponentially dividing cells were metabolically labeled first with [³H]leucine and then with [¹⁴C]leucine, and though both³H and¹⁴C were incorporated into soluble tyrosine hydroxylase, the near absence of¹⁴C in TH_i demonstrated that a lag period of at least a day exists between biosynthesis of tyrosine hydroxylase and the accumulation of measurable TH_i. The cellular accumulation of TH_i can evidently be regulated by the cell, since upon nerve growth factor (NGF) treatment of cells the total content of tyrosine hydroxylase increased and the content of TH_i decreased to yield, overall, a fivefold lower proportion of TH_i after 4 days. A large increase in urea-insoluble enzyme was found upon sublethal exposure of cells to ferrous ion and hydrogen peroxide, indicating that oxidative damage via metal-ion-catalyzed formation of hydroxide free radical can yield an enzyme that is similar in its insolubility to TH_i.Abbreviations DOPA 3,4-dihydroxyphenylalanine - NGF nerve growth factor - TH_i denaturant-insoluble tyrosine hydroxylase - EDTA ethylene diamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)-aminomethane - LLPM low-leucine pulse medium - WS water-solubilized protein - US 6 M urea-solubilized protein - UI 6 M urea-insoluble protein     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

The influence of neuropeptides on amine synthesis: Cholecystokinin-8 and neurotensin reduce striatal tyrosine hydroxylase activity

Cholecystokinin-8 and neurotensin, neuropeptides found in relatively high concentrations in some catcholaminergic neurons and/or terminal or cell body areas, reduced native and polyanion-activated rat striatal tyrosine hydroxylase activity, but not that of the enzyme activated by phosphorylating conditions. Substance P, γ₃-melanocyte-stimulating hormone, [d-ala², d-leu⁵]-enkephalin, or thyrotropin-releasing hormone had no effect on the native enzyme or on either activated form. Cholecystokinin-8 and neurotensin may interact with polyanion-activated or membrane bound-activated tyrosine hydroxylase to modulate its activity.     

Effects of subchronic nicotine administration on central dopaminergic mechanisms in the rat

Nicotine was administered acutely and subchronically (14 days) to determine whether various synaptic mechanisms are selectively altered in the nigrostriatal and mesolimbic dopaminergic systems in the rat. When added to tissue preparations in vitro, nicotine had no effects on tyrosine hydroxylase, synaptosomal uptake of [³H]dopamine or binding of [³H]spiperone to D₂ receptors in either system. However, acute treatment in vivo stimulated tyrosine hydroxylase activity in the nucleus accumbens. This effect was prevented by pretreatment with a nicotinic antagonist, suggesting that it was mediated by nicotinic receptors. Since subchronic exposure to nicotine had no effect on tyrosine hydroxylase, it appears that tolerance develops to this action. In vivo treatment with nicotine did not alter dopamine uptake or receptor binding. The results suggest that, in doses which result in moderate plasma levels, nicotine has selective stimulant actions on nerve terminals of the mesolimbic system.