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L -Tyrosine derivatives are widely applied in the pharmaceutical, food, and chemical industries. Their production is mainly confined to chemical synthesis and plant extract. Microorganisms, as cell factories, exhibit promising advantages for valuable chemical production to fulfill the increase in the demand of global markets. Yeast has been used to produce natural products owing to its robustness and genetic maneuverability. Focusing on the progress of yeast cell factories for the production of L -tyrosine derivatives, we summarized the emerging metabolic engineering approaches in building L -tyrosoine-overproducing yeast and constructing cell factories of three typical chemicals and their derivatives: tyrosol, p-coumaric acid, and L -DOPA. Finally, the challenges and opportunities of L -tyrosine derivatives production in yeast cell factories were also discussed., (© 2023 John Wiley & Sons Ltd.)

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP receptor) contribute to Parkinson's disease (PD) and motor complications associated with levodopa therapy. The N/OFQ-NOP receptor system is expressed in cortical and subcortical motor areas and, notably, in dopaminergic neurons of the substantia nigra compacta. Dopamine depletion, as in rodent models of PD results in up-regulation of N/OFQ transmission in the substantia nigra and down-regulation of N/OFQ transmission in the striatum. Consistent with this, NOP receptor antagonists relieve motor deficits in PD models by reinstating the physiological balance between excitatory and inhibitory inputs impinging on nigro-thalamic GABAergic neurons. NOP receptor antagonists also counteract the degeneration of nigrostriatal dopaminergic neurons, possibly by attenuating the excitotoxicity or modulating the immune response. Conversely, NOP receptor agonists attenuate levodopa-induced dyskinesia by attenuating the hyperactivation of striatal D 1 receptor signalling in neurons of the direct striatonigral pathway. The N/OFQ-NOP receptor system might represent a novel target in the therapy of PD., (© 2019 The British Pharmacological Society.)

Insect prophenoloxidase (PPO) induces melanization around pathogens. Before melanization, PPO is cleaved into phenoloxidase (PO) by serine proteases. Insect PPO can also be activated by exogenous proteases secreted by pathogens as well as by other compounds, such as ethanol and cetylpyridinium chloride (CPC). However, the effect of these activators on the activity of PO is unclear. In this study, the insect endogenous serine protease AMM1, α-chymotrypsin, and ethanol were used to activate recombinant Drosophila PPO1 (rPPO1), and the PO activity differed depending on the activator applied. The PO-induced intermediates during melanization also varied markedly in their numbers and abundances. Therefore, this study indicates that the mechanism of PPO activation influences PO activity. It also suggests that PO-induced different intermediates may affect the antibacterial activity during melanization due to their toxicity., (© 2018 Wiley Periodicals, Inc.)

We used in vivo proton ((1)H) Magnetic Resonance Spectroscopy (MRS) to measure the levels of the main excitatory amino acid, glutamate (Glu) and also glutamine (Gln) and GABA in the striatum and cerebral cortex in the MPTP-intoxicated mouse, a model of dopaminergic denervation, before and after dopamine (DA) replacement. The study was performed at 9.4T on control mice (n = 8) and MPTP-intoxicated mice (n = 8). In vivo spectra were acquired in a voxel (8 microL) centered in the striatum, and in the cortex (4.6 microL). Three days after basal MRS acquisitions new spectra were acquired in the striatum and cortex, after levodopa (200 mg.kg(-1)). Glu, Gln and GABA concentrations obtained in the basal state were significantly increased in the striatum of MPTP-lesioned mice (Glu: 20.2 +/- 0.8 vs 11.4 +/- 0.9 mM, p < 0.001; Gln: 5.4 +/- 1.6 vs 2.0 +/- 0.6 mM, p < 0.05; GABA: 3.6 +/- 0.8 vs 1.6 +/- 0.2 mM, p < 0.05). Levodopa lowered metabolites concentrations in the striatum of MPTP-lesioned mice (Glu: 20.2 +/- 0.8 vs 11.2 +/- 0.4 mM (+ Ldopa), p < 0.001; Gln: 5.4 +/- 1.6 vs 1.6 +/- 0.4 mM (+ Ldopa), p < 0.05; GABA: 3.6 +/- 0.8 vs 1.7 +/- 0.4 mM (+ Ldopa), p < 0.01). Metabolite levels in the striatum of MPTP-intoxicated mice + levodopa were not significantly different from those in the striatum of controls. No change was found in the cortex after DA denervation and after DA replacement between the two animals groups. These results strongly support a predominant change in striatal Glu synaptic activity in the cortico-striatal pathway. Acute levodopa administration reverses the increase of metabolites in the striatum.

Background and Purpose: This study investigates the role of alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), on catecholamine synthesis and catabolism in the central nervous system of mice., Experimental Approach: Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice., Key Results: Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in alpha(2A)- and alpha(2C)-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in alpha(2A) and alpha(2C)KOs compared with WT [WT: 2.8 +/- 0.5, 1.1 +/- 0.1; alpha(2A)KO: 6.9 +/- 0.7, 1.9 +/- 0.1; alpha(2B)KO: 2.3 +/- 0.2, 1.0 +/- 0.1; alpha(2C)KO: 4.6 +/- 0.8, 1.5 +/- 0.2 nmol.(g tissue)(-1), for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in alpha(2A) and alpha(2C)KO [WT: 40 +/- 1; alpha(2A): 77 +/- 2; alpha(2B): 40 +/- 1; alpha(2C): 50 +/- 1, maximum velocity (V(max)) in nmol.(mg protein)(-1).h(-1)], but no significant differences were found in dopamine beta-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three alpha(2)KO mice [WT: 2.0 +/- 0.0; alpha(2A): 2.4 +/- 0.1; alpha(2B): 2.2 +/- 0.0; alpha(2C): 2.2 +/- 0.0 nmol.(mg protein)(-1).h(-1)], but no significant differences were found in monoamine oxidase activity between all alpha(2)KOs and WT mice., Conclusions and Implications: In mouse brain, deletion of alpha(2A)- or alpha(2C)-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any alpha(2)-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in alpha(2A) and alpha(2C)KO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.

In insects, eukaryotic endoparasites encounter a series of innate immune effector responses mediated in large part by circulating blood cells (hemocytes) that rapidly form multilayer capsules around foreign organisms. Critical components of the encapsulation response are chemical and enzyme-catalyzed oxidations involving phenolic and catecholic substrates that lead to synthesis of eumelanin. These responses are initiated immediately upon infection and are very site-specific, provoking no undesirable systemic responses in the host. In this study, we were interested to learn if the principal oxidation pathways leading to the synthesis of eumelanin in larvae of Drosophila melanogaster were targets for inhibition by immune suppressive factors (ISF) derived from a virulent strain of the endoparasitic wasp Leptopilina boulardi. Comparative in vitro assays monitored by sensitive electrochemical detection methods showed that ISF derived from female reproductive tissues significantly diminished the oxidations of the two diphenol eumelanin precursors, dopamine and 5,6-dihydroxyindole (DHI). The oxidations of the monophenol tyrosine, and two other related diphenols, dopa and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), were not significantly inhibited by ISF. The data suggest that melanogenesis represents at least one of the host responses suppressed by L. boulardi ISF, and that the oxidation pathways selectively targeted for inhibition are those synthesizing decarboxylated pigment precursors derived from DHI. These observations, together with previous reports of adverse effects of ISF on the ability of hemocytes to adhere to foreign surfaces, suggest a multifaceted approach by the parasitoid to circumvent the innate immune response of D. melanogaster.

We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (L-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of L-DOPA semiquinone (L-DOPA-SQ). In the present study, intrastriatal infusion of L-DOPA (1.0 microm for 200 min) increased dialysate L-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, L-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of L-DOPA were negatively correlated with those of L-DOPA-SQ. Co-infusion of N-acetylcysteine (100 microm) or melatonin (50 microm) increased L-DOPA (up to 151- and 246-fold, respectively) and decreased L-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic L-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of L-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic L-DOPA, L-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic L-DOPA induced a smaller increase in dialysate L-DOPA, a greater increase in L-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with L-DOPA, in control as well as in light-exposed rats, significantly increased dialysate L-DOPA concentrations, greatly inhibited L-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous L-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with L-DOPA markedly increases striatal L-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term L-DOPA therapy of Parkinson's disease.

Objective: Parameters of a pharmacokinetic-pharmacodynamic (PK-PD) model of levodopa have been claimed to reflect the magnitude of the dopaminergic deficit in patients with Parkinson's disease. The aim of this study was to correlate such parameters with positron emission tomography (PET) with levodopa tagged with 6-fluorine 18, an established imaging method for striatal dopaminergic neurons., Methods: Twenty-three patients in different disease stages (Hoehm and Yahr stage 2.5-5 [Hoehn MM, Yahr MD. Parkinsonism: onset, progression and mortality. Neurology 1967;4:427-42]; median duration, 12 years) were studied. PK-PD modeling followed a single oral dose of levodopa/benserazide. The sum score of the Columbia Rating Scale (CURSSigma) was used for clinical assessments. A nonparametric effect compartment approach assuming a sigmoidal E(max) model was applied to the PK-PD analysis of plasma levodopa concentrations and corresponding CURSSigma. Thereafter 6-[18F]levodopa PET was performed, and the influx rate constants (k(c)) for the putamen and the caudatus region were correlated with the median effective concentration (EC(50)) and the equilibrium half-life (T(eq)) of the PK-PD model., Results: (1) A significant correlation was observed between PK-PD parameters or with k(c) putamen as the dependent variable and the duration of the disease as the independent variable, which explains 33% of the variability of the EC(50), 42% of the variability of T(eq), and 36% of the variability of k(c). (2) Significant correlations were observed between k(c) and either EC(50) or T(eq), yielding the closest correlation for the putamen region (r = -0.47, P <.05; and r = 0.55, P <.01; respectively)., Conclusions: Our findings show that key parameters of a PK-PD model of levodopa were in fairly close agreement with imaging of dopaminergic neurons by 6-[18F]levodopa PET. However, although PK-PD modeling of levodopa has been proven as a useful investigation of approaches aimed to restore dopaminergic deficits or to monitor disease progression, this modeling cannot serve as a pathomorphologic surrogate for the loss of striatal dopaminergic neurons.

We have previously shown that manganese enhances L-dihydroxyphenylanine (L-DOPA) toxicity to PC12 cells in vitro. The supposed mechanism of manganese enhancing effect [an increase in L-DOPA and dopamine (DA) auto-oxidation] was studied using microdialysis in the striatum of freely moving rats. Systemic L-DOPA [25 mg kg(-1) intraperitoneally (i.p.) twice in a 12 h interval] significantly increased baseline dialysate concentrations of L-DOPA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid, compared to controls. Conversely, DA and ascorbic acid concentrations were significantly decreased. A L-DOPA oxidation product, presumptively identified as L-DOPA semiquinone, was detected in the dialysate. The L-DOPA semiquinone was detected also following intrastriatal infusion of L-DOPA. In rats given L-DOPA i.p. , intrastriatal infusion of N-acetylcysteine (NAC) significantly increased DA and L-DOPA dialysate concentrations and lowered those of L-DOPA semiquinone; in addition, NAC decreased DOPAC+HVA and uric acid dialysate concentrations. In rats given L-DOPA either systemically or intrastriatally, intrastriatal infusion of manganese decreased L-DOPA dialysate concentrations and greatly increased those of L-DOPA semiquinone. These changes were inhibited by NAC infusion. These findings demonstrate that auto-oxidation of exogenous L-DOPA occurs in vivo in the rat striatum. The consequent reactive oxygen species generation may account for the decrease in dialysate DA and ascorbic acid concentrations and increase in enzymatic oxidation of xanthine and DA. L-DOPA auto-oxidation is inhibited by NAC and enhanced by manganese. These results may be of relevance to the L-DOPA long-term therapy of Parkinson's disease.

By bioassay-guided fractionation using mushroom tyrosinase, p-coumaric acid was characterized as the principal tyrosinase inhibitor from the fresh leaves of Panax ginseng (Araliaceae). It inhibited the oxidation of L-tyrosine more strongly than that of L-3, 4-dihydroxyphenylalanine (L-DOPA) by this enzyme. On the basis of this finding, various related phenylpropanoid analogues were also tested in order to gain new insights into their structural criteria., (Copyright 1999 John Wiley & Sons, Ltd.)

1. The present work has examined the effects of short- (30 min) and long-term (14 h) exposure to cyclosporine A (CsA) on the uptake of L-DOPA, its decarboxylation to dopamine and the cellular extrusion of taken up L-DOPA and of newly-formed amine in monolayers of LLC-PK1 cells. 2. In the presence of benserazide (50 microM), L-DOPA was rapidly accumulated in LLC-PK1 cells (cultured in collagen-treated plastic) attaining equilibrium at 30 min of incubation. Non-linear analysis of the saturation curves revealed a Km of 113+/-16 microM and a Vmax of 5581+/-297 pmol mg(-1) protein 6 min(-1). 3. In the absence of benserazide, LLC-PK1 cells incubated with increasing concentrations of L-DOPA (10 to 500 microM) for 6 min accumulate newly-formed dopamine by a saturable process with apparent Km and Vmax values of 31+/-6 microM and 1793+/-91 pmol mg(-1) protein 6 min(-1), respectively. The fractional outflow of newly-formed dopamine was found to be 20%. Up to 200 microM of intracellular newly-formed dopamine, the outward transfer of the amine was found to be a non-saturable process. 4. Short-term exposure to CsA (0.3, 1.0 and 3.0 microg ml(-1)) was found not to change the intracellular concentrations of newly-formed dopamine, but increased the levels of dopamine in the incubation medium (143% to 224% increase) and the total amount of dopamine formed (31% to 59% increase). Long-term exposure to CsA (0.03 to 3.0 microg ml(-1)) reduced the total amount of dopamine (15% to 39% reduction) and the intracellular levels of the amine (11% to 56% reduction), without changing dopamine levels in the incubation medium. Both short- and long-term exposure to CsA resulted in a concentration-dependent increase in the fractional outflow of newly-formed dopamine. 5. Short-term exposure to CsA (3.0 microg ml(-1)) reduced the apical extrusion of intracellular L-DOPA by 15% (P<0.05), whereas long-term exposure to CsA reverted this effect and decreased its intracellular availability (15% reduction; P<0.05). 6. Detection of P-glycoprotein activity was carried out by measuring verapamil- or UIC2-sensitive rhodamine 123 accumulation. Both UIC2 (3 microg ml(-1)) and verapamil (25 microM) significantly increased the accumulation of rhodamine 123 in LLC-PK1 cells. A 30 min exposure to CsA was found not to affect the accumulation of rhodamine 123 in the presence of verapamil (25 microM), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123. 7. In conclusion, the increase and the reduction in the formation of dopamine after short- and long-term exposure to CsA, respectively, correlate with the effects of the immunosuppressant on the apical cell extrusion of taken up L-DOPA, suggesting the involvement of P-glycoprotein. The effects of CsA on the fractional outflow of newly-formed dopamine appear to be mediated by a different mechanism.

1. The accumulation of L-DOPA in suspensions of renal tubules obtained from male Wistar rats occurred through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA uptake in renal tubules was as follows: Vmax = 46.3 +/- 2.8 nmol mg-1 protein h-1 and Km = 114.4 (95% confidence limits; 83.8, 156.2) microM (n = 5). The diffusion constant (in nmol-1) of the non-saturable component for the accumulation of L-DOPA was 1.3 (1.1, 1.6; n = 8). 2. The effect of 2,4-dinitrophenol was a marked reduction in the tubular uptake of L-DOPA, with an IC50 value of 12.1 (4.0, 36.9) microM. Cocaine produced a slight (P = 0.08) decrease (22% reduction at 50 microM) in the tubular uptake of L-DOPA. Corticosterone produced a considerable inhibitory effect on the uptake of L-DOPA with an IC50 value of 11.0 (3.6, 33.5) microM. The maximal inhibitory effect of probenecid was a 24% decrease in the uptake of L-DOPA; however, the more selective organic anion transport inhibitor 4,4'-diisothiocyantostilbene-2,2'-disulphonic acid (DIDS) was, found not to affect the tubular uptake of L-DOPA. The organic cation transport inhibitor, cyanine 863, was found to produce a marked decrease in the uptake of L-DOPA (IC50 = 2.02 [1.07, 3.79]). The cyanine derivatives 1,1'-diethyl-2,2'-cyanine (decynium 22) and 1,1'-diethyl-2,4'-cyanine (decynium 24) also potently inhibited L-DOPA uptake with IC50 values (in microM) of 0.63 (0.39, 1.01) and 0.10 (0.08, 0.13), respectively, both compounds were found to be more potent than cyanine 863. 3. The inhibitory effect of decynium 24 (0.2 microM) on the tubular uptake of L-DOPA was dependent on the pH of the incubation medium; at pH = 6.5 the accumulation of L-DOPA was reduced up to 37 +/- 2% of control values, whereas at pH = 7.4 and pH = 8.2 the accumulation of L-DOPA was reduced by 56 +/- 1% and 60 +/- 6%, respectively. Cyanine 863 (2 microM), decynium 22 (1 microM) and decynium 24 (0.2 microM) were found to decrease the Vmax values for the saturable component of L-DOPA uptake without changes in Km values. 4. It is concluded that the tubular uptake of L-DOPA might be promoted through a mechanism which is dependent on the activity of the organic cation transport system.

1. The present work was designed to examine the interference of L-3,4-dihydroxyphenylalanine (L-DOPA) on the cell inward transport of L-5-hydroxytryptophan (L-5-HTP) and on its decarboxylation by aromatic L-amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2. The accumulation of both L-5-HTP and L-DOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component L-5-HTP and L-DOPA uptake in renal tubules were as follows: L-5-HTP, Vmax = 24.9 +/- 4.5 nmol mg-1 protein h-1 and Km = 121 (95% confidence limits: 75, 193) microM (n = 5); L-DOPA, Vmax = 58.0 +/- 4.3 nmol mg-1 protein h-1 and Km = 135 (97, 188) microM (n = 5). When the saturation curve of L-5-HTP tubular uptake was performed in the presence of L-DOPA (250 microM), the maximal rate of accumulation of L-5-HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 +/- 1.7 nmol mg-1 protein h-1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] microM, n = 4). 3. L-DOPA (50 to 2000 microM) was found to produce a concentration-dependent decrease (38% to 91% reduction) in the tubular uptake of 5-HTP; the Ki value (in microM) of L-DOPA for inhibition of L-5-HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4. At the highest concentration tested the organic anion inhibitor, probenecid (10 microM) produced no significant (P = 0.09) changes in L-5-HTP and L-DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1-ethyl-2-[1,4-dimethyl-2-phenyl-6-pyrimidinylidene)methyl]-quino linium) produced a potent inhibitory effect on the tubular uptake of L-5-HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L-DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1-diethyl-2,4-cyanine (decynium 24) and 1,1-diethyl-2,2-cyanine (decynium 22) potently inhibited the tubular uptake of both L-5-HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4-6, respectively) and L-DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5. The Vmax and Km values for AAAD using L-DOPA as the substrate (Vmax = 479.9 +/- 74.0 nmol mg-1 protein h-1; Km = 2380 [1630, 3476] microM; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L-5-HTP (Vmax = 81.4 +/- 5.2 nmol mg-1 protein h-1, Km = 97 [87, 107] microM, n = 10). The addition of 5 mM L-DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L-5-HTP (Vmax = 56.7 +/- 3.1 nmol mg-1 protein h-1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] microM, n = 10). 6. The results presented suggest that L-5-HTP and L-DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L-DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L-5-HTP. The decrease in the formation of 5-HT as induced by L-DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.

1. Administration of cyclosporine A (CsA; 50 mg kg-1 day-1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5-6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle-treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2. No significant difference was observed between the Vmax and Km values of renal aromatic L-amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3. Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4. Maximal catechol-O-methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle-treated rats; Km (22.3 +/- 1.5 microM) values for COMT did not differ between the three groups of rats. 5. The accumulation of newly-formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA- and vehicle-treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle-treated animals. 6. The Vmax for the saturable component of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle-treated animals. Km in CsA- and vehicle-treated rats did not differ. 7. The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L-DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA-treated animals to synthesize dopamine, when exogenous L-DOPA is provided, results from an enhanced activity of the uptake process of L-DOPA in renal tubular cells.

1. The present paper reports changes in the urinary excretion of dopamine, 5-hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long-term administration of NG-nitro-L-arginine methyl ester (L-NAME). Aromatic L-amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly-formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined. 2. Twenty four hours after exposure to L-NAME, both systolic (SBP) and diastolic (DBP) blood pressure were increased by 20 mmHg; heart rate was slightly decreased. During the next 13 days both SBP and DBP increased progressively reaching 170 +/- 3 and 116 +/- 3 mmHg, respectively. 3. Baseline urinary excretion of L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) during the 4 day period of stabilization averaged 4.4 +/- 0.5, 13.8 +/- 0.3, 37.4 +/- 0.8, 180.0 +/- 2.7 and 206.1 +/- 6.7 nmol day-1, respectively. The urinary excretion of L-DOPA, dopamine and DOPAC, but not that of 3-MT and HVA, were increased from day 6-8 of L-NAME administration onwards (L-DOPA, up to 13.4 +/- 2.1; dopamine, up to 23.0 +/- 1.6; DOPAC, up to 62.8 +/- 3.7 nmol day-1). Baseline daily urinary excretion of 5-hydroxytryptamine and 5-hydroxyindolacetic acid (5-HIAA) averaged 73.5 +/- 1.1 and 241.7 +/- 5.4 nmol day-1, respectively. During the first week of L-NAME administration, the urinary excretion of both 5-hydroxytryptamine and 5-HIAA did not change significantly; however, as was found with dopamine and DOPAC, changes in the urinary excretion of 5-hydroxytryptamine were evident during the second week of L-NAME administration. 4. In experiments performed on homogenates of isolated renal tubules, the decarboxylation of L-DOPA to dopamine was dependent on the concentration of L-DOPA used (10 to 5000 microM) and saturable at 1000 microM. AAAD activity as determined in homogenates (Vmax, in nmol mg-1 protein h-1; Km in microM) was significantly (P < 0.01) higher in rats given L-NAME for 14 days (Vmax = 25 +/- 2; Km = 72 +/- 10) than in control rats (Vmax = 14 +/- 1; Km = 63 +/- 7), rats given L-NAME for 7 days (Vmax = 15 +/- 1; Km = 69 +/- 5) and rats given L-NAME plus L-arginine (Vmax = 13 +/- 1; Km = 60 +/- 3) for 14 days. 5. A considerable amount of the total dopamine formed from added L-DOPA in kidney slices escaped into the incubation medium. The application of the Michaelis-Menten equation to the net transport of newly-formed dopamine allowed the identification of a saturable (carrier-mediated transfer) and a non-saturable component (diffusion). No significant differences in the diffusional rate of transfer(0.14 +/- 0.02 micro mol-1) were observed between the four experimental groups. However, the saturable outward transfer of dopamine (Vmax, in micromol mg-1 protein h-1; Km in microM) was higher in control animals(Vmax= 2.3 +/- 0.2; Km = 568 +/- 67) than that in rats treated with L-NAME for 14 days (Vmax = 0.8 +/- 0.02;Km = 241 +/- 21), but similar to that observed in rats receiving L-NAME plus L-arginine (Vmax= 2.4+/- 0.2; Km= 618 +/- 61); the saturable dopamine outward rate of transfer in rats given L-NAME for 7days (Vmax = 3.9 +/- 0.2; Km = 1006 +/- 32) was higher than in controls.6. In conclusion, the present studies show that the hypertensive response resulting from the long-term administration of L-NAME is accompanied by an increased urinary excretion of dopamine and 5-hydroxytryptamine, which appears to follow an enhanced activity of renal AAAD. The observation that the increased AAAD activity can be reversed by the administration of L-arginine to L-NAME treated rats favours the view that the adaptational response which results in an enhanced AAAD activity probably involves a decrease in the generation of nitric oxide.

1. The influence of pargyline and of selective inhibitors of type A and B monoamine oxidase (MAO), Ro 41-1049 and Ro 19-6327 respectively, on the outflow of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in slices of rat renal cortex loaded with exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) was examined. Dopamine and DOPAC in the tissues and in the effluent were assayed by means of h.p.l.c. with electrochemical detection. 2. The levels of newly-formed dopamine and DOPAC in the perifusate decreased progressively with time. In control conditions, DOPAC/dopamine ratios in the perifusate were 3 to 5 fold those in the tissue and were found to increase progressively with time. The addition of pargyline (100 microM), produced a marked decrease in the outflow levels of DOPAC (45 to 54% reduction) and significantly increased the levels of dopamine in the effluent (102 to 158% increase); DOPAC/dopamine ratios in the perifusate remained stable throughout the perifusion and were similar to those found in the tissues. The addition of the MAO-A inhibitor Ro 41-1049 to the perifusion fluid also significantly decreased DOPAC outflow (41% to 54% reduction) and increased dopamine outflow (19% to 80% increase). In the presence of Ro 41-1049 DOPAC/dopamine ratios in the perifusate were lower (P < 0.01) than in controls; in contrast with the effect of pargyline, this ratio was found to increase (P < 0.01) throughout the perifusion period. Ro 19-6327 did not reduce the outflow of DOPAC, but significantly increased (by 40-60%) that of dopamine. In the presence of Ro 19-6237, the proportion of DOPAC to dopamine in the perifusate was similar to that of controls and significantly increased throughout the perifusion; however, this increase was less than that observed in the control group.3. When benserazide (50 microM) was added to the perifusion fluid, the levels of both dopamine and DOPAC in the effluent were similar to those observed in the absence of benserazide. However, in the presence of benserazide, DOPAC/dopamine ratios in the perifusate did not increase with time. In conditions of decarboxylase inhibition, the effects of pargyline, Ro 41-1049 and Ro 19-6327 on dopamine and DOPAC outflow were less pronounced than in experiments conducted in the absence of benserazide.4. In conclusion, the results presented here show that the fraction of newly-formed dopamine which leaves the compartment where the synthesis has occurred is a constant source for deamination into DOPAC. The results provide evidence favouring the view that MAO-A is the main form of the enzyme involved in this process; however, the data described here suggest that dopamine would also have access to MAO-B.

1. The present study has determined the kinetics of the uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3-O-methyl-L-DOPA (3-OMDOPA) in rat renal tubules and examined the effect of 3-OMDOPA on the inward transport of L-DOPA and on its conversion into dopamine in kidney homogenates. 2. The accumulation of both L-DOPA and 3-OMDOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA and 3-OMDOPA uptake in renal tubules were as follows: L-DOPA, Vmax = 11.1 nmol mg-1 protein h-1 and Km = 216 microM (n = 6); 3-OMDOPA, Vmax = 8.1 nmol mg-1 protein h-1 and Km = 231 microM (n = 5). The diffusion constant of the non-saturable component for the accumulation of L-DOPA and 3-OMDOPA was 0.0010 and 0.0014 mumol-1, respectively. 3. 3-OMDOPA (100 to 2000 microM) was found to produce a concentration-dependent decrease (29% to 81% reduction) of the saturable component of the tubular uptake of L-DOPA; the Ki value of 3-OMDOPA for inhibition of L-DOPA uptake was found to be 181 microM (n = 5). The accumulation of L-DOPA obtained in experiments conducted at 4 degrees C was not affected by 3-OMDOPA. 4. In experiments conducted in kidney homogenates only L-DOPA (10 to 5000 microM) was found to be decarboxylated. The Vmax and Km values for aromatic L-amino acid decarboxylase determined in the absence of 3-OMDOPA (Vmax = 14.1 nmol mg-1 protein h-1; Km =62 MicroM) were not significantly different from those observed when the decarboxylation of L-DOPA was carried out in the presence of 1000 MicroM 3-OMDOPA (Vmax = 15.7 nmol mg-1 protein h-1; Km = 68 MicroM).5. It is concluded that the tubular uptake of both L-DOPA and 3-OMDOPA occur through nonsaturable and saturable mechanisms; only the saturable tubular uptake of L-DOPA was found to be inhibited by 3-OMDOPA. It is further shown that 3-OMDOPA neither undergoes decarboxylation into 3-MT nor affects the decarboxylation of L-DOPA.

1. Effects of two new inhibitors of catechol O-methylation (CGP 28014 and entacapone; 30 mg kg-1, i.p.) were compared by means of brain microdialysis in rats treated with L-3,4-dihydroxyphenylalanine (L-dopa)/carbidopa (50/50 mg kg-1, i.p., respectively) or saline. 2. In saline-treated rats, CGP 28014 maximally (max) increased striatal dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) effluxes by 41% and 49%, respectively, whereas homovanillic acid (HVA) levels were decreased by 71%. 3. In the presence of L-dopa/carbidopa, a peripherally active inhibitor of catechol O-methyltransferase (COMT) entacapone had a short-lasting increasing effect on L-dopa efflux. Compared to the effects of L-dopa/carbidopa alone 3-O-methyldopa (3-OMD) levels were effectively reduced (max 79%) by entacapone, but not by CGP 28014. 4. Entacapone, in contrast to CGP 28014, increased striatal dopamine efflux (max 492% of that after L-dopa/carbidopa alone). Also DOPAC levels were increased by entacapone (255% at 180 min), but not significantly by CGP 28014 (159% at 180 min). 5. Both compounds initially decreased HVA efflux. The effect of CGP 18014 was longer-lasting. By the end of the measurement, entacapone even increased HVA levels (max 259%). 6. Our results demonstrate that entacapone is a peripheral COMT inhibitor and support the view that CGP 18014 is mainly a centrally acting inhibitor of O-methylation.

1. The present study has examined the influence of alpha-human atrial natriuretic peptide (alpha-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2. alpha-hANP (3.3 and 330 nM) was found to produce a marked reduction (63-78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 microM L-DOPA. alpha-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45-66% reduction) and DOPAC (38-61% reduction) in experiments in which increasing concentrations (1-100 microM) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10 microM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3. In kidney homogenates, alpha-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4. Both alpha-hANP (330 nM) and zaprinast (10 microM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used.5. The results obtained suggest that the inhibitory effect of alpha-hANP on the renal synthesis of dopamine is dependent on the activation of a membrane-operated mechanism, coupled to the enzyme guanylate cyclase, controlling the entry of L-DOPA into the cells.

1. The present study has examined the influence of sodium chloride (0-160 mM) and ouabain (100 and 500 microM), an inhibitor of the enzyme Na(+)-K+ ATPase, on the synthesis of dopamine in slices of rat renal cortex loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). The deamination of newly-formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also examined. The assay of L-DOPA, dopamine and DOPAC in kidney slices was performed by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. The accumulation of newly-formed dopamine and DOPAC in kidney slices loaded with L-DOPA (50 and 100 microM) was found to be dependent on the concentration of NaCl in the medium. A similar picture could be observed for DOPAC. The fractional rate of accumulation (k; mM NaCl-1) was at 50 and 100 microM L-DOPA, respectively, 0.00305 +/- 0.00036 and 0.00328 +/- 0.00029 for dopamine and 0.00672 +/- 0.00072 and 0.00641 +/- 0.00069 for DOPAC. The sodium-dependent formation of dopamine was completely abolished when the experiments were performed in the absence of oxygen. 3. In experiments performed in the presence of 120 mM NaCl, but not in conditions of low sodium (20 mM NaCl in the medium), ouabain (100 and 500 microM) was found to inhibit the accumulation of newly-formed dopamine and DOPAC (14-57% reduction; P less than 0.05); this effect was more marked at 50 and 100 microM L-DOPA. When the experiments were performed in the absence of oxygen the renal production of dopamine and DOPAC became markedly decreased (respectively, 40% and 77% reduction; P<0.05) and ouabain (100 and 500 microM) was no longer able to reduce the accumulation of both newly-formed dopamine and DOPAC.4. The formation of dopamine and its deamination to DOPAC in kidney homogenates closely depended on the concentration of L-DOPA added to the medium; ouabain (100, 500 and 1000 microM) was found to affect neither the formation of dopamine nor its deamination to DOPAC when homogenates were used instead of kidney slices.5. In conclusion, the results presented here show that the formation of dopamine in kidney slices loaded with L-DOPA is sodium-dependent and ouabain-sensitive suggesting the involvement of a co-transport system of sodium and DOPA into the tubular epithelial cell.

1. The present study has examined the formation of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in slices of the rat renal cortex and the renal medulla loaded with exogenous L-beta-3,4-dihydroxyphenylalanine (L-DOPA). The effects of pargyline and of two selective inhibitors of monoamine oxidase (MAO) types A and B, respectively Ro 41-1049 and Ro 19-6327, on the deamination of newly-synthesized dopamine in kidney slices incubated with exogenous L-DOPA were also tested. The assay of L-DOPA, dopamine, noradrenaline and DOPAC was performed by means of h.p.l.c. with electrochemical detection. 2. Incubation of renal slices with exogenous L-DOPA resulted in a concentration-dependent accumulation of dopamine and DOPAC; the tissue levels of newly-formed dopamine and DOPAC in slices of the renal medulla were 6-8% of those in cortical slices. 3. Pargyline (0.1 mM) produced a marked decrease (84% reduction) in the formation of DOPAC in kidney slices loaded with 1.0 mM L-DOPA; this effect was accompanied by a 17% increase in the accumulation of dopamine. Similar effects were obtained at higher concentrations of pargyline (0.5 and 1.0 mM). At 5.0 and 10.0 mM pargyline, a marked decrease (46 and 76% reduction) in the accumulation of newly-formed dopamine was observed. 4. The accumulation of dopamine and DOPAC was found to be time-dependent in experiments in which tissues were incubated with 5 and 10 microM L-DOPA for 5, 10, 20 and 30 min. Pargyline (0.1 mM) produced an increase in the accumulation of dopamine at all incubation periods and decreased the formation of DOPAC. 6. It is concluded that deamination of newly-formed dopamine in kidney slices loaded with L-DOPA constitutes an important mechanism of amine inactivation. The results presented also suggest that most of the MAO, located inside the compartment where the synthesis of dopamine occurs, is of the A type.

Catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activities were determined in human gastrointestinal samples. L-dopa was used as the substrate and the reaction products 3-O-methyldopa (3OMD) and dopamine were separated by reversed phase HPLC and detected by electrochemical or UV detection. COMT activities varied between 40-350 pmol/mg/min and AADC activities between 100-3300 pmol/mg/min in different parts of the gastrointestinal tract. COMT inhibitors nitecapone (OR-462) and OR-611 effectively inhibited human gastrointestinal COMT activity in vitro, the IC50 values ranging from 10-20 nM and 5-75 nM, respectively. In vitro carbidopa inhibited AADC slightly more effectively than benserazide.

1. The effects of levodopa alone (50 mg kg-1) and levodopa (10 mg kg-1) plus benserazide (50 mg kg-1) were tested on the levels of dopa, dopamine, 3-methoxytyrosine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), measured by h.p.l.c. with electrochemical detection, in samples of plasma, CSF, urine, striatum and hypothalamus of rats taken 30 min after injection. Levodopa plus benserazide produced significantly higher levels of dopa in plasma and brain than levodopa alone and reduced the peripheral synthesis and metabolism of dopamine. 2. When given chronically over 6 weeks the advantages of adding benserazide (50 mg kg-1 day-1) to levodopa (40 mg kg-1 day-1) were less marked and although more dopamine was present in the striatum than with levodopa given alone (200 mg kg-1 day-1) there was no evidence of any increase in its metabolites (HVA and DOPAC) and therefore of its turnover and utilisation. 3. The most striking effect of chronic treatment with levodopa plus benserazide was the appearance of large quantities of 3-MT in plasma, CSF and brain. 4. When levodopa alone, or levodopa plus benserazide, was given as an acute challenge to animals receiving the same treatment chronically, it was found that levodopa alone still produced increases in striatal dopamine, DOPAC and HVA in those animals dosed chronically on levodopa, but it was less effective in this respect when given with benserazide to the animals dosed with levodopa plus benserazide. 5. It is concluded that this difference in levodopa distribution may depend on the persistence in benserazide-treated animals of 3-MT, which has a long half-life and may compete with dopa for transport into the blood and brain. 6. The implication of these findings to the treatment of Parkinsonism is discussed.

In 14 patients with Parkinson's disease on long-term therapy the erythrocyte catechol-O-methyltransferase activity was found to correlate with the average plasma concentration ratio of 3-O-methyldopa to levodopa and with the fasting plasma concentration ratio of 3-O methyldopa to levodopa. Patients with the higher erythrocyte catechol-O-methyltransferase activities were those with less favorable clinical responses to levodopa. Since erythrocyte catechol-O-methyltransferase activity may reflect the activity of that enzyme in the major metabolizing tissues, catechol-O-methyltransferase activity would seem to be a significant determinant of response to levodopa.

The biosynthetic pathway melanin is present in many melanomas. Previous investigations have suggested that pharmacologic levels of intermediates in this pathway (L-dopa, dopamine, and their analogues) may inhibit macromolecular synthesis in some tissue culture melanoma cell lines and prolong survival in tumor-bearing mice. Recently, a potent antidopamine drug (pimozide) has been developed. This study was designed to investigate the effects of these drugs on murine melanomas and to correlate effects on macromolecular synthesis with competitive dopamine binding activity (receptors) and melanin synthesis. Three murine melanomas (F1, F10, B16) were studied. The amelanotic B16 cell line showed no inhibition by dopa, dopamine, or pimozide when assayed for 14C-leu or 3H-TdR incorporation. Using a competitive binding assay, only low levels of dopamine binding were present. The very melanotic F1 cell line showed no inhibition by dopa or dopamine, but pimozide inhibited 14C-leu and 3H-TdR incorporation in a dose-response fashion; 50% inhibition was noted at 10(-9) M concentration with no loss in cell viability as tested by trypan blue exclusion or cell counting. Competitive dopamine binding was present (19 pmoles per g of wet tissue) with a Kd of 0.2 nM, figures approximating those seen in normal dog caudate nucleus controls. The F10 line, with melanin production between the B16 and F1, was intermediate in terms of inhibition of 14C-leu and 3H-TdR incorporation and dopamine binding. Purified melanin did not bind and, thus, does not appear to explain these binding results. These data suggest that pimozide, a drug which is a potent dopamine antagonist, may inhibit 14C-leu and 3H-TdR incorporation in murine melanoma cells and that competitive dopamine binding (receptor) appears to correlate with this inhibition. This drug and the dopamine binding assay may be useful in the study of human melanoma.

Criteria for distinguishing dopamine autoreceptor agonism from other mechanisms of inhibiting locomotion were examined, together with the relationship between inhibition of locomotion and dopamine synthesis. ED50 potencies to inhibit locomotion of mice were established for drugs from a number of categories. Spiperone 0.02 mg kg-1 significantly (P less than 0.05) reversed inhibition of locomotion by known dopamine agonists but not that by the other types of drug. Idazoxan antagonized inhibition of locomotion due to alpha 2-agonists but not dopamine agonists. RU 24926 (N-propyl-N,N-di[2-(3-hydroxyphenyl)ethyl]amine) was antagonized by both spiperone and idazoxan. Only for dopamine agonists was there good correlation (r = 0.97) between potencies to inhibit locomotion in mice and L-dihydroxyphenylalanine (L-DOPA) accumulation in the nucleus accumbens of rats treated with gamma-butyrolactone and 3-hydroxybenzylhydrazine. The specific dopamine D1-agonist, SK&F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine), was inactive in both tests at doses up to 10 mg kg-1. The mixed dopamine agonist/antagonist, (-)-3-(3-hydroxyphenyl)-N-propylpiperidine, commonly known as (-)-3-PPP, acted as a dopamine agonist in both tests but inhibited locomotion more potently than L-DOPA accumulation. The inhibitory effects of dopamine agonists on locomotion were not prevented by alpha-methyl-p-tyrosine pretreatment. The data suggest that spiperone-reversible inhibition of locomotion in mice is a good criterion for dopamine autoreceptor agonists. The receptors involved are affected by low doses of both dopamine agonists and antagonists and seem similar to those involved in the autoreceptor mediated inhibition of dopamine synthesis. However, inhibition of locomotion is not due simply to suppression of dopamine release brought about as a secondary consequence of effects on synthesis; a separate mechanism for inhibiting dopamine release is probably involved.

Melanomas from 20 patients were evaluated for estrogen and progesterone receptors. No restriction as to patient's age, sex, race, or menstrual status was made. Fifteen of the tumors were melanin producing. Of the 20 tumors examined, seven contained more than 3 fm/mg protein of specifically inhibitable estrogen binding. None of the tumors reported here demonstrated either a 4S or 8S binding protein as shown by sucrose density gradient analysis. All tumors in which estradiol binding was observed were melanin producing, whereas none of the amelanotic melanomas (five tumors) bound this steroid. Purified tyrosinase appeared to mimic the estrogen binding detected in melanoma cytosols, as demonstrated with the dextran-coated charcoal technique. Although the binding of estradiol to tyrosinase was inhibited by DOPA, the binding of estradiol to estrogen receptor preparations was not. These studies represent an extensive of previous studies of sex steroid binding in melanomas and suggest that gradient analysis and DOPA inhibition studies should be included in the evaluation of the estrogen binding phenomenon in human melanoma.

The microinjection of macromolecules into living somatic cells has been used to assess the biological activity of a wide variety of macromolecules. This article describes and evaluates the methodology of microinjection with micropipettes and reviews information on the expression of globin mRNAs injected into normal and transformed cultured cells. A brief account is also given of other mRNAs that have been successfully translated in cultured cells.

Two patients in hepatic coma were treated with L-dopa. The first patient showed clear clinical improvement, but the second patient did not. Analyses of urinary metabolites indicated that L-dopa was not absorbed by the second patient. There was evidence that L-dopa had the following beneficial effects in the first patient: (1) increased production of urine, which could have been accompanied by increased excretion of toxins; (2) displacement of tyramine from transmitter sites (because increased excretion of p-hydroxyphenylacetic acid, a major metabolite of tyramine, occurred during L-dopa treatment in patient 1); (3) replenishment of dopamine, and to a much lesser extent, norepinephrine, at central or peripheral neuroeffector junctions; and (4) scavenging of methyl groups by L-dopa, because ratio of methylated amines to catecholamines was higher than normal in both comatose patients before L-dopa treatment, and this ratio decreased during L-dopa treatment in patient 1.

The permeability of L-dopa across the blood-brain barrier was studied in twelve patients by means of the indicator dilution method. The extraction of [1-14C]L-dopa and [3-14C]L-dopa was 11.7% and 10.4%, respectively. After pretreatment with carbidopa the extraction was unchanged (13.5% and 11.6%). The permeability coefficient (P) was calculated to 0.9 x 10(-5) cm s-1.