HR/MS-based lipidome analysis of rat brain modulated by tolcapone.

Lipids play key roles in the body, influencing cellular regulation, function, and signalling. Tolcapone, a potent catechol-O-methyltransferase (COMT) inhibitor described to enhance cognitive performance in healthy subjects, was previously shown to impact fatty acid ß-oxidation and oxidative phosphorylation. However, its impact on the brain lipidome remains unexplored. Hence, this study aimed to assess how tolcapone affects the lipidome of the rat pre-frontal cortex (PFC), a region of the brain highly relevant to tolcapone therapeutic effect, while evaluating its influence on operant behaviour. Tolcapone at 20 mg/kg was chronically administered to Wistar rats during a behavioural task and an untargeted liquid chromatography high-resolution mass spectrometry (LC-HR/MS) approach was employed to profile lipid species. The untargeted analysis identified 7227 features, of which only 33% underwent statistical analysis following data pre-processing. The results revealed an improved cognitive performance and a lipidome remodelling promoted by tolcapone. The lipidomic analysis showed 32 differentially expressed lipid species in tolcapone-treated animals (FC ≥ 1.2, p-value ≤ 0.1), and among these several triacylglycerols, cardiolipins and N-acylethanolamine (NAE 16:2) were found upregulated whereas fatty acids, hexosylceramides, and several phospholipids including phosphatidylcholines and phosphatidylethanolamines were downregulated. These preliminary findings shed light on tolcapone impact on lipid pathways within the brain. Although tolcapone improved cognitive performance and literature suggests the significance of lipids in cognition, this study did not conclusively establish that lipids directly drove or contributed to this outcome. Nevertheless, it underscores the importance of lipid modulation and encourages further exploration of tolcapone-associated mechanisms in the central nervous system (CNS).

Predictors of psychotherapy dropout in patients with borderline personality disorder: A systematic review.

INTRODUCTION: Borderline personality disorder (BPD) is a highly debilitating psychiatric condition. Despite the expansion of new BPD specific forms of psychotherapy in the last few decades, high dropout rates have been reported in these treatments. Treatment discontinuation is associated with poor patient outcomes, inefficient resource utilization and the demoralization of healthcare providers. METHODS: In order to identify predictors of psychotherapy dropout among patients with BPD, a systematic search of Medline, the Cochrane Library, PsycInfo and PsycArticles was conducted. Studies included were randomized-controlled trials in which patients diagnosed with BPD were exposed to a therapeutic intervention consisted of an evidence-based psychotherapy. The quality of evidence in the studies was assessed through the use of revised Cochrane risk of bias tool. RESULTS: Six articles, incorporating four types of psychotherapy programmes, were included. Overall, the studies present low risk of attrition and reporting bias and unclear risk of selection, performance and detection bias. Patients with weaker therapeutic alliance scores and higher hostility presented with higher dropout rates. In contrast, better mindfulness skills and greater performance in specific neuropsychological domains, such as memory and executive control, were identified as predictive of lower risk of dropout. Sociodemographic variables and treatment history did not influence treatment retention. CONCLUSIONS: Factors that influence discontinuation should be taken into consideration in future treatment programmes, in an effort to optimize retention. Qualitative assessments of patients' reasons for dropping out may also help guide adjustments.

Catechol-O-methyltransferase activity in erythrocytes from patients with eating disorders.

PURPOSE: Abnormal feeding has been linked to disruptions in brain dopaminergic activity and recent studies have assessed the role of catechol-O-methyltransferase (COMT) in eating disorders. This is the first study to quantify the soluble catechol-O-methyltransferase (S-COMT) activity in erythrocytes from patients with anorexia nervosa (AN), bulimia nervosa (BN) and binge-eating disorder (BED) and the first study at all to evaluate the COMT on patients with BED. METHODS: Forty blood samples from patients with AN, BN and BED and healthy controls were drawn to evaluate S-COMT activity in erythrocytes by high-performance liquid chromatography and mass spectrometry. Since several patients were being treated with fluoxetine 20 mg, they were included in a different group (BN MED and BED MED). Liver homogenates from rats were used to evaluate baseline S-COMT activity in the presence of fluoxetine by the same in vitro procedures and assays. RESULTS: Erythrocyte S-COMT activity (pmol/mg prt/h) was significantly increased in patients with BN and BED (41.3 ± 6.8 and 41.4 ± 14, respectively) compared to control group (25.3 ± 9.7). In fluoxetine-treated patients with BN, S-COMT activity (15.9 ± 8.8) was decreased compared to the other BN group; however, in BED group, the difference between BED MED and BED was not observed. In patients with AN, no significant difference was found compared to controls. CONCLUSION: Patients with BN and BED presented higher S-COMT activity in erythrocytes, which is in agreement with previous studies on the literature addressing the high-activity COMT allele, Val158, as risk factor for eating disorders. Although in fluoxetine-treated patients with BN the activity of S-COMT was similar to the controls, this is not explained by a direct interaction between fluoxetine and S-COMT as verified in in vitro assays.

Urinary profile of catecholamines and metabolites in Parkinson patients with deep brain stimulation.

BACKGROUND AND PURPOSE: Deep brain stimulation of the subthalamic nucleus (DBS-STN) is thought to continuously alter the activity of STN neurons in Parkinson's disease (PD). A chronic decrease in the levodopa dose with continuous STN stimulation may induce plastic neuronal changes. OBJECTIVE: The objective of this work was to study urinary excretion of catecholamines in patients with PD before and after DBS-STN. METHODS: Twenty-three patients were submitted to DBS-STN, and evaluated before and after surgery with respect to catecholamines and metabolites in 24-h urine measured by high-performance liquid chromatography with electrochemical detection. RESULTS: Of the 23 patients evaluated, a significant decrease of about 60% in the urinary excretion of L-3,4-dihydroxyphenylalanine (L-DOPA; in nmol/mg creatinine/24 h) was observed 1 week after DBS-STN. Moreover, in 17 patients with a follow-up of 8 weeks after surgery, there was a further 50% decrease in urinary L-DOPA levels, dropping to about 75% of the values before surgery. There was also a significant decrease in dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels 1 week after DBS-STN that was no longer present 8 weeks after. A significant increase in the DA/l-DOPA ratio was observed 1 week after surgery, with a further increase 8 weeks after surgery. CONCLUSION: After DBS-STN, the DA/l-DOPA ratio, an indirect measure of DA synthesis, increased. These results show that DBS-STN may improve the efficacy of oral levodopa.

Effect of (-)-Delta(9)-tetrahydrocannabinoid on the hepatic redox state of mice.

(-)-Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered Delta(9)-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: Delta(9)-THC (N = 10), treated with 10 mg/kg body weight Delta(9)-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with Delta(9)-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Delta(9)-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Delta(9)-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Delta(9)-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL.

Effect of (-)- Δ9 -tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered (∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: (∆9-THC (N = 10), treated with 10 mg/kg body weight (∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (∆9-THC: 8 percent; VCtrl: 23 percent increase) and the GSH/oxidized GSH ratio (∆9-THC: 61 percent; VCtrl: 96 percent increase), caused by treatment with the vehicle. ∆9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

Catecholamine synthesis and metabolism in the central nervous system of mice lacking alpha-adrenoceptor subtypes.

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.

Alpha2-adrenoceptor subtypes involved in the regulation of catecholamine release from the adrenal medulla of mice.

BACKGROUND AND PURPOSE: This study was carried out to elucidate which alpha(2)-adrenoceptor subtypes mediated the inhibition of noradrenaline and adrenaline release from the adrenal medulla of mice. EXPERIMENTAL APPROACH: Isolated adrenal medullae from wild-type and alpha(2A), alpha(2B) and alpha(2C)-adrenoceptor knockout (KO) mice were placed in superfusion chambers. Catecholamine overflow was evoked by 1,1-dimethyl-4-phenylpiperazinium (500 microM) in absence or in presence of the alpha(2)-adrenoceptor agonist medetomidine. The effect of medetomidine was tested in presence of the alpha-adrenoceptor antagonists rauwolscine, WB 4101, spiroxatrine, phentolamine and prazosin. KEY RESULTS: In wild-type mice, medetomidine reduced noradrenaline and adrenaline overflow in a concentration-dependent manner (EC(50) in nM: 1.54 and 1.92; E(max) in % of inhibition: 91 and 94, for noradrenaline and adrenaline, respectively). The pK (D) values of the antagonists for noradrenaline overflow did not correlate with pK(D) values at alpha(2A), alpha(2B), or alpha(2C) binding sites. The pK (D) values of the antagonists for adrenaline overflow correlated positively with pK(D) values at alpha(2C) binding sites (opossum kidney cells). The effect of medetomidine (100 nM) on noradrenaline overflow was significantly reduced in all three alpha(2)KO mice (57, 54, 44 % inhibition, for alpha(2A), alpha(2B), and alpha(2C), respectively), whereas the effect of medetomidine on adrenaline overflow was greatly reduced in alpha(2C)KO mice (14 % inhibition). CONCLUSIONS AND IMPLICATIONS: In the adrenal medulla of mice, all three alpha(2)-adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)) play an equal role in the inhibition of noradrenaline overflow, whereas the alpha(2C)-adrenoceptor is the predominant alpha(2)-adrenoceptor subtype involved in the inhibitory mechanism controlling adrenaline overflow.

Renal dopaminergic system activity in the rat remnant kidney.

BACKGROUND: Renal dopamine exerts natriuretic and diuretic effects by activating D1-like receptors. Uninephrectomy results in increased renal dopaminergic activity and dopamine-sensitive enhanced natriuresis. METHODS: The present study evaluated renal adaptations in sodium handling and the role of dopamine in rats submitted to (3/4) nephrectomy: right nephrectomy and excision of both poles of the left kidney ((3/4)nx rats). RESULTS: Two weeks after surgery the absolute urinary levels of dopamine were markedly reduced in (3/4)nx rats whereas the urinary dopamine excretion per % of residual nephrons was significantly increased in the remnant kidney of (3/4)nx rats. The V(max) values for renal aromatic L-amino acid decarboxylase, the enzyme responsible for the synthesis of renal dopamine, were decreased in (3/4)nx rats. Renal catechol-O-methyltransferase activity, the enzyme responsible for the methylation of dopamine, was increased in (3/4)nx rats whereas the renal activities of monoamine oxidases A and B did not differ between (3/4)nx and Sham animals. Volume expansion (5% body weight) resulted in similar natriuretic responses in (3/4)nx and Sham rats. During D1 antagonist administration (Sch-23390, 30 microg x h(-1) x kg(-1)) the natriuretic response to volume expansion was reduced in (3/4)nx rats more pronouncedly than in Sham animals. CONCLUSION: The decrease in absolute renal dopamine output in (3/4)nx rats is related with reduced renal synthesis and enhanced O-methylation of the amine. However, this is accompanied in (3/4)nx rats by increased renal dopamine excretion per residual nephrons and dopamine-sensitive enhanced natriuresis.

In vitro and in vivo effect of fluoxetine on the permeability of 3H-serotonin across rat intestine.

The aim of this work was to characterize the mucosal-to-serosal (apical to basolateral; AP-BL) and serosal-to-mucosal (basolateral to apical; BL-AP) transport of serotonin (5-HT) across rat jejunum, ileum, and colon, and to determine the influence of serotonin neuronal transporter inhibitors on this transport. The AP-BL apparent permeability (Papp) of 3H-5-HT increased in the order colon = jejunum < ileum, and the BL-AP Papp of 3H-5-HT increased in the order colon < jejunum = ileum. In vitro, neither fluoxetine (0.02 or 0.2 micromol/L) nor desipramine (0.4 or 4 micromol/L) had a significant effect upon the AP-BL or BL-AP Papp of 3H-5-HT in any of the intestinal regions. However, fluoxetine (0.2 micromol/L) decreased the accumulation of 3H-5-HT in the ileum (to 65% of control) in the BL-AP experiments. In vivo, chronic fluoxetine (10 mg/kg daily administered orally for 15 days), as assessed in the ileum, significantly increased (to +/-180% of control levels) the BL-AP Papp of 3H-5-HT and tended to increase the AP-BL Papp of 3H-5-HT. In conclusion, the increase in the Papp of 3H-5-HT after chronic administration of fluoxetine suggests that this treatment is able to increase the extracellular concentration of 3H-5-HT at the intestinal level.

Differences in the renal dopaminergic system activity between Wistar rats from two suppliers.

AIM: Dopamine of renal origin reduces tubular sodium reabsorption and controls blood pressure. The present study evaluated renal dopaminergic activity and its response to uninephrectomy in Wistar Han rats from two suppliers, Harlan (W-H) and Charles River (W-CR). RESULTS: After uninephrectomy, the fractional excretion of sodium (FENa+) increased in both W-CR and W-H rats (W-CR: from 0.17 +/- 0.01 to 0.27 +/- 0.02%; W-H: from 0.39 +/- 0.04 to 0.54 +/- 0.04%, P < 0.05); however, in W-CR rats the FENa+ was lower than in W-H rats in both Sham and uninephrectomized (Unx) animals (P < 0.05). Systolic blood pressure in Unx W-CR rats was higher than in Unx W-H animals (131 +/- 3 vs. 122 +/- 2 mmHg, P < 0.05). Uninephrectomy was accompanied in W-H rats by increases in urinary levels (nmol g kidney(-1) day(-1)) of dopamine (10.3 +/- 0.5 vs. 8.3 +/- 0.7, P < 0.05) and 3,4-dihydroxyphenylacetic acid (DOPAC) (30.5 +/- 3.7 vs. 21.3 +/- 1.4, P < 0.05) and increases (P < 0.05) in maximal velocity values (Vmax in nmol mg prot(-1) 15 min(-1), 325 +/- 12 vs. 265 +/- 3) for renal aromatic L-amino acid decarboxylase (AADC), the enzyme responsible for the synthesis of renal dopamine. By contrast, in W-CR rats uninephrectomy did not change either the urinary levels of dopamine (7.1 +/- 0.5 vs. 7.6 +/- 0.7) and DOPAC (25.0 +/- 1.9 vs. 24.8 +/- 4.1) or AADC activity (Vmax 199 +/- 3 vs. 193 +/- 9). The Vmax values for renal AADC in W-CR rats were lower than those found in corresponding W-H animals. CONCLUSION: Wistar rats from different suppliers represent an important source of variability in the renal dopaminergic system activity. This may contribute to differences in sodium balance and blood pressure control in response to uninephrectomy.

Impaired synthesis or cellular storage of norepinephrine, dopamine, and 5-hydroxytryptamine in human inflammatory bowel disease.

The present study was aimed at evaluating the extent of dysfunction of the enteroendocrine and enteric nervous system, as indicated by changes in tissue levels of monoamines (dopamine, DA; norepinephrine, NE; 5-hydroxytryptamine, 5-HT) and their precursors and metabolites in the colonic mucosa of patients afflicted with ulcerative colitis (UC, N = 21) and Crohn's disease (CD, N = 22). In CD, but not in UC, NE tissue levels in both the noninflamed and inflamed colonic mucosa were markedly lower than in control subjects (N = 16). In the inflamed mucosa of CD and in UC patients levels of L-DOPA were twice those in controls. DA levels in the inflamed mucosa of CD and UC patients were markedly lower than in controls. This resulted in significant reductions in DA/L-DOPA tissue ratios, a rough measure of L-amino acid decarboxylase activity. 5-HT levels in the inflamed mucosa of CD and UC patients were markedly lower than in controls. In conclusion, intestinal cellular structures responsible for the synthesis and storage of DA, NE, and 5-HT may have been affected by the associated inflammatory process in both CD and UC.

Comparative study on sodium transport and Na+,K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine.

The present study reports on the effects of dopamine on sodium transepithelial transport and Na+,K+-ATPase activity in Caco-2 cells, a human epithelial intestinal cell line which undergoes enterocyte differentiation in culture, and jejunal epithelial cells from 20 day old Wistar rats. Addition of amphotericin B to the mucosal side stimulated Isc in a concentration dependent manner (Caco-2 cells, EC50=0.9 [0.5, 1.7] microM; rat jejunum, EC50=7.4 [0.8; 70.1] microM). The presence of 1 microM dopamine did not change the effect of amphotericin B in Caco-2 cells, but produced a significant (P<0.05) decrease in the maximal effect of amphotericin B in the rat jejunum. Dopamine (1 microM), added to the serosal side, did not change the Isc profile in Caco-2 cells, but produced a significant increase in the rat jejunum. This effect was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). Basal Na+,K+-ATPase activity (in nmol Pi mg protein(-1) min(-1)) in Caco-2 cells (49.5+/-0.2) was similar to that observed in isolated rat jejunal epithelial cells (52.3+/-3.4). Dopamine (1 microM) significantly (P<0.05) decreased Na+,K+-ATPase activity in rat jejunal epithelial cells, but failed to inhibit Na+,K+-ATPase in Caco-2 cells. This effect of dopamine was antagonized by SKF 83566 (1 microM), but not S-sulpiride (1 microM), and was mimicked by SKF 38393 (10 nM), but not by quinerolane (10 nM). The specific binding of [3H]-Sch 23390 to the rat intestinal mucosa was saturable with an apparent dissociation constant (KD) of 2.4 (0.4; 4.5) nM and maximum receptor density of 259.8+/-32.6 fmol/mg protein. No significant specific binding of [3H]-Sch 23390 was observed in membranes from Caco-2 cells. In conclusion, the results obtained show that D1-like receptor mediated effects of dopamine in the rat jejunum on sodium absorption are absent in Caco-2 cells, most probably because this cell line does not express D1-like dopamine receptors, which ultimately are responsible for the inhibitory effect of the amine upon intestinal Na+,K+-ATPase.

Salt intake and intestinal dopaminergic activity in adult and old Fischer 344 rats.

We have earlier shown that the renal dopaminergic system failed to respond to high salt (HS) intake in old (24-month-old) Fisher 344 rats (Hypertension 1999;34:666-672). In the present study, intestinal Na+,K+-ATPase activity and intestinal dopaminergic tonus were evaluated in adult and old Fischer 344 rats during normal salt (NS) and HS intake. Basal intestinal Na+,K+-ATPase activity (nmol Pi/mg protein/min) in adult rats (142+/-6) was higher than in old Fischer 344 rats (105+/-7). HS intake reduced intestinal Na+,K+-ATPase activity by 20% (P<0.05) in adult, but not in old rats. Dopamine (1 microM) failed to inhibit intestinal Na+,K+-ATPase activity in both adult and old Fischer 344 rats (NS and HS diets). In adult animals, co-incubation of pertussis toxin with dopamine (1 microM) produced a significant inhibitory effect in the intestinal Na+,K+-ATPase activity. L-DOPA and dopamine tissue levels in the intestinal mucosa of adult rats were higher (45+/-9 and 38+/-4 pmol/g) than those in old rats (27+/-9 and 14+/-1 pmol/g). HS diet did not change L-DOPA and DA levels in both adult and old rats. DA/L-DOPA tissue ratios, an indirect measure of dopamine synthesis, were higher in old (1.1+/-0.2) than in adult rats (0.6+/-0.1). Aromatic L-amino acid decarboxylase (AADC) activity in the intestinal mucosa of old rats was higher than in adult rats. HS diet increased the AADC activity in adult rats, but not in old rats. It is concluded that intestinal dopaminergic tonus in old Fisher 344 rats is higher than in adult rats and is accompanied by lower basal intestinal Na+,K+-ATPase activity. In old rats, HS diet failed to alter the intestinal dopaminergic tonus or Na+,K+-ATPase activity, whereas in adult rats increases in AADC activity were accompanied by decreases in Na+,K+-ATPase activity. The association between salt intake, increased dopamine formation and inhibition of Na+,K+-ATPase at the intestinal level was not as straightforward as that described in renal tissues.

Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein.

Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag. The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E. coli, was purified by calmodulin affinity chromatography and gel filtration. Up to 16 mg of pure recombinant enzyme was recovered per liter of culture. Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 [246, 609] microM and 531 [330, 732] microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 [9, 45] microM and 38 [21, 55] microM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 [44, 397] nM and 74 [38, 143] nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1). The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 [75, 597] microM and 439 [168, 711] microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 [19, 101] nM and 61 [33, 111] nM, respectively). This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies.

BIA 3-202, a novel catechol-O-methyltransferase inhibitor, enhances the availability of L-DOPA to the brain and reduces its O-methylation.

1-[3,4-Dihydroxy-5-nitrophenyl]-2-phenyl-ethanone (BIA 3-202) is a new long-acting catechol-O-methyltransferase (COMT) inhibitor with limited access to the brain. The present study evaluated the interference of BIA 3-202 upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in plasma (3-O-methyl-L-DOPA) and brain [3-O-methyl-L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] in rats orally treated with L-DOPA (20 mg/kg) plus benserazide (30 mg/kg). At different time points (1, 3 and 6 h) after the administration of BIA 3-202 (0, 3, 10 and 30 mg/kg) or L-DOPA plus benserazide, rats were sacrificed and the right striatum was quickly dissected out and stored for the assay of L-DOPA, 3-O-methyl-L-DOPA, dopamine and amine metabolites. Levels of L-DOPA, 3-O-methyl-L-DOPA, dopamine, DOPAC and HVA in the striatum in L-DOPA plus benserazide-treated rats were higher than in vehicle-treated rats. However, this increase in striatal L-DOPA, dopamine, DOPAC and HVA was, in a dose- and time-dependent manner, even higher (P<0.05) in rats given BIA 3-202 (3, 10 and 30 mg/kg). This effect was accompanied by a marked decrease in 3-O-methyl-L-DOPA levels in the striatum of L-DOPA plus benserazide-treated rats. Increases in levels of L-DOPA and decreases in 3-O-methyl-L-DOPA levels in plasma also accompanied the administration of BIA 3-202. BIA 3-202 did not significantly affect levels of DOPAC and HVA in the striatum in vehicle-treated rats. It is concluded that administration of BIA 3-202 enhances the availability of L-DOPA to the brain by reducing its O-methylation in the periphery, which may prove beneficial in parkinsonian patients treated with L-DOPA plus an aromatic amino acid decarboxylase inhibitor.

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger.

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.

Renal dopaminergic mechanisms in renal parenchymal diseases and hypertension.

The present report addresses the status of the renal dopaminergic system activity in patients afflicted with different renal disorders and in the remnant kidney of uninephrectomized (UNX) rats, based on the urinary excretion of L-DOPA, dopamine and amine metabolites. In renal transplant recipients with good recovery of graft function (group 1, n=11), the daily urinary excretion of DOPAC, but not that of HVA, was found to increase progressively throughout the first 12 days post-transplantation from 698+/-57 nmol in the first day to 3498+/-414 nmol on day 9, and then remained constant until day 12. This resulted in a 6-fold increase in the urinary DOPAC/dopamine ratios. In renal transplant recipients with acute tubular necrosis (group 2, n=8), the urinary levels of dopamine, DOPAC and HVA were approximately 30% of those in group 1. In a group of 28 patients with chronic renal parenchymal disorders, the daily urinary excretion of L-DOPA, free dopamine and dopamine metabolites (DOPAC and HVA) correlated positively with the degree of deterioration of renal function (P<0.01). However, the U(Dopamine/(L)-DOPA) and U(DOPAC/Dopamine) ratios in patients with chronic renal insufficiency were found to be similar to those observed in patients with normal renal function. In 14 IgA nephropathy (IgA-N) patients with near normal renal function, the changes in 24 h mean blood pressure when going from 20 to 350 mmol/day sodium intake correlated negatively with the daily urinary excretion of dopamine (r(2)=0.597, P<0.01). The urinary excretion of L-DOPA and dopamine in IgA-N patients with salt-sensitive (SS) blood pressure was lower than in salt-resistant (SR) patients (P<0.05), irrespective of their daily sodium intake. However, the rise in urinary dopamine output during salt loading (from 20 to 350 mmol/day) was greater (P<0.05) in IgA-N SS patients (21.2+/-2.5% increase) than in SR patients (6.3+/-1.4% increase). Fifteen days after the surgery, uninephrectomy (UNX) in the rat was accompanied by an enhanced (P<0.05) urinary excretion of dopamine (36+/-3 vs 26+/-2), DOPAC (124+/-11 vs 69+/-6) and HVA (611+/-42 vs 354+/-7) (nmol/g kidney/kg body weight). This was accompanied by an increase in V(max) values for renal aromatic L-amino acid decarboxylase in the remnant kidney of UNX rats (P<0.05). Sch 23390, a D1 dopamine receptor antagonist, produced a marked reduction in the urinary excretion of sodium in UNX rats, whereas in sham-operated rats the decrease in urinary sodium did not attain a significant difference. It is concluded that the study of the renal dopaminergic system in patients afflicted with renal parenchymal disorders should address parameters other than free urinary dopamine, namely the urinary excretion of L-DOPA and dopamine metabolites (DOPAC and HVA). It is also suggested that in SS hypertension of chronic renal parenchymal diseases, renal dopamine produced in the residual tubular units may be enhanced during a sodium challenge, thus behaving appropriately as a compensatory natriuretic hormone.