Maria Augusta Vieira-Coelho

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 Crohns 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.

Effects of tolcapone upon soluble and membrane-bound brain and liver catechol-O-methyltransferase

The present study was aimed to evaluate the sensitivity of soluble (S) and membrane bound (MB) catechol-O-methyltransferase (COMT) from rat brain and liver to inhibitors which interact with the enzyme as competitive (tropolone), non-competitive (S-adenosyl-l-homocysteine; SAHC) and tight-binding (tolcapone and 3,5-dinitrocatechol) inhibitors. COMT activity was evaluated by the ability to methylate adrenaline (0.1 to 2000 microM) to metanephrine in the presence of a saturating concentration of the methyl donor (S-adenosyl-l-methionine). When using a fixed amount of total protein (2 micrograms/ml), but variable concentrations of COMT, the inhibitory potency of tolcapone upon S- and MB-COMT activity in the brain was in the low nM range (IC50s of 2 and 3 nM, respectively), whereas in liver the IC50 values for tolcapone against liver MB- and S-COMT (IC50s of 123 and 795 nM, respectively) were markedly higher than those observed in the brain. By contrast, when inhibition studies were performed with a fixed concentration of COMT (15 nM), as determined by the Ackermann-Potter equation, tolcapone was found to be endowed with the same potency (in the low nM range) in inhibiting S- and MB-COMT from both brain and liver. As for tolcapone, 3,5-dinitrocatechol was more potent against MB- than against S-COMT when a fixed amount of total protein was used, but showed the same potency when a fixed concentration of COMT was used. Tropolone, a competitive inhibitor, was much less potent than tolcapone and 3,5-dinitrocatechol in inhibiting S- and MB-COMT from both brain and liver and its potency was found not to depend on enzyme concentration. SAHC, a non-competitive inhibitor, behaved similarly to tight-binding inhibitors when a fixed amount of total protein was used. By contrast, when a fixed amount of enzyme was used, SAHC was found to be endowed with the same potency against S- and MB-COMT from brain and liver. In the final series of experiments the inhibitory effect of tolcapone was examined under in vitro ex vivo conditions, using the same concentration of COMT (15 nM). One hour after its oral administration, tolcapone (0.3 to 30 mg/kg) was found to be much more potent against MB-COMT than against S-COMT. In the liver, 0.3 mg/kg tolcapone resulted in 82% inhibition of MB-COMT and 31% inhibition of S-COMT. In the brain, 3.0 mg/kg tolcapone inhibited 78% MB-COMT, whereas S-COMT activity was reduced by 38% only. In conclusion, the results reported here show that tolcapone is particularly potent in inhibiting MB-COMT from liver and brain under in vivo experimental conditions, though it does not discriminate between MB- and S-COMT under in vitro experimental conditions when using the same amount of enzyme in the assay.

Assessment of renal dopaminergic system activity in the nitric oxide‐deprived hypertensive rat model

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 NGnitro‐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.

Aging, High Salt Intake, and Renal Dopaminergic Activity in Fischer 344 Rats

The present study examined renal dopaminergic activity and its response to high salt (HS) intake in adult (6-month-old) and old (24-month-old) Fischer 344 rats. Daily urinary excretion of L-3, 4-dihydroxyphenylalanine (L-DOPA), dopamine, and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid was similar in adult and old rats; by contrast, daily urinary excretion of norepinephrine in old rats was almost twice that in adult animals. HS intake (1% NaCl) over a period of 24 hours resulted in a 2-fold increase in the urinary excretion of dopamine, DOPAC, and norepinephrine in adult animals but not in old animals. Norepinephrine and L-DOPA plasma levels did not change during HS intake and were similar in both groups of rats. The natriuretic response to an HS intake in old rats (from 4.7+/-0.4 to 10.7+/-2.0 nmol. kg(-1). d(-1); Delta=6.0+/-0.9 nmol. kg(-1). d(-1)) was less than in adult rats (from 5.2+/-0.4 to 13.5+/-2.5 nmol. kg(-1). d(-1); Delta=8.3+/-0.8 nmol. kg(-1). d(-1)). A diuretic response to HS intake was observed in adult rats (from 20.9+/-2.3 to 37.6+/-2.8 mL. kg(-1). d(-1)) but not in old rats (from 37.7+/-5.7 to 42.3+/-6. 0 mL. kg(-1). d(-1)). Dopamine levels and dopamine/L-DOPA ratios in the renal cortex of old rats were greater than in adult rats. HS intake increased both dopamine levels and dopamine/L-DOPA ratios in the renal cortex of adult rats but not in old rats. Aromatic L-amino acid decarboxylase activity was higher in old rats than in adult rats; HS intake increased L-amino acid decarboxylase activity (nmol. mg protein(-1). l5 min(-1)) in adult rats (from 67+/-1 to 93+/-1) but not in old rats (from 86+/-2 to 87+/-2). Dopamine inhibited Na(+),K(+)-ATPase activity in proximal tubules obtained from adult rats, but it failed to exert such an inhibitory effect in old rats. It is concluded that renal dopaminergic tonus in old rats is higher than in adult rats but fails to respond to HS intake as observed in adult rats. This may be due in part to the inability of dopamine to inhibit Na(+),K(+)-ATPase activity in old rats.

Deletion of the neuropeptide Y (NPY) Y1 receptor gene reveals a regulatory role of NPY on catecholamine synthesis and secretion

The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y1 receptor knockout mice (Y1−/−). Compared with wild-type mice (Y1+/+), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y1−/− mice. In resting animals, catecholamine plasma concentrations were higher in Y1−/− mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y1−/− mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y1 receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y1 receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone.

α2-Adrenoceptor subtypes involved in the regulation of catecholamine release from the adrenal medulla of mice

This study was carried out to elucidate which α2‐adrenoceptor subtypes mediated the inhibition of noradrenaline and adrenaline release from the adrenal medulla of mice.

Ontogenic aspects of liver and kidney catechol-O-methyltransferase sensitivity to tolcapone.

1 The present work describes the catechol‐O‐methyltransferase (COMT) activities in the liver and kidney of developing and adult rats (aged 3, 6, 9, 18, 30 and 60 days; n = 5 per group) and evaluates the enzyme sensitivity to inhibition by tolcapone, a reversible COMT inhibitor. 2 COMT activity, evaluated by the ability to methylate adrenaline to metanephrine, was determined in liver and kidney homogenates prepared in 0.5 mM phosphate buffer (pH = 7.8) containing pargyline (0.1 mM), MgC12 (0.1 mM), EGTA (1 mM) and S‐adenosyl‐L‐methionine (0.1 mM). Vmax (in nmol mg−1 protein h−1) of liver COMT was found to decrease gradually with age, from 5.3±0.5 at the age of 3 days up to 2.9±0.2 at the age of 60 days; for the same age range, Km values (in μm; geometric means with 95% confidence limits) increased from 3.3 (1.0, 7.5) up to 13.1 (2.1, 24.1). At the age of 3 days, Vmax values for kidney COMT (2.6±0.1) were lower than those for the liver COMT. However, Vmax values for kidney COMT were found to increase up to 6.2±0.6 at the age of 18 days and then declined by 44% at the age of 30 and 60 days. In kidney, aging was also accompanied by an increase in Km values for COMT (from 2.7 [1.1, 4.3] up to 24.0 [11.7, 36.3]). 3 The sensitivity of liver and renal COMT activity to tolcapone was markedly dependent on the age, 3‐days old rats being more sensitive to tolcapone than older animals. The IC50 values (in nM) for inhibition of liver COMT by tolcapone increased gradually with age, from 41 (26, 65) at the age of 3 days up to 720 (640, 800) at the age of 60 days. As was found in the liver, IC50 values (in nM) for inhibition of kidney COMT by tolcapone also increased with age, from 8 (6, 10) at the age of 3 days up to 177 (131, 240) at the age of 60 days. In all experimental groups, the IC50 values for inhibition of liver COMT by tolcapone was higher than those for kidney COMT. 4 In conclusion, these results suggest that aging is accompanied by a decrease in liver and kidney COMT affinity for the substrate (evidenced by the increase in Km values) and a decrease in sensivity towards inhibition by tolcapone (evidenced by the increase in IC50 values). Furthermore, kidney COMT is shown to be more sensitive to inhibition by tolcapone than liver COMT, irrespective of the age of the animal.

Apical and basolateral uptake and intracellular fate of dopamine precursor l-dopa in LLC-PK1 cells

The present study was aimed at the uptake ofl-3,4-dihydroxyphenylalanine (l-dopa) and its intracellular decarboxylation to dopamine. The accumulation ofl-dopa from the apical side in cells cultured in collagen-treated plastic was found to be a saturable process with a Michaelis constant ( K m) of 123 ± 17 μM and a maximal velocity ( V max) of 6.0 ± 0.2 nmol ⋅ mg protein-1 ⋅ 6 min-1. The uptake ofl-dopa applied from either the apical or basal cell borders in cells cultured in polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed K m values of 63.8 ± 17.0 and 42.5 ± 9.6 μM and V maxvalues of 32.0 ± 5.8 and 26.2 ± 3.4 nmol ⋅ mg protein-1 ⋅ 6 min-1, respectively. Cell monolayers incubated withl-dopa, applied from either the apical or the basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly formed dopamine was a saturable process with apparent K m values of 20.5 ± 8.2 and 247.3 ± 76.8 μM when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamine escaped to the extracellular milieu. The basal outward transfer of dopamine was five- to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopamine; the apical outward transfer of the amine depended on the intracellular concentration of dopamine and was a nonsaturable process. The apical and basal outward transfers of dopamine were insensitive to cocaine (10 and 30 μM) and GBR-12909 (1 and 3 μM). The accumulation of exogenous dopamine in LLC-PK1 cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal application of dopamine a K m of 17.7 ± 4.3 and 96.0 ± 28.1 μM and a V max of 2.0 ± 0.1 and 2.2 ± 0.3 nmol ⋅ mg protein-1 ⋅ 6 min-1, respectively. However, both cocaine (10, 30, or 100 μM) and GBR-12909 (1 or 3 μM) were found not to affect the uptake of 100 μM dopamine applied from either the apical or the basal cell border. In conclusion, the data presented here show that LLC-PK1cells are endowed with considerable aromaticl-amino acid decarboxylase (AADC) activity and transportl-dopa quite efficiently through both the apical and basal cell borders. On the other hand, our observations support the possibility of a basal-to-apical gradient of AADC activity and the possibility that LLC-PK1 cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.

Dopamine formation, from its immediate precursor 3,4‐dihydroxyphenylalanine, along the rat digestive tract

Summary— The formation of dopamine, from L‐3,4‐dihydroxyphenylalanine (l–dopa), in fragments of non‐glandular and glandular stomach, duodenum, jejunum, ileum and proximal and distal colon of the rat was examined. The deamination of newly‐formed dopamine into 3,4‐dihydroxyphenylacetic acid (dopac) was also studied. The synthesis of dopamine in tissues incubated with 500 μM l–dopa for 20 min in conditions of catechol‐O‐methyltransferase (Comt) inhibition was found to be in the duodenum, jejunum and ileum 2‐fold that in the proximal colon, 6‐fold that in the glandular stomach and 120‐fold that in the non‐glandular stomach and distal colon. The formation of dopac in these tissues followed the pattern of amine formation. In the jejunum, the formation of dopamine and dopac was found to be dependent on the concentration of l–dopa (50 to 5000 μM) used. In another set of experiments, it was found that the formation of dopamine in jejunal segments loaded with increasing concentrations of l–dopa (50, 100 and 500 μM) was a time‐dependent process. The rate constant (k) of formation of dopamine as a function of time was found to be similar (0.050 ± 0.005) with either concentration of l–dopa; the rate constant of dopac formation in these experiments was, in contrast, found to be greater at the highest concentrations of l‐dopa (100 and 500 μM). Aromatic l–amino acid decarboxylase (Aaad) activity determined in homogenates of the jejunal mucosa was found to be twice that observed in homogenates of the remaining jejunal wall (muscular). The Vmax (nmol/g/h) and Km (μM) of Aaad was, respectively, in the jejunal mucoase 1324 ± 206 and 124 ± 13 and in the jejunal muscular wall 680 ± 35 and 136 ± 25. The results presented here show that the formation of dopamine along the digestive tract of the rat presents a heterogeneous pattern, the small intestine and the proximal colon being the areas where this reaction appears to be more important.

Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice

Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individuals lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc-/- mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc-/- mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc-/- mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.