Maria Paula Serrão

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.

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.

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.

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H(2)O(2)) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H(2)O(2) production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22(phox), SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.

Organ-Specific Overexpression of Renal LAT2 and Enhanced Tubular L-DOPA Uptake Precede the Onset of Hypertension

Abstract—Spontaneously hypertensive rats (SHR) might have increased renal production of dopamine. l-3,4-Dihydroxyphenylalanine (l-DOPA) uptake in renal epithelial cells is promoted through the type 2 L-type amino acid transporter (LAT2), and this might rate-limit the synthesis of renal dopamine. The present study evaluated l-DOPA uptake in isolated renal proximal tubules of SHR and normotensive controls (Wistar-Kyoto rats [WKY]). Expression of LAT1 and LAT2 in the renal cortex and intestinal mucosa was also evaluated. Tubular uptake of l-DOPA in WKY and SHR was a saturable process, being greater in the latter than the former at both 4 and 12 weeks of age. cDNA fragments (LAT1, 688 bp; LAT2, 729 bp) labeled with 32P were used as probes for Northern blot analysis. Expression of LAT2 in SHR kidneys was higher than in WKY kidneys. This increase was more marked at 4 than at 12 weeks of age. Intestinal LAT2 expression, however, was identical in SHR and WKY at both 4 and 12 week of age. By Northern blot analysis, the LAT1 transcript was not identified in either the kidney or intestine. Kidney total RNA was then reverse-transcribed and amplified by polymerase chain reaction with specific primers for LAT1. The presence of a fragment of the expected size for LAT1 led to the conclusion that LAT1 mRNA is a rare message in kidney. We conclude that overexpression of LAT2 in the SHR kidney might contribute to the enhanced l-DOPA uptake, which is organ specific and precedes the onset of hypertension.

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger.

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.

Opossum kidney (OK) cells in culture synthesize and degrade the natriuretic hormone dopamine: A comparison with rat renal tubular cells

To explore further the usefulness of opossum kidney (OK) cells in the study of renal dopaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AAAD), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B), the main enzymes involved in the synthesis and degradation of dopamine. The Vmax values for AAAD, using L-DOPA as the substrate, in rat renal tubular cells were found to be significantly (P < 0.01) higher (120-fold) than in OK cells. However, K(m) values in OK cells (1.1 mM [0.3, 1.9]) were similar to those observed in rat renal tubular cells (K(m) = 1.0 mM [0.8, 1.2]). The Vmax values for COMT (in nmol/mg protein/30 min) in OK cells (2.1 +/- 0.2) were similar to those in the rat renal tubular cells (1.6 +/- 0.1), whereas K(m) values in OK cells (2.3 microM [0.1, 4.5]) differ considerably (4.8-fold, P < 0.01) from those in rat renal tubular cells (11.2 microM [9.2, 13.1]). The Vmax values (in nmol/mg protein/20 min) for deamination of [3H]-5-hydroxytryptamine, the specific MAO-A substrate, was similar in rat renal tubular cells (12.4 +/- 1.0) and OK cells (12.9 +/- 1.1); K(m) values also did not differ between these two preparations. In contrast to rat renal tubular cells, deamination of [14C]-beta-phenylethylamine, the substrate for MAO-B, in OK cells was found to be non-saturable and to represent less than 10% of that observed in homogenates of rat tubular cells. In conclusion, OK cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade dopamine. The amounts of the enzymes AAAD, COMT and MAO-A found in this cell line are likely to be sufficient to reproduce, under in vitro conditions, the environment in which the renal dopaminergic system normally operates.

Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (l-DOPA) transporter, in pig renal LLC-PK1 epithelial cells

Organ‐specific overexpression of type 2 l‐amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SKR), accompanied by an enhanced ability to take up l‐DOPA, may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine‐mediated natriuresis. To understand the physiological role of LAT2‐mediated l‐DOPA handling, we used 21‐nucleotide small interfering RNA duplexes (siRNA) to specifically suppress LAT2 expression in LLC‐PK1 cells, a cell line that retains several properties of proximal tubular epithelial cells and takes up l‐DOPA largely through Na+‐independent transporters. After cloning the LLC‐PK1 LAT2 gene, one target region of LAT2 mRNA (nt 97‐117) was selected by scanning the length of the LAT2 gene for AA‐dinucleotide sequences and downstream 19 nucleotides. Levels of LAT2 cDNA, determined by real‐time quantitative RT‐PCR, were markedly (P<0.05) reduced by LAT2 siRNA but not by the mismatch LAT2 siRNA. The LAT2 siRNA but not the mismatch LAT2 siRNA, reduced by 85% [14C]‐l‐DOPA accumulation, a time‐ and concentration‐dependent effect. The efflux of intracellular [14C]‐l‐DOPA was markedly increased (P<0.05) by l‐DOPA and l‐leucine. The [14C]‐l‐DOPA outward transport was decreased 90% by LAT2 siRNA, but not by the mismatch LAT2 siRNA. However, treatment with the siRNA LAT2 did not affect the l‐DOPA‐induced fractional outflow of [14C]‐l‐DOPA. The Na+‐independent and pH‐sensitive l‐DOPA transporter may include the hetero amino acid exchanger LAT2, whose activation results in irans‐stimulation of l‐DOPA outward transfer.—Soares‐da‐Silva, P., Serrao, M. P., João Pinho, M., Bonifacio, M.J. Cloning and gene silencing of LAT2, the L‐3,4‐dihydroxyphenylalanine (l‐DOPA) transporter, in pig renal LLC‐PK1 epithelial cells. FASEB J. 18, 1489–1498 (2004)

Caco-2 cells in culture synthesize and degrade dopamine and 5-hydroxytryptamine: a comparison with rat jejunal epithelial cells.

To explore the usefulness of Caco-2 cells in the study of intestinal dopaminergic and 5-hydroxytryptaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AADC), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B) activities in these cells using specific substrates. The activity of these enzymes was also evaluated in isolated rat jejunal epithelial cells. The results showed that Vmax values (in nmol mg protein(-1) h(-1)) for AADC, using L-DOPA as the substrate, in rat jejunal epithelial cells (127.3+/-11.4) were found to be 6-fold higher than in Caco-2 cells (22.5+/-2.6). However, Km values in Caco-2 cells (1.24+/-0.37 mM) were similar to those observed in rat jejunal epithelial cells (1.30+/-0.29 mM). Similar results were obtained when AADC activity was evaluated using L-5HTP as substrate; in rat jejunal epithelial cells Vmax values (in nmol mg prot(-1) h(-1)) were found to be 5-fold that in Caco-2 cells (16.3+/-1.0 and 3.0+/-0.2, respectively), and Km values in Caco-2 cells (0.23+/-0.08 mM) were again similar to those observed in rat intestinal epithelial cells (0.09+/-0.03 mM). Caco-2 cells were not able to O-methylate dopamine, in contrast to rat jejunal epithelial cells (Vmax = 8.6+/-0.4 nmol mg protein(-1)(h-1); Km = 516+/-57 microM). Vmax values (in nmol mg protein(-1)(h-1)) for type A and B MAO in Caco-2 cells (19.0+/-0.6 and 5.4+/-0.6, respectively) were found to be significantly lower (P<0.05) than those in rat jejunal epithelial cells (46.9+/-3.1 and 9.6+/-1.2, respectively); however, no significant differences in the Km values were observed between Caco-2 and rat jejunal epithelial cells for both type A and B MAO. In conclusion, Caco-2 cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade DA and 5-HT, though, no COMT activity could be detected in these cells.

Evidence for the involvement of P-glycoprotein on the extrusion of taken up L-DOPA in cyclosporine A treated LLC-PK1 cells.

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. In the presence of benserazide (50 μM), 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 μM and a Vmax of 5581±297 pmol mg−1 protein 6 min−1. In the absence of benserazide, LLC‐PK1 cells incubated with increasing concentrations of L‐DOPA (10 to 500 μM) for 6 min accumulate newly‐formed dopamine by a saturable process with apparent Km and Vmax values of 31±6 μM 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 μM of intracellular newly‐formed dopamine, the outward transfer of the amine was found to be a non‐saturable process. Short‐term exposure to CsA (0.3, 1.0 and 3.0 μg 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 μg 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. Short‐term exposure to CsA (3.0 μg 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). Detection of P‐glycoprotein activity was carried out by measuring verapamil‐ or UIC2‐sensitive rhodamine 123 accumulation. Both UIC2 (3 μg ml−1) and verapamil (25 μM) 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 μM), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123. 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.