Uptake of l-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells

doi:10.1016/S0006-2952(97)00318-3

Biochemical Pharmacology

Volume 54, Issue 9, 1 November 1997, Pages 1037-1046

https://doi.org/10.1016/S0006-2952(97)00318-3 Get rights and content

Abstract

In the presence of benserazide (50 μM), l-3,4-dihydroxyphenylalanine (l-DOPA) was rapidly accumulated in both LLC-PK₁ and OK cells; equilibrium was attained at 30 min of incubation. For these LLC-PK₁ and OK cells, the analysis revealed a rate constant of inward transport (k_in in pmol/mg protein/min) of 3.6 ± 0.4 and 18.1 ± 0.3 and a rate constant of outward transport (k_out in pmol/mg protein/min) of 1.0 ± 0.1 and 5.2 ± 0.1, respectively. Nonlinear analysis of the saturation curves for LLC-PK₁ and OK cells revealed a K_m (in μM) of 86 ± 12 and 14 ± 4, respectively. The cellular accumulation of the substrate was temperature-dependent and stereoselective. Aromatic l-amino acid decarboxylase (AAAD) activity was determined in cell homogenates; nonlinear analysis of the saturation curves revealed, for LLC-PK₁ and OK cells, a K_m (in μM) of 1866 ± 107 and 845 ± 153 and a V_max (in nmol/mg protein/15 min) of 4.4 ± 0.1 and 0.9 ± 0.1, respectively. In the absence of benserazide, only a limited amount of the l-DOPA taken up was decarboxylated to dopamine in cell monolayers; the K_m value (in μM) for decarboxylation of intracellular l-DOPA in LLC-PK₁ and OK cells was 61 ± 14 and 108 ± 36, respectively. A low amount of newly formed dopamine was found to escape to the apical bathing fluid. This outward transfer of newly formed dopamine was a nonsaturable process up to 300 μM intracellular dopamine. In conclusion, the data presented here show that OK cells are endowed with a more efficient l-DOPA uptake system than LLC-PK₁ cells, but the latter are endowed with a significantly higher AAAD activity than OK cells. In both cell lines, intracellular l-DOPA is rapidly converted to dopamine, some of which diffuses out of the cell.

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Uptake and Decarboxylation of l-3,4-Dihydroxyphenylalanine in Cultured Monkey Placenta Amniotic Epithelial Cells
2007, Placenta
Citation Excerpt :
Similar findings of DA synthesis from l-DOPA have been reported for other cells possessing AADC enzyme [6,10–12]. Incubation of cells with l-DOPA in the presence of 50 μM of the peripheral AADC inhibitor benserazide, a concentration that has been found to completely inhibit AADC [12], significantly decreased DA content, thus adding further confirmation of the presence of AADC in MAEC. Interestingly, however, cells incubated in HEPES-buffer produced more dopamine than did cells incubated in serum-free RPMI-1640 medium even though the same amount of l-DOPA was added to both media.
In this study we tested the ability of monkey amniotic epithelial cells (MAEC) to take up and decarboxylate l-3,4-dihydroxyphenylalanine (l-DOPA) by incubating the cells in buffer containing l-DOPA under different experimental conditions followed by assaying cellular dopamine (DA) content using high performance liquid chromatography with electrochemical detection. Cellular contents of DA were significantly increased in a time- and l-DOPA-concentration-dependent manner, suggesting the uptake of l-DOPA by MAEC and indicating the presence of aromatic l-amino acid decarboxylase (AADC). This was confirmed by the decreased DA content in the presence of benserazide, an AADC inhibitor. Neither d-DOPA nor DA uptake blockers such as mazindol and GBR 12935 significantly affected l-DOPA uptake and hence DA levels. Further, synthesis of DA from l-DOPA was decreased in the presence of the amino acids tyrosine, phenylalanine and tryptophan, whereas the amino acids glycine and proline were without any significant effect. These findings suggest that MAEC have the capacity to selectively take up and decarboxylate l-DOPA with subsequent production of DA.
Ca<sup>2+</sup>/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells
2000, Life Sciences
The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca²+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K_m value (in μM) of 54±2 and a V_max value (in nmol mg protein/6 min) of 34±1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC₅₀=82 μM). The Ca²+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 μM) uptake with IC₅₀ Äs of 33 and 105 μM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 μM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca²+/calmodulin mediated pathways.
A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line
1999, FEBS Letters
The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk⁻), we have characterized a putative functional dopamine transporter displaying a high affinity (K_m∼250 nM) and a low capacity (∼0.1 pmol/10⁵ cells/min) for [³H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated K_i values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [³H]WIN 35,428 binding activity were detected. A partial cDNA of ∼1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1–6, displayed a deduced amino acid sequence homology of ∼96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.
Perfused rat term placenta as a preclinical model to investigate placental dopamine and norepinephrine transport
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^☆: This work was supported by Grant Praxis/2/2.1./1386/95.

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Research paperUptake of l-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells☆

Abstract

Biochem Pharmacol

Biochem Pharmacol

Eur J Pharmacol

Anal Biochem

Biochim Biophys Acta

Int J Biochem Cell Biol

Dopamine and the kidney: ten years on

Clin Sci

Dopamine causes inhibition of Na+-K+-ATPase activity in rat proximal convoluted tubule segments

Am J Physiol

Inhibition of proximal tubule Na(+)-K(+)-ATPase activity requires simultaneous activation of DA1 and DA2 receptors

Am J Physiol

Dopamine inhibits Na(+)-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase

Am J Physiol

Regulation of dopamine synthesis in the rat kidney

J Auton Pharmacol

Source and handling of renal dopamine: Its physiological importance

News Physiol Sci

Evidence that intrarenal dopamine acts as a paracrine substance at the renal tubule

Am J Physiol

Cellular mechanisms of renal tubular transport of l-DOPA and its derivatives in the rat: microperfusions studies

J Pharmacol Exp Ther

Sodium-dependence and ouabain-sensitivity of the synthesis of dopamine in renal tissues of the rat

Br J Pharmacol

Involvement of tubular sodium in the formation of dopamine in the human renal cortex

J Am Soc Nephrol

Studies on the role of sodium on the synthesis of dopamine in the rat kidney

J Pharmacol Exp Ther

Inhibitory effects of guanosine 3′:5′-cyclic monophosphate on the synthesis of dopamine in the rat kidney

Br J Pharmacol

Research paper
Uptake of l-3,4-dihydroxyphenylalanine and dopamine formation in cultured renal epithelial cells☆

Dopamine causes inhibition of Na⁺-K⁺-ATPase activity in rat proximal convoluted tubule segments

Inhibition of proximal tubule Na(⁺)-K(⁺)-ATPase activity requires simultaneous activation of DA₁ and DA₂ receptors

Dopamine inhibits Na(⁺)-H⁺ exchanger activity in renal BBMV by stimulation of adenylate cyclase