L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser⁴⁰: An Alternative Route for Dopamine Synthesis and Modulation of Na⁺/K⁺-ATPase in Kidney Cells

Abstract

Background/Aims: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na⁺/K⁺-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). Methods: Experiments involved cultured LLCPK₁ cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na⁺/K⁺-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D₁R in LLCPK₁ cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na⁺/K⁺-ATPase activity, which can be ascribed to the synthesis of dopamine. Results: LLCPK₁ cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D₁R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na⁺/K⁺-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na⁺/K⁺-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser⁴⁰ by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D₁R antagonist, totally suppressed the inhibition of Na⁺/K⁺-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. Conclusion: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na⁺/K⁺-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.

Tandem Requirement for Full Renal D1R and D5R Activity

“…Both the D 1 R and D 5 R are expressed in cultured hRPTCs (12,20,22,24,37,43), similar to the porcine RPTCs (LLCPK1) (46), both of which are fully differentiated renal epithelial cells. Both receptors are functional in hRPTCs, and share some common signaling cascades.…”

Down-regulation of long non-coding RNA XIST aggravates sepsis-induced lung injury by regulating miR-16-5p

“…Additionally, miRNAs are also involved in sepsis-mediated lung injury. Reportedly, in septic shock, miR-203 can alleviate lung injury by inhibiting VNN1 [ 27 ]. In previous studies, miR-16-5p is mainly considered as a tumor suppressor, and it can induce cell apoptosis by repressing its target genes such as SMAD3 and Akt3, both of which were crucial modulators in inflammation and lung injury [ 17 , 28 – 30 ].…”

Kidney dopamine D1-like receptors and angiotensin 1–7 interaction inhibits renal Na+ transporters

“…Activation of tyrosine hydroxylase converts tyrosine to L-DOPA, a rate-limited step in dopamine synthesis. Interestingly, both PKG and tyrosine hydroxylase are highly expressed in epithelial cells (1,16,20,29,52). Therefore, in the present study, we tested the hypothesis that ANG 1-7-MasR signaling could increase renal dopamine production via tyrosine hydrox-ylase stimulation, which would activate renal DR and induce natriuresis and diuresis.…”

“…The involvement of transporters belonging to the solute carrier superfamily has also been suggested (30,31,55). However, the role of tyrosine hydroxylase, which is highly expressed in epithelial cells, has not been fully assessed in renal dopamine synthesis (1,20,29,52). The activity of tyrosine hydroxylase is highly regulated by serine phosphorylation, involving various serine/threonine kinases and phosphatases (22).…”

“…It is widely perceived that L-DOPA, freely filtered by the glomerulus, is transported into proximal tubules mainly by Na ϩ -independent L-amino acid transporter (LAT)2, making it a rate-limiting step in dopamine synthesis (3,11,27,45). However, recent studies have shown that an alternative pathway involving tyrosine hydroxylase, an enzyme that converts tyrosine to L-DOPA, a rate-limiting step in neuronal dopamine synthesis, could be contributing to tubular dopamine production (52).…”