A major proportion of urinary dopamine derives from the renal decarboxylation of circulating dopa. This study evaluates the effects of aging on renal production of dopamine using 3- and 12-mo-old male Wistar rats. Urinary excretion of Na+, norepinephrine (NE), 3,4-dihydroxyphenylglycol, and dopa were similar in the two groups. Urinary dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were lower in older animals (dopamine, 20 +/- 6 vs. 47 +/- 7 nmol/24 h, P < 0.001; DOPAC, 142 +/- 36 vs. 304 +/- 56 nmol/24 h, P < 0.03). Urinary 3-O-methyldopa (OM-dopa) was higher in 12-mo-old rats (6.2 +/- 2.0 vs. 3.3 +/- 0.20 nmol/24 h, P < 0.03). Levels of dopa and NE in renal cortex from 12-mo-old rats were higher (P < 0.001) than in younger animals. Dopamine content in renal cortex from 3-mo-old rats was 295 +/- 64 pmol/g, whereas it was undetectable in 12-mo-old animals. Aromatic-L-amino-acid decarboxylase and monoamine oxidase activities were higher (P < 0.001) in renal cortex from 12-mo-old animals. Catechol-O-methyltransferase activity was similar in both groups. The uptake of dopa by the luminal membrane was explored using brush-border membrane vesicles. The Na(+)-gradient-driven (100 mM) uptake of dopa into vesicles from 3-mo-old animals showed at 10 s an overshoot threefold greater than the equilibrium uptake. The overshoot was blunted in 12-mo-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Short-term regulation of sodium metabolism is dependent on the modulation of the activity of sodium transporters by first and second messengers. In understanding diseases associated with sodium retention, it is necessary to identify the coupling between these messengers. We have examined whether dopamine, an important first messenger in tubular cells, activates and translocates various protein kinase C (PKC) isoforms. We used a proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was found to inhibit Na(+)-K(+)-ATPase in a PKC-dependent manner. Translocation of PKC isoforms was studied with both subcellular fractionation and confocal microscopy. Both techniques revealed a dopamine-induced translocation from cytosol to plasma membrane of PKC-alpha and -epsilon, but not of PKC-delta, -gamma, and -zeta. The process of subcellular fractionation resulted in partial translocation of PKC-epsilon. This artifact was eliminated in confocal studies. Confocal imaging permitted detection of translocation within 20 s. Translocation was abolished by a phospholipase C inhibitor and by an antagonist against the dopamine 1 subtype (D(1)) but not the 2 subtype of receptor (D(2)). In conclusion, this study visualizes in renal epithelial cells a very rapid activation of the PKC-alpha and -epsilon isoforms by the D(1) receptor subtype.
Purpose-Recent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects.Methods-The expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells.
Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of cytochrome P-450 (CYP), as a possible mediator of Na(+)-K(+)-ATPase inhibition by dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10(-5) M) inhibited Na(+)-K(+)-ATPase activity in microdissected tubular segments to 59.4 +/- 3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10(-6) M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na(+)-K(+)-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D(1) or D(2) receptors. Neither fenoldopam nor quinpirole (D(1) and D(2) agonists, respectively, both 10(-5) M) modified Na(+)-K(+)-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10(-9) M) with fenoldopam resulted in a synergistic inhibition of Na(+)-K(+)-ATPase activity (66 +/- 2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10(-9) M) synergized with forskolin (10(-5) M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG; 10(-11) M; 62.0 +/- 5.3 and 69.9 +/- 2.0% of control activity, respectively), indicating a cooperative role of 20-HETE with the D(1)-triggered pathways. In line with these results, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10(-6) M). These results demonstrate that the inhibition of Na(+)-K(+)-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D(1) signaling pathway.
20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10 μM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 μM, 23 ± 3%*; 10 μM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 μM, 16 ± 4%*; 10 μM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*-42%* by 1-10 μM HET0016. Incubation with 20-HETE (5-1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 μM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.