Source of dopamine in gastric juice and luminal dopamine-induced duodenal bicarbonate secretion via apical dopamine D2 receptors

Abstract

Background and purpose: Dopamine protects the duodenal mucosa. Here we have investigated the source of dopamine in gastric juice and the mechanism underlying the effects of luminal dopamine on duodenal bicarbonate secretion (DBS) in rodents.

Experimental approach: Immunofluorescence, UPLC-MS/MS, gastric incubation and perfusion were used to detect gastric-derived dopamine. Immunofluorescence and RT-PCR were used to examine the expression of dopamine receptors in the duodenal mucosa. Real-time pH titration and pH_i measurement were performed to investigate DBS.

Key results: H⁺ -K⁺ -ATPase was co-localized with tyrosine hydroxylase and dopamine transporters in gastric parietal cells. Dopamine was increased in in vivo gastric perfusate after intravenous infusion of histamine and in gastric mucosa incubated, in vitro, with bethanechol chloride or tyrosine. D₂ receptors were the most abundant dopamine receptors in rat duodenum, mainly distributed on the apical membrane of epithelial cells. Luminal dopamine increased DBS in a concentration-dependent manner, an effect mimicked by a D₂ receptor agonist quinpirole and inhibited by the D₂ receptor antagonist L741,626, in vivo D₂ receptor siRNA and in D₂ receptor ^-/- mice. Dopamine and quinpirole raised the duodenal enterocyte pH_i . Quinpirole-evoked DBS and PI3K/Akt activity were inhibited by calcium chelator BAPTA-AM or in D₂ receptor^-/- mice.

Conclusion and implications: Dopamine in the gastric juice is derived from parietal cells and is secreted along with gastric acid. On arrival in the duodenal lumen, dopamine increased DBS via an apical D₂ receptor- and calcium-dependent pathway. Our data provide novel insights into the protective effects of dopamine on the duodenal mucosa.

FIGURE 1

Parietal cells are the main source of dopamine in the gastric mucosa. (a) Dopamine content of gastric incubation fluid and plasma and of gastric mucosa and substantia nigra (SN) measured by UPLC‐MS/MS in rats. *P < .05, significantly different from plasma (left) or SN (right); Student's unpaired t test. (b) The labelling immunofluorescence of TH and DDC in the gastric corpus mucosa of rat. Images are representative of three different animals. A nuclear marker, DAPI (blue), was used in the present study. Scale bars: 25 μm. (c) Dopamine content measured by UPLC‐MS/MS after application of tyrosine (.05, .1, and .5 mM) or pretreatment with TH inhibitor α‐methyl‐DL‐tyrosine methyl ester hydrochloride (α‐MDL, .05 mM). *P < 0.05, significantly different from control, ^# P < .05, significantly different from .5‐mM tyrosine; one‐way ANOVA with Tukey's test. (d) The labelling immunofluorescence of H⁺‐K⁺‐ATPase and TH/DAT in the gastric corpus mucosa of rat. Images are representative of three different animals. A nuclear marker, DAPI (blue), was used in the present study. Scale bars: 25 μm. (e) Dopamine content measured by UPLC‐MS/MS after application of bethanechol chloride (10 μM) and omeprazole (10 μM). *P < .05, significantly different from control; one‐way ANOVA with Tukey's test. (f) The pH of gastric perfusate after intravenous (i.v.) infusion of histamine (2 mg·kg⁻¹·h⁻¹). *P < .05, significantly different from control by Student's paired t test. (g) Dopamine content of effluent perfusate measured by UPLC‐MS/MS after intravenous infusion of histamine (2 mg·kg⁻¹·h⁻¹). *P < .05, significantly different from control; paired Student's t test. The data are expressed as mean ± SEM; n = 8 per group

FIGURE 2

Dopamine increases duodenal HCO₃ ⁻ secretion (DBS). (a) Apical (A) and basolateral (B) addition of dopamine (DA; 10 μM) produced DBS after routine basolateral addition of indomethacin (10 μM) and TTX (1 μM). (b) Apical addition of dopamine (DA; 0.01–100 μM) induced DBS in rats. (c) Dopamine (DA; 10 μM) increased intracellular pH in the chorionic epithelial cells of the rat duodenum. (d) Distribution of the pH‐sensitive microfluorometry probe BCECF‐AM after addition of dopamine (DA; 10 μM). Differential interference contrast (DIC) microscopic image showing the three‐dimensional morphology of a duodenal villus. Images are representative of seven different animals. Scale bars: 100 μm. *P < .05, significantly different from baseline; paired Student's t test. The data are expressed as mean ± SEM; n = 7 per group

FIGURE 3

Apical D₂ receptors (D₂Rs) mediate dopamine‐induced DBS in normal rats. (a) Effects of dopamine receptor (DAR) antagonists on the response to apical addition of dopamine (DA; 10 μM) induced DBS. SCH‐23390 (10 μM) (a non‐selective D₁‐like receptor antagonist), apical addition; Sulpiride (10 μM) (a non‐selective D₂‐like receptor antagonist), apical addition; L741,626 (10 μM) (the specific D₂ receptor antagonist), apical addition; forskolin (FSK; 10 μM) (the positive control for DBS), basolateral addition. (b) Summary of the effects of dopamine receptor antagonists on dopamine‐induced DBS. (c) Changes in DBS after apical addition of the D₂‐like receptor agonist quinpirole (0.01–100 nM). (d) Apical addition of quinpirole (1–100 nM) increased the intracellular pH of chorionic epithelial cells of the rat duodenum. (e) mRNA expression of dopamine receptors in the duodenal epithelium as analysed by real‐time RT‐PCR. β‐Actin was used as an internal control. (f) Haematoxylin–eosin (HE) staining and immunofluorescence of D₂ receptors in the rat duodenum. Images are representative of three different animals. Scale bars: 150 μm (upper) and 50 μm (lower). *P < .05, significantly different from control (a–d) or from D₂ receptors (e), ^# P < .05, significantly different from dopamine (b); one‐way ANOVA with Tukey's test. The data are expressed as mean ± SEM. n = 7 per group

FIGURE 4

Apical D₂ receptors (D₂R) mediate dopamine‐induced DBS in model mice. (a) Characterization of in vivo D₂ receptor siRNA knockdown mice by western blotting (GAPDH, d‐glyceraldehyde‐3‐phosphate dehydrogenase). (b) Characterization of the D₂ receptor knockout mice by PCR. (c) Distribution of the pH‐sensitive microfluorometry probe BCECF‐AM in the chorionic epithelial cells of D₂ receptor knockout (D₂R^−/−) mice after addition of quinpirole (100 nM). Images are representative of seven pairs of different animals. (d) DBS in the control (scramble siRNA) and in vivo D₂ receptor siRNA knockdown mice after apical addition of quinpirole (10 and 100 nM). (e) DBS in the wild‐type (WT) and D₂R^−/− mice after apical addition of quinpirole (10 and 100 nM). (f) Intracellular pH in the chorionic epithelial cells of D₂R^−/− mice after addition of quinpirole (100 nM). *P < .05, significantly different from control; unpaired Student's t test (a); *P < .05, significantly different from control/WT; paired Student's t test (d, e); *P < .05, significantly different from baseline, ^# P < .05, significantly different from WT; two‐way ANOVA with Tukey's test (f). The data are expressed as mean ± SEM. n = 7 per group

FIGURE 5

The calcium/PI3K/Akt pathway is involved in dopamine‐induced DBS. (a) Effects of the adenylate cyclase inhibitor MDL12330A (10 μM, apical addition) on dopamine‐ (DA; 10 μM, apical addition), or quinpirole‐ (10 nM, apical addition) induced DBS. (b) Intracellular cAMP levels after apical addition of dopamine (DA; 10 μM) or quinpirole (10 nM). (c) Effects of the intracellular calcium chelator BAPTA‐AM (10 μM, apical addition) on dopamine (DA)‐induced DBS (apical addition, 10 μM). (d) Protein levels of PI3K and p‐PI3K in the duodenal mucosa preparations of WT and D₂R^−/− mice after treatment with quinpirole (100 nM, apical addition). (e) The ratio of phosphorylated Akt (S473) to Akt in the duodenal mucosa preparations of WT and D₂R^−/− mice measured by elisa after treatment with quinpirole (100 nM, apical addition). (f) The ratio of phosphorylated Akt (S473) to Akt in the duodenal mucosa preparations after pretreatment with BAPTA‐AM (10 μM, apical addition) and LY294002 (a selective PI3K inhibitor, 20 μM, apical addition). *P < .05, significantly different from vehicle; one‐way ANOVA with Tukey's test (b); *P < .05, significantly different from dimethyl sulphoxide (DMSO) by unpaired Student's t test (c); *P < .05, significantly different from WT, ^# P < .05, significantly different from WT + quinpirole; one‐way ANOVA with Tukey's test (d, e); *P < .05, significantly different from vehicle, ^# P < .05, significantly different from quinpirole; one‐way ANOVA with Tukey's test (f). The data are expressed as mean ± SEM. n = 6–7 per group

FIGURE 6

Diagram of the working hypothesis of gastric dopamine‐induced duodenal bicarbonate secretion via an apical D₂ receptors‐ and calcium‐dependent pathway. Dopamine (DA) in the gastric juice is derived from parietal cells and is secreted along with gastric acid. Histamine stimulates parietal cells to release additional dopamine, which arrives at the duodenal lumen and binds to D₂ receptors (D₂R) on the duodenal apical epithelium, thereby increasing bicarbonate secretion through a calcium‐dependent pathway