Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA

Abstract

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

Keywords: dopamine; gastrin; hypertension; l-DOPA; l-type amino acid transporter.

Fig. 1.

Gastrin stimulates the uptake of l-DOPA in human and mouse renal proximal tubule cells. A: dopamine production in response to incubation (20 min) of varying concentrations of gastrin (1 to 1,000 nM, n = 6, *P < 0.05 vs. control, one-way factorial ANOVA, Newman-Keuls test) and l-3,4-dihydroxyphenylalanine (l-DOPA; 100 μM); con = control (l-DOPA 100 μM alone). The minimum effective concentration of gastrin was 50 nM. Lines above bar graphs represent SE. B: effect of gastrin, L365,260 (CCK2 receptor inhibitor), and BCH (LAT inhibitor) on uptake of l-3,4-dihydroxyphenylalanine (l-DOPA) in human renal proximal tubule cells (hRPTCs). hRPTCs (passage 10) were incubated for 15 min at 37°C with vehicle or l-DOPA (100 μM), with or without gastrin (25 or 50 nM, preincubation for 5 min), and found the effective concentration of gastrin (50 nM) we used. For inhibitor studies, hRPTCs were preincubated for 6 min at 37°C with L365,260 (1 µM) or BCH (1 mM) with or without l-DOPA (100 μM) and gastrin (50 nM) treatment; n = 6/group, *P < 0.05 vs. control (first group); #P < 0.05 vs. l-DOPA and gastrin (50 nM) treatment, one-way factorial ANOVA, Newman-Keuls test. C: effect of gastrin, L365,260, and BCH on uptake of l-DOPA) in mouse (C57Bl/6J) renal proximal tubule cells (mRPTCs, passage 18). Treatments for mRPTCs were the same as in Fig. 1B, n = 6/group, *P < 0.05 vs. control (first group); #P < 0.05 vs. l-DOPA and gastrin (50 nM) treatment, one-way factorial ANOVA, Newman-Keuls test. D: total LAT-1 protein was quantified by immunoblotting in hRPTCs and mRPTCs treated with l-DOPA(100 μM) and gastrin (50 nM) with or without BCH (1 mM). Results were corrected for expression of GAPDH protein; n = 6/group. E: plasma membrane LAT-1 protein was quantified by immunoblotting in plasma membranes of hRPTCs and mRPTCs treated with l-DOPA(100 μM) and gastrin (50 nM) with or without BCH (1 mM). Protein concentrations in loaded samples were quantified by BCA assay; n = 6/group, *P < 0.05 vs. others; #P < 0.05 vs. l-DOPA and gastrin treatment; &P < 0.05 vs. hRPTC control group, one-way factorial ANOVA, Newman-Keuls test.

Fig. 2.

Gastrin stimulates uptake of l-DOPA in BALB/c and C57Bl/6J mouse kidney cortical slices. A: effect of gastrin on uptake of l-DOPA in renal cortical slices from BALB/c mice. Freshly harvested kidney was separated into cortex and medulla. The renal cortex was chopped into small pieces and incubated in DMEM. Cortical slices were incubated with l-DOPA (100 μM)with or without gastrin (25 and 50 nM); n = 6/group, *P < 0.05 vs. others, one-way factorial ANOVA, Newman-Keuls test. B: effect of gastrin on uptake of l-DOPA in renal cortical slices from C57Bl/6J mice. Preparation and treatment of renal cortical slices were as described in Fig. 2A; n = 6/group, *P < 0.05 vs. others, one-way factorial ANOVA, Newman-Keuls test.

Fig. 3.

Gastrin stimulates renal cortical uptake of l-DOPA via the l-amino acid transporter. A: effect of BCH (LAT inhibitor) on gastrin-stimulated uptake of l-DOPA in renal cortical slices from BALB/c and C57Bl/6J mice. Renal cortical slices were prepared as described in Fig. 2 and treated as in Fig. 1A, except that only one concentration of gastrin (50 nM) was used; n = 6/group, *P < 0.05 vs. control; #P < 0.05 vs. l-DOPA and gastrin treatment, one-way factorial ANOVA, Newman-Keuls test. B: total LAT-1 protein was quantified by immunoblotting in kidneys of BALB/c and C57BL/6J mice treated with l-DOPA and gastrin with or without BCH as in Fig. 3A.Results were corrected for expression of GAPDH protein. n = 6/group. Lines above the bar graphs represent the standard error from the mean. C. Plasma membrane LAT-1 protein was quantified by immunoblotting in kidneys of BALB/c and C57Bl/6J mice treated with l-DOPA and gastrin with or without BCH as Fig. 3A. Protein concentrations in loaded samples were quantified by BCA assay; n = 6/group, *P < 0.05 vs. others; #P < 0.05 vs. l-DOPA and gastrin treatment; &P < 0.05 vs. BALB/c mice, one-way factorial ANOVA, Newman-Keuls test.

Fig. 4.

Colocalization of gastrin and aquaporin 1 in BALB/c mouse kidney. A–D: gastrin immunofluorescence is absent in the cortex of mouse kidney; aquaporin 1 is expressed in the renal proximal convoluted tubule (PCT). E–H: gastrin and aquaporin 1 colocalize in the thin descending limb (TDL) but not in the proximal straight tubule (PST) or thick ascending limb (TAL) in the outer medulla of mouse kidney. I–L: gastrin and aquaporin 1 colocalize in the TDL in the inner medulla (original magnification, × 400). M: Gast mRNA (arrow), determined by in situ fluorescence hybridization, is present in the TDL. N: Gast mRNA expression in different regions of mouse kidney was determined using specific primers for Gast. The product was identified in the outer medulla, inner medulla, and papilla; no band was seen in the cortex. Mouse stomach mucosa was used as positive control for Gast mRNA. The housekeeping gene β-actin product was detected in all tested samples. Sequencing analysis of RT-PCR products identified mouse Gast mRNA (NM_010257.3). Similar results were obtained in ≥3 separate experiments.

Fig. 5.

Renal-selective silencing of Gast decreases urinary dopamine and increases blood pressure in BALB/c mice. Gast mRNA was quantified by qRT-PCR in kidneys of BALB/c mice that received a renal subcapsular infusion of Gast-specific (delivery rate: 3 µg/day) or Mock (nonsilencing) siRNA. Results were corrected for expression of Gapdh mRNA; n = 5/group, *P < 0.05 vs. Mock, t-test. GAST protein was quantified by immunoblotting in kidneys of BALB/c mice that received a renal subcapsular infusion of Gast-specific (delivery rate: 3 µg/day) or Mock siRNA. Results were corrected for expression of GAPDH protein; n = 5/group, *P < 0.05 vs. Mock, t-test. C: renal dopamine production was quantified by ELISA in urine of BALB/c mice that received a renal subcapsular infusion of Gast-specific (delivery rate: 3 µg/day) or Mock siRNA; n = 5/group, *P < 0.05 vs. Mock, t-test. D: plasma membrane LAT-1 protein was quantified by immunoblotting in kidneys of BALB/c mice that received a renal subcapsular infusion of Gast-specific(delivery rate: 3 µg/day) or Mock siRNA. Protein concentrations in loaded samples were quantifiedby BCA assay; n = 5/group, *P < 0.05 vs. Mock, t-test. E: total LAT-1 protein was quantified by immunoblotting in kidneys of BALB/c mice that received a renal subcapsular infusion of Gast-specific(delivery rate: 3 µg/day) or Mock (nonsilencing) siRNA. Results were corrected for expression of GAPDH protein; n = 5/group. F: blood pressure was measured from the aorta, via the femoral artery, under pentobarbital sodium anesthesia, in BALB/c mice that received a renal subcapsular infusion of Gast-specific (delivery rate: 3 µg/day) or Mock siRNA; n = 5/group, *P < 0.05 vs. Mock systolic blood pressure; $P < 0.05 vs. Mock mean arterial pressure (MAP); #P < 0.05 vs. Mock diastolic blood pressure; one-way factorial ANOVA, Newman-Keuls.

Fig. 6.

Colocalization of gastrin and aquaporin 1 in BALB/c mouse kidney that received a renal subcapsular infusion of Gast-specific or nonsilencing (Mock) siRNA. A–C: gastrin and aquaporin 1 colocalize in the TDL but not in TAL of mouse kidney subcapsularly infused with Mock siRNA. D–F: gastrin immunofluorescence is very weak in the TDL, but aquaporin 1 is well expressed in the TDL of mouse kidney subcapsularly infused with Gast-specific siRNA (Gastrin siRNA). Similar results were obtained in ≥3 separate experiments.