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. 2016 Apr 22;5(4):e002448.
doi: 10.1161/JAHA.115.002448.

Inhibition of D4 Dopamine Receptors on Insulin Receptor Expression and Effect in Renal Proximal Tubule Cells

Ye Zhang  1 , Hongmei Ren  1 , Xi Lu  1 , Duofen He  1 , Yu Han  1 , Hongyong Wang  1 , Chunyu Zeng  2 , Weibin Shi  2
Affiliations

Inhibition of D4 Dopamine Receptors on Insulin Receptor Expression and Effect in Renal Proximal Tubule Cells

Ye Zhang et al. J Am Heart Assoc. .

Abstract

Background: Ion transport in the renal proximal tubule (RPT), which is increased in essential hypertension, is regulated by numerous hormones and humoral factors, including insulin and dopamine. Activation of dopamine receptor inhibits sodium reabsorption, whereas activation of insulin receptor increases sodium reabsorption in RPTs, and hyperinsulinemic animals and patients have defective renal dopaminergic system. We presume that there is an inhibition of D4 receptor on insulin receptor expression and effect, and the regulation is lost in spontaneously hypertensive rats (SHRs).

Methods and results: Insulin receptor expression was determined by immunoblotting, and Na(+)-K(+)-ATPase activity was detected in both Wistar-Kyoto (WKY) and SHR RPT cells. Stimulation of D4 receptor with PD168077 decreased expression of insulin receptors, which was blocked in the presence of the calcium-channel blocker, nicardipine (10(-6) mol/L per 24 hours), in cell culture medium without calcium or in the presence of inositol 1,4,5-trisphosphate (IP3) receptor blocker (2-aminoethyl diphenylborinate [2-ADB]; 10(-6) mol/L per 24 hours), indicating that extracellular calcium entry and calcium release from the endoplasmic reticulum were involved in the signal pathway. Stimulation of the insulin receptor stimulated Na(+)-K(+)-ATPase activity, whereas pretreatment with PD168077 for 24 hours decreased the inhibitory effects of insulin receptor on Na(+)-K(+)-ATPase activity in WKY cells. However, in SHR cells, inhibition of D4 receptor on insulin receptor expression and effect were lost.

Conclusions: Activation of D4 receptor inhibits insulin receptor expression in RPT cells from WKY rats. The aberrant inhibition of D4 receptor on insulin receptor expression and effect might be involved in the pathogenesis of essential hypertension.

Keywords: dopamine receptor; hypertension; insulin; renal proximal tubule cells.

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Figures

Figure 1
Figure 1
Effect of D4 receptor on insulin receptor expression in Wistar‐Kyoto (WKY) renal proximal tubule (RPT) cells. A, Concentration response of insulin receptor protein expression in WKY cells treated with different concentrations of the D4 receptor agonist, PD168077, for 24 hours. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=6; *P<0.05 vs control). B, Time course of insulin receptor protein expression in WKY cells treated with D4 receptor agonist, PD168077 (10−7 mol/L) for different periods. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=6; *P<0.05 vs control). C, Effect of a D4 receptor agonist (PD168077) and a D4 receptor antagonist (L745870) on insulin receptor expression in WKY cells. Cells were incubated with the indicated reagents (PD168077, 10−7 mol/L; L745870, 10−6 mol/L) for 24 hours. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=9; *P<0.05 vs others). D and E, Effect of a D4 receptor agonist (ABT724) and a D4 receptor antagonist (L750667) on insulin receptor expression in WKY cells. Cells were incubated with the indicated reagents (ABT724, 10−7 mol/L; L750667, 10−6 mol/L) for 24 hours. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=7; *P<0.05 vs others). F and G, Effect of D4 receptor siRNA on regulation of D4 receptor on insulin receptor expression in WKY cells. WKY cells were incubated with D4 receptor siRNA (50 nm, 24 hours), D4 receptor expressions were determined by immunoblotting (F, n=5; *P<0.05 vs others). Cells were incubated with the indicated reagents (PD168077, 10−7 mol/L) for 24 hours with or without the presence of D4 receptor siRNA. Insulin receptor expressions were determined by immunoblotting. Results are expressed as the ratio of insulin receptor and α‐actin densities (G, n=8; *P<0.05 vs others).
Figure 2
Figure 2
Effect of D4 receptor on insulin receptor phosphorylation in Wistar‐Kyoto (WKY) renal proximal tubule (RPT) cells. A, Effect of a D4 receptor agonist (PD168077) and a D4 receptor antagonist (L745870) on insulin receptor phosphorylation in WKY cells. Cells were incubated with the indicated reagents (PD168077, 10−7 mol/L; L745870, 10−6 mol/L) for 15 minutes. Results are expressed as the ratio of phosphorylated insulin receptor and α‐actin densities (n=9; *P<0.05 vs control). B, Effect of a D4 receptor agonist (PD168077, 10−7 mol/L per 15 minutes) on insulin receptor phosphorylation in WKY and SHR RPT cells (n=9; *P<0.05 vs control). SHR indicates spontaneously hypertensive rat.
Figure 3
Figure 3
Extracellular and intracelluar calcium mediates the inhibitory effect of D4 receptor on insulin receptor expression in renal proximal tubule (RPT) cells. A, Effect of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), and the calcium‐channel blocker, nicardipine (10−6 mol/L per 24 hours), on insulin receptor protein in Wistar‐Kyoto (WKY) RPT cells. Cells were incubated with the indicated reagents. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=6; *P<0.05 vs others). B, Effect of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), on insulin receptor expression in cell culture medium with or without calcium in WKY RPT cells. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=6; *P<0.05 vs others). C, Effect of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), and inositol 1,4,5‐trisphosphate (IP3) receptor blocker (2‐aminoethoxydiphenyl borate [2‐ADB]; 10−6 mol/L per 24 hours) on insulin receptor protein in WKY RPT cells. Cells were incubated with the indicated reagents. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=9; *P<0.05 vs others). D, Effect of calcium‐channel activator BAY‐K8644 (10−6 mol/L per 24 hours) (I) or IP3 (10−6 mol/L per 24 hours) (II) on insulin receptor protein expression in WKY RPT cells. Cells were incubated with the indicated reagents. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=9; *P<0.05 vs others). E and F, Effect of the D4 receptor agonist, PD168077, on intracellular calcium concentration in the presence or absence of pharmacological agents in WKY and spontaneously hypertensive rats (SHRs) cells. Intracellular calcium concentration of RPT cells with or without PD168077 (10−7 mol/L per 15 minutes) treatment were determined by laser confocal microscopy. The stimulatory effect of PD168077 on intracellular calcium concentration was also tested in the presence of nicardipine (10−6 mol/L per 15 minutes) or 2‐ADB (10−6 mol/L per 15 minutes). Representative tracings are shown (E) and graphs of the data are shown (F) (Δ[Ca2+]i shows the difference in calcium concentration between 0 and 60 seconds). (n=6; *P<0.05 vs others).
Figure 4
Figure 4
Differential effects of D4 receptor on insulin receptor expression in renal proximal tubule (RPT) cells from Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHRs). A, Differential effects of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), on insulin receptor protein expression in RPT cells from WKY and SHRs. Cells were incubated at the indicated time points and concentrations. Results are expressed as the ratio of insulin receptor and α‐actin densities (n=6; *P<0.05 vs control). B, Differential effects of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), on insulin receptor mRNA expression in RPT cells from WKY and SHRs. Cells were incubated at indicated time points and concentrations. Results are expressed as the ratio of insulin receptor and β‐actin densities (n=9; *P<0.05 vs control). C, Effect of the D4 receptor agonist, PD168077 (10−7 mol/L per 24 hours), in the presence of calcium‐channel blocker (nicardipine, 10−6 mol/L per 24 hours) (I) or inositol 1,4,5‐trisphosphate (IP3) receptor blocker (2‐ADB; 10−6 mol/L per 24 hours) (II) on insulin receptor mRNA expression in WKY RPT cells. Cells were incubated with the indicated reagents. Real‐time quantitative polymerase chain reaction data were analyzed using the comparative CT method (n=9; *P<0.05 vs others). CT indicates threshold cycle.
Figure 5
Figure 5
Effect of pretreatment with D4 receptor on the stimulatory effect of insulin receptor on Na+‐K+‐ATPase activity in Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHRs) cells. A, Renal proximal tubule (RPT) cells from WKY and SHRs were treated with PD168077 (10−7 mol/L) or vehicle (dH2O) for 24 hours. Na+‐K+‐ATPase activity were investigated in each group. Results are expressed as percent change of control (*P<0.05 vs control; # P<0.05 vs insulin; n=7). B, RPT cells from WKY and SHRs were treated with PD168077 (10−7 mol/L) or vehicle (dH 2O) for 24 hours. Then, cells were washed 3 times (15 min/times) with serum‐free culture medium to remove all the added PD168077 and kept in serum‐free culture medium for 2 hours before Na+‐K+‐ATPase activity was investigated (*P<0.05 vs control; # P<0.05 vs insulin; n=8). C, Cells were pretreated with PD168077 (10−7 mol/L) or vehicle (dH2O) for 24 hours. After PD168077 pretreatment, cells were washed 3 times (15 min/times) with serum‐free culture medium to remove all the added PD168077, kept in serum‐free culture medium for 2 hours, and then treated with insulin (10−7 mol/L) for 15 minutes. Results are expressed as percent change of control (*P<0.05 vs control; # P<0.05 vs insulin; n=8).
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References

    1. Rosendorff C. Hypertension and coronary artery disease: a summary of the American Heart Association scientific statement. J Clin Hypertens (Greenwich). 2007;9:790–795. - PubMed
    1. McDonough AA, Biemesderfer D. Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule? Curr Opin Nephrol Hypertens. 2003;12:533–541. - PubMed
    1. Hussain T, Lokhandwala MF. Renal dopamine receptors and hypertension. Exp Biol Med (Maywood). 2003;228:134–142. - PubMed
    1. Sowers JR. Insulin resistance and hypertension. Am J Physiol Heart Circ Physiol. 2004;286:H1597–H1602. - PubMed
    1. Wei Y, Chen K, Whaley‐Connell AT, Stump CS, Ibdah JA, Sowers JR. Skeletal muscle insulin resistance: role of inflammatory cytokines and reactive oxygen species. Am J Physiol Regul Integr Comp Physiol. 2008;294:R673–R680. - PubMed

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