Antihypertensive effect of etamicastat in dopamine D2 receptor-deficient mice

Abstract

Abnormalities of the D₂R gene (DRD2) play a role in the pathogenesis of human essential hypertension; variants of the DRD2 have been reported to be associated with hypertension. Disruption of Drd2 (D₂^-/-) in mice increases blood pressure. The hypertension of D₂^-/- mice has been related, in part, to increased sympathetic activity, renal oxidative stress, and renal endothelin B receptor (ETBR) expression. We tested in D₂^-/- mice the effect of etamicastat, a reversible peripheral inhibitor of dopamine-β-hydroxylase that reduces the biosynthesis of norepinephrine from dopamine and decreases sympathetic nerve activity. Blood pressure was measured in anesthetized D₂^-/- mice treated with etamicastat by gavage, (10 mg/kg), conscious D₂^-/- mice, and D₂^+/+ littermates, and mice with the D₂R selectively silenced in the kidney, treated with etamicastat in the drinking water (10 mg/kg per day). Tissue and urinary catecholamines and renal expression of selected G protein-coupled receptors, enzymes related to the production of reactive oxygen species, and sodium transporters were also measured. Etamicastat decreased blood pressure both in anesthetized and conscious D₂^-/- mice and mice with renal-selective silencing of D₂R to levels similar or close to those measured in D₂^+/+ littermates. Etamicastat decreased cardiac and renal norepinephrine and increased cardiac and urinary dopamine levels in D₂^-/- mice. It also normalized the increased renal protein expressions of ETBR, NADPH oxidase isoenzymes, and urinary 8-isoprostane, as well as renal NHE3 and NCC, and increased the renal expression of D₁R but not D₅R in D₂^-/- mice. In conclusion, etamicastat is effective in normalizing the increased blood pressure and some of the abnormal renal biochemical alterations of D₂^-/- mice.

Figure 1.. Effect of etamicastat on blood pressure in anesthetized and conscious D₂^+/+ and D₂^−/−mice.

A. Etamicastat (Etam, 1O mg/kg) or vehicle was administered by gavage. Blood pressure was measured under pentobarbital anesthesia 9 and 18 h after etamicastat administration in different groups of D₂^+/+ and D₂^−/− mice. n=4–5/group; *P<O.O5 vs all others, one-way ANOVA followed by Holm-Sidak test B. Etamicastat (Etam, 1O mg/kg/day) or vehicle was added to the drinking water. Blood pressure was measured by telemetry in conscious mice. Values are means of 6 h measurements (one per h). D₂^+/+ n=5; D₂^−/− n=4. *P<O.O5 vs D₂^+/+ one-way ANOVA followed by Holm-Sidak test. C. Etamicastat (1O or 5O mg/kg/day) was added to the drinking water. Blood pressure was measured by telemetry in conscious mice. Values shown are means of 6 hr measurements during the day and night (one reading per h after 5 days on each treatment). D₂^+/+ n=4; D₂^−/− n=3. * P<O.O5 vs D₂^−/− vehicle or same treatment D₂^+/+, two-way ANOVA followed by Holm-Sidak test.

Figure 2.. Effect of etamicastat on blood pressure in mice with renal-selective silencing of the D₂R.

Renal cortical Drd2 was silenced by the renal subcapsular infusion of Drd2 siRNA, via an osmotic minipump for seven days in uninephrectomized adult male C57BL/6J mice (see Methods). Etamicastat (Etam, 1O mg/kg/day in the drinking water) was started immediately after pump implantation. Blood pressure was measured under anesthesia before and 7 days after pump implantation. n=5/group; *P<O.O5 vs all others; one-way ANOVA followed by Holm-Sidak test. Two-way ANOVA positive for effect of D₂R siRNA and etamicastat.

Figure 3.. Effect of etamicastat on norepinephrine (NE) and dopamine (DA) content in heart, and urine of D₂^+/+ and D₂^−/− mice.

Etamicastat (Etam, 10 mg/kg/dose) or vehicle was administered by gavage. Urine collection was started immediately after gavage and lasted for 18 h. Tissues were collected after ending the urine collection. Urine and tissue NE (A) and DA (B) were measured by HPLC-ED, n=5–8/group. The tissue or urine DA/NE ratio (C) was calculated. *P<0.05 vs all others; **P<0.05 vs same group vehicle-treated mice; one-way ANOVA followed by Holm-Sidak test.

Figure 4.. Effect of etamicastat on the renal expression of dopamine, angiotensin II type 1, and endothelin B receptors.

Etamicastat (Etam, 10 mg/kg/day) was added to the drinking water for 5 days. Receptor expression was determined by immunoblotting. A. Receptor expression in vehicle-treated mice; n=8– 10/group; B. Receptor expression in etamicastat-treated mice; n=4–5/group. *P<0.05 vs D₂^+/+ mice; one-way ANOVA followed by Holm-Sidak test.

Figure 5.. Effect of etamicastat on the expression of ROS-related enzymes.

Etamicastat (Etam, 10 mg/kg/day) was added to the drinking water for 5 days. Expression of HO-1, HO-2, NOX1, NOX2, and NOX4 was determined by immunoblotting. A. Expression of HO and NOX isoforms in vehicle-treated mice; n=5/group; B. Expression of HO and NOX isoforms in etamicastat-treated mice; n=5/group. *P<0.05 vs D₂^+/+ mice; one-way ANOVA followed by Holm-Sidak test.

Figure 6.. Effect of etamicastat on the expression of renal sodium transporters, exchangers, channels, and pump.

Etamicastat (Etam, 10 mg/kg/day) was added to the drinking water for 5 days. Renal expression of sodium transporters, exchangers, channels, and pump was determined by immunoblotting. A. Expression in vehicle-treated mice; n=4–5/group; B. Expression in etamicastat-treated mice; n=4/group. *P<0.05 vs D₂^+/+ mice; one-way ANOVA followed by Holm-Sidak test.