Inhibition of the adenosine2A receptor-epoxyeicosatrienoic acid pathway renders Dahl salt-resistant rats hypertensive

Abstract

Adenosine-induced renovasodilation in Dahl rats is mediated via activation of adenosine(2A) receptors (A(2A)Rs) and stimulation of epoxyeicosatrienoic acid (EET) synthesis. Unlike Dahl salt-resistant rats, salt-sensitive rats exhibit an inability to upregulate the A(2A)R-EET pathway with salt loading; therefore, we examined the effect of in vivo inhibition of the A(2A)R-EET pathway on blood pressure and the natriuretic response to salt-loading in Dahl salt-resistant rats. N-Methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 20 mg/kg per day), an epoxygenase inhibitor, or ZM241385 (ZM; 5 mg/kg per day), an A(2A)R antagonist, was given daily as an IV bolus dose for 3 days before and after placing rats on high salt intake (2% saline). After 3 days of high salt, systolic blood pressure per 24 hours increased from 108+/-2 mm Hg to 136+/-5 mm Hg and 140+/-4 mm Hg when treated with MS-PPOH or ZM, respectively (P<0.001). Plasma levels of EETs and dihydroxyeicosatrienoic acids during salt loading and MS-PPOH (29.3+/-1.8 ng/mL) or ZM treatment (9.8+/-0.5 ng/mL) did not increase to the same extent as in vehicle-treated rats (59.4+/-1.7 ng/mL; P<0.001), and renal levels of EETs+dihydroxyeicosatrienoic acids were 2-fold lower with MS-PPOH or ZM treatment. On day 3 of the high salt intake, MS-PPOH- and ZM-treated rats exhibited a positive Na(+) balance, and plasma Na(+) levels were significantly increased (163.3+/-1.2 and 158.1+/-4.5 mEq/L, respectively) compared with vehicle-treated rats (142.1+/-1 mEq/L), reflecting a diminished natriuretic capacity. These data support a role for the A(2A)R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading.

Figure 1. Effect of epoxygenase inhibition or A_2AR antagonism on systolic blood pressure (BP) of Dahl SR rats after 3 days treatment (A) and on systolic, diastolic and mean BP and heart rate on Days 1-3 of HS intake (B)

Dahl SR rats on NS diet were pretreated with the epoxygenase inhibitor, MS-PPOH (20 mg/Kg/day), for 3 days prior to switching rats to a HS (2% saline drinking water) intake. After 3 days of HS intake with MS-PPOH treatment, MS-PPOH was withdrawn while rats remained on HS intake for 3 more days. Rats were then switched to NS diet for 7 days. The selective A_2AR antagonist, ZM 241385 (5 mg/Kg/day), was then given for 3 days prior to switching rats back to HS intake. ZM 241385 treatment continued during 3 days of HS intake. Data are expressed as means ± SEM; n = 6-7; * p<0.05, ** p<0.005, *** p<0.001 vs. NS; ^# p<0.05, ^## p<0.005, ^### p<0.001 vs. control on each day. Note scale differences.

Figure 2. Effect of epoxygenase inhibition or A_2AR antagonism on the natriuretic response to salt-loading in Dahl SR rats

The daily difference between sodium intake and urinary sodium excretion was compared between Dahl SR rats placed on NS (0.4% NaCl) diet, HS (2% saline drinking water) intake for 3 days, and HS intake for 3 days with either epoxygenase inhibition (HS + MS-PPOH; 20 mg/Kg/day) or A_2AR antagonism (HS + ZM 241385; 5 mg/Kg/day). Data are expressed as means ± SEM; n = 6-9; * p<0.05, *** p<0.001 vs. control.

Figure 3. Effect of epoxygenase inhibition or A_2AR antagonism on plasma levels of Na⁺ after 3 days of HS intake in Dahl SR rats

Plasma levels of Na⁺ were measured after 3 days of HS (2% saline drinking water) intake or 3 days of HS intake with either epoxygenase inhibition (HS + MS-PPOH; 20 mg/Kg/day) or A_2AR antagonism (HS + ZM 241385; 5 mg/Kg/day). Data are expressed as means ± SEM; n = 4-6; * p<0.01, ** p<0.001 vs. HS.

Figure 4. Effect of epoxygenase inhibition or A_2AR antagonism on plasma levels of CYP-AA metabolites in Dahl SR rats

Plasma levels of CYP-AA metabolites were measured rats during NS (0.4% NaCl) diet, and after 3 days of HS (2% saline drinking water) intake or 3 days of HS intake with either epoxygenase inhibition (HS + MS-PPOH; 20 mg/Kg/day) or A_2AR antagonism (HS + ZM 241385; 5 mg/Kg/day). Data are expressed as means ± SEM; n = 4-6; *** p<0.001 vs. NS, †† p<0.001 vs. HS.

Figure 5. Effect of epoxygenase inhibition or A_2AR antagonism on renal levels of CYP-AA metabolites in Dahl SR rats

Renal levels of epoxyeicosatrienoic acid (EET) and dihydroxyeicosatrienoic acid (DHTs) were measured rats after 3 days of HS (2% saline drinking water) intake or 3 days of HS intake with either epoxygenase inhibition (HS + MS-PPOH; 20 mg/Kg/day) or A_2AR antagonism (HS + ZM 241385; 5 mg/Kg/day). Data are expressed as means ± SEM; n = 4; * p<0.05, ** p<0.01 vs. HS.