Role of the adenosine(2A) receptor-epoxyeicosatrienoic acid pathway in the development of salt-sensitive hypertension

Abstract

Activation of rat adenosine(2A) receptors (A(2A) R) dilates preglomerular microvessels, an effect mediated by epoxyeicosatrienoic acids (EETs). High salt (HS) intake increases epoxygenase activity and adenosine levels. A greater vasodilator response to a stable adenosine analog, 2-chloroadenosine (2-CA), was seen in kidneys obtained from HS-fed rats which was mediated by increased EET release. Because this pathway is antipressor, we examined the role of the A(2A) R-EET pathway in a genetic model of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rats. Dahl salt resistant (SR) rats fed a HS diet demonstrated a greater renal vasodilator response to 2-CA. In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed normal salt (NS) or HS diet. In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A) R and cytochrome P450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake. In vivo inhibition of the A(2A) R-EET pathway in Dahl SR rats fed a HS diet results in reduced renal EETs levels, diminished natriuretic capacity and hypertension, thus supporting a role for the A(2A) R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading. An inability of Dahl SS rats to upregulate the A(2A) R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.

Figure 1. Responsiveness of isolated, perfused rat kidneys to 10 µg 2-chloroadenosine (2-CA)

Changes in perfusion pressure and area of response to a bolus injection of 10 µg 2-CA were compared in kidneys obtained from normal salt (NS)-fed rats vs. high salt (HS)-fed rats for 7 days in the absence and presence of MS-PPOH (12 µM). Data expressed as means ± SE; n = 4. *P < 0.05 vs. control (i.e., NS vs. NS + MS-PPOH or HS vs. HS + MS-PPOH). #P < 0.05 NS vs. HS. *#P < 0.05 HS vs. NS + MS-PPOH.

Figure 2. Renal release of CYP epoxygenase metabolites in response to 10 µg 2-CA

Release of total EETs + DHTs in response to 10 µg 2-CA was compared in NS- and HS-fed rats in the absence and presence of MS-PPOH. Total EET + DHT release in response to sodium nitroprusside (SNP; 25 ng) in HS-fed rats was also measured. Data are expressed as means ± SE; n = 4–5. *P < 0.05 vs. control (i.e., HS vs. HS + MS-PPOH). #P < 0.05 NS vs. HS.

Figure 3. Effect of A_2A R inhibition on the responsiveness of isolated, perfused rat kidneys to 10 µg 2-CA under NS intake

A: vascular response to 10 µg 2-CA was assessed before and after A_2AR blockade by ZM-241385 (10 µM). B: total release of EET + DHT in response to 10 µg 2-CA was compared in the absence and presence of ZM-241385. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. control.

Figure 4. Urinary purine levels after 7 days of HS diet in Dahl SR and SS rats

Levels of urinary purines (i.e., adenosine, hypoxanthine, xanthine, and inosine) were measured in Dahl SR and SS rats on NS diet and after 7 days of HS diet. Data are means ± SE; n = 5–7. *P < 0.05.

Figure 5. Renal release of cytochrome P-450 (CYP) epoxygenase metabolites in response to 2-CA in Dahl SR and SS rats

Data are means ± SE; n = 4–5. *P < 0.05 vs. control (i.e., HS vs. HS + MS-PPOH). #P < 0.05 NS vs. HS.

Figure 6. Effect of epoxygenase inhibition or A_2A R antagonism on systolic BP of Dahl SR rats after 3 days treatment

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_2A R 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<0.001 vs. NS.

Figure 7. Effect of epoxygenase inhibition or A_2A R 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_2A R 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.