Exercise training attenuates renovascular hypertension partly via RAS- ROS- glutamate pathway in the hypothalamic paraventricular nucleus

Abstract

Exercise training (ExT) has been reported to benefit hypertension; however, the exact mechanisms involved are unclear. We hypothesized that ExT attenuates hypertension, in part, through the renin-angiotensin system (RAS), reactive oxygen species (ROS), and glutamate in the paraventricular nucleus (PVN). Two-kidney, one-clip (2K1C) renovascular hypertensive rats were assigned to sedentary (Sed) or treadmill running groups for eight weeks. Dizocilpine (MK801), a glutamate receptor blocker, or losartan (Los), an angiotensin II type1 receptor (AT1-R) blocker, were microinjected into the PVN at the end of the experiment. We found that 2K1C rats had higher mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). These rats also had excessive oxidative stress and overactivated RAS in PVN. Eight weeks of ExT significantly decreased MAP and RSNA in 2K1C hypertensive rats. ExT inhibited angiotensin-converting enzyme (ACE), AT1-R, and glutamate in the PVN, and angiotensin II (ANG II) in the plasma. Moreover, ExT attenuated ROS by augmenting copper/zinc superoxide dismutase (Cu/Zn-SOD) and decreasing p^47phox and gp^91phox in the PVN. MK801or Los significantly decreased blood pressure in rats. Together, these findings suggest that the beneficial effects of ExT on renovascular hypertension may be, in part, through the RAS-ROS-glutamate pathway in the PVN.

Figure 1. Effects of exercise training on mean arterial pressure in 2K1C rats.

MAP was increased gradually after 2K1C-surgery compared with SHAM groups. ExT attenuated 2K1C-induced hypertension compared with 2K1C + Sed rats. MAP: mean arterial pressure; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 2. Effects of eight-week exercise training or PVN microinjection of MK801 or Los on RSNA in 2K1C rats and SHAM rats.

RSNA was increased in 2K1C rats compared with SHAM rats. ExT attenuated RSNA compared with 2K1C + Sed rats. Treatment with PVN microinjection of MK801 or Los attenuated RSNA of 2K1C rats. PVN microinjection of Los exhibited lower RSNA (% of max) compared with PVN microinjection of MK801 in 2K1C rats. RSNA: renal sympathetic nerve activity; PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary; MK801: dizocilpine; Los: losartan. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 vs 2K1C groups (2K1C + ExT or 2K1C + Sed); ^#P < 0.05 vs 2K1C+PVN microinjection of MK801 (2K1C + ExT+ PVN microinjection of MK801 or 2K1C + Sed + PVN microinjection of MK801.

Figure 3. Effects of exercise training on the level of the ACE in the PVN in 2K1C rats by immunofluorescence staining.

2K1C rats had higher levels of ACE compared with SHAM rats. ExT decreased ACE expression in the PVN compared with 2K1C + Sed rats. (a) A representative immunofluorescence staining of ACE. (b) Densitometric analysis of immunofluorescent intensity of ACE. ACE: angiotensin-converting enzyme; PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 4. Effects of exercise training on the levels of the AT1-R in the PVN in 2K1C rats by immunofluorescence staining.

2K1C rats had higher levels of AT1-R compared with SHAM rats. ExT decreased AT1-R expression in the PVN compared with 2K1C + Sed rats. (a) A representative immunofluorescence staining of AT1-R. (b) Densitometric analysis of immunofluorescent intensity of AT1-R. AT1-R: angiotensin II type1 receptor; 2K1C: two-kidney, one-clip; PVN: hypothalamic paraventricular nucleus; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. 2K1C *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 5. Effects of exercise training on the level of the AT1-R in the PVN and ANGII in the plasma in 2K1C rats.

2K1C rats had higher protein expression of AT1-R in the PVN and higher level of ANGII in the plasma compared with SHAM rats. ExT significantly decreased AT1-R expression in the PVN and ANGII in the plasma compared with 2K1C + Sed rats. AT1-R: angiotensin II type1 receptor; ANG II: angiotensin II; PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed. (a) Levels of ANGII in the plasma in different groups. (b) Representative immunoblot of AT1-R. (c) Densitometric analysis of protein expression of AT1-R.

Figure 6. Effects of exercise training on the levels of the superoxide in the PVN in 2K1C rats.

ROS activity was measured by fluorescent-labeled DHE staining. 2K1C rats had stronger fluorescence intensity labeled with DHE compared with SHAM rats. ExT significantly decreased DHE staining in the PVN of 2K1C rats. (a) A representative immunofluorescence image of DHE. (b) Densitometric analysis of immunofluorescent intensity of DHE in the PVN in different groups. DHE: dihydroethidium; PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 7. Effects of exercise training on gp^91phox and p^47phox in the PVN in 2K1C rats by immunofluorescence staining.

2K1C rats had higher levels of gp^91phox and p^47phox compared with SHAM rats. ExT decreased gp^91phox and p^47phox expression in the PVN compared with 2K1C + Sed rats. (a) A representative immunofluorescence staining of gp^91phox. (b) A representative immunofluorescence staining of p^47phox. (c) Densitometric analysis of immunofluorescent intensity of gp^91phox in the PVN in different groups. (d) Densitometric analysis of immunofluorescent intensity of p^47phox in the PVN in different groups. PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 8. Effects of exercise training on the levels of p^47phox and Cu/Zn-SOD in the PVN in 2K1C rats by Western blotting.

2K1C rats had a lower level of Cu/Zn-SOD and a higher level of p^47phox compare with SHAM rats. ExT enhanced the expression of Cu/Zn-SOD and attenuated the expression of p^47phox. (a) Representative immunoblot; (b) Densitometric analysis of protein expression of Cu/Zn-SOD and p^47phox in the PVN in different groups. Cu/Zn-SOD: copper/zinc superoxide dismutase; PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM+ Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.

Figure 9. Effects of exercise training on the levels of glutamate in the PVN in 2K1C rats.

2K1C rats had higher levels of glutamate in the PVN compare with SHAM rats. ExT attenuated the level of glutamate compared with 2K1C + Sed rats. PVN: hypothalamic paraventricular nucleus; 2K1C: two-kidney, one-clip; ExT: exercise training; Sed: sedentary. Values are expressed as means ± SE. *P < 0.05 vs SHAM groups (SHAM + Sed or SHAM + ExT); ^†P < 0.05 2K1C + ExT vs 2K1C + Sed.