Chronic AT1 receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1 expression within the PVN in rats with heart failure

Abstract

Exercise training normalizes enhanced glutamatergic mechanisms within the paraventricular nucleus (PVN) concomitant with the normalization of increased plasma ANG II levels in rats with heart failure (HF). We tested whether ANG II type 1 (AT(1)) receptors are involved in the normalization of PVN glutamatergic mechanisms using chronic AT(1) receptor blockade with losartan (Los; 50 mg.kg(-1).day(-1) in drinking water for 3 wk). Left ventricular end-diastolic pressure was increased in both HF + vehicle (Veh) and HF + Los groups compared with sham-operated animals (Sham group), although it was significantly attenuated in the HF + Los group compared with the HF + Veh group. The effect of Los on cardiac function was similar to exercise training. At the highest dose of N-methyl-d-aspartate (NMDA; 200 pmol) injected into the PVN, the increase in renal sympathetic nerve activity was 93 +/- 13% in the HF + Veh group, which was significantly higher (P < 0.05) than the increase in the Sham + Veh (45 +/- 2%) and HF + Los (47 +/- 2%) groups. Relative NMDA receptor subunit NR(1) mRNA expression within the PVN was increased 120% in the HF + Veh group compared with the Sham + Veh group (P < 0.05) but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). NR(1) protein expression increased 87% in the HF + Veh group compared with the Sham + Veh group but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). Furthermore, in in vitro experiments using neuronal NG-108 cells, we found that ANG II treatment stimulated NR(1) protein expression and that Los significantly ameliorated the NR(1) expression induced by ANG II. These data are consistent with our hypothesis that chronic AT(1) receptor blockade normalizes glutamatergic mechanisms within the PVN in rats with HF.

Fig. 1.

Injection sites within and around the paraventricular nucleus (PVN). Schematic representations of serial coronal sections from the rostral (−1.4) to caudal (−2.1) regions of the PVN are shown. The distance (in mm) is shown for each section according to the Paxinos and Watson atlas (25). Each solid square represents the site of termination of an injection within the PVN. Each solid triangle represents the site of termination of an injection outside of the PVN, the data from which were not included for analysis. Inset: representative photomicrograph of an injection site within the PVN of a rat. AH, anterior hypothalamus; f, fornix; 3V, third ventricle; SO, supraoptic nucleus; ox, optic tract.

Fig. 2.

Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate [HR; in beats/min (bpm)] responses to N-methyl-d-aspartate (NMDA) injected into the PVN. Segments of original recordings from individual rats from each experimental group [sham operated (Sham) + vehicle (Veh), HF + Veh, Sham + losartan (Los), and HF + Los groups] show the responses of RSNA, integrated (Int)RSNA, MAP, and HR to different doses of NMDA injected into the PVN.

Fig. 3.

Mean RSNA, MAP, and HR responses to NMDA injected into the PVN. A: mean changes in RSNA after injections of NMDA into the PVN. B: mean changes in MAP after injections of NMDA into the PVN. C: mean changes in HR after injections of NMDA into the PVN. *P < 0.05 vs. Sham groups within the same dose of NMDA; #P < 0.05 vs. Veh groups within the same dose of NMDA.

Fig. 4.

mRNA expression of the NMDA receptor NR₁ subunit in punched PVN samples measured by real-time RT-PCR. Expression was measured by determining at which cycle the fluorescence of each sample rose above threshold (C_t). Lower C_t values correspond to higher expression. Shown are examples of original real-time RT-PCR traces comparing the Sham + Veh and HF + Veh groups (top) and the HF + Veh and HF + Los groups (bottom). Insets: rpl19 (reference gene) traces.

Fig. 5.

mRNA expression of the NMDA receptor NR₁ subunit in punched PVN samples measured by real-time RT-PCR. Mean data of the relative expression of NR₁ mRNA to rpl19 mRNA are shown. *P < 0.05 vs. Sham groups; #P < 0.05 vs. Veh groups.

Fig. 6.

Protein expression of the NMDA receptor NR₁ subunit in punched PVN samples measured by Western blot analysis. A: example of visualized electrophoresis bands of NR₁ protein. B: mean data of band densities of NR₁ relative to β-tubulin. *P < 0.05 vs. Sham groups; #P < 0.05 vs. Veh groups.

Fig. 7.

Control protein expression of NMDA receptor subunits. A: protein expression of the NR₁ subunit in punched supraoptic nucleus (SON), lateral hypothalamus (LH), and cortex samples measured by Western blot analysis. There were no significant differences between groups. B: NMDA receptor subunit NR_2B protein expression in punched PVN samples measured by Western blot analysis. There were no significant differences between groups.

Fig. 8.

A: protein expression of NMDA receptor subunit NR₁ (Western blot and densitometric analysis) in ANG II-treated NG-108 cells. B: effect of Los on the expression of NMDA receptor subunit NR₁ protein in ANG II-treated NG-108 cells. *P < 0.05 vs. control; #P < 0.05 vs. ANG II.