Effects of Inducible Nitric Oxide Synthase Inhibition on Cardiovascular Risk of Adult Endotoxemic Female Rats: Role of Estrogen

Abstract

Aim: Autonomic modulation responds to ovarian hormones and estrogen increases nitric oxide bioavailability. Also, females have minor susceptibility to sepsis and a higher survival rate. However, few studies have evaluated the role of estrogen in cardiovascular, autonomic, and oxidative parameters during initial endotoxemia and under inducible nitric oxide synthase (iNOS) inhibition in female rats. Methods: Female wistar rats were subjected to ovariectomy and divided into three groups: OVX (ovariectomized), OVX+E (OVX plus daily estradiol) and SHAM (false surgery). After 8 weeks, mean arterial pressure (MAP) and heart rate (HR) were recorded in non-anesthetized catheterized rats, before and after intravenous LPS injection, preceded by S-methylisothiourea sulfate (SMT) injection, or sterile saline. Cardiovascular recordings underwent spectral analysis for evaluation of autonomic modulation. Two hours after LPS, plasma was collected to assess total radical-trapping antioxidant (TRAP), nitrite levels (NO2), lipoperoxidation (LOOH), and paraoxonase 1 (PON1) activity. Results: Two hours after LPS, females treated with SMT presented a decrease of MAP, when compared to saline-LPS groups. At this same time, all SMT+LPS groups presented an increase of sympathetic and a decrease of parasympathetic modulation of HR. Two hours after saline+LPS, OVX presented decreased total radical-trapping antioxidant (TRAP) compared to SHAM. When treated with SMT+LPS, OVX did not altered TRAP, while estradiol reduced LOOH levels. Conclusion: iNOS would be responsible for sympathetic inhibition and consumption of antioxidant reserves of females during endotoxemia, since iNOS is inhibited, treatment with estradiol could be protective in inflammatory challenges.

Keywords: blood pressure; endotoxemia; heart rate; lipopolysaccharide; nitric oxide; ovariectomy.

FIGURE 1

Experimental protocol. (I) and (II) sham or ovariectomized -operated rats were bred for 8 weeks and submitted to catheterization of femoral artery and vein. (III) OVX+E: OVX rats were treated with estradiol valerate (1 mg kg^-1, gavage, once daily) for 8 weeks, beginning 24 h after ovariectomy, and submitted to catheterization of femoral artery and vein. 24 h after catheterization, blood pressure was recording for 10 min and saline (0.9%, i.v.) or SMT (3 mg kg^-1, i.v.) was injected. After 10 min, LPS was injected (5 mg kg^-1, i.v.) and blood pressure recording continued for 2 h. LPS, lipopolysaccharide; SMT, S-methylisothiourea.

FIGURE 2

Effects of daily estradiol treatment (1 mg kg^-1) during 8 weeks on body weight gain (A), tibia length (B) and relation between uterus weight and tibia length (C). Sham-operated (SHAM), ovariectomized (OVX), or estrogen-treated OVX (OVX+E) groups. Graph (A) represents mean ± SEM, (B,C) are bar graphs showing median ± interquartile ranges. ^∗P < 0.01 vs. SHAM; ^#P < 0.01 vs. OVX. Weight gain = body weight at the time of euthanasia – body weight on the day of surgery. The number of rats per group is indicated in the figure.

FIGURE 3

Typical recordings of pulsatile arterial pressure (PAP, mmHg), mean arterial pressure (MAP, mmHg) and heart rate (HR, bpm) illustrating the cardiovascular responses to intravenous LPS injection (5 mg kg^-1) in non-anesthetized rats, preceded by saline or SMT (3 mg kg^-1) injection, 8 weeks post sham-operation (SHAM), bilateral ovariectomy (OVX), or OVX plus daily estradiol treatment (OVX+E). Arrows indicate the time of injections.

FIGURE 4

Time-course response to LPS (5 mg kg^-1) of MAP (A) and HR (B) after saline (0.9%) injection, and MAP (C) and HR (D) after SMT (3 mg kg^-1) injection. Mean ± SEM of five minutes before and the respective minute after LPS administration. Comparison of area under the curves (AUC) of MAP (E) and HR (F). Differences of corresponding saline group: (S) SHAM, (O) OVX, and (E) OVX+E. Different from SHAM (^∗) inside the treatment, (P < 0.05). The number of rats per group is indicated in the (A,C).

FIGURE 5

Cardiovascular and spectral hemodynamic data 2 h after LPS injection (5 mg kg^-1) for sham-operated (SHAM), ovariectomized (OVX), or estrogen-treated OVX (OVX+E) groups treated with SMT (3 mg kg^-1) or vehicle (saline 0.9%, 1 ml kg^-1) 10 min before LPS. (A) Mean arterial pressure (MAP), (B) spectral density of systolic blood pressure in the low-frequency (SBP-LF), (C) heart rate (HR), (D) spectral densities of interbeat intervals (IBI) in the low-frequency (IBI-LFnu), (E) IBI in the high-frequency (IBI-HFnu), (F) LF HF^-1 ratio (IBI-LF HF^-1), (G) total baroreflex gain. Data are represented in % of time zero. Differences of corresponding saline group: (S) SHAM, (O) OVX and (E) OVX+E, P ≤ 0.05. Values are means ± S.E.M. The number of rats per group is indicated in the (A,C).

FIGURE 6

Plasma biochemical parameters, 2 h after LPS (5 mg kg^-1), for sham-operated (SHAM), ovariectomized (OVX), or estrogen-treated OVX (OVX+E) groups pre-treated with SMT (3 mg kg^-1) or vehicle (saline 0.9%, 1 ml kg^-1) 10 min before LPS. (A) Nitrite levels, (B) total radical-trapping antioxidant parameter (TRAP), (C) paraoxonase 1 (PON1) activity and (D) lipoperoxidation. Values are means ± SEM. Differences of corresponding saline group: (S) SHAM, (O) OVX, and (E) OVX+E, P ≤ 0.05. ^∗P < 0.01 vs. corresponding SHAM; ^#P < 0.05 vs. corresponding OVX. The number of rats per group is indicated in the figure.