Downregulation of carbon monoxide as well as nitric oxide contributes to peripheral chemoreflex hypersensitivity in heart failure rabbits

Abstract

Peripheral chemoreflex sensitivity is potentiated in clinical and experimental chronic heart failure (CHF). Downregulation of nitric oxide (NO) synthase (NOS) in the carotid body (CB) is involved in this effect. However, it remains poorly understood whether carbon monoxide (CO) also contributes to the altered peripheral chemoreflex sensitivity in CHF. This work highlights the effect of NO and CO on renal sympathetic nerve activity (RSNA) in response to graded hypoxia in conscious rabbits. Renal sympathetic nerve responses to graded hypoxia were enhanced in CHF rabbits compared with sham rabbits. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1.2 microg x kg(-1) x min(-1)) and the CO-releasing molecule tricarbonyldichlororuthenium (II) dimer {[Ru(CO)(3)Cl(2)](2), 3.0 microg x kg(-1) x min(-1)} each attenuated hypoxia-induced RSNA increases in CHF rabbits (P < 0.05), but the degree of attenuation of RSNA induced by SNAP or [Ru(CO)(3)Cl(2)](2) was smaller than that induced by SNAP + [Ru(CO)(3)Cl(2)](2). Conversely, treatment with the NOS inhibitor N(omega)-nitro-L-arginine (30 mg/kg) + the heme oxygenase (HO) inhibitor Cr (III) mesoporphyrin IX chloride (0.5 mg/kg) augmented the renal sympathetic nerve response to hypoxia in sham rabbits to a greater extent than treatment with either inhibitor alone and was without effect in CHF rabbits. In addition, using immunostaining and Western blot analyses, we found that expression of neuronal NOS, endothelial NOS, and HO-2 protein (expressed as the ratio of NOS or HO-2 expression to beta-tubulin protein expression) was lower in CBs from CHF (0.19 +/- 0.04, 0.17 +/- 0.06, and 0.15 +/- 0.02, respectively) than sham (0.63 +/- 0.04, 0.56 +/- 0.06, and 0.27 +/- 0.03, respectively) rabbits (P < 0.05). These results suggest that a deficiency of NO and CO in the CBs augments peripheral chemoreflex sensitivity to hypoxia in CHF.

Fig. 1.

A and C: renal sympathetic nerve activity (RSNA)-arterial Po₂ (Pa_O2) relationships before and after intravenous administration of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in sham rabbits and rabbits with chronic heart failure (CHF). B and D: RSNA-Pa_O2 relationships before and after intravenous administration of the NO synthase (NOS) inhibitor N^ω-nitro-l-arginine (l-NNA). Hydralazine (0.01–0.06 mg·kg⁻¹·min⁻¹) or phenylephrine (0.2–1.0 μg·kg⁻¹·min⁻¹) was infused as appropriate to maintain mean arterial pressure (MAP) at control levels and minimize baroreflex effects (see Table 3). Values are means ± SE; n, number of animals. % of max, percentage of maximum. *P < 0.05 vs. control (before drug administration).

Fig. 2.

A and C: RSNA-Pa_O2 relationships before and after intravenous administration of the carbon monoxide (CO)-releasing molecule tricarbonyldichlororuthenium (II) dimer {[Ru(CO)₃Cl₂]₂} in sham and CHF rabbits. B and D: RSNA-Pa_O2 relationships before and after intravenous administration of the heme oxygenase (HO) inhibitor Cr (III) mesoporphyrin IX chloride (CrMP) in sham and CHF rabbits. Values are means ± SE; n, number of animals. *P < 0.05 vs. control.

Fig. 3.

A and C: RSNA-Pa_O2 relationships before and after intravenous administration of SNAP + [Ru(CO)₃Cl₂]₂ in sham and CHF rabbits. B and D: RSNA-Pa_O2 relationships before and after intravenous administration of l-NNA + CrMP. Hydralazine (0.01–0.06 mg·kg⁻¹·min⁻¹) or phenylephrine (0.2–1.0 μg·kg⁻¹·min⁻¹) was infused as appropriate to normalize MAP (see Table 3). Values are means ± SE; n, number of animals. *P < 0.05 vs. control.

Fig. 4.

A: changes in RSNA response to hypoxia after manipulation with l-NNA (30 mg/kg), CrMP (0.5 mg/kg), or l-NNA + CrMP in sham rabbits. B: changes in RSNA response to hypoxia after manipulation with SNAP (1.2 μg·kg⁻¹·min⁻¹), Ru(CO)₃Cl₂]₂ (3.0 μg·kg⁻¹·min⁻¹), or SNAP + Ru(CO)₃Cl₂]₂ in CHF rabbits. Hydralazine (0.01–0.06 mg·kg⁻¹·min⁻¹) was infused after l-NNA injection to maintain MAP in sham rabbits. Normoxia (∼90 Torr Pa_O2), mild hypoxia (∼65 Torr Pa_O2), and severe hypoxia (∼42 Torr Pa_O2) as illustrated in Figs. 1–3. Values are means ± SE; n, number of animals. *P < 0.05 vs. l-NNA. #P < 0.05 vs. CrMP. $P < 0.05 vs. SNAP. &P < 0.05 vs. Ru(CO)₃Cl₂]₂.

Fig. 5.

Protein expression of heme oxygenase-2 (HO-2) in carotid bodies (CBs) from sham and CHF rabbits. A: representative bands of HO-2 and β-tubulin proteins. B: relative HO-2 protein expression in sham and CHF CBs. Values are means ± SE; n = 5 in each group. *P < 0.05 vs. sham. C: colocalization of tyrosine hydroxylase (TH) and HO-2 in CBs from a sham (a–c) and a CHF (d–f) rabbit. a and d: Green immunofluorescent image for TH; b and e: red immunofluorescent image for HO-2; c and f: merged image (yellow) for overlap of TH and HO-2.

Fig. 6.

Protein expression of neuronal NO synthase (nNOS) in CBs from sham and CHF rabbits. A: representative bands of nNOS and β-tubulin proteins. B: relative nNOS protein expression in sham and CHF CBs. Values are means ± SE; n = 5 in each group. *P < 0.05 vs. sham. C: colocalization of TH and nNOS in CBs from a sham (a–c) and a CHF (d–f) rabbit. a and d: Green immunofluorescent image for TH; b and e: red immunofluorescent image for nNOS; c and f: merged image (yellow) for overlap of TH and nNOS.

Fig. 7.

Protein expression of endothelial NO synthase (eNOS) in CBs from sham and CHF rabbits. A: representative bands of eNOS and β-tubulin proteins. B: relative eNOS protein expression in sham and CHF CBs. Values are means ± SE; n = 5 in each group. *P < 0.05 vs. sham. C: colocalization of TH and eNOS in CBs from a sham (a–c) and a CHF (d–f) rabbit. a and d: Green immunofluorescent image for TH; b and e: red immunofluorescent image for eNOS; c and f: merged image (yellow) for overlap of TH and eNOS.