The results from the present study indicate that: (1) in sham rabbits, Ang II perfusion to the isolated CB preparation enhances the CB chemoreceptor sensitivity to hypoxia, and L158,809 (a selective AT1 receptor antagonist), PAO and APO (NADPH oxidase inhibitors), and tempol (a SOD mimetic) significantly blunts the Ang II-enhanced CB chemoreceptor sensitivity to hypoxia; (2) similarly, L158,809, PAO, APO, and tempol also decrease CB chemoreceptor responses to hypoxia in CHF rabbits; (3) mRNA and protein expressions of NADPH oxidase components (gp91phox, p47phox and p40phox) in the CB are markedly increased in CHF rabbits, compared to that in sham rabbits; (4) L158,809, PAO, APO, and tempol effectively attenuate Ang II- and CHF-induced superoxide anion production in the CB homogenates. These data suggest that an endogenous Ang II-NADPH oxidase-superoxide signaling pathway in the CB mediates the enhanced CB chemoreceptor activity to hypoxia in CHF.
Sympathetic hyperactivity in CHF contributes to late-stage deterioration of cardiac function [2
]. Previous studies have shown that an enhanced peripheral chemoreflex sensitivity contributes significantly to the generalized sympathetic over activation in CHF patients and experimental animals [4
]. The CB is the primary sensor site of the peripheral chemoreflex. Our recent study revealed that elevation of endogenous Ang II and upregulation of AT1
receptors in the CB contributed to the enhanced CB chemoreceptor sensitivity to hypoxia and to the enhanced peripheral chemoreflex function in CHF rabbits [13
]. However, the mechanism by which Ang II augments the CB chemoreceptor sensitivity to hypoxia was not known prior to this study.
Ang II activates NADPH oxidase [14
], which serves as a significant source of intracellular reactive oxygen species (ROS) in many tissues [15
]. Gao, et al. found that Ang II-NADPH oxidase-derived radical stress in autonomic areas of the brain was involved in the sympathoexcitation in rabbits with pacing-induced CHF [20
], the same animal model as in the present study. Furthermore, a significant body of evidence indicates that the components of NADPH oxidase are present in the peripheral chemoreceptors including pulmonary neuroepithelial bodies and CB [21
Our present results confirmed our previous study showing that endogenous Ang II and the AT1
receptor in the CB plays a major role in enhancing CB chemoreceptor sensitivity to hypoxia in CHF rabbits [13
]. We further found that the Ang II- and CHF-enhanced CB chemoreceptor sensitivity to hypoxia is blunted by NADPH oxidase inhibitors (PAO and APO) and superoxide scavenger (tempol) (Figs. and ). At the same time, mRNA and protein expressions of NADPH oxidase components (gp91phox
) and superoxide anion production are elevated in the CB from CHF rabbits (Figs. -). An AT1
receptor antagonist, NADPH oxidase inhibitors, and superoxide scavenger attenuate the superoxide anion production induced by Ang II and CHF (Figs. and ). These results clearly indicate that Ang II-NADPH oxidase-derived superoxide anion signaling contributes to the enhanced CB chemoreceptor sensitivity to hypoxia in CHF rabbits.
The present study also confirms our previous observation [13
] that neither Ang II nor L-158,809 affects CB chemoreceptor activity under normoxic conditions in sham and CHF rabbits, respectively. Consistent with this, the present study demonstrates that perfusion of the CB with either a NADPH oxidase inhibitor (PAO or APO) or tempol does not affect the baseline CB chemoreceptor activity in either sham or CHF rabbits (). These results, taken together, suggest that NADPH oxidase-derived superoxide anion signaling pathway mediates the Ang II-enhanced sensitivity of CB chemoreceptors to hypoxia in CHF, but does not contribute to the elevated basal chemoreceptor activity observed under normoxic conditions in our CHF animals.
In previous studies [6
], we have shown that downregulation of nNOS expression with decreased nitric oxide (NO) production in the CB is responsible for the elevated resting CB chemoreceptor discharge under normoxic conditions in CHF rabbits. The two constitutive isomers of NO synthase (NOS), neuronal NOS (nNOS) and endothelial NOS (eNOS), are localized to nerve fibers and vascular endothelium in the CB, and NO is inhibitory to the CB chemoreceptor activity [34
]. We have also found that the NO donor, Snitroso-N-acetylpenicillamine or gene transfer of nNOS using an adenoviral vector to the CB in CHF rabbits reverses the enhanced CB chemoreceptor activity seen in the CHF state [6
]. Our present observation that NADPH oxidase-derived superoxide anion mediates the enhanced CB chemoreceptor sensitivity to hypoxia in CHF rabbits raises the question whether there may be an important interaction between these two signaling pathways (upregulation of Ang II-NADPH oxidase-superoxide anion pathway and downregulation of NOS-NO pathway) in CHF. Ang II may contribute to depressed bioavailable NO in the CB by suppressing nNOS gene expression [35
] and/or increased scavenging of NO through superoxide anion production. Conversely, the downregulation of NO production in the CB in CHF may act to enhance the effects of Ang II by reduced scavenging of superoxide anion by NO. The relationship among NO, Ang II and superoxide anion on CB chemoreceptor function is not yet clear and deserves further study. But it is clear that upregulation of Ang II-NADPH oxidase-superoxide anion pathway and downregulation of NOS-NO pathway in the CB in CHF state have complementary effects on the CB chemoreceptor activity.
Our previous study has shown that endogenous Ang II modulates the CB chemoreceptor sensitivity to hypoxia via its inhibitory effects on O2
channels in CB glomus cells from CHF rabbits [25
]. In the present study, NADPH oxidase-derived superoxide anion mediated the Ang II- and CHF-induced enhancement of the CB chemoreceptor sensitivity to hypoxia. Therefore, It is possible that NADPH oxidase-derived superoxide anion is also involved in the inhibitory effect of Ang II on O2
channels in CB glomus cells from CHF rabbits. Further studies are needed to define the involvement of NADPH oxidase-derived superoxide anion on the modulation of the O2
channels in CB glomus cells from CHF rabbits.
NADPH oxidase is a complex enzyme consisting of two membrane-bound components (gp91phox
) and three cytosolic components (p67phox
, and p40phox
], which was first found in phagocytic cells [30
]. Studies have shown that gp91phox
, and p47phox
subunits of the NADPH oxidase exist in the CB glomus cells in rats [21
]. However, we found the mRNA of gp91phox
, and p40phox
but not p22phox
are expressed in the rabbit CBs () although we could detect the mRNA expression of p67phox
in liver and kidney from sham rabbits as a positive control. Similarly, Gao, et al. also found that p22phox
cannot be detected in the rabbit rostral ventrolateral medulla (RVLM) neurons [20
]. Our present study cannot explain this discrepancy. One possibility is that p22phox
in the CB of rabbits may be unique homologues of the proteins that are undetectable using standard primers and antibodies. By analogy, recent expansion of information available in genome databases has led to identification of several novel homologues of gp91phox
in animals, which forms the Nox family [36
In the present study, superoxide anion production in the CB was increased in CHF rabbits compared to sham rabbits. Exogenous Ang II similarly enhanced the superoxide anion in the CB from sham rabbits (Fig. and ). Conversely, L158,809 and PAO markedly reversed the elevated superoxide anion production induced by Ang II and CHF to levels observed at rest in sham rabbits (Fig. and ). These results indicate that Ang II-NADPH oxidase is a main source of the elevated superoxide anion production in the CB in CHF. It also appears that superoxide anion production in the CB is limited by the availability of cofactor NADPH under normal (sham) conditions, but to a much smaller extent in the CHF condition. Thus both increased NADPH oxidase expression and increased NADPH availability play a role in the enhanced superoxide production in the CB in CHF.
We also found that the superoxide scavenger, tempol, nearly abolished superoxide anion production in CBs from both sham and CHF animals (), whereas L-158,809 or PAO decreased elevated Ang II and CHF induced superoxide levels to the control level observed in sham rabbits. Therefore, it seems that a small level of superoxide anion production arises from other sources (perhaps xanthine-xanthine oxidase, mitochondrial complex enzymes, etc) in the CB at rest in sham rabbits, and that this level of alternatively derived superoxide production is not altered by Ang II or by the induction of CHF (i.e. the level of superoxide production after NADPH oxidase inhibition was the same in CBs from sham and CHF rabbits and was comparable to the control level in sham rabbits, ).
To what extent does superoxide production apart from NADPH oxidase activity influence CB chemoreceptor sensitivity in CHF? Although the inhibitory effect of tempol on superoxide anion production was greater than that of either L158,809 or PAO in CHF rabbits (), there was no difference among these three inhibitors on their ability to inhibit enhanced CB chemoreceptor activity in the CHF rabbits (). From these results, we assume that the superoxide anions produced by the Ang II-NADPH oxidase pathway, but not other superoxide ion sources, largely mediate the enhancement of the CB chemoreceptor activity in CHF rabbits.
Although our present study indicates that the Ang II-NADPH oxidase-superoxide anion signaling pathway mediates the enhanced peripheral chemoreceptor sensitivity in pacing-induced CHF rabbits, this signaling pathway has not been tested or confirmed in CHF patients with exaggerated peripheral chemoreflex function. In addition, the present study was performed in the isolated and perfused CB preparations, which could directly and indirectly affect the characteristics of the CB chemoreceptor in response to hypoxia. Therefore, care should be taken when extrapolating the data obtained here to the clinical phenomena observed in the CHF patients.
In conclusion, the expression of NADPH oxidase and the production of superoxide anion are elevated in the CBs from rabbits with CHF. This Ang II-NADPH oxidase-superoxide anion signaling pathway is involved in the enhanced CB chemoreceptor sensitivity to hypoxia in the CHF state.