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. 2011 Oct;111(4):964-70.
doi: 10.1152/japplphysiol.00022.2011. Epub 2011 Jun 2.

Angiotensin II evokes sensory long-term facilitation of the carotid body via NADPH oxidase

Ying-Jie Peng  1 , Gayatri Raghuraman, Shakil A Khan, Ganesh K Kumar, Nanduri R Prabhakar
Affiliations

Angiotensin II evokes sensory long-term facilitation of the carotid body via NADPH oxidase

Ying-Jie Peng et al. J Appl Physiol (1985). 2011 Oct.

Abstract

We previously reported that reactive oxygen species generated by NADPH oxidase 2 (Nox2) induces sensory plasticity of the carotid body, manifested as a progressive increase in baseline sensory activity or sensory long-term facilitation (sLTF). ANG II, a peptide generated within the carotid body, is a potent activator of Nox2. In the present study, we tested the hypothesis that ANG II evokes sLTF of the carotid body via Nox2 activation. Experiments were performed on carotid bodies ex vivo from adult rats and mice. Sensory activity was recorded from the carotid sinus nerve. Repetitive (5 times for 30 s each at 5-min intervals), but not continuous (for 150 s), application of 60 pM ANG II evoked robust sLTF of the carotid body. ACh, ATP, substance P, and KCl, when applied repetitively, stimulated the carotid body but did not evoke sLTF. Reactive oxygen species levels increased in response to repetitive applications of ANG II, and this effect was blocked by apocynin, an inhibitor of Nox2, as well as losartan, an angiotensin type 1 (AT(1)) receptor antagonist. Losartan, apocynin, and 4-(2-aminoethyl)benzenesulfonyl fluoride prevented ANG II-induced sLTF, which was absent in mice deficient in gp91(phox), the catalytic subunit of the Nox2 complex. These results demonstrate that repetitive application of ANG II induces sLTF of the carotid body via activation of Nox2 by AT(1) receptors.

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Figures

Fig. 1.
Fig. 1.
Effect of ANG II on carotid body sensory activity. A and B: sensory responses [impulses (imp)/s] of the carotid body to repetitive (5 times for 30 s each at 5-min intervals, arrows in A) and continuous (for 150 s, arrows in B) applications of 60 pM ANG II. Insets: superimposed action potentials of a “single” fiber from which data were derived. White line represents baseline activity. C: sensory activity. Values are means ± SE from 11 carotid bodies each. Significant difference from baseline: *P < 0.05; **P < 0.01. For clarity, statistical significance is presented only during poststimulation period.
Fig. 2.
Fig. 2.
Effects of repetitive applications of excitatory modulators on carotid body sensory activity. A: carotid body sensory responses to repetitive applications (5 times for 30 s each at 5-min intervals at arrows) of ACh (10 μM), ATP (100 μM), substance P (SP, 1 μM), and KCl (40 mM). Insets: superimposed action potentials of a single fiber from which data were derived. White line represents baseline activity. B: average data of the sensory long-term facilitation (LTF; sensory activity averaged over the entire 60 min of the postapplication period). Values are means ± SE; n = 13 for ACh and ATP, n = 12 for SP, and n = 11 for KCl. NS, P > 0.05 compared with prestimulation baseline activity.
Fig. 3.
Fig. 3.
Effect of losartan on ANG II-evoked carotid body sensory LTF (sLTF). A: carotid body sensory response to repetitive application of 60 pM ANG II (5 times for 30 s each at 5-min intervals at arrows) in the presence of vehicle (top) and 3 μM losartan (bottom). Insets: superimposed action potentials of a single fiber from which data were derived. White line represents baseline activity. B: sensory activity. Values are means ± SE from 11 carotid bodies each of vehicle and losartan. Statistical significance: *P < 0.05; **P < 0.01 vs. baseline activity. For clarity, statistical significance is presented only during poststimulation period.
Fig. 4.
Fig. 4.
Effect of ANG II on reactive oxygen species (ROS) levels in the carotid body. Malondialdehyde (MDA) levels were analyzed in carotid body extracts as an index of ROS. Top: protocols for MDA analysis with repetitive and continuous applications of ANG II. Analysis of MDA at the end of 60-min superfusion without ANG II served as controls. A: carotid bodies were challenged with repetitive application of 60 pM ANG II (5 times for 30 s each at 5-min intervals), and MDA levels were determined at minute 60 of the poststimulation period. Protocols were repeated in the presence of 3 μM losartan, an angiotensin type 1 (AT1) receptor antagonist, or 500 μM apocynin, an inhibitor of NADPH oxidase 2 (Nox2). B: effect of continuous application of 60 pM ANG II for 150 s on MDA levels in the carotid body. MDA levels were determined at 5 min (ANG II ×1, 5 min) or 60 min (ANG II ×1, 60 min). MDA levels were elevated when measured at 5 min, but not 60 min, after termination of continuous application of ANG II and losartan (3 μM) or apocynin (500 μM) blocked this effect. Values are means ± SE from 4 individual experiments performed in triplicate. ***P < 0.001. NS, P > 0.05 compared with vehicle-treated controls.
Fig. 5.
Fig. 5.
NADPH oxidase inhibitors prevent ANG II-induced sLTF of the carotid body. A: sensory responses of the carotid body to repetitive application of 60 pM ANG II (5 times for 30 s each at 5-min intervals at arrows) in the presence of vehicle (top) and apocynin (bottom). Insets: superimposed action potentials of a single fiber from which data were derived. White line represents baseline activity. B: sensory activity in response to repetitive applications of ANG II in the presence of vehicle, apocynin (Apo, 500 μM), or 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, 500 μM). Values are means ± SE; n = 14 carotid bodies in each group. Statistical significance: *P < 0.05; **P < 0.01 compared with baseline activity. For clarity, statistical significance is presented only during poststimulation period.
Fig. 6.
Fig. 6.
Absence of ANG II-induced sLTF in mice deficient in gp91phox. A: carotid body sensory response to repetitive applications of 60 pM ANG II (5 times for 30 s each at 5-min intervals at arrows) in wild-type (WT; top) and gp91phox−/Y (bottom) mice. Insets: superimposed action potentials of a single fiber from which data were derived. White line represents baseline activity. B: sensory activity in response to repetitive applications of ANG II in both genotypes. Values are means ± SE; n = 11 carotid bodies each of WT and gp91phox−/Y. **P < 0.01 compared with WT. For clarity, statistical significance is presented only during the poststimulation period.
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