H2S mediates O2 sensing in the carotid body

Abstract

Gaseousmessengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine gamma-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.

Fig. 1.

CSE localization in the mouse carotid body and carotid body responses to hypoxia and hypercapnia in CSE^+/+ and CSE^−/− mice. (A) CSE expression in carotid bodies from CSE^+/+ and CSE^−/− mice. Carotid body sections were stained with antibodies specific for CSE or tyrosine hydroxylase (TH), a marker of glomus cells. (Scale bar: 20 μm.) (B) Sensory response of isolated carotid bodies to hypoxia (Hx) ( ∼ 39 mmHg; at black bar) in CSE ^+/+ and CSE^−/− mice. Integrated carotid body sensory activity (CB activity) is presented as impulses per second (imp/s). Superimposed action potentials from the single fiber are presented in Inset. (C) Carotid body responses to graded hypoxia from CSE^+/+ and CSE^−/− mice, measured as the difference in response between baseline and hypoxia (Δimp/s). Data are mean ± SEM of n = 24 (CSE^+/+) and n = 23 (CSE^−/−) fibers from eight mice each. (D) H₂S levels (mean ± SEM) in carotid bodies from CSE^+/+ and CSE^−/− mice under normoxia (NOR) and hypoxia (Hx) ( ∼ 40 mmHg) from four independent experiments. (E) Example illustrating carotid body responses to CO₂ in CSE^+/+ and CSE^−/− mice. (F) Average data (mean ± SEM) of CO₂ response from n = 24 (CSE ^+/+) and n = 19 (CSE^−/−) fibers from eight mice in each group. *** and **, P < 0.001 and P < 0.01, respectively; n.s. (not significant), P > 0.05.

Fig. 2.

Ventilatory responses to hypoxia and hypercapnia in CSE^+/+ and CSE^−/− mice. Ventilation was measured in unsedated mice by whole body plethysmography under normoxia (21% O₂), hypoxia (12% O₂), and hypercapnia (5% CO₂). Hypoxia and hypercapnia lasted for 5 min. Representative tracings of breathing are shown in A and average data of minute ventilation (V_E) in response to 12% O₂, i.e., hypoxia (B) and 5% CO₂ i.e., hypercapnia (C). The data presented are mean ± SEM from eight CSE^+/+ and CSE^−/− mice each. **, P < 0.01; n.s. (not significant), P > 0.05.

Fig. 3.

CSE localization in the rat carotid body and rat carotid body responses to hypoxia and hypercapnia in the absence and presence of DL-propargylglycine. (A) Cystathionine γ-lyase (CSE) expression in rat carotid body. Carotid body sections were stained with antibodies specific for CSE and tyrosine hydroxylase (TH), a marker of glomus cells (Left). Effects of graded hypoxia on H₂S levels in vehicle-(PAG−) and DL-propargylglycine (PAG+)-treated carotid bodies. Data are mean ± SEM from five individual experiments (Right). (B) Examples of carotid body response to hypoxia (Hx) ( = 38 mmHg; at black bar) in vehicle- and PAG-treated rats (Left). Average (mean ± SEM) data of sensory response to graded hypoxia (Right), PAG− n = 12 fibers from six rats; PAG+ n = 10 fibers from six rats. (C) Example of carotid body response to CO₂ in the same rats as in B ( ∼ 68 mmHg; at black bar (Left)] and average (mean ± SEM) data of CO₂ response (Right). Data derived from n = 9 fibers (PAG−) and n = 10 (PAG+) fibers from six rats each. In B and C, integrated carotid body sensory activity (CB activity) is presented as impulses per second (imp/s). Superimposed action potentials from the single fiber are presented in Inset. **, P < 0.01.

Fig. 4.

Effect of NaHS on rat carotid body sensory activity. (A) Example of rat carotid body response to increasing concentrations of NaHS, an H₂S donor (at black bar; Left). Average (mean ± SEM) data of dose–response to NaHS (Center) and time course of sensory response to NaHS (100 μM) and hypoxia [ = 42 mmHg (Right)]. Data in middle and right panels were obtained from n = 13 fibers from six rats. (B) Effect of Ca²⁺ free medium on rat carotid body responses to 100 μM NaHS and hypoxia (Hx) ( = 42 mmHg; at black bar). CaCl₂ was replaced by 3 mM MgCl₂ and 5 mM EGTA was added to the medium. (Left) Example and Right average (mean ± SEM) data from five rats (n = 8 fibers). In A and B, Integrated carotid body sensory activity (CB activity) is presented as impulses per second (imp/s). Superimposed action potentials from the single fiber are presented in the inset. **, P < 0.01; n.s. (not significant), P > 0.05.

Fig. 5.

Effect of HO-2 inhibition on carotid body H₂S generation and sensory activity in CSE^+/+ and CSE^−/− mice. (A) Effect of Cr(III)MP (1 μM), an inhibitor of heme oxygenase 2 on H₂S generation in carotid bodies under normoxia ( ∼ 146 mmHg) from CSE^+/+ and CSE^−/− mice. Data presented are mean ± SEM from three experiments. Examples of baseline and hypoxic response (Hx) ( ∼ 40 mmHg; at black bar) of carotid bodies from vehicle- and Cr(III)MP-treated CSE^+/+ and CSE^−/− mice (B) and average data (mean ± SEM) from six mice in each group (n = 8–12 fibers) in C and D. In B, Integrated carotid body sensory activity (CB activity) is presented as impulses per second (imp/s). Superimposed action potentials from the single fiber are presented in Inset. *** and *, P < 0.001 and P < 0.05, respectively; n.s. (not significant), P > 0.05.

Fig. 6.

CSE localization in the mouse and rat neonatal adrenal medullary chromaffin cells (AMC) and the effect of CSE disruption on mouse and rat AMC hypoxia sensing. (Top) Cystathionine γ-lyase (CSE) expression in AMC from mice (A) and rat pups (B). (Middle) Examples of catecholamine secretion from AMC from neonatal mice (A) and rats (B) in response to hypoxia (Hx) ( = 36 mmHg) or high K⁺ (40 mM). Black bar represents the duration of hypoxia or K⁺ application. (Bottom) Average data (mean ± SEM) of total catecholamine (CA) secreted during Hx or K⁺ (CA molecules 10⁷ i.e., number of secretory events multiplied by catecholamine molecules secreted per event). n = 9 cells each from CSE^+/+ and CSE^−/− and n = 10–12 cells from rat pups. **, P < 0.01; n.s. (not significant), P > 0.05 compared with CSE^+/+ mice or vehicle-treated rat cells.