Introduction
Carotid bodies are sensory organs for monitoring arterial blood oxygen levels and stimulate breathing in response to hypoxia
. They are small clusters of chemoceptors located near the bifurcation of the carotid artery and are made up of primarily two cell types:
- glomus or type I cells: are derived from neural crest and are the main responsible of oxygen sensing
- type II cells: resembling glial cells of the nervous system
The carotid body contains the most vascular tissue in the human body, providing a perfect report of the oxygen levels in arterial blood.
Hypoxia triggers an action potential in glomus cells through the afferent fibers of the glossopharyngeal nerve, which relays the information to the central nervous system. But who are the real oxygen sensors and what is the pathway involved?
Historically, it has been known that brief inhalation of carbon monoxide(CO) inhibits ventilator stimulation by hypoxia(Santiago T V,1976) while in 1922 it was reported that inhalation of hydrogen sulfide(H 2 S) stimulates breathing. These two gaseous messengers are tightly linked together and the aim of this work is to outline the pathway involved.
CO is endogenously generated during degradation of heme by heme oxyhenases (HOs) utilizing cytocrome P-450 reductase, NADPH and molecular 0 2 as co-factors (Maines,1997) . HOs use as metabolic substrates alpha and beta chains of hemoglobin, denatured myoglobin, met-hemoglobin. It is known there are two OHs isoforms, but under basal conditions HO-2 is the only heme oxygenase expressed in carotid body.
Hypoxia decreases HO activity in a stimulus-dependent way in placenta and in cerebral vasculature( Morikawa, 2012 ). Under basal conditions it has been shown relatively high levels of CO and hypoxia(P0 2=40mmHg) reduced impressively CO generation(4nmol/min/mg protein). These evidences suggest physiological levels of hypoxia inhibit HO activity and reduce CO generation. Furthermore endogenous CO is a physiological inhibitor of the carotid body sensory activity as administration of zinc-protoporphirin-9, a HO inhibitor, increases the carotid body sensory activity in a dose-dependent manner(Prabhakar,1995":http://www.ncbi.nlm.nih.gov/pubmed/?term=7892214 )
THE HEME OXYGENASE SYSTEM:A Regulator of Second Messenger Gases
Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
Carbon monoxide: a role in carotid body chemoreception.
Example illustrating the effect of hypoxia (Hx; PO2 ~36 mmHg) on carotid body ex vivo sensory activity in a 8 week old, male wild type (HO-2+/+) and age and gender matched heme oxygenase-2 (HO-2) knockout mice (HO-2−/−). Black bar represents the duration of the hypoxic challenge. Inset represents the single unit action potential from which the data are derived. Note the elevated baseline activity and augmented sensory response to hypoxia in HO-2 knockout carotid body.
Effects of CO levels on ion channels
Hypoxia inhibits some K + channels in type I cells resulting in depolarization, leading to activation of voltage-gated Ca 2+ channels and calcium influx(Peers and Wyatt). Maxi K channels( that are Ca 2+ activated K + channel) are targets of hypoxia in glomus cells. CO enhances maxi K channel activity in glomus cells; on the other hand, disrupting HO-2 activity , like hypoxia, inhibits the maxi k channel activity. Anyway the nature and identity of the Ca 2+ channels activated by hypoxia remains to be investigated.
The role of maxiK channels in carotid body chemotransduction.
Glomus cells express cystathionine gamma-lyase(CSE) and cystathionine beta-sinthase(CBS), two major enzymes which catalyze endogenous H 2 S formation. CSE contributes for the major share of hydrogen sulfide basal levels, the residual part being provided by CBS.
CSE preponderates in the peripheral tissues whose H 2 S levels are reduced 90% in CSE knock out mice. CSE knock out mice shows exhibits severely impaired response to hypoxia, with basal H 2 S generation under normoxia significantly reduced compared to wild type mice. Finally carotid bodies from knockout and wild type mice respond to CO 2 with a comparable increase in sensory discharge.
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) (P0 2 ∼ 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. © 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) (P0 2 ∼ 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
H 2 S stimulates carotid body sensory activity
The effects of exogenous administration of hydrogen sulfide on the sensory activity have been investigated. NaHS, a donor of H 2 S, enhanced carotid body activity CSE knockout and wild type mice. Carotis body response to NaHS is stimulus-dependent, occurring within seconds after its application, and sensory activity promptly returned to baseline after termination of the stimulus.
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 [P0 2 = 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) P0 2 (= 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.
Mechanism of increased H 2 S generation by hypoxia
Hypoxia increases H 2 S levels in a stimulus-dependent manner and this response is absent in CSE knockout mice. But how might hypoxia increase H 2 S generation? Considered that CO inhibits carotid body sensory activity it can be evicted that it inhibits H 2 S generation by CSE. Indeed HO inhibitor markedly increases basal H 2 S levels in the carotid body under normoxia and increased baseline sensory activity in wild type mice. These evidences suggest that CO is a physiological inhibitor of CSE-dependent H 2 S generation. So, the low sensory activity of the carotid body under normoxia is due to inhibition of H 2 S generation by CO, and hypoxia reduces HO-2 activity to reverse the inhibition and augment H 2 S formation(Peng et al., 2010) .
H2S mediates O2 sensing in the carotid body.
Conclusions
There are important evidences that the interaction between OHs and CSE provides an useful and sensitive sensory mechanism for oxygen sensing in the carotid body. OH-2 satisfies some of the criterion of the heme protein that initiates the hypoxic response of the carotid body: first of all it is expressed in glomus cells, and has a great affinity for oxygen, as little variations in oxygen levels lead to substantial loss of its activity. Infact HO-2 is inhibited by physiological levels of hypoxia (P0 2=40mmHg). Inhibition of HO-2 activity by hypoxia results in reduced generation of CO, an inhibitory gas messenger, which by removing inhibition on CSE increases H 2 S generation, an excitatory gas messenger. It has been proposed that interaction between HO-2-CO and CSE- H 2 S systems in concert with K + channels constitute major components of a larger sensory system.
