Eric W. Kostuk

The human carotid body transcriptome with focus on oxygen sensing and inflammation – a comparative analysis

•  The carotid body (CB) is the key oxygen sensor and governs the ventilatory response to hypoxia. •  CB oxygen sensing and signalling gene expression is well described in animals whereas human data are absent. •  Here we have characterized the human CB global gene expression in comparison with functionally related tissues and mouse CB gene expression. •  We show that the human CB expresses oxygen sensing genes in common with mice but also differs on key genes such as certain K+ channels. There is moreover increased expression of inflammatory response genes in human and mouse CBs in comparison with related tissues. •  The study establishes similarities but also important differences between animal and human CB gene expression profiles and provides a platform for future functional studies on human CBs.

Inflammation in the carotid body during development and its contribution to apnea of prematurity

Breathing is a complex function that is dynamic, responsive, automatic and often unstable during early development. The carotid body senses dynamic changes in arterial oxygen and carbon dioxide tension and reflexly alters ventilation and plays an essential role in terminating apnea. The carotid body contributes 10-40% to baseline ventilation in newborns and has the greatest influence on breathing in premature infants who characteristically have unstable breathing leading to apnea of prematurity. In this review, we will discuss how both excessive and minimal contributions from the carotid body destabilizes breathing in premature infants and how exposures to hypoxia or infection can lead to changes in the sensitivity of the carotid body. We propose that inflammation/infection during a critical period of carotid body development causes acute and chronic changes in the carotid body contributing to a protracted course of intractable and severe apnea known to occur in a subset of premature infants.

The impact of hypoxia and low glucose on the release of acetylcholine and ATP from the incubated cat carotid body

The carotid body (CB) is a polymodal sensor which increases its neural output to the nucleus tractus solitarii with a subsequent activation of several reflex cardiopulmonary responses. Current reports identify acetylcholine (ACh) and adenosine triphosphate (ATP) as two essential excitatory neurotransmitters in the cat and rat CBs. This study explored the impact of hypoxia, low glucose, and the two together on the release of both ACh and ATP from two incubated cat CBs. The CBs were prepared with standard procedures in accordance with the policies and regulations of the Institutional Animal Care and Use Committee. When normalized to their controls, a significant increase of ACh in the incubation medium was measured in response to hypoxia, low glucose, and the combined stimuli. When normalized to their controls, a significant increase in ATP in the incubation medium was measured in response to hypoxia and to the combined stimuli. Low glucose generated an increase in ATP which was not statistically significant (P>0.05). Second, normalizing the initial 3-4 or 2-3 min Time Segment of the challenge Stage to the final 3-4 or 2-3 min Time Segment of the control Stage for both ACh and ATP generated significant increases in response to hypoxia, low glucose (ACh only), and the combined stimuli. The data suggested the possibility that in the cat the increased CB neural output in response to low glucose might be due primarily to ACh.

Differential Expression of Large-Conductance Ca2+-Activated K Channels in the Carotid Body between DBA/2J and A/J Strains of Mice

The carotid body (CB) is a primary chemosensory organ for arterial hypoxia. Inhibition of K channels in chemosensory glomus cells (GCs) are considered to be responsible for hypoxic chemoreception and/or chemotransduction of the CB. Hypoxic sensitivity of large-conductance calcium-activated K (BK) channels has been established in the rat CB. Our previous work has shown the BK channel β2 subunits are more expressed in the CB of the DBA/2J mouse than that of the A/J mouse. Because the DBA/2J mouse is more sensitive to hypoxia than the A/J mouse, our general hypothesis is that BK channels play a role in the sensitivity of the mouse CB to mild hypoxia. We performed vigorous analysis of the gene expression of α, β2, and β4 subunits of BK channels in the CB. We found that α and β2 subunits were expressed more in the CB of the DBA/2J mice than that of the A/J mice. No differences were found in the β4 subunit expression. These differences were not seen in the neighboring tissues, the superior cervical ganglion and the carotid artery, suggesting that the differences are CB specific. Further, the sensitivity of BK channels in GCs to mild hypoxia was examined in patch clamp experiments using undissociated CBs. Iberiotoxin significantly inhibited K current of GCs in the DBA/2J mice, but not in the A/J mice. When reducing PO2 to ∼70 mmHg, K current reversibly decreased in GCs of the DBA/2J, but not of the A/J mice. In the presence of iberiotoxin, mild hypoxia did not inhibit K current in either strains. Thus, the data suggest that BK channels in GCs of the DBA/2J mice are sensitive to mild hypoxia. Differential expression of BK channel β subunits in the CBs may, at least in part, explain the different hypoxic sensitivity in these mouse strains.

Carotid chemoreceptor development in mice.

Mice are the most suitable species for understanding genetic aspects of postnatal developments of the carotid body due to the availability of many inbred strains and knockout mice. Our study has shown that the carotid body grows differentially in different mouse strains, indicating the involvement of genes. However, the small size hampers investigating functional development of the carotid body. Hypoxic and/or hyperoxic ventilatory responses have been investigated in newborn mice, but these responses are indirect assessment of the carotid body function. Therefore, we need to develop techniques of measuring carotid chemoreceptor neural activity from young mice. Many studies have taken advantage of the knockout mice to understand chemoreceptor function of the carotid body, but they are not always suitable for addressing postnatal development of the carotid body due to lethality during perinatal periods. Various inbred strains with well-designed experiments will provide useful information regarding genetic mechanisms of the postnatal carotid chemoreceptor development. Also, targeted gene deletion is a critical approach.

Divergent postnatal development of the carotid body in DBA/2J and A/J strains of mice

We have previously shown that the adult DBA/2J and A/J strains of mice differ in carotid body volume and morphology. The question has arisen whether these differences develop during the prenatal or postnatal period. Investigating morphological development of the carotid body and contributing genes in these mice can provide further understanding of the appropriate formation of the carotid body. We examined the carotid body of these mice from 1 day to 4 wk old for differences in volume, morphology, and gene expression of Gdnf family, Dlx2, Msx2, and Phox2b. The two strains showed divergent morphology starting at 1 wk old. The volume of the carotid body increased from 1 wk up to 2 wk old to the level of 4 wk old in the DBA/2J mice but not in the A/J mice. This corresponds with immunoreactivity of LC3, an autophagy marker, in A/J tissues at 10 days and 2 wk. The differences in gene expression were examined at 1 wk, 10 days, and 2 wk old, because divergent growth occurred during this period. The DBA/2Js carotid body at 1 wk old showed a greater expression of Msx2 than the A/Js carotid body. No other candidate genes showed consistent differences between the ages and strains. The difference was not seen in sympathetic cervical ganglia of 1 wk old, suggesting that the difference is carotid body specific. The current study indicates the critical postnatal period for developing distinctive morphology of the carotid body in these mice. Further studies are required to further elucidate a role of Msx2 and other uninvestigated genes.

A Short-Term Fasting in Neonates Induces Breathing Instability and Epigenetic Modification in the Carotid Body.

The respiratory control system is not fully developed in newborn, and data suggest that adequate nutrition is important for the development of the respiratory control system. Infants need to be fed every 2-4 h to maintain appropriate energy levels, but a skip of feeding can occur due to social economical reasons or mild sickness of infants. Here, we asked questions if a short-term fasting (1) alters carotid body (CB) chemoreceptor activity and integrated function of the respiratory control system; (2) causes epigenetic modification within the respiratory control system. Mouse pups (<P14) were fasted for 3-6 h. Breathing became irregular and slow. The number and duration of apnea increased. Ventilatory response to hypoxia was also depressed even after the pups were returned to own dams. These effects were more prominent when the pups were younger and fasting time was longer. The hypoxic response of the carotid sinus nerve activity appeared to be depressed after fasting. Moreover, fasting increased global 5mC and 5-hmC content in DNA isolated from the CB but not DNA in the superior cervical ganglion (SCG). Methylation specific PCR (MSPCR) revealed that fasting increased methylation of leptin and socs3 genes. The results suggest fasting inhibits CB activity leading to hypoventilation, and low glucose does not compensate the low CB activity. Epigenetic effect on CB function/activity may be related to the prolonged effect of fasting on ventilation.

Role of BK Channels in Murine Carotid Body Neural Responses in vivo

The aim of this study was to explore the role of BK channels in the hypoxic sensitivity of the in vivo murine carotid body (CB). Four strains of mice (DBA/2J, A/J, BKα1 knockout and BKα1 wild type - FVB background) were used. The mice were anesthetized, paralyzed and mechanically ventilated (PaCO(2) ~ 35 mmHg, PO(2) > 300 mmHg). We measured carotid sinus nerve (CSN) activity during three gas challenges (F(I)O(2): 0.21, 0.15 and 0.10). CSN activity was analyzed with time-variant spectral analysis with frequency domain conversion (Fast Fourier Transforms). Afferent CSN activity increased with lowering F(I)O(2) in the DBA/2J, BKKO and BKWT mice with the most robust response in 600-800 frequencies. No substantial changes were observed in the A/J mice. Although maximal neural output was similar between the BKKO and BKWT mice, the BKWT had a higher early response compared to BKKO. Thus, BK channels may play a role in the initial response of the CB to hypoxia. The contribution of BKβ subunits or the importance of frequency specific responses was unable to be determined by the current study.

ATP Release from the Carotid Bodies of DBA/2J and A/J Inbred Mouse Strains

The purposes of this study were to: (1) establish an effective method to measure the release of ATP from the mouse carotid body (CB) and (2) determine the release of ATP from the CB of the DBA/2 J (high hypoxic responder) and A/J (low hypoxic responder) mouse in response to hypoxia and hypercapnia. An incubation chamber was constructed utilizing a Costar® Spin-X Centrifuge Tube Filter. The filter was coated with low melting point agarose to hold 4 CBs or 4 superior cervical ganglia (SCG). Hypoxia did not increase ATP release from the CB of either strain. ATP increased in response to a normoxic/hypercapnic challenge in the DBA/2 Js CB but not in the A/Js CB. ATP release from the SCG was affected by neither hypoxia nor hypercapnia in both strains. Thus, we have concluded: (1) we successfully established a chamber system to measure ATP released from the mouse CB; (2) ATP may not be an excitatory neurotransmitter in the CB of these mice under hypoxia; (3) ATP may be a neurotransmitter in the CB of the DBA/2 J mouse strain during hypercapnia.

Hydrogen sulfide acting at the carotid body and elsewhere in the organism

H2S, the most recently explored gasotransmitter, has been found to have actions in at least three types of tissues.