Alexander Balbir

Role of acetylcholine in neurotransmission of the carotid body

Acetylcholine (ACh) has been considered an important excitatory neurotransmitter in the carotid body (CB). Its physiological and pharmacological effects, metabolism, release, and receptors have been well documented in several species. Various nicotinic and muscarinic ACh receptors are present in both afferent nerve endings and glomus cells. Therefore, ACh can depolarize or hyperpolarize the cell membrane depending on the available receptor type in the vicinity. Binding of ACh to its receptor can create a wide variety of cellular responses including opening cation channels (nicotinic ACh receptor activation), releasing Ca(2+) from intracellular storage sites (via muscarinic ACh receptors), and modulating activities of K(+) and Ca(2+) channels. Interactions between ACh and other neurotransmitters (dopamine, adenosine, nitric oxide) have been known, and they may induce complicated responses. Cholinergic biology in the CB differs among species and even within the same species due to different genetic composition. Development and environment influence cholinergic biology. We discuss these issues in light of current knowledge of neuroscience.

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.

The impact of adenosine on the release of acetylcholine, dopamine, and norepinephrine from the cat carotid body.

Exogenously administered adenosine provokes an increase in respiration in both animal models and in man. Administered near the carotid body adenosine increases neural output from the carotid body in rats and cats. Hypoxia has the same effect. Hypoxia also provokes a release of acetylcholine (ACh), dopamine (DA), and norepinephrine (NE) from the carotid body. The present study aimed to determine the effect of exogenous adenosine on the release of ACh, DA, and NE from the carotid bodies of cats. After a recovery period (from surgery) carotid bodies were first incubated for 10 (DA, NE) or 15 (ACh) min in Eppendorf tubes containing 85 microL of a physiological salt solution equilibrated with 40% O2/5% CO2 at 37 degrees C (hyperoxia). At the end of the incubation period the medium was drawn off, and measured for ACh, DA, and NE using HPLC-ECD methods. Next 85 microL of the medium and the tubes were equilibrated with a hypoxic gas mixture (4% O2/5% CO2) and the carotid bodies were incubated for 10 (DA, NE) or 15 (ACh) min, at the end of which the medium was drawn off and measured for ACh, DA, and NE. In the ACh studies there followed a post-hypoxic hyperoxic exposure (40% O2/5% CO2). ACh tubes were then made 100 microM with respect to adenosine, and the hyperoxic, hypoxic, and post-hypoxic hyperoxic challenges were repeated. One of the two DA, NE tubes had the 100 microM adenosine from the start. Adenosine significantly increased the release of ACh, but significantly decreased the hypoxia-induced release of DA. Potential mechanisms for these changes are reviewed.

Identification of M1 and M2 muscarinic acetylcholine receptors in the cat carotid body chemosensory system.

The carotid body is a major arterial chemoreceptor that senses low O2 tension, high CO2 tension and low pH in the arterial blood. It is generally believed that neurotransmitters, including acetylcholine (ACh), participate in the genesis of afferent neural output from the carotid body and modulate the function of chemoreceptor cells (glomus cells). Previous pharmacological studies suggest that M1 and M2 muscarinic ACh receptors (mAChRs) are involved in these processes. This study was designed to demonstrate the presence and localization of M1 and M2 mAChRs in the carotid body and in the petrosal ganglion of the cat. Since DNA sequences of the cat M1 and M2 mAChRs were not known, we first determined partial DNA sequences. These sequences and deduced amino acid sequences highly resembled those of human and the rat. Subsequent reverse transcription-polymerase chain reaction (RT-PCR)analysis has demonstrated that mRNAs for M1 and M2 mAChRs are present in the carotid body and the petrosal ganglion of the cat. Immunohistochemistry has indicated that the localization of these receptors appears different. Immunoreactivity for M1 mAChR was strong in nerves in the carotid body. Nerve endings positively stained for M1 mAChR appear to innervate glomus cells. Weak staining for M1 mAChRs was seen in glomus cells. On the other hand, M2 receptor protein seems to be present in glomus cells but not on nerve endings. One third of the neurons in the petrosal ganglion showed immunoreactivity for M1 mAChR. Many neurons and nerve fibers in the petrosal ganglion expressed M2 mAChR immunoreactivity. The results were consistent with previous pharmacological studies. Thus, activation of M1 mAChRs on afferent nerve endings may be linked to the increase in neural output during hypoxia. Further, M1 and M2 mAChRs on glomus cells modulate the release of neurotransmitters.

Behavioral and respiratory characteristics during sleep in neonatal DBA/2J and A/J mice

The ventilatory response to hypoxia depends on the carotid body function and sleep-wake states. Therefore, the response must be measured in a consistent sleep-wake state. In mice, EMG with behavioral indices (coordinated movements, CMs; myoclonic twitches, MTs) has been used to assess sleep-wake states. However, in neonatal mice EMG instrumentation could induce stress, altering their behavior and ventilation. Accordingly, we examined: (1) if EMG can be eliminated for assessing sleep-wake states; and (2) behavioral characteristics and carotid body-mediated respiratory control during sleep with EMG (EMG+) or without EMG (EMG-). Seven-day-old DBA/2J and A/J mice were divided into EMG+ and EMG- groups. In both strains, CMs occurred when EMG was high; MTs were present during silent/low EMG activity. The durations of high EMG activity and of CMs were statistically indifferent. Thus, CMs can be used to indicate wake state without EMG. The stress caused by EMG instrumentation may be distinctively manifested based on genetic background. Prolonged agitation was observed in some EMG+ DBA/2J (5 of 13), but not in A/J mice. The sleep time and MT counts were indifferent between the groups in DBA/2J mice. The EMG+ A/J group showed longer sleep time and less MT counts than the EMG- A/J group. Mean respiratory variables (baseline, hyperoxic/hypoxic responses) were not severely influenced by EMG+ in either strain. Individual values were more variable in EMG+ mice. Carotid body-mediated respiratory responses (decreased ventilation upon hyperoxia and increased ventilation upon mild hypoxia) during sleep were clearly observed in these neonatal mice with or without EMG instrumentation.

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.

Genetic regulation of chemoreceptor development in DBA/2J and A/J strains of mice.

The role of the carotid body (CB) in response to hypoxia is very well defined (Fitzgerald and Shirahata, 1997). The hypoxic ventilatory response (HVR) is characterized by an increase in ventilation, but this response remains variable among individuals (Eisele et al., 1992; Vizek et al., 1987; Weil 1970). Genetics may play a critical role in explaining this variability. Indeed, longitudinal and twin studies do demonstrate the role of genetics in the HVR (Collins et al., 1978; Kawakami et al., 1982). Studies utilizing inbred strains of mice have also demonstrated the effect of genetics on the response to hypoxia (Tankersley et al., 1994 & 2000). Two strains of mice in these studies were identified as having extreme responses to hypoxia. The DBA/2J strain demonstrated the highest HVR, whereas the A/J strain demonstrated the lowest HVR (Tankersley et al., 1994). In another study analyzing the role of genetics in a mouse model of sleepinduced hypoxia, the DBA/2J strain demonstrated an increased sensitivity to hypoxia during sleep, compared to that of the A/J strain (Rubin et al., 2004). In order to elucidate a potential explanation that may contribute to this difference in hypoxic sensitivity, we examined CB morphology and volume in adult DBA/2J and A/J strains (Yamaguchi et al., 2003). Results demonstrated a significantly larger volume as well as an increased glomus cell quantity in the CB of DBA/2J strain compared to that of the A/J strain. A question arises whether these differences exist from early neonatal ages. Or, developmental plasticity may contribute to these strain differences. In this study, we analyzed CB volume, glomus cell quantity, and ventilation during development in the DBA/2J and A/J strains of mice. We also introduced a glimpse into the genetic influence on chemoreceptor development with emphasis on the role of glial-cell-line-derived neurotrophic factor (GDNF).

Genetic Influence on Carotid Body Structure in DBA/2J and A/J Strains of Mice

The carotid body is a major chemosensory organ for hypoxia, hypercapnia and acidosis in the arterial blood (Fitzgerald and Shirahata 1997; Gonzalez et al., 1994). During hypoxia, the neural output from the carotid body increases and reflexly modifies several variables in the respiratory system. A prominent response is an increase in ventilation, but the hypoxic ventilatory response (HVR) among individuals varies widely (Eisele et al., 1992; Vizek et al., 1987). Studies in humans (Collins et al., 1978; Kawakami et al., 1982; Nishimura et al., 1991; Thomas et al., 1993) suggest that genetic factors significantly contribute to those differences. Similarly, studies in mice (Tankersley et al., 1994) and rats (Weil et al., 1998) clearly indicate that genetic determinants robustly influenced HVR. Among several inbred strains of mice the DBA/2J mice demonstrated the highest HVR and the A/J mice the lowest HVR (Tankersley et al., 1994). The differences in HVR between these two strains of mice may be closely related to the structural differences of the carotid body (Yamaguchi et al., 2003). The size of the carotid body and the quantity of glomus cells in the DBA/2J mice are significantly larger than those in the A/J mice. Those differences were clearly segregated between the strains, suggesting that genetic factors strongly influence the observed phenotypic differences between the DBA/2J and A/J mice. The purpose of the current study was to confirm that the morphological characteristic differences in the carotid body between the DBA/2J and A/J mice are controlled by genetic factors. Thus, we generated the first-filial progeny (F1) by a crossing the DBA/2J (female) and A/J (male) strains of mice, and examined the morphological differences of the carotid body in the DBA/2J, A/J and their F1 mice.

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.

Modulators of Cat Carotid Body Chemotransduction

The Carotid Body (CB) senses hypoxia, hypercapnia, and acidosis in the arterial blood. The resulting increase in CB neural output (CBNO) to the nucleus tractus solitarius in the medulla promotes reflex responses in the respiratory, circulatory, renal, and endocrine systems. Increases in CBNO are commonly thought to be due to the release of neurotransmitters from glomus cells in the CB. Additional to the action of these released transmitters on the postsynaptic afferent neurons which abut on the glomus cells the transmitters act presynaptically on glomus cell autoreceptors. Among the several transmitters contained in the glomus cells there now exists considerable evidence supporting excitatory roles for both acetylcholine (ACh) and ATP and an inhibitory role for dopamine (DA) and norepinephrine (NE) (Fitzgerald, 2000). The release of ACh (Fitzgerald et al., 1999; Kim et al., 2004) and catecholamines (Wang and Fitzgerald, 2002) appears to be influenced by modulators. The present study investigated the action of adenosine (ADO) on the release of ACh, DA, and NE since it has been reported that ADO influences CBNO (McQueen and Ribeiro, 1981) and CB-mediated increases in ventilation (Monteiro and Ribeiro, 1987). The study further investigated the action of nitric oxide (NO) on the release of ACh since NO has been reported to reduce the hypoxia-induced increase in CBNO (Wang, et al., 1994).