Machiko Shirahata

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.

Presence of nicotinic acetylcholine receptors in cat carotid body afferent system.

With immunocytochemical techniques using a monoclonal antibody for alpha7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that alpha7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed alpha7 subunits. These data support a role for acetylcholine in carotid body neurotransmission.

Acetylcholine release from cat carotid bodies.

Hypoxia, hypercapnia and acidosis stimulate the carotid body (CB) sending increased neural activity via a branch of the glossopharyngeal nerve to nucleus tractus solitarius; this precipitates an impressive array of cardiopulmonary, endocrine and renal reflex responses. However, the cellular mechanisms by which these stimuli generate the increased CB neural output are only poorly understood. Central to the understanding of these mechanisms is the determination of which agents are released within the CB in response to hypoxia, and serve as the stimulating transmitter(s) for chemosensory nerve endings. Acetylcholine (ACh) has been proposed as such an agent from the outset, but this proposal has been, and remains, controversial. The present study tests two hypotheses: (1) The CB releases ACh under normoxic/normocapnic conditions; and (2) The amount released increases during hypoxia and other conditions known to increase neural output from the CB. These hypotheses were tested in 12 experiments in which both CBs were removed from the anesthetized cat and incubated at 37 degrees C in a physiological salt solution while the solution was bubbled with four different concentrations of oxygen and carbon dioxide. The incubation medium was exchanged at 10 min intervals for 30 min (three periods of incubation). The medium was analyzed with high performance liquid chromatography-electrochemical detection for ACh content. Normoxic/normocapnic conditions (21% O2/6% CO2) produced a total of 0.639 +/- 0.106 pmol/150 microl (mean +/- S.E.M.; n = 12). All stimulating conditions produced larger total outputs: 4% O2/2% CO2 produced 1.773 +/- 0.46 pmol/150 microl; 0% O2/5% CO2, 0.868 +/- 0.13 pmol/150 microl; 4% O2/10% CO2, 1.077 +/- 0.21 pmol/150 microl. These three amounts were significantly greater than the normoxic/normocapnic condition, but indistinguishable among themselves. Further, the amount of ACh released did not diminish over the 30 min of stimulation. These data support the concept that during hypoxia ACh functions as a stimulating transmitter in the CB, and are consistent with the earlier reports of cholinergic enzymes and receptors found in the CB.

Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support of this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

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.

EFFECTS OF A CONTINUOUS INFUSION OF DOPAMINE ON THE VENTILATORY AND CAROTID BODY RESPONSES TO HYPOXIA IN CATS

1. We investigated how a continuous infusion of dopamine (DA; 5μg/kg per min), which is often used clinically, would affect the ventilation and carotid chemoreceptor neural activity in anaesthetized cats.

The presence of CO2/HCO3- is essential for hypoxic chemotransduction in the in vivo perfused carotid body

Carotid chemoreceptor activity was increased by the perfusion of the carotid body in vivo with hypoxic HEPES-buffered solution (HBS) containing CO2/HCO3- (HBA+), but not with hypoxic HBS without CO2/HCO3- (HBS-). When the perfusate was switched to hypoxic HBS+ during hypoxic HBS-perfusions, chemoreceptor activity increased immediately. Thus, CO2/HCO3- played a critical role in the hypoxic chemotransduction of the in vivo perfused carotid body.

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.

Carotid body denervation prevents fasting hyperglycemia during chronic intermittent hypoxia

Obstructive sleep apnea causes chronic intermittent hypoxia (IH) and is associated with impaired glucose metabolism, but mechanisms are unknown. Carotid bodies orchestrate physiological responses to hypoxemia by activating the sympathetic nervous system. Therefore, we hypothesized that carotid body denervation would abolish glucose intolerance and insulin resistance induced by chronic IH. Male C57BL/6J mice underwent carotid sinus nerve dissection (CSND) or sham surgery and then were exposed to IH or intermittent air (IA) for 4 or 6 wk. Hypoxia was administered by decreasing a fraction of inspired oxygen from 20.9% to 6.5% once per minute, during the 12-h light phase (9 a.m.-9 p.m.). As expected, denervated mice exhibited blunted hypoxic ventilatory responses. In sham-operated mice, IH increased fasting blood glucose, baseline hepatic glucose output (HGO), and expression of a rate-liming hepatic enzyme of gluconeogenesis phosphoenolpyruvate carboxykinase (PEPCK), whereas the whole body glucose flux during hyperinsulinemic euglycemic clamp was not changed. IH did not affect glucose tolerance after adjustment for fasting hyperglycemia in the intraperitoneal glucose tolerance test. CSND prevented IH-induced fasting hyperglycemia and increases in baseline HGO and liver PEPCK expression. CSND trended to augment the insulin-stimulated glucose flux and enhanced liver Akt phosphorylation at both hypoxic and normoxic conditions. IH increased serum epinephrine levels and liver sympathetic innervation, and both increases were abolished by CSND. We conclude that chronic IH induces fasting hyperglycemia increasing baseline HGO via the CSN sympathetic output from carotid body chemoreceptors, but does not significantly impair whole body insulin sensitivity.

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.