Nikol A. Piskuric

Signal processing at mammalian carotid body chemoreceptors.

Mammalian carotid bodies are richly vascularized chemosensory organs that sense blood levels of O(2), CO(2)/H(+), and glucose and maintain homeostatic regulation of these levels via the reflex control of ventilation. Carotid bodies consist of innervated clusters of type I (or glomus) cells in intimate association with glial-like type II cells. Carotid bodies make afferent connections with fibers from sensory neurons in the petrosal ganglia and receive efferent inhibitory innervation from parasympathetic neurons located in the carotid sinus and glossopharyngeal nerves. There are synapses between type I (chemosensory) cells and petrosal afferent terminals, as well as between neighboring type I cells. There is a broad array of neurotransmitters and neuromodulators and their ionotropic and metabotropic receptors in the carotid body. This allows for complex processing of sensory stimuli (e.g., hypoxia and acid hypercapnia) involving both autocrine and paracrine signaling pathways. This review summarizes and evaluates current knowledge of these pathways and presents an integrated working model on information processing in carotid bodies. Included in this model is a novel hypothesis for a potential role of type II cells as an amplifier for the release of a key excitatory carotid body neurotransmitter, ATP, via P2Y purinoceptors and pannexin-1 channels.

P2Y2 receptor activation opens pannexin-1 channels in rat carotid body type II cells: potential role in amplifying the neurotransmitter ATP.

Carotid body (CB) chemoreceptor complexes consist of receptor type I cells, intimately associated with glia‐like type II cells whose function is poorly understood. We show that type II cells in the rat CB express gap junction‐like proteins, pannexin‐1 (Panx‐1) channels, which form non‐selective pores permeable to ions and large molecules such as ATP, a key CB neurotransmitter. Activation of purinergic P2Y2 receptors on type II cells led to a rise in intracellular Ca2+, and a prolonged membrane depolarization due to opening of Panx‐1 channels. In a CB co‐culture model, where purinergic P2X2/3‐expressing petrosal neurones served as a reporter or biosensor of ATP release, we show that selective activation of P2Y2 receptors on type II cells can lead to ATP release via Panx‐1 channels. We propose that type II cells may function as amplifiers of the neurotransmitter ATP during chemotransduction, via the mechanism of ATP‐induced ATP release.

Effects of chemostimuli on [Ca2+]i responses of rat aortic body type I cells and endogenous local neurons: comparison with carotid body cells

Key points  •  Mammalian aortic bodies (ABs) are putative peripheral chemoreceptor cells presumed to monitor the oxygen content of arterial blood, although their direct chemosensitivity has never been previously demonstrated at the cellular level. •  We used Ca2+ imaging to show for the first time that a variety of stimuli, including hypoxia, isohydric and acidic hypercapnia, and isocapnic acidosis, caused increases in cytosolic [Ca2+] in AB chemoreceptor cells. •  We also showed that some local neurons, known to be uniquely associated with these AB paraganglia in situ, generated robust [Ca2+]i responses to these chemostimuli, suggesting that they may subserve a sensory function. •  These results will help us better understand how AB cells sense the composition of the blood in their local environment near the heart, and how they communicate with sensory neurons to initiate homeostatic reflexes during situations of low oxygen supply, like anaemia.

Expanding role of ATP as a versatile messenger at carotid and aortic body chemoreceptors

Abstract  In mammals, peripheral arterial chemoreceptors monitor blood chemicals (e.g. O2, CO2, H+, glucose) and maintain homeostasis via initiation of respiratory and cardiovascular reflexes. Whereas chemoreceptors in the carotid bodies (CBs), located bilaterally at the carotid bifurcation, control primarily respiratory functions, those in the more diffusely distributed aortic bodies (ABs) are thought to regulate mainly cardiovascular functions. Functionally, CBs sense partial pressure of O2 (), whereas ABs are considered sensors of O2 content. How these organs, with essentially a similar complement of chemoreceptor cells, differentially process these two different types of signals remains enigmatic. Here, we review evidence that implicates ATP as a central mediator during information processing in the CB. Recent data allow an integrative view concerning its interactions at purinergic P2X and P2Y receptors within the chemosensory complex that contains elements of a ‘quadripartite synapse’. We also discuss recent studies on the cellular physiology of ABs located near the aortic arch, as well as immunohistochemical evidence suggesting the presence of pathways for P2X receptor signalling. Finally, we present a hypothetical ‘quadripartite model’ to explain how ATP, released from red blood cells during hypoxia, could contribute to the ability of ABs to sense O2 content.

Confocal immunofluorescence study of rat aortic body chemoreceptors and associated neurons in situ and in vitro

Aortic bodies (ABs) are putative peripheral arterial chemoreceptors, distributed near the aortic arch. Though presumed to be analogous to the well‐studied carotid bodies (CBs), their anatomical organization, innervation, and function are poorly understood. By using multilabel confocal immunofluorescence, we investigated the cellular organization, innervation, and neurochemistry of ABs in whole mounts of juvenile rat vagus and recurrent laryngeal (V‐RL) nerves and in dissociated cell culture. Clusters of tyrosine hydroxylase‐immunoreactive (TH‐IR) glomus cells were routinely identified within these nerves. Unlike the CB, many neuronal cell bodies and processes, identified by peripherin (PR) and neurofilament/growth‐associated protein (NF70/GAP‐43) immunoreactivity, were closely associated with AB glomus clusters, especially near the V‐RL bifurcation. Some neuronal cell bodies were immunopositive for P2X2 and P2X3 purinoceptor subunits, which were also found in nerve terminals surrounding glomus cells. Immunoreactivity against the vesicular acetylcholine transporter (VAChT) was detected in local neurons, glomus cells, and apposed nerve terminals. Few neurons were immunopositive for TH or neuronal nitric oxide synthase. A similar pattern of purinoceptor immunoreactivity was observed in tissue sections of adult rat V‐RL nerves, except that glomus cells were weakly P2X3‐IR. Dissociated monolayer cultures of juvenile rat V‐RL nerves yielded TH‐IR glomus clusters in intimate association with PR‐ or NF70/GAP‐43‐IR neurons and their processes, and glial fibrillary acidic protein‐IR type II (sustentacular) cells. Cocultures survived for several days, wherein neurons expressed voltage‐activated ionic currents and generated action potentials. Thus, this coculture model is attractive for investigating the role of glomus cells and local neurons in AB function. J. Comp. Neurol. 519:856–873, 2011.

Low glucose sensitivity and polymodal chemosensing in neonatal rat adrenomedullary chromaffin cells.

Glucose is the primary metabolic fuel in mammalian fetuses, yet mammals are incapable of endogenous glucose production until several hours after birth. Thus, when the maternal supply of glucose ceases at birth there is a transient hypoglycemia that elicits a counterregulatory surge in circulating catecholamines. Because the innervation of adrenomedullary chromaffin cells (AMCs) is immature at birth, we hypothesized that neonatal AMCs act as direct glucosensors, a property that could complement their previously established roles as hypoxia and acid hypercapnia sensors. During perforated-patch, whole cell recordings, low glucose depolarized and/or excited a subpopulation of neonatal AMCs; in addition, aglycemia (0 mM glucose) caused inhibition of outward K(+) current, blunted by the simultaneous activation of glibenclamide-sensitive K(ATP) channels. Some cells were excited by each of the three metabolic stimuli, i.e., aglycemia, hypoxia (Po(2) ∼30 mmHg), and isohydric hypercapnia (10% CO(2); pH = 7.4). Using carbon fiber amperometry, aglycemia and hypoglycemia (3 mM glucose) induced robust catecholamine secretion that was sensitive to nickel (50 μM and 2 mM) and the L-type Ca(2+) channel blocker nifedipine (10 μM), suggesting involvement of both T-type and L-type voltage-gated Ca(2+) channels. Fura-2 measurements of intracellular Ca(2+) ([Ca(2+)] (i)) revealed that ∼42% of neonatal AMCs responded to aglycemia with a significant rise in [Ca(2+)] (i). Approximately 40% of these cells responded to hypoxia, whereas ∼25% cells responded to both aglycemia and hypoxia. These data suggest that together with hypoxia and acid hypercapnia, low glucose is another important metabolic stimulus that contributes to the vital asphyxia-induced catecholamine surge from AMCs at birth.

Glucosensing in an immortalized adrenomedullary chromaffin cell line: Role of ATP-sensitive K+ channels

Using an immortalized adrenal chromaffin cell line (MAH cells), we investigated the cellular mechanisms underlying sensitivity to glucose-free solution (aglycemia) using ratiometric Ca2+ imaging and whole-cell recording. Though few cells (< 15%) responded to aglycemia with an increase in intracellular-free Ca2+ concentration ([Ca2+]i), in most cells (approximately 75%), aglycemia caused > 50% suppression of the Delta[Ca2+]i induced by the depolarizing stimulus, high (10 mM) K+. Moreover, in normal K+, the average aglycemia-induced rise in Cai2+ as well as the proportion of aglycemia-responsive cells increased in the presence of the K(ATP) channel blocker, glibenclamide. During membrane potential (Vm) measurements, aglycemia induced either hyperpolarization (6/20), depolarization (4/20) or no change in Vm. RT-PCR and Western blotting confirmed the presence of K(ATP) channel subunits Kir6.2 and SUR1 in MAH cells. These findings suggest a dual inhibitory and excitatory action of aglycemia in MAH cells, where activation of K(ATP) channels effectively inhibits or blunts the Delta[Ca2+]i due to the excitatory effect. Thus, this cell line appears as an attractive model for studying the molecular mechanisms of glucosensing.

Potential roles of ATP and local neurons in the monitoring of blood O2 content by rat aortic bodies

•  What is the central question of this study? How do aortic bodies monitor blood O2 content? We investigated whether ATP, known to be released from red blood cells during hypoxia, could contribute via interactions with local neurons. •  What is the main finding and its importance? In dissociated aortic body cultures from the vagus and recurrent laryngeal nerves, some local neurons expressed functional P2X2/3 purinoceptors and were electrically coupled; there was also the potential for cholinergic neurotransmission. Large molecules, such as Evans Blue, had easy access to local neurons via the circulation. We hypothesize that sensing of low blood O2 content may involve ATP release from red blood cells, leading to stimulation of local ‘sensory’ aortic body neurons.

Developmental Regulation of Glucosensing in Rat Adrenomedullary Chromaffin Cells: Potential Role of the K ATP Channel

During birth, when the maternal supply of glucose is occluded, there is a drastic fall in blood glucose in the newborn. This stimulus triggers the non-neurogenic release of catecholamines from adrenomedullary chromaffin cells, which restores blood glucose homeostasis. In this report we present preliminary data showing that glucosensing is present in neonatal chromaffin cells from adrenal slices but absent in chromaffin cells from juvenile slices. Moreover, we show that the aglycemia-evoked rise in intracellular Ca2+ is robust in neonatal chromaffin cells but blunted in juvenile chromaffin cells. Lastly, we show that the Kir6.2 subunit of the KATP channel, is upregulated in the adrenal medulla in juvenile animals providing a potential mechanism for the developmental regulation of glucosensing.