The Responses Of Carotid Body To IL-1β And Acute Hypoxia Stimuli

Increasing evidences have indicated that the immune system does not act in isolation but strongly influenced by central nervous system (CNS) through autonomic nerves or neuroendocrine hormones2. Furthermore, the modulations between the CNS and immune system are bidirectional3,4. Recent investigations have proposed that there exists an immunosensory system, in which immune-derived cells detect infections to ensure adequate immune reactions and concomitantly, signal appropriate neural substrates including the brain.Upon detection of pathogens, immune cells release a variety of immune cytokines such as interleukin-1β(IL-1β), tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6) and interferon-gamma (IFNγ) during the early state of immune challenge. Among these particular cytokines, IL-1βis a key initiator for the febrile response, a brain-mediated early event during immune and infectious condition. Thus, IL-1βappears to play a prominent role in mediation of peripheral immune activation to the CNS3,5. However, because it is a large hydrophilic peptide and hardly across the blood brain barrier6, how the IL-1βaffects the brain still is an unsolved question. Several possible routes for the cytokine-to-brain communication have been proposed, such as via the circumventricular organs (areas in the brain are lack of blood-brain barrier), brain vascular epithelial cells by saturable transport mechanism or by production of downstream signal molecules such as prostagladin E2, and meningeal cells4,7. However, the amount of cytokines entered the brain through these proposed routes are rather small and whether such a trace amount of cytokines could effectively evoke a brain activity is uncertain. In recent years, the peripheral nerves, most notably the vagus, have been paid much attention in this respect9,10. It is hypothesized that the peripherally derived cytokines might stimulate the sensory endings of vagus or glomus cells of paraganglia, the minute dopaminergic cell clusters closely associated with the branches of the vagus and innervated by the nerve 11, and subsequently, the information are sent to the brain through firings conducted in the nerve. The experiment of Goehler et al has provided an indirect evidence that biotinylated IL-1 receptor antagonist could bind to the glomus cells of vagal paraganglia to support this hypothesis12. However, no direct electrophysiological evidences are available at present.The mammalian carotid body (CB) is a small vascular organ situated in the carotid artery bifurcation and classically recognized as a peripheral chemoreceptor responsible for detection of decreased PO2 (hypoxia), increased PCO2 (hypercapnia), and decreased pH (acidity) in the arterial blood13. Recent evidences show that the organ can also sense low blood glucose14 further support its role as a polymodal sensor to multiple stimuli from the blood. According to anatomical categorization, the CB is the largest paraganglion in the body that has the similar configuration to its abdominal partner15. For example, the CB is also composed of two types of cells, glomus cells (type I cells, principal cells) and sustentacular cells (type II cells, supporting cells), and the former is dopaminergic as well. Our previous work has demonstrated morphologically that the IL-1 receptor type I is strongly expressed in the glomus cells in the normal rat CB16. Given these, a hypothesis is raised that peripheral immune cytokines might cause depolarization of glomus cells, induce secretion of excitatory transmitters and evoke firings in the carotid sinus nerve (CSN), the afferent nerve innervating the organ. These proposed connections may be an alternative route in transmission of peripheral immune signals to the brain.The PC12 cell line was originally established from a transplantable rat adrenal pheochromocytoma. Similar to normal adrenomedullary cells, the PC12 cells can synthesize, store, and secrete catecholamine, a group of substances served as hormone in periphery and as transmitter in CNS. Since the cells undergo further differentiation to develop properties of sympathetic ganglion neurons upon application of nerve growth factor (NGF) 18, PC12 cells have become a well-used cell model in a wide variety of physiological and pharmacological studies for neuronal functions, such as regulatory mechanisms of neuronal excitability and effect of reagents on neurons 19-21. Interestingly, it has been proved recently that the PC12 cells are sensitive to hypoxia and acid, therefore, these cells could be used a cell model to study mechanisms of chemoreception 25-27.Addrenomedullary cells are belonging to the category of paraneurons according to anatomical criteria. PC12 cell share many common physiological and pharmacological properties with other paraganglionic cells such as the carotid body type I glomus cells 20,32. In ontogeny, both addrenomedullary cells and carotid body glomus cells are originated from neural crest and both of them bear O2-sensitive K+ channels22,23. They both synthesize and store catecholamines and a variety of neuropeptides such as dopamine and acetylcholine 13,24; and both of them depolarize and secrete catecholamine in response to hypoxia24,25,33,34. Furthermore, recent study 27 indicated that, similar to the CB glomus cell, acid could evoke catecholamine secretion from PC12 cells.Therefore, by using patch-clamping technology, extracellular recording technology, calcium imaging, amperometric technology etc., we investigated the effect of IL-1βon the electrophysiologic characteristics and the [Ca²⁺]i of PC12 cells and CB glomus cells and, also the effect of of IL-1βon the CSN firings and the secretion of catecholamine of CB. The results are briefly listed in the following:â‘´We demonstrated morphologically that interleukin-1 receptor type I was expressed in the PC12 cells. Extracellular application of IL-1βinhibited the outward voltage-dependent and TEA-sensitive potassium currents (IK) in the PC12 cells in a concentration-dependent manner, and pre-incubation with the interleukin-1 receptor antagonist (IL-1ra) almost completely abolished this effect. Furthermore, application of IL-1βshifted steady-state inactivation of IK in hyperpolarizing direction, but did not alter its steady-state activation. In addition, IL-1β-induced inhibition of IK leads to membrane depolarization and a transient increase of [Ca²⁺]i in PC12 cells .⑵The results from whole-cell patch clamp recordings and calcium imaging showed that extracellular application of IL-1βsignificantly decreased the outward potassium current and triggered a transient rise of [Ca2 +] i in the cultured glomus cells of rat CB. Furthermore, by using extracellular recordings and pharmacological intervention, it was found that IL-1βstimulation to CB in the anaesthetized rat in vivo significantly increased the discharge rate in the carotid sinus nerve, most likely mediated by ATP but not dopamine release.⑶In contrast to the increase of catecholamine release from the isolated CB tissue or single glomus cells, hypoxia induced a rapid decrease in catecholamine release in vivo. This evoked rapid catecholamine decrease signal was reversed by cutting the CSN, indicating that catecholamines play an important role in communication between the CB and the CSN in vivo. On the other hand, this decease of catecholamine probably excited the CSN in return because the direct application of dopamine, the most abundant catecholamine transmitter in the CB, suppressed hypoxia-induced sinus nerve activity. Blocking dopamine action in the CB with D2 receptor antagonist haloperidol increased spontaneous CSN activity. However, topic application of D1 receptor SCH23390 has no significant influence on the CSN activity. In addition, the immunohistochemical results showed that both D1 and D2 receptors express in the CB glomus cells and the terminal of CSN and, notely, the D1 is mostly located in the glomus cells and D2 is mostly located in the CSN fibers. Futermore, extracellular application of IL-1β, via both D1 and D2 receptors, induced endocytosis of CB glomus cell. In addition, adenosine attenuated or reversed hypoxia-induced CAs decrease in CB of rat in situ.The main conclusions that draw from the above results are described as the following:â‘´PC12 cell has the potential ability of sensing extracellular cytokine stimuli such as IL-1β. Although the mechanisms underlying IL-1βsensing via binding receptor have yet to be fully determined, the phenomenon is clearly of great physiological importance.⑵The response of CB to IL-1βstimulation provides evidences for a novel function of CB in perception of immune stimulation and proposes a possibility that the CB might play a role in immune-to-brain communication. However, the detailed mechanisms and the relationship between responses induced by IL-1βand other chemical stimulus in CB need to be further investigated.⑶The basal dopamine level in CB is sufficient to prevent most spontaneous firing in CSN in normoxia. During hypoxia, however, CSN fires upon removal of partial inhibitory dopamine level in the CB and, adenosine may play a key role during this process. We conclude that DA is the transducer between oxygen sensor cells and CSN in vivo.