The Effect Of Cytokines And Acute Hypoxia On Intracellular Ca²⁺ And Neurotransmitters Release In Rat Carotid Body Glomus Cells

The carotid body (CB) is a peripheral arterial chemoreceptor organ located the bifurcation of the common carotid artery. The carotid body chemoreceptor can sense PaO2, PaCO2 and pH in blood, and generate preventive respiratory and cardiovascular reflexes. The carotid body chemoreceptors consist of groups of specialized sensory glomus (type I) cells and sustenacular (type II) cells surrounded by a plexus of fenestrated capillaries and sinusoids. The most distinguishing feature of type I cell is the presence of numerous dense-core vesicles in their cytoplasm, which are known to contain ACh, catecholamines (CAs, including DA and NA), enkephalin, substance P and other putative neurotransmitters. Type II cells are thin and elongate in shape and surround clusters of type I cell in an ensheathing, capsule-like fashion.The carotid body is innervated by afferent terminals of the carotid sinus nerve (CSN), a branch of the ninth cranial nerve, whose neurons located in the petrosal ganglion. The carotid body is innervated by adrenergic nerve and parasympathetic nerve, too.This study includes two sets of experiments: (1) The effect of proinflammatory cytokine IL-6 on intracellular Ca2+ ([Ca2+]i) and neurotransmitters release in rat CB glomus cells.(2) Based on the previous work, hypoxia-induced CAs secretion in rat CB in vivo was further explored.1 The effect of IL-6 on [Ca2+]i and neurotransmitters release in rat CB glomus cellsCommunication pathways exist between the immune system and brain, allowing bidirectional regulation of immune and brain responses to infection. Detection of pathogens (e.g., bacteria) by the cells of the immune system (e.g., macrophages) triggers these immune cells to synthesize and release a series of chemical messengers, including the cytokines tumor necrosis factor-α(TNF-α), interleukin-β(IL-1β) and IL-6. A lot of evidence showed the cytokines signal the brain through several routes when infection has occurred. One important route is that cytokines stimulate vagal afferents terminals or paraganglia which form afferent synapses with vagal fibers, increase vagal afferent nerve activity, which carry these signals directly to the brain. CB, considered the largest paraganglion in the body, might play the common role with abdominal paraganglia.In a previous study, we found that interleukin 1 receptor type I (IL-1RI) and interleukin 6 receptor alpha (IL-6Rα) was strongly expressed in type I cells of the carotid body in SD rats, and this indicated there is a substance basis in the carotid body which could senses the stimulation of proinflammatory cytokine. Electrophysiologic studies confirmed extracellular application of IL-1β significantly decreased the outward potassium current, triggered a transient rise of [Ca2+]i in the cultured glomus cells of rat CB, and increased the discharge rate in the carotid sinus nerve. Above studies showed CB is not only a well-known peripheral arterial chemoreceptor organ, but also a organ sensing blood-borne immune stimulus (e.g., IL-1), result in increasing vagal afferent nerve activity, which signal to the brain. L-6 is a polyfunctional proinflammatory cytokine, which has been proposed as a mediator for immune-to-brain communication. As mentioned above, IL-6Rαis expressed in the CB of normal rats, suggesting that the CB might directly sense IL-6 levels. However, the precise effect of IL-6 on CB has not been elucidated.In this part of experiments, regarding the CB as research object, by using immunohistochemistry, western blotting, cell culture, calcium imaging, amperometric technology etc, we investigated the effect of IL-6 on [Ca2+]i and CAs release in rat CB glomus cells. We also observed the expression of dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R) in rat CB by using immunohistochemistry.The results are as follows.(1) In normal rat CB, IL-6Rαand gp130 were expressed in glomus cells, suggesting that the CB might directly sense IL-6 levels.(2) Calcium imaging showed that extracellular application of IL-6 or TNF-αinduced a rise in [Ca2+]i in cultured rat CB glomus cells.(3) Amperometry showed that local application of IL-6 evoked CAs release from isolated glomus cells or the clusters of glomus cell. CAs release from the glomus cells was dose-dependent and mediated by external Ca2+ influx.(4) The results showed D1R and D2R were expressed in normal rat CB. D1R only expressed in CB glomus cells. However, Strong immunoreactivity for D2R is distributed not only in the glomus cells but also in nerve fibers in rat CB.The study shows IL-6 receptor exist in normal rat CB glomus cells, extracellular application of IL-6 or TNF-αinduced a rise in [Ca2+]i in cultured rat CB glomus cells, and IL-6 evoked CAs release from glomus cells. These researches provided more evidence to the hypothesis that CB is a peripheral organ sensing immune information, and may guide us to recognize and research the function of the carotid body in a new view.2 hypoxia-induced CAs secretion in rat CB in vivoIt is well known that CAs is one of transmitters released by CB glomus cells. CAs including DA and NA, DA is predominant in several species. Although glomus cells also contain NA, hypoxia preferentially releases DA rather than NA. However, previous studies on hypoxia induced DA release were based on isolated single glomus cells or intact CBs, these responses in vitro don't reflect the real responses of the carotid body in vivo. In order to known CB's response to hypoxia in vivo, the release of DA was recored from rat CB in real time in vivo by amperometric technique and compared with that in vitro. The study will provide some evidence for us to understand DA's function during hypoxia processing in rats.The present work was followed SHF's previous work. In contrast to the increase of catecholamine release from the isolated CB tissue or single glomus cells, he found that hypoxia induced a significant decrease of amperometric current (Iamp) in vivo, it seems to suggest hypoxia induced a rapid decrease in CAs release in vivo. In additional, he observed this evoked rapid CAs decrease signal was reversed by cutting the CSN. By using amperometric technology in vitro and in vivo, we investigated whether the decrease of hypoxia-induced Iamp in vivo reflects the decrease of CAs release.The results are as follows.(1) Consistent with SHF's recordings, hypoxia induced a significant decrease Iamp recorded by CFE in anaesthetized rats CB in vivo. However, the tests in isolated CBs showed a significant increase in Iamp following perfusion with a hypoxic solution. These results seem to show that hypoxia induced a rapid decrease in CAs release in vivo and a rapid increase in CAs release in vitro.(2) In contrast to SHF's result, the decrease in Iamp induced by hypoxia in vivo was not reversed by denervation. This result suggests that the difference of hypoxia-induced CAs secretion between in vivo and in vitro was not resulted in by neural feedback.(3) Dopamine transporter inhibitors (cocaine and nomifensine) didn't affect hypoxia-induced Iamp in CB of rat in vivo. We measured CB response to hypoxia in vivo by CFE at different holding potentials, and found the decreased Iamp induced by hypoxia in vivo was not really due to specific oxidation of CAs. The two experiments showed that hypoxia-induced Iamp decraese in rat CB in vivo was not induced by the reduced CAs secretion.(4) The baseline of Iamp recorded in rat CB by CFE was the same as that in PBS in vivo, it indicates the baseline of Iamp recorded in CB by CFE in vivo didn't reflect the base level of CAs secretion in CB. We found the decreased Iamp induced by hypoxia in rat CB also was recorded in the area near CB of rat by CFE.(5) In additional, CO2 treatment in anaesthetized rats, a significant increase in Iamp was recorded by CFE in CB in vivo. These results seem to show that CO2 induced a rapid increase in CAs release in viv. However, it needs us to futher explore. We also observed that CO2 significantly increased the discharge rate in the CSN in vivo.This part of experiments denied SHF's conclusion. We considered that the decreased Iamp induced by hypoxia in anaesthetized rats CB in vivo didn't reflect CAs secretion decreasing. It needs to be further explored that the increased Iamp induced by CO2 reflect CAs secretion decreasing in anaesthetized rats CB in vivo.