Cultured Carotid Body Research Articles

Evidence for protein kinase involvement in the 5-HT-[Ca2+ ]i -pannexin-1 signalling pathway in type II glial cells of the rat carotid body.

What is the central question of this study? The mammalian carotid body (CB) is a peripheral chemoreceptor organ involved in O2 and CO2 /H+ homeostasis. Recent studies suggest that 5-HT, released from CB receptor cells, can stimulate adjacent glial-like typeII cells, leading to an increase in intracellular Ca2+ (Δ[Ca2+ ]i ) and activation of ATP-permeable pannexin-1 (Panx-1) channels. The aim of this study was to elucidate the role of protein kinases in the 5-HT-[Ca2+ ]i -Panx-1 signalling pathway. What is the main finding and its importance? Src family kinase and protein kinaseA, acting downstream from Δ[Ca2+ ]i , played central roles in 5-HT-mediated Panx-1 channel activation. This provides new insight into mechanisms regulating CB excitation, especially in pathophysiological conditions. Chemoreceptor (typeI) cells of the rodent carotid body (CB) synthesize and release several neurotransmitters/neuromodulators, including 5-hydroxytryptamine (5-HT), implicated in enhanced CB excitation after exposure to chronic intermittent hypoxia, e.g. sleep apnoea. However, recent studies suggest that 5-HT can robustly stimulate adjacent glial-like typeII cells via ketanserin-sensitive 5-HT2 receptors, leading to intracellular Ca2+ elevation (Δ[Ca2+ ]i ) and activation of ATP-permeable pannexin-1 (Panx-1) channels. Using dissociated rat CB cultures, we investigated the role of protein kinases in the intracellular signalling pathways in typeII cells. In isolated typeII cells, 5-HT activated a Panx-1-like inward current (I5-HT ) that was reversibly inhibited by the Src family kinase inhibitor PP2 (1μm), but not by its inactive analogue, PP3 (1μm). Moreover, I5-HT was reversibly inhibited (>90%) by H89 (1μm), a protein kinaseA blocker, whereas the protein kinaseC blocker GF109203X (2μm) was largely ineffective. In contrast, the P2Y2R agonist UTP (100μm) activated Panx-1-like currents that were reversibly inhibited (∼60%) by either H89 or GF109203X. Using fura-2 spectrofluorimetry, the 5-HT-induced Δ[Ca2+ ]i was unaffected by PP2, H89 and GF109293X, suggesting that the kinases acted downstream of the Ca2+ rise. Given that intracellular Ca2+ chelation was previously shown to block receptor-mediated Panx-1 current activation in typeII cells, these data suggest that CB neuromodulators use overlapping, but not necessarily identical, signalling pathways to activate Panx-1 channels and release ATP, a CB excitatory neurotransmitter. In conclusion, these studies provide new mechanistic insight into 5-HT signalling in the CB that has pathophysiological relevance.

Molecular Characterization of Equilibrative Nucleoside Transporters in the Rat Carotid Body and Their Regulation by Chronic Hypoxia.

The mammalian carotid body (CB) is the main peripheral arterial chemoreceptor organ that is excited by decreases in blood PO2 (hypoxia) and increases in blood PCO2/H+. An increase in CB afferent carotid sinus nerve (CSN) discharge results in respiratory and cardiovascular reflex responses that help maintain homeostasis. The CB consists mainly of innervated clusters of the chemoreceptive type I (glomus) cells that are associated with the processes of glial-like type II cells. Extracellular ATP and adenosine (ADO) levels increase in response to acute hypoxia and there is evidence that during chronic sustained hypoxia ADO elevation plays a major role in regulating CB chemosensitivity and CSN discharge. We recently characterized the molecular identities of ectonucleotidase enzymes involved in regulating extracellular ATP hydrolysis to produce ADO in the rat CB. In the present study, we focus on a molecular characterization of the equilibrative nucleoside transporter (ENT) system that is known to regulate extracellular ADO concentrations in the rat CB based on pharmacological studies. Examination of ENT expression using quantitative PCR (qPCR) analysis revealed the expression of both ENT1 and ENT2 mRNAs in whole CB extracts from ~2-week-old juvenile rats. In dissociated rat CB cultures, both ENT1 and ENT2 immunoreactivity was localized to type I cell clusters. Furthermore, we show that ENT1 and ENT2 mRNA expression is downregulated in CBs isolated from rat pups exposed to chronic hypobaric hypoxia (~1 week). These findings reveal the molecular identities of the ENT system expressed in the rat CB and are consistent with the proposed shift to ADO signaling during chronic hypoxia.

Characterization of ectonucleotidase expression in the rat carotid body: regulation by chronic hypoxia.

The carotid body (CB) chemoreflex maintains blood Po2 and Pco2/H+ homeostasis and displays sensory plasticity during exposure to chronic hypoxia. Purinergic signaling via P1 and P2 receptors plays a pivotal role in shaping the afferent discharge at the sensory synapse containing catecholaminergic chemoreceptor (type I) cells, glial-like type II cells, and sensory (petrosal) nerve endings. However, little is known about the family of ectonucleotidases that control synaptic nucleotide levels. Using quantitative PCR (qPCR), we first compared expression levels of ectonucleoside triphosphate diphosphohydrolases (NTPDases1,2,3,5,6) and ecto-5'-nucleotidase (E5'Nt/CD73) mRNAs in juvenile rat CB vs. brain, petrosal ganglia, sympathetic (superior cervical) ganglia, and a sympathoadrenal chromaffin (MAH) cell line. In whole CB extracts, qPCR revealed a high relative expression of surface-located members NTPDase1,2 and E5'Nt/CD73, compared with low NTPDase3 expression. Immunofluorescence staining of CB sections or dissociated CB cultures localized NTPDase2,3 and E5'Nt/CD73 protein to the periphery of type I clusters, and in association with sensory nerve fibers and/or isolated type II cells. Interestingly, in CBs obtained from rats reared under chronic hypobaric hypoxia (~60 kPa, equivalent to 4,300 m) for 5-7 days, in addition to the expected upregulation of tyrosine hydroxylase and VEGF mRNAs, there was a significant upregulation of NTPDase3 and E5'Nt/CD73 mRNA, but a downregulation of NTPDase1 and NTPDase2 relative to normoxic controls. We conclude that NTPDase1,2,3 and E5'Nt/CD73 are the predominant surface-located ectonucleotidases in the rat CB and suggest that their differential regulation during chronic hypoxia may contribute to CB plasticity via control of synaptic ATP, ADP, and adenosine pools.

Evidence that 5-HT stimulates intracellular Ca2+ signalling and activates pannexin-1 currents in type II cells of the rat carotid body.

5-HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH). In the present study, we show that, in addition to type I cells, adjacent glial-like type II cells express functional, ketanserin-sensitive 5-HT2 receptors, and their stimulation increases cytoplasmic Ca2+ derived from intracellular stores. In type II cells, 5-HT activated a ketanserin-sensitive inward current (I5-HT ) that was similar to that (IUTP ) activated by the P2Y2R agonist, UTP. As previously shown for IUTP , I5-HT was inhibited by BAPTA-AM and carbenoxolone (5μm), a putative blocker of ATP-permeable pannexin (Panx)-1 channels; IUTP was reversibly inhibited by the specific Panx-1 mimetic peptide channel blocker, 10 Panx peptide. Paracrine stimulation of type II cells by 5-HT, leading to ATP release via Panx-1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea). Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5-HT and increased autocrine-paracrine 5-HT2 receptor signalling contributes to sensory long-term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial-like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca2+ ]i ) and activating carbenoxolone-sensitive, ATP-permeable, pannexin (Panx)-1-like channels. In the present study, using dissociated rat CB cultures, we found that 5-HT induced Δ[Ca2+ ]i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5-HT-induced Ca2+ response in type II cells was dose-dependent (EC50 ∼183nm) and largely inhibited by the 5-HT2A receptor blocker, ketanserin (1μm), and also arose mainly from intracellular stores. 5-HT also activated an inward current (I5-HT ) in type II cells (EC50 ∼200nm) that was reversibly inhibited by ketanserin (1-10nm), the Ca2+ chelator BAPTA-AM (5μm), and low concentrations of carbenoxolone (5μm), a putative Panx-1 channel blocker. I5-HT reversed direction at approximately -11mV and was indistinguishable from the UTP-activated current (IUTP ). Consistent with a role for Panx-1 channels, IUTP was reversibly inhibited by the specific Panx-1 mimetic peptide blocker 10 Panx (100μm), although not by its scrambled control peptide (sc Panx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5-HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx-1 channels.

Angiotensin II mobilizes intracellular calcium and activates pannexin-1 channels in rat carotid body type II cells via AT1 receptors.

Key points A locally generating, angiotensin II (ANG II) system is present in the rat carotid body (CB) and up‐regulation of this system occurs in certain pathophysiological situations, enhancing sympathetic activity. Here, we show that, similar to chemoreceptor type I cells, glial‐like type II cells also express functional AT1Rs, stimulation of which causes release of Ca2+ from intracellular stores. ANG II–AT1R signalling in type II cells activates an inward current carried by pannexin‐1 (Panx‐1) channels which are known to act as conduits for release of ATP, a key CB excitatory neurotransmitter. Combined effects of ANG II and ATP, which also activates Panx‐1 currents via P2Y2 receptors, were synergistic; chelating intracellular Ca2+ with BAPTA prevented Panx‐1 current activation. We propose that the excitatory function of ANG II in the CB involves dual actions at both type I and type II cells. AbstractA local angiotensin‐generating system is present in the carotid body (CB) and increased angiotensin II (ANG II) signalling contributes to enhanced CB excitation in chronic heart failure (CHF) and after chronic or intermittent hypoxia. ANG II actions have thus far been attributed solely to stimulation of AT1 receptors (AT1Rs) on chemoreceptor type I cells. Here, we show that in dissociated rat CB cultures, ANG II also stimulates glial‐like type II cells, identified by P2Y2‐receptor‐induced intracellular Ca2+ elevation (Δ[Ca2+]i). ANG II induced a dose‐dependent (EC50 ∼8 nm), robust Δ[Ca2+]i in type II cells that was reversibly abolished by the AT1R blocker losartan (1 μm). The ANG II‐induced Δ[Ca2+]i persisted in Ca2+‐free medium but was sensitive to store depletion with cyclopiazonic acid (1 μm). Similar to P2Y2 receptor agonists, ANG II (20–1000 nm) activated pannexin‐1 (Panx‐1) current that was reversibly abolished by carbenoxolone (5 μm). This current arose with a variable delay and was reversibly inhibited by losartan. Repeated application of ANG II often led to current run‐down, attributable to AT1R desensitization. When applied to the same cell the combined actions of ANG II and ATP on Panx‐1 current were synergistic. Current induced by either ligand was inhibited by BAPTA‐AM (1 μm), suggesting that intracellular Ca2+ signalling contributed to Panx‐1 channel activation. Because open Panx‐1 channels release ATP, a key CB excitatory neurotransmitter, it is plausible that paracrine stimulation of type II cells by ANG II contributes to enhanced CB excitability, especially in pathophysiological conditions such as CHF and sleep apnoea.

Enhanced adenosine A2b receptor signaling facilitates stimulus-induced catecholamine secretion in chronically hypoxic carotid body type I cells

Chronic hypoxia (CHox) augments chemoafferent activity in sensory fibers innervating carotid body (CB) chemoreceptor type I cells; however, the underlying mechanisms are poorly understood. We tested the hypothesis that enhanced paracrine signaling via adenosine (Ado) A2b receptors is involved. Dissociated rat CB cultures were exposed for 24 h to normoxia (Nox, 21% O2) or CHox (2% O2) or treated with the hypoxia mimetic deferoxamine mesylate (DFX), and catecholamine secretion from type I cells was monitored by amperometry. Catecholamine secretion was more robust in CHox and DFX type I cells than Nox controls after acute exposure to acid hypercapnia (10% CO2, pH 7.1) and high K(+) (75 mM). Exogenous Ado increased catecholamine secretion in a dose-dependent manner, and the EC50 was shifted to the right from ∼21 μM Ado in Nox cells to ∼78 μM in CHox cells. Ado-evoked secretion in Nox and CHox cells was markedly inhibited by MRS-1754, an A2b receptor blocker, but was unaffected by SCH-58261, an A2a receptor blocker. Similarly, MRS-1754, but not SCH-58261, partially inhibited high-K(+)-evoked catecholamine secretion, suggesting a contribution from paracrine activation of A2b receptors by endogenous Ado. CB chemostimuli, acid hypercapnia, and hypoxia elicited a MRS-1754-sensitive rise in intracellular Ca(2+) that was more robust in CHox and DFX than Nox cells. Taken together, these data suggest that paracrine Ado A2b receptor signaling contributes to stimulus-evoked catecholamine secretion in Nox and CHox CB chemoreceptors; however, the effects of Ado are more robust after CHox.

Effects of low glucose on carotid body chemoreceptor cell activity studied in cultures of intact organs and in dissociated cells

The participation of the carotid body (CB) in glucose homeostasis and evidence obtained in simplified cultured CB slices or dissociated cells have led to the proposal that CB chemoreceptor cells are glucoreceptors. However, data generated in intact, freshly excised organs deny CB chemoreceptor cells' glucosensing properties. The physiological significance of the contention has prompted the present study, performed in a newly developed preparation of the intact CB organ in culture that maintains chemoreceptor cells' microenvironment. Chemoreceptor cells of intact CBs in culture retained their capacity to store, synthesize, and secrete catecholamine in response to hypoxia for at least 6 days. Aglycemia did not elicit neurosecretion in dissociated chemoreceptor cells or in intact CB in culture, but potentiated hypoxia-elicited neurosecretion, exclusively, in 1-day-old intact CB cultures and dissociated chemoreceptor cells cultured for 24 h. In fura 2-loaded cells, aglycemia (but not 1 mM) caused a slow Ca(2+)-dependent and nifedipine-insensitive increase in fluorescence at 340- to 380-nm wavelength emission ratio and augmented the fluorescent signal elicited by hypoxia. Association of nifedipine and KBR7943 (a Na(+)/Ca(2+) exchanger inhibitor) completely abolished the aglycemic Ca(2+) response. We conclude that chemoreceptor cells are not sensitive to hypoglycemia. We hypothesize that cultured chemoreceptor cells become transiently more dependent on glycolysis. Consequently, aglycemia would partially inhibit the Na(+)/K(+) pump, causing an increase in intracellular Na(+) concentration, and a reversal of Na(+)/Ca(2+) exchanger. This would slowly increase intracellular Ca(2+) concentration and cause the potentiation of the hypoxic responses. We discuss the nature of the signals detected by chemoreceptor cells for the CB to achieve its glycemic homeostatic role.

Glia-like Stem Cells Sustain Physiologic Neurogenesis in the Adult Mammalian Carotid Body

Neurogenesis is known to occur in the specific niches of the adult mammalian brain, but whether germinal centers exist in the neural-crest-derived peripheral nervous system is unknown. We have discovered stem cells in the adult carotid body (CB), an oxygen-sensing organ of the sympathoadrenal lineage that grows in chronic hypoxemia. Production of new neuron-like CB glomus cells depends on a population of stem cells, which form multipotent and self-renewing colonies in vitro. Cell fate mapping experiments indicate that, unexpectedly, CB stem cells are the glia-like sustentacular cells and can be identified using glial markers. Remarkably, stem cell-derived glomus cells have the same complex chemosensory properties as mature in situ glomus cells. They are highly dopaminergic and produce glial cell line-derived neurotrophic factor. Thus, the mammalian CB is a neurogenic center with a recognizable physiological function in adult life. CB stem cells could be potentially useful for antiparkinsonian cell therapy.

Detection of hypoxia-evoked ATP release from chemoreceptor cells of the rat carotid body

The carotid body (CB) is a chemosensory organ that detects changes in chemical composition of arterial blood and maintains homeostasis via reflex control of ventilation. Thus, in response to a fall in arterial PO 2 (hypoxia), CB chemoreceptors (type I cells) depolarize, and release neurotransmitters onto afferent sensory nerve endings. Recent studies implicate ATP as a key excitatory neurotransmitter released during CB chemoexcitation, but direct evidence is lacking. Here we use the luciferin–luciferase bioluminescence assay to detect ATP, released from rat chemoreceptors in CB cultures, fresh tissue slices, and whole CB. Hypoxia evoked an increase in extracellular ATP, that was inhibited by L-type Ca 2+channel blockers and reduced by the nucleoside hydrolase, apyrase. Additionally, iberiotoxin (IbTX; 100 nM), a blocker of O 2-sensitive Ca 2+-dependent K + (BK) channels, stimulated ATP release and largely occluded the effect of hypoxia. These data strongly support a neurotransmitter role for ATP in carotid body function.

Role of ET-1 in hypoxia-induced mitosis of cultured rat carotid body chemoreceptors.

The mammalian carotid body (CB) contains O2-chemoreceptors, i.e. glomus cells, which display increased mitoses and endothelin-1 (ET-1) expression during chronic hypoxia. To investigate whether endogenous ET-1 might mediate these mitogenic effects, we quantified bromodeoxyuridine (BrdU) uptake by tyrosine hydroxylase (TH)-positive glomus cells in rat CB cultures using double-label immunofluorescence. In normoxia (20% O2), 2-day exposure to ET-1 (10-1000 nM) caused a dose-dependent increase in BrdU uptake which peaked (approximately 55% of TH+ cells) at around 500 nM ET-1. In chronic hypoxia (5% O2) alone, BrdU uptake was stimulated (approximately 46% of TH+ cells) relative to normoxia (approximately 30%), but the effect was abolished in the presence of specific (BQ 123) or non-specific (PD 142893) ETA receptor antagonists (10(-5) M). Thus paracrine/autocrine release of ET-1 in the hypoxic carotid body may promote glomus cell mitosis via ET(A) receptors.

Effect of the removal of extracellular Ca 2+ on the response of cytosolic concentrations of Ca 2+ to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca 2+ on the response of cytosolic concentrations of Ca 2+ ([Ca 2+] i) to ouabain, an Na +/K + exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca 2+] i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells ( n=29). The ouabain-induced [Ca 2+] i increase was abolished by the removal of extracellular Na +. D600 (50 μM), an L-type voltage-gated Ca 2+ channel antagonist, inhibited the [Ca 2+] i increase by 57±7% ( n=4). Removal of extracellular Ca 2+ eliminated the [Ca 2+] i increase, but subsequent washing out of ouabain in Ca 2+-free solution produced a rise in [Ca 2+] i (62±8% increase, n=6, P<0.05), referred to as a [Ca 2+] i rise after Ca 2+-free/ouabain. The magnitude of the [Ca 2+] i rise was larger than that of ouabain-induced [Ca 2+] i increase. D600 (5 μM) inhibited the [Ca 2+] i rise after Ca 2+-free/ouabain by 83±10% ( n=4). These results suggest that ouabain-induced [Ca 2+] i increase was due to Ca 2+ entry involving L-type Ca 2+ channels which could be activated by cytosolic Na + accumulation. Ca 2+ removal might modify the [Ca 2+] i response, resulting in the occurrence of a rise in [Ca 2+] i after Ca 2+-free/ouabain which mostly involved L-type Ca 2+ channels.

Role of acetylcholine receptors and dopamine transporter in regulation of extracellular dopamine in rat carotid body cultures grown in chronic hypoxia or nicotine.

Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for approximately 12 days in normoxia (Nox; 20% O2), chronic hypoxia (CHox; 6% O2), or chronic nicotine (CNic; 10 microM nicotine, 20% O2), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (approximately 15-min) exposure to hypoxia (5% O2; approximately 6x basal), high extracellular K+ (30 mM; approximately 10x basal), nomifensine (1 microM; a selective DA uptake inhibitor; approximately 3x basal), and nicotine (100 microM; approximately 5x basal), but not methylcholine (300 microM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (approximately 9x control), the stimulatory effect of high K+ (3-4x basal) and acute hypoxia (approximately 2x basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (approximately 9x control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (approximately 2x basal), nicotine, nomifensine, and high K+ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 microM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 microM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.

Effects of the removal of extracellular Ca 2+ on [Ca 2+] i responses to FCCP and acetate in carotid body glomus cells of adult rabbits

The effects of the removal of extracellular Ca 2+ on the responses of cytosolic concentrations of Ca 2+ ([Ca 2+] i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 μM) induced an increase in [Ca 2+] i (mean±S.E.M., 108±14%). After withdrawal of the protonophore the increased [Ca 2+] i returned slowly to a resting level. The [Ca 2+] i response was attenuated by an inorganic Ca 2+ channel antagonist Ni 2+ (2 mM) by 81±4%, and by an L-type voltage-gated Ca 2+ channel antagonist D600 (10 μM) by 53±13%. The removal of extracellular Ca 2+ eliminated the [Ca 2+] i response in 71% of the tested cells ( n=17), and depressed it by 68±6% in the rest. Recovery following stimulation with FCCP in the absence of Ca 2+ reversibly produced a rapid and large rise in [Ca 2+] i, referred to as a [Ca 2+] i rise after Ca 2+-free/FCCP. The magnitude of a [Ca 2+] i rise after Ca 2+-free/FCCP (285±28%, P<0.05) was larger than that of an increase in [Ca 2+] i induced by FCCP in the presence of Ca 2+ and had a correlation with the intensity of the suppression of the [Ca 2+] i response by Ca 2+ removal. A [Ca 2+] i rise after Ca 2+-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca 2+ caused a rise in [Ca 2+] i, referred to as a [Ca 2+] i rise after Ca 2+-free/acetate which was sensitive to D600. The magnitude of the [Ca 2+] i rise was larger than that of a change in [Ca 2+] i caused by acetate in the presence of Ca 2+. These results suggest that FCCP-induced increase in [Ca 2+] i was, in most cells, due to Ca 2+ influx via L-type voltage-gated Ca 2+ channels and, in some cells, due to both Ca 2+ influx and Ca 2+ release from internal Ca 2+ pool. The removal of extracellular Ca 2+ might modify [Ca 2+] i responses to acidic stimuli, causing [Ca 2+] i rises after Ca 2+-free/acidic stimuli which involve mostly L-type Ca 2+ channels.

Role of Basic FGF and Oxygen in Control of Proliferation, Survival, and Neuronal Differentiation in Carotid Body Chromaffin Cells

Crest-derived glomus cells of the carotid body (CB) are O2-sensitive chemoreceptors, which resemble sympathoadrenal (SA) chromaffin cells. In this study, we tested whether perinatal rat glomus cells are sensitive to basic fibroblast growth factor (bFGF)in vitroand whether their sensitivity is regulated by oxygen. In chemically defined medium, bFGF (1–100 ng/ml) caused a significant, dose-dependent increase in the number of surviving tyrosine hydroxylase-positive (TH+) glomus cells in embryonic (E17–E19) CB cultures, following a 48-hr exposure. Though basic FGF (10 ng/ml) appeared mitogenic for these cells, based on stimulation of bromodeoxyuridine (BrdU) uptake, it supported survival of only ∼60% of the initial TH+population, suggesting that significant cell death was occurring. This apparent cell loss in E17 cultures could be largely prevented by combined treatment with bFGF and low oxygen (6% O2). In contrast, in early postnatal (P1) cultures, glomus cell number was relatively unchanged over 48 hr under control conditions or in presence of mitogenic activity from either bFGF or low oxygen. However, combined treatment with both bFGF and low oxygen stimulated proliferation of P1 glomus cells such that by 48 hr the TH+population had increased to ∼1.5× the initial density. Basic FGF (10 ng/ml) also stimulated neurite outgrowth and neurofilament expression in E18–E19, but not P1–P3, glomus cells. In contrast to bFGF, treatment with nerve growth factor was ineffective. Taken together, these results suggest that bFGF and low oxygen are mitogens for perinatal CB chromaffin cells and interact cooperatively as survival factors. It is plausible that these mechanisms may operate to regulate chemoreceptor cell density, during the animal's transition fromin utero(hypoxic) toex utero(normoxic) life.

Two types of voltage-gated K channels in carotid body cells of adult cats

The purpose of this study was to investigate if the oxygen-sensitive K channel is present in the carotid body cells of adult cats, and if all carotid body cells express the oxygen-sensitive K channel. A standard patch-clamp technique with a whole-cell configuration was applied to cultured carotid body cells from adult cats. The cells were continuously perfused with Krebs equilibrated with 5% CO2/air or 5% CO2/argon at room temperature. The results showed that electrophysiologically at least two types of cells existed in cultured cat carotid body cells. One type expressed the oxygen-sensitive K channel and the other expressed the oxygen-insensitive K channel. The oxygen-sensitive K channel was voltage-dependent with a threshold potential of −30 mV. No inactivation was observed during 40 ms of stimulation. The slope of the steady-state current-voltage curve was almost linear in the range from −30 mV to + 50 mV. Hypoxia (pO2 = 25 mmHg) reversibly depressed the K current by 22%. The current was inhibited by 4-aminopyridine (10 mM) and tetraethylammonium (4–25 mM), but insensitive to charybdotoxin (100 nM). The oxygen-insensitive K channel showed similar characteristics to that of oxygen-sensitive K channel in the threshold and the speed of activation, and the shape of I–V curve. The cat is the third species in which the oxygen-sensitive K channel was found in the carotid body. The sensitivity of K channels to oxygen may be a unique feature of chemosensory cells, but the properties of the oxygen-sensitive K channels are different among cats, rats, and rabbits.

Plasticity in cultured carotid body chemoreceptors: environmental modulation of GAP-43 and neurofilament.

In this study we use dissociated cell cultures of the rat carotid body to investigate the adaptive capabilities of endogenous oxygen chemoreceptors, following chronic stimulation by various environmental factors. These oxygen chemoreceptors are catecholamine-containing glomus cells, which derive from the neural crest and resemble adrenal medullary chromaffin cells. Using double-label immunofluorescence, we found that chronic exposure of carotid body cultures to hypoxia (2% to 10% oxygen) caused a significant fraction of tyrosine hydroxylase-positive (TH+) glomus cells to acquire detectable immunoreactivity for growth-associated protein GAP-43. The effect was dose-dependent and peaked around an oxygen tension of 6%, where approximately 30% of glomus cells were GAP-43 positive. Treatment with agents that elevate intracellular cyclic adenosine monophosphate (cAMP) (i.e., dibutyryl cAMP or forskolin) also markedly stimulated GAP-43 expression. Since hypoxia is known to increase cAMP levels in glomus cells, it is possible that the effect of hypoxia on GAP-43 expression was mediated, at least in part, by a cAMP-dependent pathway. Unlike hypoxia, however, cAMP analogs also stimulated neurofilament (NF 68 or NF 160 kD) expression and neurite outgrowth in glomus cells, and these properties were enhanced by retinoic acid. Nerve growth factor, which promotes neuronal differentiation in related crest-derived endocrine cells, and dibutyryl cGMP were ineffective. Thus, it appears that postnatal glomus cells are plastic and can express neuronal traits in vitro. However, since hypoxia stimulated GAP-43 expression, without promoting neurite outgrowth, it appears that the two processes can be uncoupled. We suggest that stimulation of GAP-43 by hypoxia may be important for other physiological processes, e.g., enhancing neurotransmitter release or sensitization of G-protein-coupled receptor transduction.

Culture of arterial chemoreceptor cells from adult cats in defined medium

Recently patch clamp techniques and optical fluorometric techniques have been applied to freshly dissociated or cultured carotid body glomus cells to investigate chemosensory mechanisms of the carotid body. However, very few studies have shown the effects of the dissociation and/or culture conditions on the health and function of the cells. The purpose of this study was to develop a culture method which support healthy and functioning carotid body cells from adult cats. Carotid bodies were dissociated with 0.1–0.2% collagenase and gentle trituration. The cells were plated on glass wells coated with poly-D-lysin and Matrigel, and cultured in chemically defined medium. Culture was maintained for up to 37 days without overgrowth of fibroblasts. Glomus cells extended their processes within and from clusters. Single glomus cells acquired the shape of neurons. Glomus cells synthesized dopamine and its secretion increased during exposure of the cells to hypoxia. Tyrosine hydroxylase was expressed throughout the culture period. These results indicate that glomus cells cultured under conditions described here are healthy and function in a manner similar to that in vivo.

Effect of remving [Ca 2+] o on anoxia-induced [Ca 2+] i increase in cultured carotid body glomus cells of newborn rabbits

Effect of remving [Ca 2+] o on anoxia-induced [Ca 2+] i increase in cultured carotid body glomus cells of newborn rabbits

Plasticity in cultured arterial chemoreceptors: effects of chronic hypoxia and cyclic AMP analogs.

Exposure of humans and animals to chronic hypoxia causes hypertrophy of the chemosensory carotid body (McGregor et al., 1988), and sensitization of the ventilatory reflex (Barnard et al., 1987; Nielsen et al., 1987). The underlying mechanisms are unclear but are likely to involve the glomus or type 1 cells, which are now generally considered to be the oxygen sensors (Gonzalez et al., 1992). These structural and functional changes may be triggered by the direct, chronic stimulation of glomus cells by low PO2, perhaps acting via the intracellular second messenger cAMP (see Wang et al. 1991). To address this we investigated whether chronic exposure of carotid body cultures to hypoxia (6% O2) or cAMP analogs leads to modification in glomus cells properties that may explain carotid body plasticity during ventilatory acclimatization.

Influence of intracellular pH on the membrane potential of cultured carotid body glomus cells

The influence of acidity on the resting potential (Em) of cultured rat glomus cells was studied within a narrow range (7.32-7.54) of extracellular pH (pH0). Under these conditions, the intracellular pH (pHi) ranged from 6.3 to 7.21. Resting potentials were most negative when pHi was lowest, and least negative when pHi was highest. These effects were determined by the equilibrium potential of H+ ions (EH) since Em linearly followed EH. However, EH was little affected by pH0. Therefore, intracellular, and not extracellular, acidity was most important in contributing to the Em of glomus cells.