Rat Carotid Body Glomus Cells Research Articles

Interleukin‐6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells

Although abundant evidence indicates mutual regulation between the immune and the central nervous systems, how the immune signals are transmitted to the brain is still an unresolved question. In a previous study we found strong expression of proinflammatory cytokine receptors, including interleukin (IL)-1 receptor I and IL-6 receptor alpha in the rat carotid body (CB), a well-known arterial chemoreceptor that senses a variety of chemostimuli in the arterial blood. We demonstrated that IL-1 stimulation increases intracellular calcium ([Ca(2+)](i)) in CB glomus cells, releases ATP, and increases the discharge rate in carotid sinus nerve. To explore the effect of IL-6 on CB, here we examine the effect of IL-6 on [Ca(2+)](i) and catecholamine (CA) secretion in rat CB glomus cells. Calcium imaging showed that extracellular application of IL-6 induced a rise in [Ca(2+)](i) in cultured glomus cells. Amperometry showed that local application of IL-6 evoked CA release from glomus cells. Furthermore, the CA secretory response to IL-6 was blocked by 200 microM Cd(2+), a well-known Ca(2+) channel blocker. Our experiments provide further evidence for the responsiveness of the CB to proinflammatory cytokines and indicate that the CB might play a role in inflammation sensing and transmission of such information to the brain.

2‐APB mediated effects on hypoxic calcium influx in rat carotid body glomus cells

Hypoxia depolarizes the glomus cells of the carotid body and induces calcium rise which was reversed by 2‐APB, an inhibitor of store‐operated calcium channels situated in the endoplasmic reticulum (ER), whereas those by hypercapnia and high extracellular [K+] were not, although both hypoxia and hypercapnia depolarize the glomus cells. Hypoxia‐dependent calcium rise was triggered by a reduction of IP3‐receptors and 2‐APB significantly reversed this response. Further, mitochondrial inhibitions completely overlapped the 2‐APB inhibition, suggesting same mechanism of effects. Also, ATP elevated the [Ca2+]i in normoxia due to calcium influx and the release from the [Ca2+]i stores, both of which were inhibited by 2‐APB. This reaction, as revealed by 2‐APB, is unique and is due to possible involvement of phosphorylation mechanisms in hypoxia, and not operated in hypercapnia. Therefore we hypothesize that store‐operated calcium channels in the ER, where IP3 receptors reside, is reacted with 2‐APB and is critical for the inhibitory response. However, 2‐APB did not block hypercapnic‐ and high extracellular [K+]‐dependent calcium increase.Hypercapnia and high extracellular K+ have no access to this [Ca2+]i stores of ER, and hence have no effects.Supported by NIH grant 43413.

Store‐operated hypoxic but not hypercapnic calcium rise is reversed in rat glomus cells by 2‐APB

We studied calcium responses in the enzymatically separated rat carotid body glomus cells. Store‐operated hypoxic calcium rise in glomus cells was reversed by 2‐APB whereas those by hypercapnia and by high extracellular [K+] were not. 2‐APB and mitochondrial inhibitions completely overlapped, pointing to the same mechanism of effects. Also, ATP elevated the intracellular calcium in normoxia due to influx and to release from the intracellular stores, both of which were inhibited by 2‐APB. This distinction as revealed by 2‐APB is unique and is due to possible involvement of phosphorylation mechanisms in hypoxia. Also, the store‐operated calcium channels in the endoplsmic reticulum where IP3 receptors reside and is reacted with 2‐APB is critical for the hypoxic inhibitory response. Hypercapnia and high extracellular K+ have no access to this region of endoplasmic reticulum, and hence have no effects. The specific inhibition of calcium rise by 2‐APB is yet a novel example of cellular calcium signaling. (Supported by NIH grant R37‐HL‐43413).

IL‐1β inhibits IK and increases [Ca2+]i in the carotid body glomus cells and increases carotid sinus nerve firings in the rat

Increasing evidence indicates that there exists a reciprocal communication between the immune system and the brain. Interleukin 1beta (IL-1beta), a proinflammatory cytokine produced during immune challenge, is believed to be one of the mediators of immune-to-brain communication, but how it gets into the brain is unknown because of its large molecular weight and difficulty in crossing the blood-brain barrier. Our previous work has demonstrated that IL-1 receptor type I is strongly expressed in the glomus cells of rat carotid body (CB), a well characterized polymodal chemoreceptive organ which serves not only for the detection of hypoxia, hypercapnia and acidity, but also for low temperature and blood glucose. The present study was designed to test whether IL-1beta could stimulate the CB glomus cells and alter the discharge properties in the carotid sinus nerve, the afferent nerve innervating the organ. The results from whole-cell patch-clamp recordings and calcium imaging showed that extracellular application of IL-1beta significantly decreased the outward potassium current and triggered a transient rise in [Ca(2+)](i) in the cultured glomus cells of rat CB. Furthermore, by using extracellular recordings and pharmacological intervention, it was found that IL-1beta stimulation of the CB in the anaesthetized rat in vivo significantly increased the discharge rate in the carotid sinus nerve, most probably mediated by ATP release. This experiment provides evidence that the CB responds to cytokine stimulation and proposes the possibility that the CB might play a role in immune-to-brain communication.

Expression of histamine receptors and effect of histamine in the rat carotid body chemoafferent pathway

Chemosensory information from peripheral arterial oxygen sensors in the carotid body is relayed by petrosal ganglion neurons to the respiratory networks in the medulla oblongata. Biogenic amines, including histamine, released from glomus (type I) cells of the carotid body are considered to be primary transmitters in hypoxic chemosensitivity. Immunocytochemistry at light-and electron-microscopical levels, and RT-PCR, revealed the expression of histamine receptors 1 and 3 as well as histidine decarboxylase in the rat carotid body glomus cells and petrosal ganglion neurons. Histamine receptors 1 and 3, but not histidine decarboxylase, were also observed in the ventrolateral, intermediate and commissural subnuclei of the nucleus tractus solitarii in the medulla oblongata. In order to examine the possible role of histamine in the afferent branch of the respiratory system, we applied histamine receptor 1 and 3 agonists to the carotid body, which caused a mildly increased phrenic nerve activity in a working heart-brainstem preparation. Moreover, microinjection of antagonists of histamine receptors 1 and 3 into the nucleus tractus solitarii caused significant changes in the inspiratory timing and the chemoreceptor response. Our data show that histamine acting via histamine receptors 1 and 3 plays an important neuromodulatory role in the afferent control of chemosensitivity.

Role of mitochondria in the regulation of hypoxia-inducible factor-1α in the rat carotid body glomus cells

Hypoxia-inducible factor-1alpha (HIF-1alpha) protein, a heterodimeric transcription factor that regulates transcriptional activation of several genes, is involved in adaptive responses to hypoxia. Earlier, we have reported that in carotid body (CB), the peripheral oxygen sensing organ, HIF-1alpha is up-regulated during hypoxia. One model proposes that an intact mitochondrial respiratory chain is necessary for this regulation of HIF-1alpha. To test this hypothesis in the CB glomus cells, we studied the effect of mitochondrial electron transport chain (ETC) inhibitors: rotenone (complex I; 1 microM), malonate (complex II; 0.5 M), antimycin A (complex III; 1 microg/ml), sodium azide (complex IV; 5 mM), and uncoupler of oxidative phosphorylation: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 mM) on HIF-1alpha expression during normoxia and hypoxia. Inhibitors and uncoupler of mitochondrial ETC abrogated hypoxia-induced HIF-1alpha expression in isolated glomus cells significantly (P < 0.001). Effect of rotenone during hypoxia was abolished by succinate (4 mM), a substrate for complex II. Further, HIF-1alpha expression was not altered by any of these mitochondrial inhibitors during normoxia. Taken together, these results strongly indicate that a functional mitochondrial ETC is required for the stabilization of HIF-1alpha, and further the connection between HIF-1alpha and mitochondria in CB oxygen sensing is reiterated.

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1alpha and -1beta in the rat carotid body and glomus cells.

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1alpha and HIF-1beta proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe(2+) chelation in the rat carotid body (CB) glomus cells. HIF-1alpha bound to HIF-1beta translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1alpha was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po(2) approximately 7 Torr) and Nx with ciclopirox olamine (CPX, 5 microM) for 1 h showed a significant ( P<0.001) increase in HIF-1alpha protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1beta protein expression. HIF-1alpha subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1alpha by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1alpha in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1alpha which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells.

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O2 -regulated K+ channels. However, the mechanisms involved in the detection of changes of O2 tension are unknown. We have studied the interaction between glomus cell O2 sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca2+-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is mimicked by 1-methyl-4-phenylpyridinium ion (MPP+), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP+)-sensitive molecule critically participates in acute oxygen sensing in the carotid body.

High PCO does not alter pH i, but raises [Ca 2+] i in cultured rat carotid body glomus cells in the absence and presence of CdCl 2

We measured the effect of high P CO (500–550 Torr) on the pH i and [Ca 2+] i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca 2+] i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca 2+] release from intracellular Ca 2+ stores. The membrane potential model postulates that the rise in [Ca 2+] i comes from influx of extracellular Ca 2+ through voltage-dependent Ca 2+ channels (VDCC) of the L-type. High P CO did not change pH i at PO 2 of 120–135 Torr, showing that CO-induced changes in [Ca 2+] i are not due to changes in pH i. High P CO caused a highly significant rise in [Ca 2+] i from 90±12 nM to 675±65 nM, both in the absence and in the presence of 200 μM CdCl 2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca 2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca 2+ through VDCCs according to the membrane potential model.

Modulation of junctional conductance between rat carotid body glomus cells by hypoxia, cAMP and acidity

Short-term cultures of glomus cells (up to seven days), were employed to study intercellular electrical communications. Bidirectional electric coupling was established under current clamping after impaling two adjacent glomus cells with microelectrodes, and alternate stimulation and recording. Their resting potential ( V m) and input resistance ( R o) were thus measured. Both coupled cells were then voltage clamped at a level between their V ms. Current pulses applied to either cell elicited a transjunctional voltage ( V j) and current ( I j), used to calculate the junctional conductance ( G j). G j was 1.52±0.29 nS (mean±S.E.; n=147). V j linearly influenced G j, suggesting ohmic junctions. G j was not affected by V m in 50% of the cases. However, there was V m-dependence in the others, but voltage changes had to be large (>±40 mV from the V m). Therefore, physiologically or pharmacologically induced glomus cell depolarization or hyperpolarization may not significantly affect intercellular coupling unless there are large variations in V m. Hypoxia (induced by Na 2S 2O 4 1 mM or 100% N 2) decreased G j in 60–80% of the pairs while producing tighter coupling in the rest. Similar effects were obtained when the medium was acidified with lactic acid 1–10 mM. Cobalt chloride (3 mM) prevented, diminished or reversed the changes in G j observed during low PO 2, suggesting that [Ca 2+] i changes are important in hypoxic uncoupling. However, non-specific cationic effects of Co 2+ have not been ruled out. Applications of the membrane-permeant dB-cAMP 1 mM tightened coupling in almost all cell pairs. This is important because endogenous cAMP increases during hypoxia. Our results suggest that multiple factors modulate junctional conductance between glomus cells. Changes in G j by `natural' stimuli and/or cAMP may play an important role in chemoreception, especially in titrating the release of transmitters toward the carotid nerve terminals.

PH regulation in adult rat carotid body glomus cells. Importance of extracellular pH, sodium, and potassium.

The course of intracellular pH (pHi) was followed in superfused (36 degrees C) single glomus (type I) cells of the freshly dissociated adult rat carotid body. The cells had been loaded with the pH-sensitive fluorescent dye 2',7'-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein. The high K(+)-nigericin method was used for calibration. The pHi of the glomus cell at pHo 7.40, without CO2, was 7.23 +/- 0.02 (n = 70); in 5% CO2/25 mM HCO3-, pHi was 7.18 +/- 0.08 (n = 9). The pHi was very sensitive to changes in pHo. Without CO2, delta pHi/delta pHo was 0.85 (pHo 6.20-8.00; 32 cells), while in CO2/HCO3- this ratio was 0.82 irrespective of whether pHo (6.80-7.40; 14 cells) was changed at constant PCO2 or at constant [HCO3-]o. The great pHi sensitivity of the glomus cell to pHo is matched only by that of the human red cell. An active Na+/H+ exchanger (apparent Km = 58 +/- 6 mM) is present in glomus cells: Na+ removal or addition of the amiloride derivative 5-(N,N-hexamethylene)-amiloride induced pHi to fall by as much as 0.9. The membrane of these cells also contains a K+/H+ exchanger. Raising [K+]o from 4.7 to 25, 50, or 140 mM reversibly raised pHi by 0.2, 0.3, and 0.6, respectively. Rb+ had no effect, but in corresponding concentrations of Tl+ alkalinization was much faster than in K+. Reducing [K+]o to 1.5 mM lowered pHi by 0.1. These pHi changes were shown not to be due to changes in membrane voltage, and were even more striking in the absence of Na+. Intrinsic buffering power (amount of strong base required to produce, in the nominal absence of CO2, a small pHi rise) increased from 3 to approximately 21 mM as pHi was lowered, but remained nearly unchanged below pHi 6.60. The fitted expression assumed the presence of one "equivalent" intracellular buffer (pK 6.41, 41 mM). The exceptional pHi sensitivity to pHo suggests that the pHi of the glomus cell is a link in the chemoreceptor's response to external acidity.

Hypoxia decreases intracellular calcium in adult rat carotid body glomus cells.

1. Carotid body chemoreceptors were removed intact from adult rats and subjected to protease and collagenase enzymatic digestion of connective tissue. 2. Recordings from the sinus nerve demonstrated that chemotransduction remains intact for at least 2-3 h after isolation, enzyme exposure, and suspension in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline at room PO2. 3. After mechanical dissociation, the interrelationship between changes in extracellular PO2 and pH and relative changes in intracellular calcium (Ca2+i) were observed in glomus cells with the use of fluo-3 and confocal microscopy. 4. Brief (60-s) decreases in PO2 from 150 mmHg to near 0 mmHg, at nadir, caused a marked reduction in Ca2+i (peak delta F/F0 = -32 +/- 3%, mean +/- SE, n = 43), which rapidly recovered after reoxygenation. The decrease was reproducible from trial to trial and was also observed in HCO3(-)-buffered Ringer solution. 5. Superfusion with Ca(2+)-free HEPES saline with 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) blocked the hypoxia-induced increase in afferent chemoreceptor activity in vitro. Superfusion of the same solution over isolated cells for 15 min caused a large decrease in Ca2+i (-34 +/- 7%, n = 16). 6. In the presence of Ca(2+)-free HEPES, reoxygenation caused calcium fluorescence to increase. This suggests that the Ca2+ decrease during hypoxia is due, at least partially, to binding to an intracellular site. 7. Extracellular cobalt (1 mM, 15 min) also reversibly blocked the chemoreceptor response to hypoxia, in vitro, and caused a reduction in Ca2+i (delta F/F0 = -37 +/- 8%, n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)