Rat Carotid Body Type Research Articles

Acute Oxygen-Sensing via Mitochondria-Generated Temperature Transients in Rat Carotid Body Type I Cells.

The Carotid Bodies (CB) are peripheral chemoreceptors that detect changes in arterial oxygenation and, via afferent inputs to the brainstem, correct the pattern of breathing to restore blood gas homeostasis. Herein, preliminary evidence is presented supporting a novel oxygen-sensing hypothesis which suggests CB Type I cell “hypoxic signaling” may in part be mediated by mitochondria-generated thermal transients in TASK-channel-containing microdomains. Distances were measured between antibody-labeled mitochondria and TASK-potassium channels in primary rat CB Type I cells. Sub-micron distance measurements (TASK-1: 0.33 ± 0.04 µm, n = 47 vs TASK-3: 0.32 ± 0.03 µm, n = 54) provided evidence for CB Type I cell oxygen-sensing microdomains. A temperature-sensitive dye (ERthermAC) indicated that inhibition of mitochondrial activity in isolated cells caused a rapid and reversible inhibition of mitochondrial thermogenesis and thus temperature in these microdomains. Whole-cell perforated-patch current-clamp electrophysiological recordings demonstrated sensitivity of resting membrane potential (Vm) to temperature: lowering bath temperature from 37°C to 24°C induced consistent and reversible depolarizations (Vm at 37°C: -48.4 ± 4.11 mV vs 24°C: -31.0 ± 5.69 mV; n = 5; p < 0.01). These data suggest that hypoxic inhibition of mitochondrial thermogenesis may play an important role in oxygen chemotransduction in the CB. A reduction in temperature within cellular microdomains will inhibit plasma membrane ion channels, influence the balance of cellular phosphorylation–dephosphorylation, and may extend the half-life of reactive oxygen species. The characterization of a thermosensory chemotransduction mechanism, that may also be used by other oxygen-sensitive cell types and may impact multiple other chemotransduction mechanisms is critical if we are to fully understand how the CBs, and potentially other oxygen-sensitive cells, respond to hypoxia.

Lactate and Short‐Chain Fatty Acids on Ca2+ Oscillations in Rat Carotid Body Type 1 Cells

Carotid body (CB) Type 1 (glomus) cells express Olfr78, a Gs‐coupled receptor that binds lactate and short chain fatty acids. Lactate is produced in all cells under anaerobic conditions, and short chain fatty acids are produced by bacterial fermentation in the colon. Lactate‐Olfr78 signaling has been proposed to be involved in hypoxia‐induced excitation of CB, but this idea is currently controversial. Ca oscillations are low‐level Ca signals in glomus cells. Here we compared the effects of lactate/short chain fatty acids and hypoxia on Ca oscillations in isolated glomus cell. Lactate ≈ acetate &gt; propionate (1–20 mM) produced a concentration‐dependent increase in the frequency of Ca oscillations with no overall effects on the background TASK K current and resting cell membrane potential. Verapamil abolished Ca oscillations with or without lactate. Forskolin (2–10 μM) produced no significant effect on Ca oscillations, suggesting that adenylyl cyclase/cAMP may not be involved in lactate‐induced changes in Ca signaling. H‐89 (1 μM) did not block lactate‐induced increase in Ca oscillations, questioning the role of PKA. Comparison of lactate and hypoxia effects on Ca oscillations indicated that 5–10 mM lactate and 2–5%O2 (mild‐moderate) hypoxia produce similar effects on Ca oscillations. Thus, an increase of blood lactate to 5–10 mM may be necessary to mimic the effect of mild‐moderate hypoxia on Ca signaling in CB glomus cells.

Ca2+ oscillations in rat carotid body type 1 cells in normoxia and hypoxia.

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca2+ entry (SOCE) using 2-APB abolished Ca2+ oscillations. Inhibition of endoplasmic reticulum Ca2+-ATPase (SERCA) using thapsigargin abolished Ca2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca2+ oscillations. Inhibitors of L- and T-type Ca2+ channels (Cav; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca2+ oscillations. Thus, Ca2+ oscillations observed in normoxia were caused by cyclical Ca2+ fluxes at the ER, which was supported by Ca2+ influx via Ca2+ channels. Hypoxia (2-5% O2) increased the frequency and amplitude of Ca2+ oscillations, and Cav inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca2+ oscillations represent the basic Ca2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.

Selective mu and kappa Opioid Agonists Inhibit Voltage-Gated Ca2+ Entry in Isolated Neonatal Rat Carotid Body Type I Cells.

It is known that opioids inhibit the hypoxic ventilatory response in part via an action at the carotid body, but little is known about the cellular mechanisms that underpin this. This study's objectives were to examine which opioid receptors are located on the oxygen-sensing carotid body type I cells from the rat and determine the mechanism by which opioids might inhibit cellular excitability.Immunocytochemistry revealed the presence of μ and κ opioid receptors on type I cells. The μ-selective agonist DAMGO (10 μM) and the κ-selective agonist U50-488 (10 μM) inhibited high K(+) induced rises in intracellular Ca(2+) compared with controls. After 3 h incubation (37 °C) with pertussis toxin (150 ng ml(-1)), DAMGO (10 μM) and U50-488 (10 μM) had no significant effect on the Ca(2+) response to high K(+).These results indicate that opioids acting at μ and κ receptors inhibit voltage-gated Ca(2+) influx in rat carotid body type I cells via G(i)-coupled mechanisms. This mechanism may contribute to opioid's inhibitory actions in the carotid body.

Acutely Administered Leptin Increases [Ca2+] i and BK Ca Currents But Does Not Alter Chemosensory Behavior in Rat Carotid Body Type I Cells.

Obesity related pathologies are the health care crisis of our generation. The fat cell derived adipokine leptin has been shown to be a central stimulant of respiration. Very high levels of leptin, however, are associated with the depressed ventilatory phenotype observed in obesity hypoventilation syndrome. Leptin receptors have been identified on carotid body type I cells but how their activation might influence the physiology of these cells is not known.The acute application of leptin evoked calcium signaling responses in isolated type I cells. Cells increased their Fura 2 ratio by 0.074 ± 0.010 ratio units (n = 39, P < 0.001). Leptin also increased the peak membrane currents in 6 of 9 cells increasing the peak macroscopic currents at +10 mV by 61 ± 14 % (p < 0.02). Leptin administered in the presence of the selective BK(Ca) channel inhibitor Paxilline (0.5 μM) failed to increase membrane currents (n = 5). Interestingly, leptin did not significantly alter the resting membrane potential of isolated type I cells (n = 9) and anoxic/acidic depolarizations were unaffected by leptin (n = 7, n = 6).These data suggest that leptin receptors are functional in type I cells but that their acute activation does not alter chemosensory properties. Future studies will use chronic models of leptin dysregulation.

Acute leptin evokes physiological responses in rat carotid body type I cells (873.4)

Leptin is a central stimulant of respiration and also contributes to obesity related sympathoactivation. Recently leptin receptors have been identified on type I cells from the carotid body. Our most recent data indicate that activation of these receptors with leptin initiates calcium signaling events and increases outward macroscopic currents in isolated type I cells.The acute (10 min) application of leptin (200 ng.ml‐1) evoked calcium signaling responses in isolated type I cells. Cells significantly increased their Fura 2 F340/F380 ratio by 0.09 ± 0.015 ratio units (n =24, P &lt; 0.001). Leptin also increased the peak membrane currents in a subset (65%) of type I cells. At +10mV leptin (200ng.ml‐1) increased peak macroscopic currents from 123 ± 19 pA.pF‐1 to 198 ± 28 pA.pF‐1 (n = 6, P &lt; 0.02). The remaining 35% of type I cells were unresponsive to this concentration of leptin. Interestingly, leptin does not alter the rate of oxidative phosphorylation in isolated type I cells as measured by reduction of C12‐resazurin to C12‐resorufin.These data suggest that changes in leptin signaling may potentially influence carotid body function at the level of the type I cell. Given the involvement of the carotid bodies in the control of breathing and sympathetic tone, this finding may be especially pertinent to obesity related pathologies such as obesity hypoventilation syndrome and essential hypertension.Grant Funding Source: Supported by NIH‐1RO1HL091836 &amp; NIH‐ 1S10RR025075‐01

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

Signal processing in the carotid body (CB) is initiated at receptor glomus (or type I) cells which depolarize and release the excitatory neurotransmitter ATP during chemoexcitation by hypoxia and acid hypercapnia. Glomus cell clusters (GCs) occur in intimate association with glia-like type II cells which express purinergic P2Y2 receptors (P2Y2Rs) but their function is unclear. Here we immunolocalize the gap junction-like protein channel pannexin-1 (Panx-1) in type II cells and show Panx-1 mRNA expression in the rat CB. As expected, type II cell activation within or near isolated GCs by P2Y2R agonists, ATP and UTP (100 μm), induced a rise in intracellular [Ca(2+)]. Moreover in perforated-patch whole cell recordings from type II cells, these agonists caused a prolonged depolarization and a concentration-dependent, delayed opening of non-selective ion channels that was prevented by Panx-1 blockers, carbenoxolone (5 μm) and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS; 10 μm). Because Panx-1 channels serve as conduits for ATP release, we hypothesized that paracrine, type II cell P2Y2R activation leads to ATP-induced ATP release. In proof-of-principle experiments we used co-cultured chemoafferent petrosal neurones (PNs), which express P2X2/3 purinoceptors, as sensitive biosensors of ATP released from type II cells. In several cases, UTP activation of type II cells within or near GCs led to depolarization or increased firing in nearby PNs, and the effect was reversibly abolished by the selective P2X2/3 receptor blocker, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 μm). We propose that CB type II cells may function as ATP amplifiers during chemotransduction via paracrine activation of P2Y2Rs and Panx-1 channels.

Ion channel regulation by the Lkb1‐AMPK signalling pathway: the key to carotid body activation by hypoxia and metabolic homeostasis at the whole body level

Our recent investigations provide further support for the proposal that, consequent to inhibition of mitochondrial oxidative phosphorylation, activation of AMP‐activated protein kinase (AMPK) mediates carotid body excitation by hypoxia. Consistent with the effects of hypoxia, intracellular dialysis from a patch pipette of an active (thiophosphorylated) recombinant AMPK heterotrimer (α2β2γ1) or application of the AMPK activators AICAR and A769662: (1) Inhibited BKCa currents and TASK K+ currents in rat carotid body type I cells; (2) Inhibited whole‐cell currents carried by KCa1.1 and TASK3, but not TASK1 channels expressed in HEK293 cells; (3) Triggered carotid body activation. Furthermore, preliminary data suggest that either conditional knockout of Lkb1 in type I cells or global knockout of the catalytic α1 and α2 subunit of AMPK, respectively, markedly attenuated the ventilatory response of mice to hypoxia. Accumulating evidence therefore suggests that the Lkb1‐AMPK signalling pathway is necessary for hypoxia‐response coupling by the carotid body, and serves to regulate oxygen and therefore energy supply at the whole body level.

Selective Expression in Carotid Body Type I Cells of a Single Splice Variant of the Large Conductance Calcium- and Voltage-activated Potassium Channel Confers Regulation by AMP-activated Protein Kinase

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Inhibition of adenylate cyclase attenuates muscarinic Ca2+ signaling by a PKA-independent mechanism in rat carotid body Type I cells

Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²(+) signaling is inhibited by the activation of G(i)-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²(+) imaging techniques it was observed that inhibition of muscarinic Ca²(+) signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²(+) response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²(+) signaling whereas the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiated Ca²(+) signaling. Thus in Type I cells inhibition of adenylate cyclase inhibited muscarinic Ca²(+) signaling via a PKA-independent pathway that may rely upon modulation of Epac.

Evidence for functional, inhibitory, histamine H3 receptors in rat carotid body Type I cells

The Type I cells are the sensory elements of the carotid bodies and play a critical role in defining the ventilatory response to hypoxia and hypercapnia. Type I cells release multiple neurotransmitters during a chemosensory stimulus resulting in increased firing of the carotid sinus nerve and modification of the breathing pattern. While much is known about the actions of individual neurotransmitters in this system, very little is known about how multiple neurotransmitters may integrate to shape the output of the carotid body. Recent data has indicated that the neurotransmitter histamine does not excite isolated Type I cells despite being released during hypoxia and its receptors being present on the Type I cells. Here the hypothesis that histamine might modulate an excitatory neurotransmitter such as acetylcholine was tested. Using calcium imaging techniques it was found that histamine attenuated calcium signaling events initiated by the muscarinic acetylcholine receptor agonist acetyl-β-methylcholine via an H3 receptor mediated mechanism. In summary, these results suggest that when acetylcholine and histamine are co-released from Type I cells in response to chemostimuli, histamine may attenuate or modulate the excitatory presynaptic actions of acetylcholine.

Differential effects of halothane and sevoflurane on hypoxia-induced intracellular calcium transients of neonatal rat carotid body type I cells

The purpose of this study was to investigate the effects of halothane and sevoflurane on the magnitude of the increase in intracellular calcium with hypoxia in carotid body type I (glomus) cells. We wished to ascertain if the effects of these agents in single cells paralleled their known effects on the human hypoxic ventilatory response, where halothane depresses this response more than does sevoflurane. We studied single glomus cells from neonatal rat carotid bodies. Halothane and sevoflurane were administered in concentrations ranging from 0.18 to 1.45 and 0.1 to 2 minimum alveolar concentration (MAC), respectively (rat values). The intracellular calcium response to a approximately 90 s period of hypoxia was measured using indo-1 dye. We also assessed if these agents influenced the calcium response to exposure to potassium 100 mM. Halothane depressed the increase in intracellular calcium with hypoxia more than did sevoflurane (P=0.036). Although halothane was depressive at all concentrations tested, sevoflurane depressed the calcium response only at 2 MAC. Both agents also depressed the calcium response to elevated extracellular potassium-halothane more so than sevoflurane (P=0.004). The actions of the agents in single cells reflect their known influence on human hypoxic ventilatory response, consistent with the notion that the cellular process underlies the whole-body effect. The responses to elevated extracellular potassium, which depolarizes the cell membrane, indicate that (in addition to molecular mechanisms previously proposed), voltage-activated calcium channels may also be involved in the anaesthetic effect.

Presynaptic regulation of isolated neonatal rat carotid body type I cells by histamine

The type I cells are the chemoreceptive elements of the carotid bodies and are critical in defining the ventilatory response to hypoxia and hypercapnia. Recent evidence has suggested that histamine is released by the carotid body in response to hypoxia and acts as an excitatory neurotransmitter. Here we use isolated neonatal rat type I cells to assess the presynaptic actions of histamine and define the receptor subtypes that mediate them. All four histamine receptor subtypes are expressed on the type I cells, however activation of these receptors with histamine or selective agonists caused no rise in intracellular calcium ([Ca(2+)](i)) and histamine did not augment calcium entry or modulate macroscopic currents evoked in type I cells. Thus activation of histamine receptors on type I cells is unlikely to provide a presynaptic positive feedback mechanism during chemotransduction and any excitatory role attributed to the actions of histamine is likely to come from a postsynaptic effect on the carotid sinus nerve (CSN).

The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN(-)) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca(2+), which was inhibited on removal of extracellular Ca(2+). Under current clamp conditions rotenone and CN(-) caused a rapid membrane depolarization and elevation of [Ca(2+)](i). Voltage clamping cells to -70 mV substantially attenuated this rise in [Ca(2+)](i). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K(+) currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K(+) current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN(-) or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells.

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.

Presynaptic modulation of rat arterial chemoreceptor function by 5-HT: role of K+ channel inhibition via protein kinase C

The peripheral control of breathing is mediated by O2-sensitive carotid body (CB) type 1 cells, which express multiple neurotransmitters including the monoamines, dopamine and serotonin (5-HT). Whereas dopamine has been extensively studied, 5-HT has received little attention. Here, to elucidate the role of 5-HT in CB chemotransmission, we used perforated-patch recording from rat type 1 cell clusters and co-cultured petrosal (afferent) neurones. 5-HT induced action potentials and/or membrane depolarization associated with a conductance decrease in approximately 40% of recordings from type 1 cells (n = 78/192). These responses were markedly inhibited by the 5-HT2 receptor antagonist ketanserin (10-50 microM) and by the protein kinase C (PKC) inhibitor chelerythrine (50 microM). The PKC activator 1-oleoyl-2-acetylglycerol (OAG; 50 microM) mimicked the 5-HT-induced depolarization, and the combined effects of 5-HT and OAG were non-additive. The 5-HT-induced responses reversed near the potassium (K+) equilibrium potential (at approximately -82 mV; EK = -83 mV), suggesting inhibition of a resting K+ conductance. In type 1 cells (n = 7), voltage-activated outward K+ current was also inhibited by 1-50 microM 5-HT, an effect that was prevented by PKC inhibitors (chelerythrine and NPC 15437) and mimicked by OAG; the outward K+ current inhibited by 5-HT appeared to be predominantly a Ca(2+)-dependent K+ current. The 5-HT2 receptor blockers ketanserin and ritanserin reversibly inhibited spontaneous action potentials and the hypoxia-induced receptor potential recorded from clustered type 1 cells. Moreover, these blockers reversibly inhibited the hypoxic chemosensory response recorded postsynaptically in petrosal neurones that functionally innervated type 1 clusters in co-culture. RT-PCR and confocal immunofluorescence techniques revealed 5-HT2a receptor expression in rat CB type 1 cells. These results suggest that release of endogenous 5-HT regulates CB chemoreceptor function presynaptically, by a positive feedback mechanism involving autocrine-paracrine stimulation of 5-HT2a receptors and PKC modulation of resting and Ca(2+)-dependent K+ conductances.

Presynaptic modulation of rat arterial chemoreceptor function by 5-HT: role of K+ channel inhibition via protein kinase C.

The peripheral control of breathing is mediated by O2-sensitive carotid body (CB) type 1 cells, which express multiple neurotransmitters including the monoamines, dopamine and serotonin (5-HT). Whereas dopamine has been extensively studied, 5-HT has received little attention. Here, to elucidate the role of 5-HT in CB chemotransmission, we used perforated-patch recording from rat type 1 cell clusters and co-cultured petrosal (afferent) neurones. 5-HT induced action potentials and/or membrane depolarization associated with a conductance decrease in approximately 40% of recordings from type 1 cells (n = 78/192). These responses were markedly inhibited by the 5-HT2 receptor antagonist ketanserin (10-50 microM) and by the protein kinase C (PKC) inhibitor chelerythrine (50 microM). The PKC activator 1-oleoyl-2-acetylglycerol (OAG; 50 microM) mimicked the 5-HT-induced depolarization, and the combined effects of 5-HT and OAG were non-additive. The 5-HT-induced responses reversed near the potassium (K+) equilibrium potential (at approximately -82 mV; EK = -83 mV), suggesting inhibition of a resting K+ conductance. In type 1 cells (n = 7), voltage-activated outward K+ current was also inhibited by 1-50 microM 5-HT, an effect that was prevented by PKC inhibitors (chelerythrine and NPC 15437) and mimicked by OAG; the outward K+ current inhibited by 5-HT appeared to be predominantly a Ca(2+)-dependent K+ current. The 5-HT2 receptor blockers ketanserin and ritanserin reversibly inhibited spontaneous action potentials and the hypoxia-induced receptor potential recorded from clustered type 1 cells. Moreover, these blockers reversibly inhibited the hypoxic chemosensory response recorded postsynaptically in petrosal neurones that functionally innervated type 1 clusters in co-culture. RT-PCR and confocal immunofluorescence techniques revealed 5-HT2a receptor expression in rat CB type 1 cells. These results suggest that release of endogenous 5-HT regulates CB chemoreceptor function presynaptically, by a positive feedback mechanism involving autocrine-paracrine stimulation of 5-HT2a receptors and PKC modulation of resting and Ca(2+)-dependent K+ conductances.

Continuous, but not episodic hypoxia, induces CREB phosphorylation in rat carotid body type I cells.

The CB sensitivity to hypoxia is increased following sustained, long-term hypoxia such as in ventilatory acclimatization to high altitude (Bisgard 1994). The mechanisms underlying the cellular responses and adaptation to hypoxia are poorly understood. It is assumed that a major component of this adaptation is regulation of gene expression. cAMP response element-binding protein (CREB) is a nuclear protein. CREB is activated upon phosphorylation at Ser and modulates the transcription of genes with cAMP response elements (CRE) in their promoters (Ginty 1997). Recently, it has been shown that hypoxia induces phosphorylation of CREB at Ser in cultured PC 12 cells (Beitner-Johnson and Millhorn 1998), which are similar to the CB type I cells in several important characteristics related to oxygen-dependent gene regulation.

Identification of an oxygen-sensitive potassium channel in neonatal rat carotid body type I cells.

Identification of an oxygen-sensitive potassium channel in neonatal rat carotid body type I cells.

O(2) modulates large-conductance Ca(2+)-dependent K(+) channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism.

Hypoxic inhibition of large-conductance Ca(2+)-dependent K(+) channels (maxiK) of rat carotid body type I cells is a well-established fact. However, the molecular mechanisms of such inhibition and the role of these channels in the process of hypoxic transduction remain unclear. We have examined the mechanisms of interaction of O(2) with maxiK channels exploring the effect of hypoxia on maxiK currents recorded with the whole-cell and the inside-out configuration of the patch-clamp technique. Hypoxia inhibits channel activity both in whole-cell and in excised membrane patches. This effect is strongly voltage- and Ca(2+)-dependent, being maximal at low [Ca(2+)] and low membrane potential. The analysis of single-channel kinetics reveals a gating scheme comprising three open and five closed states. Hypoxia inhibits channel activity increasing the time the channel spends in the longest closed states, an effect that could be explained by a decrease in the Ca(2+) sensitivity of those closed states. Reducing maxiK channels with dithiothreitol (DTT) increases channel open probability, whereas oxidizing the channels with 2,2'-dithiopyridine (DTDP) has the opposite effect. These results suggest that hypoxic inhibition is not related with a reduction of channel thiol groups. However, CO, a competitive inhibitor of O(2) binding to hemoproteins, fully reverts hypoxic inhibition, both at the whole-cell and the single-channel level. We conclude that O(2) interaction with maxiK channels does not require cytoplasmic mediators. Such interaction could be mediated by a membrane hemoprotein that, as an O(2) sensor, would modulate channel activity.