Carotid Body Type Research Articles

Chronic hypoxia abolished the postnatal increase in carotid body type I cell sensitivity to hypoxia.

The O2 sensitivity of carotid chemoreceptor type I cells is low just after birth and increases with postnatal age. Chronic hypoxia during postnatal maturation blunts ventilatory and carotid chemoreceptor neural responses to hypoxia, but the mechanism remains unknown. We tested the hypothesis that chronic hypoxia from birth impairs the postnatal increase in type I cell O2 sensitivity by comparing intracellular Ca2+ concentration ([Ca2+]i) responses to graded hypoxia in type I cell clusters from rats born and reared in room air or 12% O2. [Ca2+]i levels at 0, 1, 5, and 21% O2, as well as with 40 mM K+, were measured at 3, 11, and 18 days of age with use of fura 2 in freshly isolated cells. The [Ca2+]i response to elevated CO2/low pH was measured at 11 days. Chronic hypoxia from birth abolished the normal developmental increase in the type I cell [Ca2+]i response to hypoxia. Effects of chronic hypoxia on development of [Ca2)]i responses to elevated K+ were small, and [Ca2+]i responses to CO2 remained unaffected. Impairment of type I cell maturation was partially reversible on return to normoxic conditions. These results indicate that chronic hypoxia severely impairs the postnatal development of carotid chemoreceptor O2 sensitivity at the cellular level and leaves responses to other stimuli largely intact.

Chemoreceptor discharges and cytochrome redox changes of the rat carotid body: role of heme ligands.

In superfused in vitro rat carotid body, we recorded chemoreceptor discharges and the redox state of cytochromes simultaneously to identify the primary oxygen-sensing protein controlling transmitter release and electrical activity of the carotid sinus nerve. These parameters were tested under the influence of heme ligands such as oxygen, cyanide, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and CO. During stimulation, there was an initial increase in discharge frequency followed by a decline or suppression of activity. Photometric changes lagged and were maintained as nerve activity decreased. Reducing mitochondrial cytochromes by cyanide or prolonged severe hypoxia, suppressed the chemoreceptor discharge. 4-(2-Aminoethyl)-benzenesulfonyl fluoride, a specific inhibitor of the phagocytic cytochrome b(558), also silenced the chemoreceptors after an initial excitation. CO increased the chemoreceptor discharge under normoxia, an effect inhibited by light, when the cytochromes were not reduced. When the discharges were depressed by severe hypoxia, exposure to light excited the chemoreceptors and the cytochromes were reduced. The rapidity of the chemosensory responses to light and lack of effect on dopamine release from type I cells led us to hypothesize that carotid body type I cells and the apposed nerve endings use different mechanisms for oxygen sensing: the nerve endings generate action potentials in association with membrane heme proteins whereas cytosolic heme proteins signal the redox state, releasing modulators or transmitters from type I cells.

Background leak K +-currents and oxygen sensing in carotid body type 1 cells

One model of oxygen sensing by the carotid body is that hypoxia depolarises type 1 cells leading to voltage-gated calcium entry and the secretion of neurotransmitters which then excite afferent nerves. This paper revues the mechanisms responsible for the membrane depolarisation in response to hypoxia. It concludes that depolarisation is caused not through the inhibition of calcium activated or delayed rectifier K +-channels but through the inhibition of an entirely new type of background K +-channel. This channel lacks sensitivity to the classical K +-channel inhibitors TEA and 4-AP. New evidence does however reveal that background K +-channels in the type 1 cell can be inhibited by Ba 2+ and that Ba 2+ depolarises isolated type 1 cells. Intriguingly, Ba 2+ is the only K +-channel inhibitor thus far reported to stimulate the carotid body. These studies therefore support the hypothesis that depolarisation of the type 1 cell is an integral part of the oxygen sensing pathway in the carotid body.

Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells.

1. Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4-dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mM Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. 2. In perforated-patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to -70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. 3. Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. 4. FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3-4 s. 5. We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage-gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation.

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O 2) on the properties of K + and Ca 2+ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K + current amplitudes were not affected by this period of hypoxia, but K + current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K + current density was separated into Ca 2+-dependent and Ca 2+-independent components by bath application of 200 μM Cd 2+, which blocked Ca 2+ currents and therefore, indirectly, Ca 2+-dependent K + currents. Ca 2+-dependent K + current density was not significantly different in control and chronically hypoxic type I cells. Cd 2+-resistant (Ca 2+-insensitive) K + current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po 2 15–22 mmHg) caused reversible, selective inhibition of Ca 2+-dependent K + currents in both groups of cells and Ca 2+-insensitive K + currents were unaffected by acute hypoxia. Ca 2+ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca 2+ channel current inhibition caused by nifedipine (5 μM). Acute hypoxia did not affect Ca 2+ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca 2+-insensitive, voltage-gated K + currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.

Inhibition of Ca2+-dependent K+ channels in rat carotid body type I cells by protein kinase C.

1. Whole-cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body. 2. Pretreatment of cells for 10 min at 37 C with 4alpha-phorbol 12,13-didecanoate (4alpha-PDD, 200 nM), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nM 12-O-teradecanoylphorbol-13-acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co-incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 microM). 3. The sensitivity of K+ currents to inhibition by 200 microM Cd2+ (indicative of the presence of Ca2+-dependent K+ channels) was markedly reduced in TPA-treated cells as compared with sham-treated cells, cells treated with 4alpha-PDD, and cells treated with both TPA and BIM. Cd2+-resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range -100 mV to -50 mV. 4. Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nM 4alpha-PDD as compared with cells treated with the same concentration of TPA. 5. The degree of inhibition of K+ currents caused by hypoxia (Po2 15-20 mmHg) was unaltered by pretreatment of cells with 3 microM BIM. 6. The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+-dependent K+ channel inhibitor iberiotoxin (20 nM) failed to depolarize these cells further. 7. Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+-dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.

K +-current modulated by PO 2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O 2 chemoreception, hypoxia suppresses K + currents leading to cell depolarization, [Ca 2+] i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca 2+ sensitive and other K + currents in rat carotid body type I cells, with and without low [Ca 2+] o plus high [Mg 2+] o , did not essentially influence low P O 2 effects on [Ca 2+] i and chemosensory discharge. Thus, hypoxia may suppress the K + currents in glomus cells but K + current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K +–O 2 current is linked to events in chemoreception is not substantiated. K +–O 2 current is an epiphemenon which is not directly linked with O 2 chemoreception.

Swelling‐ and cAMP‐Activated Cl− Currents in Isolated Rat Carotid Body Type I Cells

1. In the whole-cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl- currents during cell swelling effected by hypotonic extracellular solutions. 2. Hypotonic solutions evoked outwardly rectifying, non-inactivating currents which showed time-independent activation. The reversal potential (E(rev)) for the hypotonically evoked current was 1.6 +/- 0.6 mV (n = 26). Reduction of extracellular Cl- from 133 to 65.5 mM caused a shift in E(rev) of +14.7 +/- 0.4 mV (n = 5). 3. The swelling-activated Cl- current could not activate when ATP was omitted from the patch pipette or when substituted for the non-hydrolysable ATP analogues 5'-adenylylimidodiphosphate, AMP-PNP (2 mM) or beta, gamma-methylene-adenosine 5'-triphosphate. AMP-PCP (2 mM). The current also failed to activate in the absence of free intracellular Ca2+. 4. The swelling-activated Cl- current was sensitive to blockade by the Cl- channel blockers niflumic acid (300 microM) and 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS; 200 microM), although the blockade by DIDS was voltage dependent. 5. A similar, non-inactivating, outwardly rectifying Cl- current was evoked by the inclusion of cAMP (200 microM) in the patch pipette. This current could be inhibited by niflumic acid (300 microM), DIDS (200 microM) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP. 6. In conclusion, carotid body type I cells possess Cl- currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.

Synapse formation and hypoxic signalling in co-cultures of rat petrosal neurones and carotid body type 1 cells.

1. To investigate synaptic mechanisms mediating chemosensory signalling in the carotid body, we developed co-cultures of chemoreceptor type 1 cell clusters and dissociated petrosal neurones (PNs) from 7- to 14-day-old rat pups and tested for functional connectivity in CO2-HCO3(-)-or Hepes-buffered medium at approximately 35 degrees C. 2. When cultured without type 1 cells, PNs were almost always quiescent (n = 104) and unresponsive to hypoxia (Po2 = 5-25 mmHg) during perforated patch, whole-cell recordings of membrane potential or voltage-activated currents; in contrast, many PNs (77 out of 170) that were juxtaposed to type 1 cell clusters in co-culture displayed spontaneous activity, comprising spikes and subthreshold potentials (SSPs) that resembled synaptic potentials. 3. Additional tests suggested that de novo chemical synapses developed between PNs and type 1 cell clusters in vitro. For example: (i) the spontaneous activity was reversibly suppressed by substituting low calcium-high magnesium in the bath; (ii) SSPs had variable amplitudes and persisted following action potential blockade with TTX (1 microM); (iii) the interval distribution between successive spontaneous events appeared random; and (iv) the frequency of spontaneous potentials was diminished (reversibly) by the nicotinic antagonist hexamethonium (100 microM), suggesting contributions from the spontaneous release of ACh. 4. Many complexes of 'juxtaposed' PNs and type 1 clusters were physiologically functional, since exposure to hypoxia caused a reversible depolarization and/or increased spike discharge in approximately 30% of such neurones (n = 140). The hypoxia-induced spike discharge persisted in the presence of the dopamine D2 receptor blocker spiperone (10-50 microM; n = 5); however, this discharge was reversibly inhibited by 100-200 microM hexamethonium, suggesting that it was mediated, at least in part, by ACh acting through nicotinic receptors. 5. The hypoxia-induced spike discharge and frequency of spontaneous potentials in co-cultured PNs were reversibly suppressed when the buffer was switched from CO2-HCO3- to Hepes (10 mM) at pH 7.4; further, 'functional' PNs that displayed spontaneous activity and/or hypoxia-induced responses in co-culture were encountered more frequently in CO2-HCO3- (> or = 40%) than in Hepes (< or = 26%) buffer. 6. We conclude that functional chemical synapses can develop de novo in cultures of carotid body type 1 cells and PNs and that ACh is probably an important excitatory neurotransmitter secreted from type 1 cells during hypoxic chemotransduction in the rat carotid body.

Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia

Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.

A novel oxygen-sensitive potassium current in rat carotid body type I cells.

1. Hypoxic stimuli depolarize carotid body type I cells causing voltage-gated calcium influx. This study investigates the cause of this membrane depolarization. Isolated type I cells from neonatal (11-16 day) rat carotid bodies were used in the experiments. 2. Tetraethylammonium (TEA; 10 mM), 1 and 5 mM 4-aminopyridine (4-AP) and 20 nM charybdotoxin all failed to evoke a significant rise in [Ca2+]i. Similarly, in perforated patch whole-cell recordings, a combination of 10 mM TEA and 5 mM 4-AP failed to depolarize type I cells. 3. In type I cells voltage clamped at -70 mV, anoxia evoked a small inward current under control conditions, but had no effect in the absence of pipette and extracellular K+. 4. Anoxia decreased resting membrane conductance from 322 to 131 pS. The anoxia-sensitive current (measured using voltage ramps from -100 to -40 mV) had a reversal potential of -89 mV in 4.5 mM Ko+ and -66 mV in 20 mM Ko+, indicating that this current was carried principally by potassium ions. In contrast, 10 mM TEA + 5 mM 4-AP had little effect on the current-voltage relationship of the cells over the same range. 5. This O2-sensitive K+ conductance showed only mild outward rectification over the range -90 to +30 mV, which could be approximated by the Goldman-Hodgkin-Katz current equation. In addition, there was no time-dependent activation or inactivation of membrane currents elicited by voltage steps in the range -100 to -30 mV. 6. The O2-sensitive K+ conductance was inhibited by graded reductions in PO2 to 40 Torr and below, with a K1/2 of about 12 Torr. 7. The data suggest that hypoxia depolarizes type I cells principally through the inhibition of a small voltage-insensitive resting (or background) K+ conductance, and not through the inhibition of voltage-gated TEA and 4-AP-sensitive K+ channels (e.g. maxi-K or KO2 channels), as has been previously suggested.

Muscarinic and nicotinic receptors raise intracellular Ca2+ levels in rat carotid body type I cells.

1. The effects of cholinergic agonists upon intracellular free Ca2+ levels ([Ca2+]i) have been studied in enzymically isolated rat carotid body single type I cells, using indo-1. 2. Acetylcholine (ACh) dose-dependently increased [Ca2+]i in 55% of cells studied (EC50 = 13 microM). These [Ca2+]i rises were partially inhibited by atropine or mecamylamine. 3. Specific nicotinic and muscarinic agonists also elevated [Ca2+]i in a dose-dependent manner (nicotine, EC50 = 15 microM; methacholine, EC50 = 20 microM). 4. While the majority of the ACh-sensitive cells responded to both classes of cholinergic agonist, 29% responded exclusively to nicotinic stimulation and 9% responded exclusively to muscarinic stimulation. 5. In the presence of nicotinic agonists, Ca2+i responses were transient. In the presence of muscarinic agonists, Ca2+i responses consisted of an initial rise, which then declined to a lower plateau level. 6. Nicotinic responses were rapidly abolished in Ca(2+)-free medium, suggesting that they are dependent on Ca2+ influx. 7. The plateau component of the muscarinic-activated response was also abolished in Ca(2+)-free conditions. The rapid initial [Ca2+]i rise, however, could still be evoked after several minutes in Ca(2+)-free medium. Muscarine also increased Mn2+ quenching of intracellular fura-2 fluorescence. These data suggest that the full muscarinic response depends on both Ca2+ release from intracellular stores and Ca2+o influx. 8. The results indicate that, in rat carotid body type I cells, both nicotinic and muscarinic acetylcholine receptors increase [Ca2+]i, but achieve this via different mechanisms. ACh may therefore play a role in carotid body function by modulating Ca2+i in the chemosensory type I cells.

Developmental loss of hypoxic chemosensitivity in rat adrenomedullary chromaffin cells.

1. We investigated whether adrenomedullary chromaffin cells (AMCs) derived from neonatal (postnatal day (P) 1-P2) and juvenile (P13-P20) rats, and maintained in short-term culture (1-3 days), express O2-chemoreceptive properties. 2. In whole-cell recordings, the majority (approximately 70%; n = 47) of neonatal AMCs were sensitive to hypoxia. Under voltage clamp, acute hypoxia (PO2 approximately 40 mmHg) suppressed voltage-dependent K+ current by 25.1 +/- 3.4% (mean +/- S.E.M.; n = 22); under current clamp, acute hypoxia caused a membrane depolarization of 14.1 +/- 1.3 mV (n = 13) from a resting potential of -54.8 +/- 2.8 mV (n = 13), and this was often sufficient to trigger action potentials. 3. Exposure of neonatal AMC cultures to a moderate (PO2 approximately 75 mmHg) or severe (PO2 approximately 35 mmHg) hypoxia for 1 h caused a dose-dependent stimulation (approximately 3 or 6 times normoxia, respectively) of catecholamine (CA) release, mainly adrenaline, determined by HPLC. This induced CA release was abolished by the L-type calcium channel blocker, nifedipine (10 microM). 4. In contrast to the above results in neonates, hypoxia had no significant effects on voltage-dependent K+ current, membrane potential, or CA release in juvenile AMCs. 5. We conclude that rat adrenal chromaffin cells possess a developmentally regulated O2-sensing mechanism, similar to carotid body type I cells.

Regulation of tyrosine hydroxylase mRNA stability by oxygen in PC12 cells.

Mammals evolved to live in the environment that contains 21% of oxygen. In order to preserve optimal tension of oxygen (pO2) in the blood, organisms developed specialized cells that are O2-sensors and initiate adaptive and protective regulatory mechanisms during limited availability of oxygen in the environment (i.e. hypoxia). The best known of these O2-sensitive cells are the carotid body type I glomus cells that immediately detect even relatively moderate reduction in pO2 in the arterial blood and communicate this information via carotid sinus nerve to the central nervous system networks that regulate the respiratory and cardiovascular systems. A major neurotransmitter that is released during hypoxia from type I cells is catecholamine dopamine. In type I cells hypoxia stimulates the activity of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis (Gonzalez et al, 1977; Hanbauer et al, 1977), and induces dopamine synthesis and release (Fidone et al, 1982a,b; Fishman et al, 1985). Moreover, hypoxia causes a five-fold increase in TH mRNA in a mechanism that is intrinsic to the type I cells, i.e. independent from neural or hormonal inputs (Czyzyk-Krzeska et al, 1992). Further identification and characterization of the O2-dependent mechanisms that regulate TH gene expression required analysis at the molecular level. This type of studies has been, however, hindered by paucity of tissue provided by carotid body (only approximately 10,000 oxygen sensitive cells per carotid body in the rat). Our laboratory has recently reported that a clonal cell line derived from adrenal medulla tumor -pheochromocytoma- (PC12 cells) demonstrates several characteristics very similar to the carotid body type I cells. During hypoxia PC12 cells depolarize and release dopamine (Zhu et aI, 1996).

Co-cultures of rat petrosal neurons and carotid body type 1 cells. A model for studying chemosensory mechanisms.

In mammals reflex hyperventilation following acute hypoxia is mediated via impulses in the carotid sinus nerve (CSN) which supplies afferent innervation to O2- sensitive type 1 cells of the carotid body (Eyzaguirre & Zapata, 1984; Gonzalez et al, 1994). Type 1 cells contain multiple neurotransmitters, several of which are potential candidates for initiating or modulating CSN discharge during chemosensory signaling. The underlying synaptic events are poorly understood, and even controversial, in part because it is difficult to obtain reliable recordings from the afferent nerve endings in situ (see Eyzaguirre et al, 1983), and the transmitter sensitivity of these endings or of their parent cell bodies are unknown. Here we describe a preparation based on co-culture of rat type 1 cells and their afferent (petrosal) neurons which, combined with electrophysiological perforatedpatch recording from identified neurons, provide an attractive approach for studying these mechanisms. Previous attempts using co-cultures of rat type 1 cells and nodose (sensory) neurons were hampered by the paucity of successful contacts, probably due to the low frequency of ‘appropriate’ chemosensory neurons present (Alcayaga & Eyzaguirre, 1990).KeywordsTyrosine HydroxylaseCarotid BodyAfferent Nerve EndingSynaptic EventCarotid Sinus NerveThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Acetylcholine elevates intracellular Ca2+ via muscarinic and nicotinic receptors in rat carotid body type I cells.

Acetylcholine (ACh) has been proposed to act as an excitatory neurotransmitter on sensory nerve endings (reviewed in Fidone & González, 1986) in the carotid body. However, several facts undermine this hypothesis, particularly the species-dependence of its effects and the apparent absence of cholinergic receptors on afferent terminals of the carotid sinus nerve. In contrast, both nicotinic and muscarinic receptors abound in type I and type II cells (Dinger et al, 1985, 1986). Consequently, although the evidence for a post-synaptic role for ACh is questionable, there is good evidence for a pre-synaptic role, which would account for the high ACh-sensitivity of the discharge frequency of the sinus nerve (Monti-Bloch & Eyzaguirre, 1980). We have therefore investigated the effects of ACh on Ca2+ signalling in freshly isolated single type I cells.KeywordsMuscarinic ReceptorCarotid BodyCholinergic ReceptorMuscarinic AgonistSensory Nerve EndingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Distribution of epithelioid cells in the wall of the chicken aorta and their functional role as chemoreceptors.

The ultrastructural characteristics of epithelioid cells in the wall of the chicken aorta have been studied by several investigators. Their characteristics were homologous to those of carotid body type I cells and are considered to be one of the peripheral chemoreceptors. However, there are few descriptions about their location, distribution, and how they react to chemical signals, nor have there been many reports about the localization of bioactive substances in the epithelioid cells. Therefore we designed this investigation to address these problems. Wholemount immunohistochemistry using antiserotonin antiserum, scanning (SEM), and transmission (TEM) electron microscopy were used to observe the epithelioid cells and the lumen of the chicken aorta. The localizations of bioactive substances in the epithelioid cells were immunohistochemically investigated using 11 antisera. Epithelioid cells were dispersed in the wall of the aorta, forming a band approximately 1 mm in width, located 10 mm proximal to the confluence of the right and left ligamenta arteriosa. Serotonin, chromogranin, and neuron specific enolase immunoreactivities were detected in the epithelioid cells. SEM observations clearly demonstrated intraendothelial fenestrations, 1-3 microns in diameter, on the endothelial surface of the region of the band of epithelioid cells. TEM observations revealed that these fenestrations corresponded to endothelial gaps, directly beneath which epithelioid cells were sometimes located. The epithelioid cells are in direct access to the aortic lumen through endothelial fenestrations. Thus they may be able to perceive chemical signals from arterial blood directly.

Immunohistochemical demonstration of four subunits of neutrophil NAD(P)H oxidase in type I cells of carotid body

We demonstrate, by means of immunohistochemistry, that type I cells of human, guinea pig, and rat carotid bodies react with antisera raised against the subunits p22phox, gp91phox, p47phox, and p67phox of the NAD(P)H oxidase isolated from human neutrophil granulocytes. The findings support previous photometric studies that indicate that carotid body type I cells possess a putative oxygen sensor protein that is similar to the neutrophil NAD(P)H oxidase and consists of a hydrogen peroxide generating low-potential cytochrome b558 with cofactors regulating the electron transfer to oxygen.

Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells.

1. An acid-induced rise in the intracellular calcium concentration ([Ca2+]i) of type I cells is thought to play a vital role in pH/PCO2 chemoreception by the carotid body. In this present study we have investigated the cause of this rise in [Ca2+]i in enzymatically isolated, neonatal rat type I cells. 2. The rise in [Ca2+]i induced by a hypercapnic acidosis was inhibited in Ca(2+)-free media, and by 2 mM Ni2+. Acidosis also increased Mn2+ permeability. The rise in [Ca2+]i is dependent, therefore, upon a Ca2+ influx from the external medium. 3. The acid-induced rise in [Ca2+]i was attenuated by both nicardipine and methoxyverapamil (D600), suggesting a role for L-type Ca2+ channels. 4. Acidosis depolarized type I cells and often (approximately 50% of cells) induced action potentials. These effects coincided with a rise in [Ca2+]i. When membrane depolarization was prevented by a voltage clamp, acidosis failed to evoke a rise in [Ca2+]i. The acid-induced rise in [Ca2+]i is a consequence, therefore, of membrane depolarization. 5. Acidosis decreased the resting membrane conductance of type I cells. The reversal potential of the acid-sensitive current was about -75 mV. 6. A depolarization (30 mM [K+]o)-induced rise in [Ca2+]i was blocked by either the removal of extracellular Ca2+ or the presence of 2 mM Ni2+, and was also substantially inhibited by nicardipine. Under voltage-clamp conditions, [Ca2+]i displayed a bell-shaped dependence on membrane potential. Depolarization raises [Ca2+]i, therefore, through voltage-operated Ca2+ channels. 7. Caffeine (10 mM) induced only a small rise in [Ca2+]i (< 10% of that induced by 30 mM extracellular K+). Ca(2+)-induced Ca2+ release is unlikely, therefore, to contribute greatly to the rise in [Ca2+]i induced by depolarization. 8. Although the replacement of extracellular Na+ with N-methyl-D-glucamine (NMG), but not Li+, inhibited the acid-induced rise in [Ca2+]i, this was due to membrane hyperpolarization and not to the inhibition of Na(+)-Ca2+ exchange or Na(+)-dependent action potentials. 9. The removal of extracellular Na+ (NMG substituted) did not have a significant effect upon the resting [Ca2+]i, and only slowed [Ca2+]i recovery slightly following repolarization from 0 to -60 mV. Therefore, if present, Na(+)-Ca2+ exchange plays only a minor role in [Ca2+]i homeostasis. 10. In summary, in the neonatal rat type I cell, hypercapnic acidosis raises [Ca2+]i through membrane depolarization and voltage-gated Ca2+ entry.

Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)