Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.     

Function of the rat carotid body chemoreceptors in ageing

Some age-related deficits in the ventilatory responses have been attributed to a decline in the functionality of the carotid body (CB) arterial chemoreceptors, but a systematic study of the CB function in ageing is lacking. In rats aged 3-24 months, we have performed quantitative morphometry on specific chemoreceptor tissue, assessed the function of chemoreceptor cells by measuring the content, synthesis and release of catecholamines (a chemoreceptor cell neurotransmitter) in normoxia and hypoxia, and determined the functional activity of the intact organ by measuring chemosensory activity in the carotid sinus nerve (CSN) in normoxia, hypoxia and hypercapnic acidosis. We found that with age CBs enlarge, but at the same time there is a concomitant decrease in the percentage of chemoreceptor tissue. CB content and turnover time for their catecholamines increase with age. Hypoxic stimulation of chemoreceptor cells elicits a smaller release of catecholamines in rats after 12 months of age, but a non-specific depolarizing stimulus elicits a comparable release at all ages. In parallel, there was a marked decrease in the responsiveness to hypoxia, but not to an acidic-hypercapnic stimulus, assessed as chemosensory activity in the CSN. We conclude that in aged mammals chemoreceptor cells become hypofunctional, leading to a decreased peripheral drive of ventilation.     

Hypoxia induces adenosine release from the rat carotid body

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.     

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP > alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.     

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.     

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 microm) and low intensity (PO2 approximately 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5'-(N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2-p-(2-Carboxyethyl)phenethyl-amino-5'-N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 x 10(-9) m) and high (160 x 10(-6) m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.     

Ventilatory acclimatization to hypoxia is not dependent on arterial hypoxemia

Goats were prepared so that one carotid body (CB) could be perfused with blood in which the gas tensions could be controlled independently from the blood perfusing the systemic arterial system, including the brain. Since one CB is functionally adequate, the nonperfused CB was excised. To determine whether systemic arterial hypoxemia is necessary for ventilatory acclimatization to hypoxia (VAH), the CB was perfused with hypoxic normocapnic blood for 6 h [means +/- SE: partial pressure of carotid body O2 (PcbO2), 40.6 +/- 0.3 Torr; partial pressure of carotid body CO2 (PcbCO2), 38.8 +/- 0.2 Torr] while the awake goat breathed room air to maintain systemic arterial normoxia. In control periods before and after CB hypoxia the CB was perfused with hyperoxic normocapnic blood. Changes in arterial PCO2 (PaCO2) were used as an index of changes in ventilation. Acute hypoxia (0.5 h of hypoxic perfusion) resulted in hyperventilation sufficient to reduce average PaCO2 by 6.7 Torr from control (P less than 0.05). Over the subsequent 5.5 h of hypoxic perfusion, average PaCO2 decreased further, reaching 4.8 Torr below that observed acutely (P less than 0.05). Acute CB hyperoxic perfusion (20 min) following 6 h of hypoxia resulted in only partial restoration of PaCO2 toward control values; PaCO2 remained 7.9 Torr below control (P less than 0.05). The progressive hyperventilation that occurred during and after 6 h of CB hypoxia with concomitant systemic normoxia is similar to that occurring with total body hypoxia. We conclude that systemic (and probably brain) hypoxia is not a necessary requisite for VAH.     

Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons.

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.     

Central vs. peripheral chemoreceptors in ventilatory stimulation by Hacetate

Intravenous infusion of Hacetate in conscious rabbits induces a greater decrease in cerebrospinal fluid (CSF) [HCO3-] and arterial CO2 partial pressure (PaCO2) than does HCl, HNO3, or Hacetate. To test whether acetate per se can stimulate central chemoreceptors, HCl- or Hacetate-acidified mock CSF was infused via the cisterna magna in conscious rabbits with catheters preimplanted under anesthesia. HCl infusion induced a greater decrease in PaCO2 refuting this hypothesis. To evaluate the role of the carotid body HCl and Hacetate were infused intravenously in an intact (CB+) and a chemodenervated group (CB-). In CB+ rabbits Hacetate infusion produced a greater decrease in PaCO2. In CB- rabbits, the fractional decrease in arterial PaCO2 was less for both acids compared with that of the CB+ rabbits, but it was significantly greater for Hacetate infusion (21.2 +/- 2.5%, mean +/- SE) than for HCl infusion (14.5 +/- 1.8%). Thus the carotid body is not necessary for the greater Hacetate ventilatory stimulation. The working hypothesis is that nonionic diffusion of Hacetate into brain or acetate replacement of HCO3- in CSF production lowers [HCO3-] near central chemoreceptors.     

Autonomic microganglion cells: a source of acetylcholine in the rat carotid body.

Hypoxic chemosensitivity of peripheral arterial chemoreceptors and the ventilatory response to O2 deprivation increases with postnatal development. Multiple putative neurotransmitters, which are synthesized in the carotid body (CB), are thought to mediate signals generated by hypoxia. Acetylcholine (ACh) is believed to be a major excitatory neurotransmitter participating in hypoxic chemosensitivity. However, it is not known whether ACh originates from type I cells in the CB. In these studies, we tested the hypothesis that choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) mRNAs are expressed in the CB and that mRNA levels would increase with postnatal maturation or exposure to hypoxia. Semiquantitative in situ hybridization histochemistry and immunohistochemistry were used to localize cholinergic markers within neurons and cells of the rat CB, the nodose-petrosal-jugular ganglion complex, and the superior cervical ganglion up to postnatal day 28. We show that the pattern of distribution, in tissue sections, is similar for both ACh markers; however, the level of VAChT mRNA is uniformly greater than that of ChAT. VAChT mRNA and immunoreactivity are detected abundantly in the nodose-petrosal-jugular ganglion complex in a number of microganglion cells embedded in nerve fibers innervating the CB for all postnatal groups, whereas ChAT mRNA is detected in only a few of these cells. Contrary to our hypothesis, postnatal maturation caused a reduction in ACh trait expression, whereas hypoxic exposure did not induce the upregulation of VAChT and ChAT mRNA levels in the CB, microganglion, or within the ganglion complex. The present findings indicate that the source of ACh in the CB is likely within autonomic microganglion cells and cholinergic nerve terminals.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Relative latency of responses of chemoreceptor afferents from aortic and carotid bodies

Discharges from aortic and carotid body chemoreceptor afferents were simultaneously recorded in 18 anesthetized cats to test the hypothesis that aortic chemoreceptors, because of their proximity to the heart, respond to changes in arterial blood gases before carotid chemoreceptors. We found that carotid chemoreceptor responses to the onset of hypoxia and hypercapnia, and to the intravenously administered excitatory drugs (cyanide, nicotine, and doxapram), preceded those of aortic chemoreceptors. Postulating that this unexpected result was due to differences in microcirculation and mass transport, we also investigated their relative speed of responses to changes in arterial blood pressure. The aortic chemoreceptors responded to decreases in arterial blood pressure before the carotid chemoreceptors, supporting the idea that the aortic body has microcirculatory impediments not generally present in the carotid body. These findings strengthened the concept that carotid bodies are more suited for monitoring blood gas changes due to respiration, whereas aortic bodies are for monitoring circulation.     

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.     

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. © 1995 John Wiley & Sons, Inc.     

Spatial and temporal Ca2+, Mg2+, and ATP2- dynamics in cardiac dyads during calcium release

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.     

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.     

Hypoxic modulation of systolic blood pressure and urinary sodium excretion in spontaneously hypertensive rats after carotid body denervation

To explore the role of arterial chemoreceptors, the effect of hypobaric hypoxia on urinary sodium excretion and systolic blood pressure was investigated in conscious spontaneously hypertensive rats (SHR) with carotid body denervation (CBD) or after sham-operation (SO). Denervation of the carotid bodies was performed by section of the carotid sinus nerves. Exposure to hypobaric hypoxia equivalent to high altitude of 4000 m led to a more pronounced decrease in systolic blood pressure in CBD-rats than in SO-rats. The pattern of urinary sodium excretion observed on the first two days of hypoxia in both groups was not affected by the chemodenervation. It is being suggested that arterial chemoreceptors do not play a critical role in blood pressure and natriuretic responses to hypobaric hypoxia in conscious SHR.     

Fast neurogenesis from carotid body quiescent neuroblasts accelerates adaptation to hypoxia

Unlike other neural peripheral organs, the adult carotid body (CB) has a remarkable structural plasticity, as it grows during acclimatization to hypoxia. The CB contains neural stem cells that can differentiate into oxygen‐sensitive glomus cells. However, an extended view is that, unlike other catecholaminergic cells of the same lineage (sympathetic neurons or chromaffin cells), glomus cells can divide and thus contribute to CB hypertrophy. Here, we show that O₂‐sensitive mature glomus cells are post‐mitotic. However, we describe an unexpected population of pre‐differentiated, immature neuroblasts that express catecholaminergic markers and contain voltage‐dependent ion channels, but are unresponsive to hypoxia. Neuroblasts are quiescent in normoxic conditions, but rapidly proliferate and differentiate into mature glomus cells during hypoxia. This unprecedented “fast neurogenesis” is stimulated by ATP and acetylcholine released from mature glomus cells. CB neuroblasts, which may have evolved to facilitate acclimatization to hypoxia, could contribute to the CB oversensitivity observed in highly prevalent human diseases.     

Time-dependent effect of hypoxia on carotid body chemosensory function

The time-dependent effects of hypoxia on the discharge rate carotid chemoreceptors were measured in anesthetized cats. Hypoxic exposure of two different durations were used: a short-term exposure (2-3 h) was used to measure the response of the same carotid chemoreceptors; and a long-term exposure (28 days at inspired PO2 of 70 Torr) to study carotid chemoreceptor properties in one group of cats relative to those of a control group. In the chronically hypoxic and control groups, determinations were made of the 1) steady-state responses to four levels of arterial PO2 (PaO2) at constant levels of arterial PCO2; 2) steady-state responses to acute hypercapnia during hyperoxia; and 3) maximal discharge rates during anoxia. We found that the acute responses of carotid chemoreceptor afferents to a given level of hypoxia (PaO2 = 30-40 Torr) did not significantly change within 2-3 h. After long-term exposure the carotid chemoreceptor responses to hypoxia significantly increased, with no significant changes in the hypercapnic response and in the maximal discharge rate during anoxia. We conclude that isocapnic hypoxia may not elicit a sufficient cellular response within 2-3 h in the cat carotid body to sensitize the O2 responsive mechanism, but hypoxia of longer duration will sensitize such a mechanism, thereby augmenting the chemosensory activity.     

Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-alpha secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-alpha release, resulting from TNF-alpha mRNA expression in rat cultured brain microglia. The TNF-alpha release was maximally elicited by 1 mM ATP and also induced by a P2X(7) receptor-selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, suggesting the involvement of P2X(7) receptor. ATP-induced TNF-alpha release was Ca(2+)-dependent, and a sustained Ca(2+) influx correlated with the TNF-alpha release in ATP-stimulated microglia. ATP-induced TNF-alpha release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca(2+). These results indicate that extracellular ATP triggers TNF-alpha release in rat microglia via a P2 receptor, likely to be the P2X(7) subtype, by a mechanism that is dependent on both the sustained Ca(2+) influx and ERK/p38 cascade, regulated independently of Ca(2+) influx.