L. Almaraz

Oxygen and acid chemoreception in the carotid body chemoreceptors

The carotid bodies are arterial chemoreceptors that are sensitive to blood PO2, PCO2 and pH. They are the origin of reflexes that are crucial for maintaining PCO2 and pH in the internal milieu and for adjusting the O2 supply according to the metabolic needs of the organism in situations of increased demand, such as exercise and while breathing at decreased O2 partial pressures during ascent or when living at high altitude. Chemoreceptor cells of the carotid body transduce the blood-borne stimuli into a neurosecretory response that is dependent on external Ca2+. These cells have an O2-sensitive K+ current that is reversibly inhibited by low PO2. It is proposed that the depolarization produced by inhibition of this K+ current activates Ca2+ channels; Ca2+ influx and neurosecretion follow. The cells have also a potent Na(+)-Ca2+ antiporter that could be responsible for the intracellular Ca2+ rise required to trigger the release of neurotransmitters during high PCO2 or low pH stimulation.

Effects of Different Types of Stimulation on Cyclic AMP Content in the Rabbit Carotid Body: Functional Significance

Abstract: Cyclic AMP levels in rabbit carotid bodies incubated under control conditions, 100% O2‐ or 95% O2/5% CO2‐equilibrated medium, are close to 1 pmol/mg wet tissue (range 0.4–2.43 pmol/mg). Isobutylmethylxanthine (0.5 mM) increases cyclic AMP levels by a factor of 14 and 8 in HEPES‐and CO2/CH3O−‐buffered medium, respectively. Forskolin (0.5–10 μM) applied during 30 min increases cyclic AMP levels in a dose‐dependent manner. Incubation of carotid bodies at low O2 tensions resulted in an elevation of cyclic AMP levels both in the absence and in the presence of isobutylmethylxanthine. In the latter conditions cyclic AMP increase was maximum at an O2 tension of 46 mm Hg and tended to decrease at extremely low Po2. In isobutylmethylxanthine‐containing Ca2+‐free medium, cyclic AMP increased linearly with decreasing Po2 from 66 to 13 mm Hg; the absolute cyclic AMP levels attained in Ca2+‐free medium were smaller than those observed in Ca2+‐containing medium at any Po2. The differences between Ca2+‐free and Ca2+‐containing media appear to be due to the action of released neurotransmitters in the latter conditions, because dopamine and norepinephrine, which are known to be released by hypoxia in a Ca2+‐dependent manner, increase cyclic AMP in the carotid body. Low pH/high Pco2 and high [K+]e increase cyclic AMP levels only in Ca2+‐containing medium. Forskolin potentiates the release of catecholamines induced by low Po2. These results suggest that cyclic AMP plays an important role in the modulation of the chemoreception process.

Effects of cyanide and uncoupler on chemoreceptor activity and ATP content of the cat carotid body

In cat carotid bodies (c.b.s) incubated in vitro with [3H]tyrosine to label the stores of catecholamines, it was found that CN promotes dose- and Ca2+-dependent release of [3H]dopamine (DA) from c.b. tissues in parallel to the increased electrical activity recorded from the carotid sinus nerve (c.s.n.). Two different uncouplers, dinitrophenol (DNP) and carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), both activate also in a dose-dependent fashion, release of DA and electrical activity in the c.s.n. However, while cyanide (CN) (10(-4) M) applied during 5 min reduced the adenosine triphosphate (ATP) content of the c.b. by 45%, DNP (2.5 x 10(-4) M) and CCCP (10(-6) M) applied for the same period of time did not modify the ATP levels of the organ. At the above concentrations, the 3 agents increased about 8-fold the electrical activity recorded from the c.s.n. Thus, contrary to the postulates of the metabolic hypotheses, our findings indicate that the decrease in the ATP content in the c.b. is not a prerequisite for the activation of the chemoreceptors. We propose alternative mechanisms to explain the chemostimulant action of the metabolic poisons.

Cyclic AMP Modulates Differentially the Release of Dopamine Induced by Hypoxia and Other Stimuli and Increases Dopamine Synthesis in the Rabbit Carotid Body

We have investigated the effects of different treatments that increase cyclic AMP levels on the in vitro synthesis and release of catecholamines in the rabbit carotid body. We also measured the rate of 45Ca2+ efflux from previously loaded carotid bodies under different conditions. Forskolin produced a dose‐dependent increase in the release of [3H]dopamine elicited by a hypoxic stimulus of medium intensity (Po2= 33 mm Hg) without altering basal [3H]dopamine release (100% O2‐equilibrated medium). At a concentration of 5 × 10‐‐6M, forskolin increased the release of [3H]dopamine induced by hypoxic stimuli of different intensities; the increase was maximal (498%) at the lowest intensity of hypoxic stimuli (Po2= 66 mm Hg), averaged 260% for hypoxic stimuli of intermediate intensity and 2 × 10‐‐4M cyanide, and was 150% under anoxia. Dibutyryl cyclic AMP (2 mM) and 3‐isobutyl‐1‐methylxanthine (0.5 mM) mimicked forskolin effects under hypoxic stimulation. Forskolin (5 × 10‐‐6M) also increased (180%) the release of [3H]dopamine induced by 20% CO2/pH 6.6, 2.5 × 10‐‐4M dinitrophenol, and 3 × 10‐‐5M ionomycin. Forskolin and 3‐isobutyl‐1‐methylxanthine were without effect on the release of [3H]dopamine elicited by 30 mM extracellular K+. Forskolin (5 × 10‐‐6M) augmented significantly the rate of 45Ca2+ efflux induced by hypoxic stimuli (Po2 of 33 and 66 mm Hg) and 2 × 10‐‐4M cyanide and showed a tendency to increase (20%) the 45Ca2+ efflux induced by dinitrophenol and low pH and to decrease (21%) the efflux induced by 30 mM K+ without altering the rate of efflux under basal conditions. Finally, forskolin (10‐‐5M) produced a 40% increase in the rate of [3H]dopamine synthesis from [3H]tyrosine. From the present and previously published observations, two physiological roles emerge for cyclic AMP in the carotid body: (a) The release of dopamine elicited by natural stimuli (low Po2 and low pH/high Pco2) is positively modulated by the associated increase in cAMP levels. In the case of the hypoxic stimulus, cyclic AMP may act on the O2‐sensing machinery and the exocytotic process, whereas in the case of the acidic stimuli, it may affect only the exocytotic process. (b) The activation of dopamine synthesis observed during hypoxic stimulation may be produced. at least in part, by the concomitant increase in level of cyclic AMP. via a cyclic AMP‐dependent phosphorylation of tyrosine hydroxylase.

Effects of high potassium on the release of [3H]dopamine from the cat carotid body in vitro.

Using an in vitro preparation of the cat carotid body, we have characterized the release of [3H]dopamine (DA) induced by high extracellular K+. Pulse superfusion (3 min) with high K+ Tyrode solution (20‐80 mM) evoked a concentration‐dependent release of [3H]DA from type I cells with a threshold at about 20 mM‐extracellular K+ and a plateau at about 60 mM‐extracellular K+. Equivalent low extracellular Na+ concentration ([Na+]o) solutions osmotically balanced with sucrose did not induce release. The high extracellular K+ concentration ([K+]o)‐evoked release of [3H]DA by type I cells was dependent on the presence of Ca2+ in the superfusion media. On prolonged (10‐14 min) superfusion with high K+ Tyrode solution, the [3H]DA release evoked by 60 mM‐extracellular K+ was transient, while that evoked by 30 mM‐extracellular K+ was sustained. In preparations superfused for 6 min with 60 mM‐extracellular K+ and zero extracellular Ca2+ concentration ([Ca2+]o) Tyrode solution, reintroduction of Ca2+ did not elicit a secretory response. Ba2+ was a potent secretagogue of [3H]DA in preparations superfused with normal and zero [Ca2+]o Tyrode solution. Additionally, Ba2+ was capable of eliciting a secretory response from type I cells in preparations previously exposed (6 min) to 60 mM‐extracellular K+, whether or not [Ca2+]o was present. With regards to stimulus‐secretion coupling, our results indicate that high [K+]o probably depolarizes type I cells. This effect would, in turn, activate voltage‐dependent Ca2+ channels, allowing the entrance of this ion to activate the neurosecretory response.

Effects of 2-deoxy-d-glucose on in vitro cat carotid body

The process of chemosensory transduction in the arterial chemoreceptors is not well understood. According to the metabolic hypothesis of chemoreception, a decrease in arterial pO2 will produce a decrease in the adenosine triphosphate (ATP) content in the chemosensory type I cells, leading to release of a neurotransmitter and increased sensory neural activity. There is a paucity of direct experimental support for this hypothesis, and in the present work, we have tested the postulates of the metabolic hypothesis in an in vitro preparation of cat carotid body using 2-deoxy-D-glucose as an ATP-depleting agent. This preparation, when superfused with Tyrode containing 5 mM Na+-pyruvate instead of glucose, responds normally to hypoxia, low pH and acetylcholine, and maintains normal ATP levels. Under these conditions, 2-deoxy-D-glucose is a chemostimulant, i.e. electrical activity in the carotid sinus nerve is increased, with a threshold concentration of 0.25 mM and a maximum response at about 2-4 mM. These concentrations of 2-deoxyglucose evoke a dose-dependent release of [3H]dopamine (synthesized from [3H]tyrosine) from the carotid bodies which parallels the electrical activity. The 2-deoxy-D-glucose-evoked release and electrical activity is dependent on the presence of extracellular Ca2+. These same concentrations of 2-deoxy-D-glucose significantly reduce the ATP content of the carotid bodies. The events postulated by the metabolic hypothesis, i.e. decrease in ATP content, release of a putative neurotransmitter and activation of the sensory nerve endings are found to occur simultaneously. A possible cause-effect relationship between these three events is discussed.

Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Current concepts on the location and functional significance of nicotinic receptors in the carotid body rest on alpha-bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by prior incubation with the tritiated natural precursor [3H]tyrosine, we have found that nicotine induces release of [3H]catecholamines in a dose-dependent manner (IC50 = 9.81 microM). We also found that mecamylamine (50 microM) completely abolished the nicotine-induced release, while alpha-bungarotoxin (100 nM; approximately 20 times its binding Kd) only reduced the release by 56%. These findings indicate that chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to alpha-bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception.

Presence of D1 receptors in the rabbit carotid body

Rabbit carotid bodies incubated in vitro in the presence of 5 x 10(-4) M 3-isobutyl 1-methylxanthine have cyclic AMP levels of 13.1 +/- 1.6 pmol/mg fresh tissue (means +/- S.E.M; n = 11). Dopamine (10(-6) and 10(-5) M) increased the cyclic AMP content to 24.4 +/- 2.85 and 40.6 +/- 3.29 pmol/mg tissue. The specific D1 agonist SKF38393 at 10(-6) M increased the cyclic AMP content to 23.9 +/- 2.3 pmol/mg fresh tissue and the specific D1 antagonist SCH23390 at 10(-6) M completely blocked the effect of 10(-5) M dopamine. dBcAMP (2 x 10(-3) M) potentiated the release of dopamine induced by a mild hypoxic stimulus and SKF38393 did not modify it. It is concluded that the carotid body has D1 receptors positively coupled to adenylate cyclase that seem to be located in the vasculature of the organ. This set of receptors, when activated by injected dopamine, produced vasodilatation leading to a decrease in carotid sinus nerve activity.

Oxygen Sensing in the Carotid Body

In the present article we review in a concise manner the literature on the mechanisms of O2 chemoreception in the carotid body of adult mammals. In the first section we describe the basic structure of the carotid body, and define this organ as a secondary sensory receptor. In the second section is presented the most relevant literature on the O2 metabolism in the carotid body to define the parameters of O2 chemoreception, including hypoxic thresholds and P50 of the hypoxic responses. The final section is devoted to the mechanisms of detection of the hypoxic stimulus. We provide the data in favor and against each of the current three models on O2 chemoreception: the membrane model, the metabolic hypothesis with its different versions and the NAD(P)H oxidase model.

In vitro activation of cyclo-oxygenase in the rabbit carotid body: effect of its blockade on [3H]catecholamine release.

The release of prostaglandin E2 (PGE2) from rabbit carotid bodies (CBs) incubated in basal conditions (PO2 approximately 132 mmHg; PCO2 approximately 33 mmHg; pH = 7.42) amounts to 94.4 +/‐ 10.1 pg (mg protein)‐1 (10 min)‐1 (mean +/‐ S.E.M.). Incubation of the CB in a hypoxic solution (PO2 approximately 46 mmHg) produced a significant 40% increase (P < 0.05) in the release of PGE2. Indomethacin (2 microM) prevented the hypoxia‐induced release of PGE2. Sensory plus sympathetic denervation of the CB 4 days prior to the experiments did not modify either basal or low PO2‐induced PGE2 release, indicating that intraglomic nerve endings are not significant sources for the PGE2 released. Incubation of the CB in an acidic‐hypercapnic solution (PO2 approximately 132 mmHg; PCO2 approximately 132 mmHg; pH = 6.60) or in a high K(+)‐containing solution (35 mM) was also effective in promoting an increase in the outflow of PGE2 from the organs. The release of [3H]catecholamines ([3H]CA) from the CB elicited by incubating the organs in low PO2 solutions (PO2 ranged between 66 and 13 mmHg) was potentiated by two inhibitors of cyclo‐oxygenase, acetylsalicylic acid (ASA, 100 microM) and indomethacin (2 microM). The effect persisted after chronic denervation of the organ. The secretory response elicited by acidic stimuli was also augmented by cyclo‐oxygenase inhibitors. Thus, [3H]CA release elicited by incubating the CBs in the acidic‐hypercapnic solution increased by 300% in the presence of indomethacin (2 microM), and ASA (100 microM) more than doubled the release induced by dinitrophenol (100 microM), a protonophore that mimics an acidic stimulus. Indomethacin, but not ASA, moderately increased the high K(+)‐evoked [3H]CA release. The effect of indomethacin on the release of [3H]CA elicited by acidic and hypoxic stimuli was reversed by PGE2 in a dose‐dependent manner (0.3‐300 nM). These results show that low PO2 and high PCO2‐low pH, the natural stimuli to the CB, as well as high extracellular [K+], activate the cyclo‐oxygenase pathway in the CB, promoting an increase in the outflow of PGE2. The data also show that the blockade of this pathway activates the stimulus‐induced [3H]CA release from the CB, indicating that naturally released prostanoids exert an inhibitory control on chemoreceptor cells. The data lend support to the notion that the hyper‐reactivity of the ventilatory response to hypoxia in subjects under anti‐inflammatory drug treatment results from CB cycloxygenase inhibition.