A revisit to O2 sensing and transduction in the carotid body chemoreceptors in the context of reactive oxygen species biology.

Oxygen-sensing and transduction in purposeful responses in cells and organisms is of great physiological and medical interest. All animals, including humans, encounter in their lifespan many situations in which oxygen availability might be insufficient, whether acutely or chronically, physiologically or pathologically. Therefore to trace at the molecular level the sequence of events or steps connecting the oxygen deficit with the cell responses is of interest in itself as an achievement of science. In addition, it is also of great medical interest as such knowledge might facilitate the therapeutical approach to patients and to design strategies to minimize hypoxic damage. In our article we define the concepts of sensors and transducers, the steps of the hypoxic transduction cascade in the carotid body chemoreceptor cells and also discuss current models of oxygen- sensing (bioenergetic, biosynthetic and conformational) with their supportive and unsupportive data from updated literature. We envision oxygen-sensing in carotid body chemoreceptor cells as a process initiated at the level of plasma membrane and performed by a hemoprotein, which might be NOX4 or a hemoprotein not yet chemically identified. Upon oxygen-desaturation, the sensor would experience conformational changes allosterically transmitted to oxygen regulated K+ channels, the initial effectors in the transduction cascade. A decrease in their opening probability would produce cell depolarization, activation of voltage dependent calcium channels and release of neurotransmitters. Neurotransmitters would activate the nerve endings of the carotid body sensory nerve to convey the information of the hypoxic situation to the central nervous system that would command ventilation to fight hypoxia.

Effects of intermittent hypoxia on blood gases plasma catecholamine and blood pressure.

Obstructive sleep apnoea syndrome (OSAS) is a disorder characterized by repetitive episodes of complete (apnoea) or partial (hypopnoea) obstruction of airflow during sleep. The severity of OSAS is defined by the apnoea hypopnoea index (AHI) or number of obstructive episodes. An AHI greater than 30 is considered severe, but it can reach values higher than 100 in some patients. Associated to the OSA there is high incidence of cardiovascular and neuro-psychiatric pathologies including systemic hypertension, stroke, cardiac arrhythmias and atherosclerosis, diurnal somnolence, anxiety and depression. In the present study we have used a model of intermittent hypoxia (IH) of moderately high intensity (30 episodes/h) to evaluate arterial blood gases and plasma catecholamines as main effectors in determining arterial blood pressure. Male rats were exposed toIH with a regime of 80s, 20% O(2) // 40s, 10%O(2), 8 h/day, 8 or 15 days.Lowering the breathing atmosphere to 10% O(2) reduced arterial blood PO(2) to 56.9 mmHg (nadir HbO(2) 86, 3%). Plasma epinephrine (E) and norepinephrine (NE) levels at the end of 8 and 15 days of IH showed a tendency to increase, being significant the increase of norepinephrine (NE) levels in the group exposed to intermittent hypoxia during 15 days. We conclude that IH causes an increase in sympathetic activity and a concomitant increase in NE levels which in turn would generate an increase in vascular tone and arterial blood pressure.

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity.

Low oxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K(+) channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K(+) channel inhibition, subsequent cell depolarization and activation of neurotransmitter release. We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (E(GSH)) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB). We found that rotenone (1 microM), antimycin A (0.2 microg/ml) and sodium azide (5mM) decreased E(GSH); NAC restored E(GSH) to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between E(GSH) and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrial-generated modifications of E(GSH) and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia.

Chemoreception in the context of the general biology of ROS.

Superoxide anion is the most important reactive oxygen species (ROS) primarily generated in cells. The main cellular constituents with capabilities to generate superoxide anion are NADPH oxidases and mitochondrial respiratory chain. The emphasis of our article is centered in critically examining hypotheses proposing that ROS generated by NADPH oxidase and mitochondria are key elements in O(2)-sensing and hypoxic responses generation in carotid body chemoreceptor cells. Available data indicate that chemoreceptor cells express a specific isoform of NADPH oxidase that is activated by hypoxia; generated ROS acting as negative modulators of the carotid body (CB) hypoxic responses. Literature is also consistent in supporting that poisoned respiratory chain can produce high amounts of ROS, making mitochondrial ROS potential triggers-modulators of the CB activation elicited by mitochondrial venoms. However, most data favour the notion that levels of hypoxia, capable of strongly activating chemoreceptor cells, would not increase the rate of ROS production in mitochondria, making mitochondrial ROS unlikely triggers of hypoxic responses in the CB. Finally, we review recent literature on heme oxygenases from two perspectives, as potential O(2)-sensors in chemoreceptor cells and as generators of bilirubin which is considered to be a ROS scavenger of major quantitative importance in mammalian cells.

Ventilatory responses and carotid body function in adult rats perinatally exposed to hyperoxia.

Hypoxia increases the release of neurotransmitters from chemoreceptor cells of the carotid body (CB) and the activity in the carotid sinus nerve (CSN) sensory fibers, elevating ventilatory drive. According to previous reports, perinatal hyperoxia causes CSN hypotrophy and varied diminishment of CB function and the hypoxic ventilatory response. The present study aimed to characterize the presumptive hyperoxic damage. Hyperoxic rats were born and reared for 28 days in 55%-60% O2; subsequent growth (to 3.5-4.5 months) was in a normal atmosphere. Hyperoxic and control rats (born and reared in a normal atmosphere) responded with a similar increase in ventilatory frequency to hypoxia and hypercapnia. In comparison with the controls, hyperoxic CBs showed (1) half the size, but comparable percentage area positive to tyrosine hydroxylase (chemoreceptor cells) in histological sections; (2) a twofold increase in dopamine (DA) concentration, but a 50% reduction in DA synthesis rate; (3) a 75% reduction in hypoxia-evoked DA release, but normal high [K+]0-evoked release; (4) a 75% reduction in the number of hypoxia-sensitive CSN fibers (although responding units displayed a nearly normal hypoxic response); and (5) a smaller percentage of chemoreceptor cells that increased [Ca2+]1 in hypoxia, although responses were within the normal range. We conclude that perinatal hyperoxia causes atrophy of the CB-CSN complex, resulting in a smaller number of chemoreceptor cells and fibers. Additionally, hyperoxia damages O2-sensing, but not exocytotic, machinery in most surviving chemoreceptor cells. Although hyperoxic CBs contain substantially smaller numbers of chemoreceptor cells/sensory fibers responsive to hypoxia they appear sufficient to evoke normal increases in ventilatory frequency.

Reduced to oxidized glutathione ratios and oxygen sensing in calf and rabbit carotid body chemoreceptor cells.

1. The aim of this work was to test the redox hypotheses of O(2) chemoreception in the carotid body (CB). They postulate that hypoxia alters the levels of reactive oxygen species (ROS) and the ratio of reduced to oxidized glutathione (GSH/GSSG), causing modifications to the sulfhydryl groups/disulfide bonds of K+ channel proteins, which leads to the activation of chemoreceptor cells. 2. We found that the GSH/GSSG ratio in normoxic calf CB (30.14 +/- 4.67; n = 12) and hypoxic organs (33.03 +/- 6.88; n = 10), and the absolute levels of total glutathione (0.71 +/- 0.07 nmol (mg tissue)(-1), normoxia vs. 0.76 +/- 0.07 nmol (mg tissue)(-1), hypoxia) were not statistically different. 3. N-Acetylcysteine (2 mM; NAC), a precursor of glutathione and ROS scavenger, increased normoxic glutathione levels to 1.03 +/- 0.06 nmol (mg tissue)(-1) (P < 0.02) and GSH/GSSG ratios to 59.05 +/- 5.05 (P < 0.001). 4. NAC (20 microM-10 mM) did not activate or inhibit chemoreceptor cells as it did not alter the normoxic or the hypoxic release of (3)H-catecholamines ((3)H-CAs) from rabbit and calf CBs whose CA deposits had been labelled by prior incubation with the natural CA precursor (3)H-tyrosine. 5. NAC (2 mM) was equally ineffective in altering the release of (3)H-CAs induced by stimuli (high external K+ and ionomycin) that bypass the initial steps of the hypoxic cascade of activation of chemoreceptor cells, thereby excluding the possibility that the lack of effect of NAC on normoxic and hypoxic release of (3)H-CAs results from a concomitant alteration of Ca(2+) channels or of the exocytotic machinery. 6. The present findings do not support the contention that O(2) chemoreception in the CB is linked to variations in the GSH/GSSG quotient as the redox models propose.

Protective effect of melatonin against adriamycin toxicity in the rat.

Adriamycin, an anthracyclinic antibiotic frequently used in quimioterapeutic treatments is highly toxic; it inhibits protein synthesis and provokes prooxidant effects. Melatonin has recently been shown to have high antioxidative properties. We tested if melatonin is able to neutralize the oxidative damage induced by a single dose (20 mg/kg, i.p.) of adriamycin preceded (3 days) and followed (7 days) by a low pharmacological dose (50 microg/kg, i.p.) of melatonin. After the administration of a single dose of adriamycin (20 mg/kg i.p.) to male Wistar rats, the reduced to oxidized glutathione (GSH/GSSG) ratio and the glutathione peroxidase (GPx, E.C. 1.11.1.9.) activity in the brain, intestine, heart, kidney, and lung were significantly reduced. When the treatment of adriamycin was preceded and followed by low pharmacological doses of melatonin, the decrease in the GSH/GSSG ratio was significantly reduced but the reduction in GPx activity was not attenuated. A significant increase in lipid peroxidation products was observed in brain, heart, and kidney tissues after a single administration of adriamycin, which was attenuated by pre- and post-treatment with a low pharmacological dose of melatonin. Our results demonstrate that oxidative damage induced by the antitumor drug, adriamycin, can be reduced by low pharmacological doses of melatonin.

Endogenous rhythms of melatonin, total antioxidant status and superoxide dismutase activity in several tissues of chick and their inhibition by light.

Melatonin was recently shown to be a component of the antioxidative defense system of organisms due to its free radical scavenging ability and to its capacity to stimulate several antioxidant enzymes. In this report, we studied the endogenous rhythm of the antioxidant enzyme superoxide dismutase (SOD) in three different tissues (cerebral cortex, liver and lung) of chick (Gallus domesticus) (three weeks, at age and sacrificed every 2 hr). During the study the chicks were under a light:dark cycle of 12:12. Total antioxidant status of the plasma was correlated with physiological blood melatonin concentrations. Superoxide dismutase activity exhibited a marked 24 hr rhythm in cerebral cortex, lung and liver, with peak activity coincident with the melatonin and total antioxidant status peaks. The exposure of chicks to constant light for 7 days eliminated the melatonin rhythm as well as the peaks in superoxide dismutase activity and the total antioxidant status. These findings suggest that the melatonin rhythm may be related to the nighttime increase in the superoxide dismutase activity and to total antioxidant capacity of the blood.

Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo.

Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time-dependent manner; these changes correlate well with the half-life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 microg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria-modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug-induced mitochondrial damage in vivo.

Relationships between melatonin, glutathione peroxidase, glutathione reductase, and catalase. Endogenous rhythms on cerebral cortex in Gallus domesticus.

In this report, we studied the endogenous rhythms of three antioxidant enzymes: glutathione peroxidase (E.C.1.11.1.9), glutathione reductase (E.C.1.6.4.2) and catalase (E.C.1.11.1.6) in cortex of chick brain and correlate them with physiological blood melatonin concentrations.

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.).

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.

Effects of ethylene glycol tetraacetic acid, A23187 and calmodulin, calcium activated neutral proteinase antagonists on melatonin secretion in perifused chick pineal gland.

We have recently described, using perifused pineal glands, that calcium influx participates in the activation of chick pineal gland. This study shows that the loss of perifused chick pineal gland activity is a complex process which seems to involve the release of calcium from intracellular stores, calmodulin and calcium-activated neutral protease (CANP). Pineal glands were perifused with Krebs medium (controls) or with Krebs medium plus the drugs ethylene glycol tetraacetic acid (EGTA; calcium chelator), A23187 (calcium ionophore), EGTA plus A23187 (extra-intra cellular calcium chelation), trifluoperazine and CGS9343B (calmodulin inhibitors), and E-64 (CANP inhibitor) at the time of the natural peak of melatonin release. When EGTA or A23187 were added to the perifusion medium, no effects were observed. On the other hand, when the calcium chelator EGTA plus A23187 (free extra and intracellular calcium levels were dramatically decreased), trifluoperazine, CGS 9343B or E-64 were added to the perifusion medium melatonin synthesis increased significantly and was sustained for 8 h. We propose a prominent role for calcium output from intracellular stores in regulating melatonin production primarily by acting on Ca-calmodulin and calcium-activated neutral protease.

Rhythms of glutathione peroxidase and glutathione reductase in brain of chick and their inhibition by light.

Melatonin was recently shown to be a component of the antioxidative defense system of organisms due to its free radical scavenging and antioxidant activities. Pharmacologically, melatonin stimulates the activity of the peroxide detoxifying enzyme glutathione peroxidase in rat brain and in several tissues of chicks. In this report, we studied the endogenous rhythm of two antioxidant enzymes, glutathione peroxidase and glutathione reductase, in five regions (hippocampus, hypothalamus, striatum, cortex and cerebellum) of chick brain and correlated them with physiological blood melatonin concentrations. Glutathione peroxidase exhibited a marked 24 h rhythm with peak activity in each brain region which had acrophases about 8 h after lights off and about 4 h after the serum melatonin peak was detected. Glutathione reductase activity exhibited similar robust rhythms with the peaks occurring roughly 2 h after those of glutathione peroxidase. We suggest that neural glutathione peroxidase increases due to the rise of nocturnal melatonin levels while glutathione reductase activity rises slightly later possibly due to an increase of its substrate, oxidized glutathione. The exposure of chicks to constant light for 6 days eliminated the melatonin rhythm as well as the peaks in both glutathione peroxidase and glutathione reductase activities. These findings suggest that the melatonin rhythm may be related to the nighttime increases in the enzyme activities, although other explanations cannot be excluded.

Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen.

Hyperbaric oxygen exposure rapidly induces lipid peroxidation and cellular damage in a variety of organs. In this study, we demonstrate that the exposure of rats to 4 atmospheres of 100% oxygen for 90 min is associated with increased levels of lipid peroxidation products [malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA)] and with changes in the activities of two antioxidative enzymes [glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels of total glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and cerebellum) but not in liver. When animals were injected with melatonin (10 mg/kg) immediately before the 90-min hyperbaric oxygen exposure, all measurements of oxidative damage were prevented and were similar to those in untreated control animals. Melatonin's actions may be related to a variety of mechanisms, some of which remain to be identified, including its ability to directly scavenge free radicals and its induction of antioxidative enzymes via specific melatonin receptors.

Nocturnal decreases in nitric oxide and cyclic GMP contents in the chick brain and their prevention by light.

The diurnal variations in the contents of nitric oxide (NO) and cyclic GMP were studied in the chick brain. NO and cyclic GMP contents in the chick brain were lower at night than during the day and were inversely correlated with high night-time tissue melatonin levels. Furthermore, when animals were kept in light at night, tissue melatonin levels remained at low diurnal values, whereas NO and cyclic GMP contents remained high. Since we have previously shown that physiological concentrations of melatonin inhibit nitric oxide synthase (NOS) activity in different brain areas, the nocturnal decrease in brain NO and cyclic GMP contents may be, in part, a consequence of the nocturnal inhibitory effect of melatonin on NOS activity.

Effect of calcium on melatonin secretion in chick pineal gland I.

Melatonin is the neurohormone which is synthesized by the pineal gland and secreted rhythmically. The role of calcium in the activation of melatonin production remains unknown. In this study, we demonstrated that calcium input participates in the regulation of chick pineal gland. Pineal glands from Gallus domesticus were perifuse with Krebs medium (controls) or with Krebs medium plus drugs (ethylene glycol tetraacetic acid (EGTA) or calcium ionophore A23187). When EGTA was added to the perifusion medium, free extracellular calcium concentrations were dramatically decreased and melatonin synthesis was decreased. On the other hand, when the calcium ionophore A23187 was added to the perifusion medium, chick pineal glands exhibited a marked increase in secretion of melatonin. No effects were observed when chick pineal glands were treated with drugs during or after the time of the natural peak levels. We propose that calcium input from extracellular medium and output from intracellular calcium reserves are primary mechanisms in the activation of melatonin synthesis in the chick pineal gland.

Iron decreases the nuclear but not the cytosolic content of the neurohormone melatonin in several tissues in chicks.

This paper describes the influence of iron on both nuclear and cytosolic melatonin contents in several tissues of chicks. The neurohormone melatonin was estimated by means of radioimmunoassay. Iron, administered as FeCl3, decreased the nuclear melatonin level in a variety of tissues, including brain, heart, lung, kidney, and erythrocytes (nucleated cells in chicks) but was not seen in either the liver or gut. All variations related with iron were seen in the nuclear fraction, while only in the pineal gland did the melatonin content of the cytosol change as a result of iron treatment. We also observed a day-night rhythm in the nuclear melatonin: high nuclear levels of melatonin at night and low levels during the light period. This is the first report of nuclear localization of melatonin in any avian cell.

Melatonin stimulates the activity of the detoxifying enzyme glutathione peroxidase in several tissues of chicks.

The pineal hormone melatonin has been shown to directly scavenge free radicals and to stimulate, in the mammalian brain, at least one enzyme, glutathione peroxidase, which reduces free radical generation. In the present studies, we examined the effect of melatonin on glutathione peroxidase activity in several tissues of an avian species. Melatonin (500 micrograms/kg), when injected into chicks, increased glutathione peroxidase activity within 90 min in every tissue examined. Tissue melatonin levels, measured by radioimmunoassay, also increased following its peripheral administration. Depending on the tissue, the measured increases in melatonin varied from 75% to 1,300% over the control values. The melatonin-induced increases in glutathione peroxidase activity varied with the tissue and were between 22% and 134%. These percentage increases in glutathione peroxidase activity were directly correlated with tissue melatonin content. These results suggest that melatonin induces the activity of the detoxifying enzyme, glutathione peroxidase, in several tissues in the chick. The findings also suggest that melatonin would reduce the generation of highly toxic hydroxyl radicals by metabolizing its precursor, hydrogen peroxide. Because of this ability to stimulate glutathione peroxidase activity, melatonin should be considered as a component of the antioxidative defense system in this avian species.