Asunción Rocher

γ-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm

Two homologous sulfur‐rich basic polypeptides form wheat endosperm, so‐called γ1‐purothionin and γ2‐purothionin, are described. Purification involves extraction with volatile solvents and ammonium bicarbonate fractionation followed by reversed‐phase high‐performance liquid chromatography. The complete primary structure of these two polypeptides has been determined by automatic degradation of the intact, S‐carboxymethylated γ‐purothionins and peptides obtained by enzymatic cleavage. γ1‐Purothionin and γ2‐purothionin consist of 47 amino acids with an assessed molecular weight of 5239 and 5151 Da, respectively and 8 cysteines organized in 4 disulfide bridges. They present a high degree of homology among themselves (89% of identity) and are the first two thionin‐like polypeptides, so‐called y‐thionins, described from wheat endosperm.

Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia

Reactive oxygen species (ROS) are oxygen-containing molecular entities which are more potent and effective oxidizing agents than is molecular oxygen itself. With the exception of phagocytic cells, where ROS play an important physiological role in defense reactions, ROS have classically been considered undesirable byproducts of cell metabolism, existing several cellular mechanisms aimed to dispose them. Recently, however, ROS have been considered important intracellular signaling molecules, which may act as mediators or second messengers in many cell functions. This is the proposed role for ROS in oxygen sensing in systems, such as carotid body chemoreceptor cells, pulmonary artery smooth muscle cells, and erythropoietin-producing cells. These unique cells comprise essential parts of homeostatic loops directed to maintain oxygen levels in multicellular organisms in situations of hypoxia. The present article examines the possible significance of ROS in these three cell systems, and proposes a set of criteria that ROS should satisfy for their consideration as mediators in hypoxic transduction cascades. In none of the three cell types do ROS satisfy these criteria, and thus it appears that alternative mechanisms are responsible for the transduction cascades linking hypoxia to the release of neurotransmitters in chemoreceptor cells, contraction in pulmonary artery smooth muscle cells and erythropoietin secretion in erythropoietin producing cells.

Ionic mechanisms for the transduction of acidic stimuli in rabbit carotid body glomus cells.

1. The release of [3H]dopamine (DA) in response to inhibition of the Na+ pump or to intracellular acid load was studied in rabbit carotid bodies (CB) previously incubated with the precursor [3H]tyrosine. The ionic requirements of the release response and the involvement of specific ion transport systems were investigated. 2. Inhibition of the Na+ pump, by incubating the CB with ouabain or in K(+)‐free medium, evokes a DA release response which requires the presence of Na+ and Ca2+ in the medium and is insensitive to nisoldipine. This suggests that the response is triggered by entry of external Ca2+ through Na(+)‐Ca2+ exchange, a consequence of the increase in intracellular Na+ resulting from inhibition of the pump. 3. Incubation of the CB in medium equilibrated with 20% CO2 at pH 6.6, or in medium containing the protonophore dinitrophenol (DNP) or the weak acid propionate, elicits a DA release response which requires also the presence of Na+ and Ca2+ in the medium and is insensitive to dihydropyridines. 4. Ethylisopropylamiloride (EIPA), an inhibitor of the Na(+)‐H+ exchanger, markedly decreases the release response elicited by DNP or propionate in bicarbonate‐free medium, but has not any effect in bicarbonate‐buffered medium. In the latter condition, the EIPA‐insensitive release of DA is inhibited by reducing the HCO3‐ concentration in the medium to 2 mM or by removal of Cl‐, suggesting that in bicarbonate‐buffered medium a Na(+)‐dependent HCO3(‐)‐Cl‐ exchanger is involved in the release response. 5. It is concluded that the release of DA by the chemoreceptor cells in response to acidic stimulation is triggered by entry of external Ca2+ through Na(+)‐Ca2+ exchange. This exchange is promoted by the increase of intracellular Na+ that results from the operation of Na(+)‐coupled H(+)‐extruding mechanisms activated by the acid load.

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 µm) and low intensity (PO2 ≈ 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 × 10−9 m) and high (160 × 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.

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.

CELLULAR MECHANISMS OF OXYGEN CHEMORECEPTION IN THE CAROTID BODY

The carotid bodies (CB) are arterial chemoreceptors that by sensing changes of arterial PO2, PCO2 and pH can initiate and modify ventilatory and cardiovascular reflexes in order to maintain PO2, PCO2 and pH within physiological levels. It is now generally accepted that the glomus or type I cells of the CB are the transducers of hypoxic stimuli, and relay chemosensory information to the brainstem via neurotransmitter release at synaptic contacts with afferent terminals of the carotid sinus nerve. This article reviews the mechanisms of the O2-sensing process at the cellular level. We consider first the transduction of the hypoxic stimulus, in which most of the experimental evidence currently favors a mechanism involving modulation of the electrical properties of type I cells. The last part of the article deals with the transmission of the stimulus between type I cells and afferent nerve terminals, and we present an overview on the issue of neurotransmission in the CB, summarizing the actions of the main neurotransmitters present in the organ.

Intermittent hypoxia and diet-induced obesity: effects on oxidative status, sympathetic tone, plasma glucose and insulin levels, and arterial pressure

Obstructive sleep apnea (OSA) consists of sleep-related repetitive obstructions of upper airways that generate episodes of recurrent or intermittent hypoxia (IH). OSA commonly generates cardiovascular and metabolic pathologies defining the obstructive sleep apnea syndrome (OSAS). Literature usually links OSA-associated pathologies to IH episodes that would cause an oxidative status and a carotid body-mediated sympathetic hyperactivity. Because cardiovascular and metabolic pathologies in obese patients and those with OSAS are analogous, we used models (24-wk-old Wistar rats) of IH (applied from weeks 22 to 24) and diet-induced obesity (O; animals fed a high-fat diet from weeks 12 to 24) to define the effect of each individual maneuver and their combination on the oxidative status and sympathetic tone of animals, and to quantify cardiovascular and metabolic parameters and their deviation from normality. We found that IH and O cause an oxidative status (increased lipid peroxides and diminished activities of superoxide dismutases), an inflammatory status (augmented C-reactive protein and nuclear factor kappa-B activation), and sympathetic hyperactivity (augmented plasma and renal artery catecholamine levels and synthesis rate); combined treatments worsened those alterations. IH and O augmented liver lipid content and plasma cholesterol, triglycerides, leptin, glycemia, insulin levels, and HOMA index, and caused hypertension; most of these parameters were aggravated when IH and O were combined. IH diminished ventilatory response to hypoxia, and hypercapnia and O created a restrictive ventilatory pattern; a combination of treatments led to restrictive hypoventilation. Data demonstrate that IH and O cause comparable metabolic and cardiovascular pathologies via misregulation of the redox status and sympathetic hyperactivity.

Identification of major rye secalins as coeliac immunoreactive proteins.

Six distinct gamma- and omega-type secalins, together with two new low molecular mass glycoproteins, have been identified as the major coeliac immunoreactive proteins from a chloroform/methanol soluble extract from rye endosperm. These components were characterized by a combination of reverse-phase high-performance liquid chromatography, immunoblotting using a coeliac serum and microsequencing analysis. This allowed the identification of a group of secalins with different molecular masses according to their N-terminal amino-acid sequence: one omega-type secalin of 40 kDa (omega 1-40); three gamma-type secalins, one of 70 kDa (gamma-70) and two of 35 kDa (gamma-35); as well as two low molecular mass glycoproteins of 15 and 18 kDa, all exhibiting coeliac serum antigenicity. Moreover, four additional rye components, including two low molecular mass proteins, which did not react with coeliac sera, have also been identified. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the three main purified coeliac immunogenic secalins, gamma-70, gamma-35 and omega 1-40, indicated molecular masses of 71457, 32240 and 39117 Da, respectively. The omega 1-40 secalin displays a significant absorption in the visible region which could be related to its peculiar low capacity to bind both coeliac sera antibodies and Coomassie brilliant blue dye.

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.

Role of voltage‐dependent calcium channels in stimulus–secretion coupling in rabbit carotid body chemoreceptor cells

We have defined Ca2+ channel subtypes expressed in rabbit carotid body (CB) chemoreceptor cells and their participation in the stimulus‐evoked catecholamine (CA) release. Ca2+ currents (ICa) activated at –30 mV, peaked at +10 mV and were fully blocked by 200 μm Cd2+. L‐type channels (sensitive to 2 μm nisoldipine) activated at –30 mV and carried 21 ± 2% of total ICa. Non‐L‐type channels activated at potentials positive to –10 mV and carried: N channels (sensitive to 1 μmω‐conotoxin‐GVIA) 16 ± 1% of total ICa, P/Q channels (sensitive to 3 μmω‐conotoxin‐MVIIC after nisoldipine plus GVIA) 23 ± 3% of total ICa and R channels (resistant to all blockers combined) 40 ± 3% of total ICa. CA release induced by hypoxia, hypercapnic acidosis, dinitrophenol (DNP) and high K+o in the intact CB was inhibited by 79–98% by 200 μm Cd2+. Hypoxia, hypercapnic acidosis and DNP, depolarized chemoreceptor cells and eventually generated repetitive action potential discharge. Nisoldipine plus MVIIC nearly abolished the release of CAs induced by hypoxia and hypercapnic acidosis and reduced by 74% that induced by DNP. All these secretory responses were insensitive to GVIA. 30 and 100 mm K+o brought resting membrane potential (Em) of chemoreceptor cells (–48.1 ± 1.2 mV) to –22.5 and +7.2 mV, respectively. Thirty millimolar K+o‐evoked release was abolished by nisoldipine but that induced by 100 mm K+o was mediated by activation of L, N, and P/Q channels. Data show that tested stimuli depolarize rabbit CB chemoreceptor cells and elicit CA release through Ca2+ entry via voltage‐activated channels. Only L and P/Q channels are tightly coupled to the secretion of CA.