Hypercapnic Conditions Research Articles

Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats.

Control of breathing and blood pressure by parafacial neurons in conscious rats

The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n = 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABA(A) agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min(-1) kg(-1) with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats.

Regional pulmonary blood flow in the lung of the chicken

It is known that alterations in respiratory gases in birds can cause a nonhomogenous redistribution of pulmonary blood flow between the 2 separate gas-exchanging regions of the avian lung, the paleopulmo (PALEO) and neopulmo (NEO); however, the effect of alterations in respired gas content on the distribution of pulmonary blood flow in birds, such as the chicken, that possess a highly developed NEO is not known. This study used a colorimetric microsphere method to determine the effects of hypoxia and hypercapnia on the relative distribution of pulmonary blood flow in anesthetized chickens (Gallus domesticus) during control (normoxic) and experimental (hypoxic or hypercapnic) conditions, where the relative regional distribution of blood flow in the lung is expressed as the ratio NEO/PALEO. Administration of a hypoxic gas mixture (16.0% O(2)) produced a 13.4% increase in NEO/PALEO, and, administration of a hypercapnic gas mixture (5.0% CO(2)) resulted in a 27.8% increase in NEO/PALEO. Our results are consistent with a mechanism in which the regional redistribution of pulmonary blood flow is mediated by local intrapulmonary factors.

Linear and Nonlinear Modeling of Cerebral Flow Autoregulation Using Principal Dynamic Modes

Cerebral Flow Autoregulation (CFA) is the dynamic process by which cerebral blood flow is maintained within physiologically acceptable bounds during fluctuations of cerebral perfusion pressure. The distinction is made with “static” flow autoregulation under steady-state conditions of perfusion pressure, described by the celebrated “autoregulatory curve” with a homeostatic plateau. This paper studies the dynamic CFA during changes in perfusion pressure, which attains critical clinical importance in patients with stroke, traumatic brain injury and neurodegenerative disease with a cerebrovascular component. Mathematical and computational models have been used to advance our quantitative understanding of dynamic CFA and to elucidate the underlying physiological mechanisms by analyzing the relation between beat-to-beat data of mean arterial blood pressure (viewed as input) and mean cerebral blood flow velocity(viewed as output) of a putative CFA system. Although previous studies have shown that the dynamic CFA process is nonlinear, most modeling studies to date have been linear. It has also been shown that blood CO2 tension affects the CFA process. This paper presents a nonlinear modeling methodology that includes the dynamic effects of CO2 tension (or its surrogate, end-tidal CO2) as a second input and quantifies CFA from short data-records of healthy human subjects by use of the modeling concept of Principal Dynamic Modes (PDMs). The PDMs improve the robustness of the obtained nonlinear models and facilitate their physiological interpretation. The results demonstrate the importance of including the CO2 input in the dynamic CFA study and the utility of nonlinear models under hypercapnic or hypocapnic conditions.

Linear and Nonlinear Modeling of Cerebral Flow Autoregulation Using Principal Dynamic Modes

Cerebral Flow Autoregulation (CFA) is the dynamic process by which cerebral blood flow is maintained within physiologically acceptable bounds during fluctuations of cerebral perfusion pressure. The distinction is made with “static” flow autoregulation under steady-state conditions of perfusion pressure, described by the celebrated “autoregulatory curve” with a homeostatic plateau. This paper studies the dynamic CFA during changes in perfusion pressure, which attains critical clinical importance in patients with stroke, traumatic brain injury and neurodegenerative disease with a cerebrovascular component. Mathematical and computational models have been used to advance our quantitative understanding of dynamic CFA and to elucidate the underlying physiological mechanisms by analyzing the relation between beat-to-beat data of mean arterial blood pressure (viewed as input) and mean cerebral blood flow velocity(viewed as output) of a putative CFA system. Although previous studies have shown that the dynamic CFA process is nonlinear, most modeling studies to date have been linear. It has also been shown that blood CO2 tension affects the CFA process. This paper presents a nonlinear modeling methodology that includes the dynamic effects of CO2 tension (or its surrogate, end-tidal CO2) as a second input and quantifies CFA from short data-records of healthy human subjects by use of the modeling concept of Principal Dynamic Modes (PDMs). The PDMs improve the robustness of the obtained nonlinear models and facilitate their physiological interpretation. The results demonstrate the importance of including the CO2 input in the dynamic CFA study and the utility of nonlinear models under hypercapnic or hypocapnic conditions.

Participation of Locus coeruleus (LC) noradrenergic neurons on breathing in female rats

LC has been reported to regulate CO2‐drive to breathing in males. Since, sex hormones have a significant impact on ventilation; the present study examined the role of LC noradrenergic neurons in estradiol‐treated ovariectomized (OVX+E2) rats in response to hypercapnia. To selectively destroy LC noradrenergic neurons we administered 6‐hydroxydopamine (6‐OHDA, 8 μg in 1 μL of vehicle) bilaterally or vehicle (ascorbic acid) into LC of OVX+E2 rats. Bilateral inhibition of LC neurons with 6‐OHDA reduced resting respiratory frequency (57±3.53, vs. vehicle: 93.5±3.75 breaths/min) and the increase in ventilation produced by hypercapnia (7% CO2) (1052.5±11.5, vs. vehicle: 1625±89.7 mL/min/kg). Our results also suggest that noradrenergic LC neurons play an important modulation in the control of ventilation during resting or hypercapnic conditions in female rats. Financial support: FAPESP, CNPq and INCT of Comparative Physiology

GABAergic neurons in the medullary raphé possess network independent chemosensitivity in situ

The identity and location of central pH/CO2 sensitive chemoreceptors are not fully understood. Serotonin (5‐HT) and γ‐aminobutyric acid (GABA) synthesizing neurons in the medullary raphé have demonstrated intrinsic chemosensitivity in vitro. This evidence forms the basis for our “push‐pull” model of raphé contributions to chemosensitivity. We have previously shown that CO2‐stimulated 5‐HT neurons occur in the medullary raphé in situ. Here, we test the hypothesis that the medullary raphé contains GABA synthesizing CO2‐inhibited neurons that retain their chemosensitivity after pharmacological blockade of major fast synaptic inputs. To assess chemosensitivity, we record extracellular single neuron discharge during normocapnic and hypercapnic conditions within the medullary raphé of the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. Network dependence of chemosensitivity is assessed by application of antagonists for AMPA, NMDA, glycine, and GABAa receptors that disrupt fast‐synaptic network properties. Juxtacellular labeling and immunohistochemistry establish neurotransmitter phenotypes of recorded neurons. Results support independence of CO2‐inhibited GABA neuron chemosensitivity from fast synaptic inputs. Supported by NIH 54NS041069‐06A1.

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3β or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Farbduplexsonographie der retrobulbären Gefäße und Hyperkapnie bei Normaldruckglaukom

The aim of the study was to investigate retrobulbar flow velocities during hypercapnia in patients with normal tension glaucoma (NTG) without systemic vasospasm and jn controls. A total of 16 NTG patients (mean age 58 ± 14 years) and 16 control subjects (mean age 50 ± 13 years, p = 0.10) were enrolled in this study. Flow velocities, peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistive indices (RI) of the ophthalmic (OA) and central retinal arteries (CRA) were assessed using color Doppler imaging. Blood flow velocities were measured under normocapnic and hypercapnic conditions (increasing the end-tidal pCO(2) by 15%). Blood pressure, ventilation rate and oxygen saturation were monitored simultaneously. During hypercapnia, velocity responses of the PSV (p = 0.044) and EDV (p = 0.037) of the CRA were significantly different in NTG patients and healthy controls, showing a greater increase of flow velocities in control subjects. Flow velocities of the OA increased significantly in both groups (PSV p = 0.039, EDV p = 0.003) during hypercapnia. Blood pressure, oxygen saturation and intraocular pressure changed similarly in both study groups with carbon dioxide provocation. Velocity response to hypercapnia was reduced in the CRA of NTG patients compared to controls. This may indicate a faulty vasodilatory response in NTG patients without vasospastic disease.

Abstract 2762: Asymmetry of The Blood Oxygen Level-Dependent fMRI Response During Hypercapnia Is Reliable to Evaluate Cerebrovascular Reactivity in Patients with Severe Unilateral Carotid Stenosis

Background and Purpose: Previous transcranial Doppler studies have suggested that, in patients with severe carotid stenosis, the lack of cerebrovascular reactivity (CVR) is an independent predictor of ipsilateral stroke. The BOLD (Blood Oxygenation Level-Dependent) fMRI contrast can be used to assess the CVR during normal condition and during hemodynamic stress, like hypercapnia. The purpose of this study is to evaluate the BOLD signal parameters on the Middle Cerebral Artery (MCA) territory, induced by auditory stimulus, during different levels of hypercapnia in patients with severe unilateral carotid stenosis, comparing the ipsilateral hemisphere (IH) to the contralateral hemisphere (CH). Methods: The images were obtained from 16 patients with severe unilateral carotid stenosis. Patients were submitted to an auditory stimulus (3s) in three different conditions: normocapnia and at EtCO2 increase of 5 and 10mmHg. The images were acquired with a 3T Philips MR, preprocessed and analyzed using an autoregressive method. Results: The BOLD signal from the IH was different from the CH during the basal condition and at 5mmHg (p<0.0001), but not at the 10mmHg ETCO2 increase. For each BOLD parameter, the major differences between the hemispheres were seen on the onset time (p<0.0001) and amplitude of BOLD signal (p<0.0001). The width difference was significant between the basal and 10mmhHg increase (p<0.01). In these three parameters, the BOLD signal of the IH presented no significant variations with the CO2 increment. However, the BOLD signal for CH showed an increase on the time-to-onset and width and amplitude decrease. The time to peak parameters of the BOLD signal showed no differences between the hemispheres and at hypercapnic conditions. Conclusion: In our results the IH did not respond to the hypercapnic stress as the CH. Therefore, among patients with severe carotid stenosis, BOLD can reliably identify some that have an exhausted CVR, which cannot respond to a vasodilatory stress like hypercapnia. Future studies using this technique may help to select patients for recanalization procedures.

Resource allocation and extracellular acid–base status in the sea urchin Strongylocentrotus droebachiensis in response to CO2 induced seawater acidification

Anthropogenic CO2 emission will lead to an increase in seawater pCO2 of up to 80–100Pa (800–1000μatm) within this century and to an acidification of the oceans. Green sea urchins (Strongylocentrotus droebachiensis) occurring in Kattegat experience seasonal hypercapnic and hypoxic conditions already today. Thus, anthropogenic CO2 emissions will add up to existing values and will lead to even higher pCO2 values >200Pa (>2000μatm). To estimate the green sea urchins’ potential to acclimate to acidified seawater, we calculated an energy budget and determined the extracellular acid base status of adult S. droebachiensis exposed to moderately (102–145Pa, 1007–1431μatm) and highly (284–385Pa, 2800–3800μatm) elevated seawater pCO2 for 10 and 45 days.A 45-day exposure to elevated pCO2 resulted in a shift in energy budgets, leading to reduced somatic and reproductive growth. Metabolic rates were not significantly affected, but ammonium excretion increased in response to elevated pCO2. This led to decreased O:N ratios. These findings suggest that protein metabolism is possibly enhanced under elevated pCO2 in order to support ion homeostasis by increasing net acid extrusion. The perivisceral coelomic fluid acid–base status revealed that S. droebachiensis is able to fully (intermediate pCO2) or partially (high pCO2) compensate extracellular pH (pHe) changes by accumulation of bicarbonate (maximum increases 2.5mM), albeit at a slower rate than typically observed in other taxa (10-day duration for full pHe compensation). At intermediate pCO2, sea urchins were able to maintain fully compensated pHe for 45 days. Sea urchins from the higher pCO2 treatment could be divided into two groups following medium-term acclimation: one group of experimental animals (29%) contained remnants of food in their digestive system and maintained partially compensated pHe (+2.3mM HCO3−), while the other group (71%) exhibited an empty digestive system and a severe metabolic acidosis (−0.5 pH units, −2.4mM HCO3−). There was no difference in mortality between the three pCO2 treatments.The results of this study suggest that S. droebachiensis occurring in the Kattegat might be pre-adapted to hypercapnia due to natural variability in pCO2 in its habitat. We show for the first time that some echinoderm species can actively compensate extracellular pH. Seawater pCO2 values of >200Pa, which will occur in the Kattegat within this century during seasonal hypoxic events, can possibly only be endured for a short time period of a few weeks. Increases in anthropogenic CO2 emissions and leakages from potential sub-seabed CO2 storage (CCS) sites thus impose a threat to the ecologically and economically important species S. droebachiensis.

Modeling of Pressure-Flow Relationship in the Eye

The blood supply to the human eye is complex. The posterior pole is nourished by 2 different vascular beds. The retinal circulation supplies the inner retina including the retinal ganglion cells. For this vascular bed the autoregulatory capacity is well documented and has been proven many years ago. The choroidal circulation supplies the outer retina including the photoreceptors. The autoregulatory capacity of the choroid has been debated for many years. Data from rabbit experiments have shown that the choroid also shows autoregulatory properties. In this manuscript we present modeling of the pressure-flow relationship and explain several approaches for studying the autoregulatory behaviour of the ocular vasculature, including static autoregulation, dynamic autoregulation and correlation of group data. A study investigating static autoregulation under conditions of normocapnia and hypercapnia as well as different experiments investigating the mechanisms underlying the response of the ocular vascular beds to changes in perfusion pressure are presented.

Noninvasive Measurements of Cerebral Blood Flow, Oxygen Extraction Fraction, and Oxygen Metabolic Index in Human with Inhalation of Air and Carbogen using Magnetic Resonance Imaging

Noninvasive magnetic resonance (MR) methods have been explored to provide quantitative measurements of cerebral blood flow (CBF), oxygen extraction fraction (OEF), and oxygen metabolic index (OMI = CBF × OEF). In this study, we sought to evaluate whether MR measured OEF, CBF, and OMI can consistently detect the expected physiological changes in humans under normal and hyperoxic hypercapnic conditions. Nine healthy human subjects were scanned while breathing through a mask, alternating inhaled gas in a sequential order as room air, carbogen (3% CO2 mixed with 97% O2), room air, carbogen, and room air. OEF, CBF, and OMI were obtained from the whole brain, gray matter (GM), and white matter (WM) at each gas inhalation state. Similar to previous positron emission tomography findings, our study consistently demonstrated a 10-12% decrease in OEF with a 10% increase of CBF and a stable OMI during carbogen inhalation. Moreover, GM/WM ratio in CBF and OMI remained constant during air and carbogen breathing. In addition, OEF, CBF, and OMI were highly reproducible if the same inhaled gas was used. In summary, our results demonstrate that noninvasive MR measurements can provide reproducible measurements of OEF, CBF, and OMI in normal subjects under normal and altered physiological conditions.

Early and Late Stimulus-Evoked Cortical Hemodynamic Responses Provide Insight into the Neurogenic Nature of Neurovascular Coupling

Understanding neurovascular coupling is a prerequisite for the interpretation of results obtained from modern neuroimaging techniques. This study investigated the hemodynamic and neural responses in rat somatosensory cortex elicited by 16 seconds electrical whisker stimuli. Hemodynamics were measured by optical imaging spectroscopy and neural activity by multichannel electrophysiology. Previous studies have suggested that the whisker-evoked hemodynamic response contains two mechanisms, a transient 'backwards' dilation of the middle cerebral artery, followed by an increase in blood volume localized to the site of neural activity. To distinguish between the mechanisms responsible for these aspects of the response, we presented whisker stimuli during normocapnia ('control'), and during a high level of hypercapnia. Hypercapnia was used to 'predilate' arteries and thus possibly 'inhibit' aspects of the response related to the 'early' mechanism. Indeed, hemodynamic data suggested that the transient stimulus-evoked response was absent under hypercapnia. However, evoked neural responses were also altered during hypercapnia and convolution of the neural responses from both the normocapnic and hypercapnic conditions with a canonical impulse response function, suggested that neurovascular coupling was similar in both conditions. Although data did not clearly dissociate early and late vascular responses, they suggest that the neurovascular coupling relationship is neurogenic in origin.

Acidosis Dilates Brain Parenchymal Arterioles by Conversion of Calcium Waves to Sparks to Activate BK Channels

Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BK(Ca)) channels by local Ca(2+) signals (Ca(2+) sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known. The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BK(Ca) channels in this response. Internal diameter and vascular smooth muscle cell Ca(2+) signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), vascular smooth muscle cells exhibited primarily RyR-dependent Ca(2+) waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca(2+) wave activity, and dramatically increased Ca(2+) spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BK(Ca) channel or RyR blockers, in a nonadditive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BK(Ca) channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of K(ATP) channels or nitric oxide synthase. These results support the novel concept that acidification, by converting Ca(2+) waves to sparks, leads to the activation of BK(Ca) channels to induce dilation of cerebral PAs.

The potential of spatially resolved spectroscopy for monitoring angiogenesis in the chorioallantoic membrane

Over the last decade, the poultry sector has sought to develop ways to monitor chicken embryonic development as to optimize the incubation conditions. One of the parameters of development which may change under different incubation conditions is the angiogenesis in the chorioallantoic membrane (CAM). To be able to quantify these changes in the angiogenesis and detect long-term effects on health, a non-destructive technique is necessary. In this article, the first steps toward such a non-destructive technique are successfully taken. A spatially resolved spectroscopy set-up is built and tested for its potential to measure changes in angiogenesis with incubation time, and differences between a normal and hypercapnic incubation. In this first study, reflectance measurements are performed directly on the CAM as the eggshell considerably complicates the analysis. This issue should be addressed in future research to come to a really non-destructive technique. An experiment was conducted in which one group was incubated under normal conditions, and another under early prenatal hypercapnic conditions (i.e., increased CO(2) concentrations). The angiogenesis in the CAM was measured at embryonic day (ED) 10, 13, and 16. The measurements showed a clear blood spectrum with an increasing amount of blood in time, and significant differences in the reflectance as function of the source-detector distances. However, no significant differences between the hypercapnia and the control group could be detected.

Carbon dioxide enhances substance P-induced epithelium-dependent bronchial smooth muscle relaxation in Sprague–Dawley rats

Hypocapnia and hypercapnia constrict and relax airway smooth muscle, respectively, through pH- and calcium (Ca(2+))-mediated mechanisms. In this study we explore a potential role for the airway epithelium in these responses to carbon dioxide (CO(2)). Contractile and relaxant responses of isolated rat bronchial rings were measured under hypocapnic, eucapnic, and hypercapnic conditions. Substance P was added to methacholine precontracted bronchial rings with and without epithelium. The role of Ca(2+) was assessed using Ca(2+)-free solutions and a Ca(2+) channel blocker, nifedipine. The effects of pH were assessed in solutions with HEPES buffer. Hypocapnic challenge increased the organ bath's pH and increased bronchial smooth muscle resting tension. This effect was abolished with HEPES buffer and partially inhibited by nifedipine. Hypocapnic conditions suppressed substance P-induced epithelium-dependent relaxation, whereas hypercapnia augmented the response. The epithelial hypocapnic effect was pH dependent, whereas the hypercapnic effect was pH independent. CO(2) had no effect on the epithelial independent smooth muscle agonists methacholine and isoproterenol. In conclusion our data indicate that, in addition to the effects of pH and Ca(2+), CO(2) affects airway smooth muscle by a pH-independent, epithelium-mediated mechanism. These findings could potentially lead to new treatments for asthma involving CO(2)-sensing receptors in the airways.

Effect of long-standing hypoxic and/or hypercapnic conditions on the sensory epithelia and the neurons in the inner ear of humans

Effect of long-standing hypoxic and/or hypercapnic conditions on the sensory epithelia and the neurons in the inner ear of humans

Effects of ocean acidification on trace element accumulation in the early-life stages of squid Loligo vulgaris

The anthropogenic release of carbon dioxide (CO 2) into the atmosphere leads to an increase in the CO 2 partial pressure ( pCO 2) in the ocean, which may reach 950 μatm by the end of the 21st century. The resulting hypercapnia (high pCO 2) and decreasing pH (“ocean acidification”) are expected to have appreciable effects on water-breathing organisms, especially on their early-life stages. For organisms like squid that lay their eggs in coastal areas where the embryo and then paralarva are also exposed to metal contamination, there is a need for information on how ocean acidification may influence trace element bioaccumulation during their development. In this study, we investigated the effects of enhanced levels of pCO 2 (380, 850 and 1500 μatm corresponding to pH T of 8.1, 7.85 and 7.60) on the accumulation of dissolved 110mAg, 109Cd, 57Co, 203Hg, 54Mn and 65Zn radiotracers in the whole egg strand and in the different compartments of the egg of Loligo vulgaris during the embryonic development and also in hatchlings during their first days of paralarval life. Retention properties of the eggshell for 110mAg, 203Hg and 65Zn were affected by the pCO 2 treatments. In the embryo, increasing seawater pCO 2 enhanced the uptake of both 110mAg and 65Zn while 203Hg showed a minimum concentration factor (CF) at the intermediate pCO 2. 65Zn incorporation in statoliths also increased with increasing pCO 2. Conversely, uptake of 109Cd and 54Mn in the embryo decreased as a function of increasing pCO 2. Only the accumulation of 57Co in embryos was not affected by increasing pCO 2. In paralarvae, the CF of 110mAg increased with increasing pCO 2, whereas the 57Co CF was reduced at the highest pCO 2 and 203Hg showed a maximal uptake rate at the intermediate pCO 2. 54Mn and 65Zn accumulation in paralarvae were not significantly modified by hypercapnic conditions. Our results suggest a combined effect of pH on the adsorption and protective properties of the eggshell and of hypercapnia on the metabolism of embryo and paralarvae, both causing changes to the accumulation of metals in the tissues of L. vulgaris.

Proteomic response to elevated PCO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress

Estuaries are characterized by extreme fluctuations in CO(2) levels due to bouts of CO(2) production by the resident biota that exceed its capacity of CO(2) consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO(2) (P(CO(2)); i.e. environmental hypercapnia) decrease the pH of estuarine waters and, ultimately, extracellular and intracellular pH levels of estuarine organisms such as mollusks that have limited capacity for pH regulation. We analyzed proteomic changes associated with exposure to elevated P(CO(2)) in the mantle tissue of eastern oysters (Crassostrea virginica) after 2 weeks of exposure to control (∼39 Pa P(CO(2))) and hypercapnic (∼357 Pa P(CO(2))) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high P(CO(2)) resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high P(CO(2)) compared with control conditions. Of the 54 differentially expressed proteins, we were able to identify 17. Among the identified proteins, two main functional categories were upregulated in response to hypercapnia: those associated with the cytoskeleton (e.g. several actin isoforms) and those associated with oxidative stress (e.g. superoxide dismutase and several peroxiredoxins as well as the thioredoxin-related nucleoredoxin). This indicates that exposure to high P(CO(2)) (∼357 Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO(2) levels may cause oxidative stress by increasing the production of reactive oxygen species (ROS) either indirectly by lowering organismal pH, which may enhance the Fenton reaction, and/or directly by CO(2) interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO(2) than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.