Acute Oxygen-Sensing via Mitochondria-Generated Temperature Transients in Rat Carotid Body Type I Cells

The Carotid Bodies (CB) are peripheral chemoreceptors that detect changes in arterial oxygenation and, via afferent inputs to the brainstem, correct the pattern of breathing to restore blood gas homeostasis. Herein, preliminary evidence is presented supporting a novel oxygen-sensing hypothesis which suggests CB Type I cell “hypoxic signaling” may in part be mediated by mitochondria-generated thermal transients in TASK-channel-containing microdomains. Distances were measured between antibody-labeled mitochondria and TASK-potassium channels in primary rat CB Type I cells. Sub-micron distance measurements (TASK-1: 0.33 ± 0.04 µm, n = 47 vs TASK-3: 0.32 ± 0.03 µm, n = 54) provided evidence for CB Type I cell oxygen-sensing microdomains. A temperature-sensitive dye (ERthermAC) indicated that inhibition of mitochondrial activity in isolated cells caused a rapid and reversible inhibition of mitochondrial thermogenesis and thus temperature in these microdomains. Whole-cell perforated-patch current-clamp electrophysiological recordings demonstrated sensitivity of resting membrane potential (Vm) to temperature: lowering bath temperature from 37°C to 24°C induced consistent and reversible depolarizations (Vm at 37°C: -48.4 ± 4.11 mV vs 24°C: -31.0 ± 5.69 mV; n = 5; p \u3c 0.01). These data suggest that hypoxic inhibition of mitochondrial thermogenesis may play an important role in oxygen chemotransduction in the CB. A reduction in temperature within cellular microdomains will inhibit plasma membrane ion channels, influence the balance of cellular phosphorylation–dephosphorylation, and may extend the half-life of reactive oxygen species. The characterization of a thermosensory chemotransduction mechanism, that may also be used by other oxygen-sensitive cell types and may impact multiple other chemotransduction mechanisms is critical if we are to fully understand how the CBs, and potentially other oxygen-sensitive cells, respond to hypoxia

Ethanol and Opioids Do Not Act Synergistically To Depress Excitation in Carotid Body Type I Cells

The combination of opioids and ethanol can synergistically depress breathing and the acute ventilatory response to hypoxia. Multiple studies have shown that the underlying mechanisms for this may involve calcium channel inhibition in central neurons. But we have previously identified opioid receptors in the carotid bodies and shown that their activation inhibits calcium influx into the chemosensitive cells. Given that the carotid bodies contribute to the drive to breathe and underpin the acute hypoxic ventilatory response, we hypothesized that ethanol and opioids may act synergistically in these peripheral sensory organs to further inhibit calcium influx and therefore inhibit ventilation. Methods Carotid bodies were removed from 56 Sprague–Dawley rats (1021 days old) and then enzymatically dissociated to allow calcium imaging of isolated chemosensitive type I cells. Cells were stimulated with high K+ in the presence and absence of the µ-opioid agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) (10 µM), a maximal sublethal concentration of ethanol (3 g L-1, 65.1 mM) or a combination of both. Results DAMGO alone significantly inhibited Ca2+ influx but this effect was not potentiated by the high concentration of ethanol. Conclusion These results indicate for the first time that while opioids may suppress breathing via an action at the level of the carotid bodies, ethanol is unlikely to potentiate inhibition via this pathway. Thus, the synergistic effects of ethanol and opioids on ventilatory parameters are likely mediated by central rather than peripheral actions

Sequential and Batch Processing Methods of the EBP Learning Algorithm

Placental abnormalities can cause Pregnancy-Associated Disorders, including preeclampsia, intrauterine growth restriction, and placental insufficiency, resulting in complications for both the mother and fetus. Trophoblast cells within the labyrinthine layer of the placenta facilitate the exchange of nutrients, gases, and waste between mother and fetus; therefore, the development of this cell layer is critical for fetal development. As trophoblast cells differentiate, it is assumed their metabolism changes with their energy requirements. We hypothesize that proper regulation of trophoblast metabolism is a key component of normal placental development; therefore, we examined the role of AMP-activated kinase (AMPK, PRKAA1/2), a sensor of cellular energy status. Our previous studies have shown that AMPK knockdown alters both trophoblast differentiation and nutrient transport. In this study, AMPKα1/2 shRNA was used to investigate the metabolic effects of AMPK knockdown on SM10 placental labyrinthine progenitor cells before and after differentiation. Extracellular flux analysis confirmed that AMPK knockdown was sufficient to reduce trophoblast glycolysis, mitochondrial respiration, and ATP coupling efficiency. A reduction in AMPK in differentiated trophoblasts also resulted in increased mitochondrial volume. These data indicate that a reduction in AMPK disrupts cellular metabolism in both progenitors and differentiated placental trophoblasts. This disruption correlates to abortive trophoblast differentiation that may contribute to the development of Pregnancy-Associated Disorders

Discovery and Use of a Natural Mutation that Results in Severe Combined Immuno Deficiency in Pigs

Piglets from the low residual feed intake (RFI) line at ISU were found to be affected with a lethal autosomal recessive mutation that causes Severe Combined Immunodeficiency (SCID). Bone marrow allotransplantation rescued the immune deficiency in four of nine attempted transfers; the other five exhibited signs of severe graft versus host disease and were euthanized. A genome wide association study identified a 5.6 Mb region that contained the causative mutation. Affected haplotypes were traced back to the founders of the RFI population, who were sourced from the purebred Yorkshire population. The SCID pigs will be useful as a biomedical model, as pigs are anatomically and genetically more similar to humans than SCID mice, which are now widely used. Development of a genetic test for the causative mutation will be valuable to the swine industry, allowing breeders to identify carriers

Adrenaline release evokes hyperpnoea and an increase in ventilatory CO2 sensitivity during hypoglycaemia: a role for the carotid body

KEY POINTS: Hypoglycaemia is counteracted by release of hormones and an increase in ventilation and CO(2) sensitivity to restore blood glucose levels and prevent a fall in blood pH. The full counter‐regulatory response and an appropriate increase in ventilation is dependent on carotid body stimulation. We show that the hypoglycaemia‐induced increase in ventilation and CO(2) sensitivity is abolished by preventing adrenaline release or blocking its receptors. Physiological levels of adrenaline mimicked the effect of hypoglycaemia on ventilation and CO(2) sensitivity. These results suggest that adrenaline, rather than low glucose, is an adequate stimulus for the carotid body‐mediated changes in ventilation and CO(2) sensitivity during hypoglycaemia to prevent a serious acidosis in poorly controlled diabetes. ABSTRACT: Hypoglycaemia in vivo induces a counter‐regulatory response that involves the release of hormones to restore blood glucose levels. Concomitantly, hypoglycaemia evokes a carotid body‐mediated hyperpnoea that maintains arterial CO(2) levels and prevents respiratory acidosis in the face of increased metabolism. It is unclear whether the carotid body is directly stimulated by low glucose or by a counter‐regulatory hormone such as adrenaline. Minute ventilation was recorded during infusion of insulin‐induced hypoglycaemia (8–17 mIU kg(−1) min(−1)) in Alfaxan‐anaesthetised male Wistar rats. Hypoglycaemia significantly augmented minute ventilation (123 ± 4 to 143 ± 7 ml min(−1)) and CO(2) sensitivity (3.3 ± 0.3 to 4.4 ± 0.4 ml min(−1) mmHg(−1)). These effects were abolished by either β‐adrenoreceptor blockade with propranolol or adrenalectomy. In this hypermetabolic, hypoglycaemic state, propranolol stimulated a rise in [Formula: see text] , suggestive of a ventilation–metabolism mismatch. Infusion of adrenaline (1 μg kg(−1) min(−1)) increased minute ventilation (145 ± 4 to 173 ± 5 ml min(−1)) without altering [Formula: see text] or pH and enhanced ventilatory CO(2) sensitivity (3.4 ± 0.4 to 5.1 ± 0.8 ml min(−1) mmHg(−1)). These effects were attenuated by either resection of the carotid sinus nerve or propranolol. Physiological concentrations of adrenaline increased the CO(2) sensitivity of freshly dissociated carotid body type I cells in vitro. These findings suggest that adrenaline release can account for the ventilatory hyperpnoea observed during hypoglycaemia by an augmented carotid body and whole body ventilatory CO(2) sensitivity

The Carnegie Supernova Project: First Near-Infrared Hubble Diagram to z~0.7

The Carnegie Supernova Project (CSP) is designed to measure the luminosity distance for Type Ia supernovae (SNe Ia) as a function of redshift, and to set observational constraints on the dark energy contribution to the total energy content of the Universe. The CSP differs from other projects to date in its goal of providing an I-band {rest-frame} Hubble diagram. Here we present the first results from near-infrared (NIR) observations obtained using the Magellan Baade telescope for SNe Ia with 0.1 < z < 0.7. We combine these results with those from the low-redshift CSP at z <0.1 (Folatelli et al. 2009). We present light curves and an I-band Hubble diagram for this first sample of 35 SNe Ia and we compare these data to 21 new SNe Ia at low redshift. These data support the conclusion that the expansion of the Universe is accelerating. When combined with independent results from baryon acoustic oscillations (Eisenstein et al. 2005), these data yield Omega_m = 0.27 +/- 0.0 (statistical), and Omega_DE = 0.76 +/- 0.13 (statistical) +/- 0.09 (systematic), for the matter and dark energy densities, respectively. If we parameterize the data in terms of an equation of state, w, assume a flat geometry, and combine with baryon acoustic oscillations, we find that w = -1.05 +/- 0.13 (statistical) +/- 0.09 (systematic). The largest source of systematic uncertainty on w arises from uncertainties in the photometric calibration, signaling the importance of securing more accurate photometric calibrations for future supernova cosmology programs. Finally, we conclude that either the dust affecting the luminosities of SNe Ia has a different extinction law (R_V = 1.8) than that in the Milky Way (where R_V = 3.1), or that there is an additional intrinsic color term with luminosity for SNe Ia independent of the decline rate.Comment: 44 pages, 23 figures, 9 tables; Accepted for publication in the Astrophysical Journa

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.Early Career Fellowship from the National Health and Medical Research Council GNT1012521(A.R.W.); Wellcome Trust Programme Grant (J.R.K., C.M.D.) 094425/Z/10/Z; Samsung GRO Grant (M.R.W.)This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pone.013003