Zeitschriften

Journal of Molecular Medicine -     

Zeitschriften

Journal of Molecular Medicine -     

Zeitschriften

Journal of Molecular Medicine -     

THE HISTOPATHOLOGY OF PROGRESSIVE MUSCULAR DYSTROPHY AS REVEALED BY ULTRAVIOLET PHOTOMICROGRAPHY

1. In a series of rats subjected to hemorrhage and shock a high negative correlation was found between the portal and peripheral venous oxygen saturations and the arterial blood pressure on the one hand, and the blood amino nitrogen levels on the other, and a high positive correlation between the portal and the peripheral oxygen saturations and between each of these and the blood pressure. 2. In five cats subjected to hemorrhage and shock the rise in plasma amino nitrogen and the fall in peripheral and portal venous oxygen saturations were confirmed. Further it was shown that the hepatic vein oxygen saturation falls early in shock while the arterial oxygen saturation showed no alteration except terminally, when it may fall also. 3. Ligation of the hepatic artery in rats did not affect the liver''s ability to deaminate amino acids. Hemorrhage in a series of hepatic artery ligated rats did not produce any greater rise in the blood amino nitrogen than a similar hemorrhage in normal rats. The hepatic artery probably cannot compensate to any degree for the decrease in portal blood flow in shock. 4. An operation was devised whereby the viscera and portal circulation of the rat were eliminated and the liver maintained only on its arterial circulation. The ability of such a liver to metabolize amino acids was found to be less than either the normal or the hepatic artery ligated liver and to have very little reserve. 5. On complete occlusion of the circulation to the rat liver this organ was found to resist anoxia up to 45 minutes. With further anoxia irreversible damage to this organ''s ability to handle amino acids occurred. 6. It is concluded that the blood amino nitrogen rise during shock results from an increased breakdown of protein in the peripheral tissues, the products of which accumulate either because they do not circulate through the liver at a sufficiently rapid rate or because with continued anoxia intrinsic damage may occur to the hepatic parenchyma so that it cannot dispose of amino acids.     

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Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH

Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H⁺ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H⁺ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H⁺ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H⁺ load and are likely to be most vulnerable to future decreases in ocean pH.

Anthropogenic CO₂ emissions and the subsequent dissolution of CO₂ in seawater have resulted in substantial changes in ocean carbonate chemistry (1). The resultant decrease in seawater pH, termed ocean acidification, is predicted to be particularly detrimental for calcifying organisms (2). Mean global surface ocean pH is currently around 8.2 but is predicted to fall as low as 7.7 by 2100 (3) and is likely to continue to fall further in the following centuries. Present-day marine organisms can experience significant fluctuations in seawater pH, particularly in coastal and upwelling regions (4, 5). Ocean acidification is therefore predicted to have an important influence not only on mean surface ocean pH but also on the extremes of pH experienced by marine organisms (6, 7).Coccolithophores (Haptophyta) are a group of globally distributed unicellular phytoplankton that are characterized by their covering of intricately formed calcite scales (coccoliths). Coccolithophores account for a significant proportion of ocean productivity and are the main producers of biogenic calcite, making major contributions to global biogeochemical cycles, including the long-term export of both inorganic and organic carbon from the ocean photic zone to deep waters (8, 9). Unlike the vast majority of calcifying organisms, coccolithophore calcification occurs in an intracellular compartment, the Golgi-derived coccolith vesicle (CV), effectively isolating the calcification process from direct changes in seawater carbonate chemistry. Nevertheless, extensive laboratory observations indicate that ocean acidification may negatively impact coccolithophore calcification, albeit with significant variability of responses between species and strains (10–14). The negative impact on calcification rates occurs at calcite saturation states (Ω_calcite) >1, indicating that it results primarily from impaired cellular production rather than dissolution (10, 15). However, prediction of how natural coccolithophore populations may respond to future changes in ocean pH are hampered by lack of mechanistic understanding of pH impacts at the cellular level (10).As calcification occurs intracellularly using external HCO₃⁻ as the primary dissolved inorganic carbon (DIC) source (16–18), coccolith formation is not directly dependent on external CO₃²⁻ concentrations. However, the uptake of HCO₃⁻ as a substrate for calcification results in the equimolar production of CaCO₃ and H⁺ in the CV (18). In order to maintain saturation conditions for calcite formation, H⁺ produced by the calcification process must be rapidly removed from the CV, placing extraordinary demands for cellular pH regulation to prevent cellular acidosis (18).Lower calcification rates under ocean acidification conditions appear to be primarily due to decreased pH rather than other aspects of carbonate chemistry (10, 19, 20). Coccolithophores exhibit highly unusual membrane physiology, including the presence of voltage-gated H⁺ channels in the plasma membrane (21) and a high sensitivity of cytosolic pH (pH_cyt) to changes in external pH (pH_o) (21, 22). Voltage-gated H⁺ channels are associated with rapid H⁺ efflux in a number of specialized animal cell types (23) and contribute to effective pH regulation in coccolithophores (21). As H⁺ channel function is dependent on the electrochemical gradient of H⁺ across the plasma membrane, this mechanism could be impaired under lower seawater pH. However, it remains unknown whether H⁺ channels play a direct role in removal of calcification-derived H⁺ or contribute to the sensitivity of coccolithophores to ocean acidification.Coccolithophores exhibit significant diversity in their extent of calcification (SI Appendix, Fig. S1). The ratio of particulate inorganic carbon to particulate organic carbon (PIC/POC) of a coccolithophore culture is a measure of the relative rates of inorganic carbon fixation by calcification and organic carbon fixation by photosynthesis, respectively, and is commonly used as a simple metric to define the degree of calcification. The abundant bloom-forming species Emiliania huxleyi is moderately calcified (PIC/POC of around 1) and has been the focus of the vast majority of the studies into the effects of environmental change in coccolithophores (13). Coastal species belonging to the Pleurochrysidaceae and Hymenomonadaceae are lightly calcified, commonly exhibiting a PIC/POC of less than 0.5 (24–27). Species such as Coccolithus braarudii, Calcidiscus leptoporus, and Helicosphaera carteri exhibit much higher PIC/POC ratios and contribute the majority of carbonate export to the deep ocean in many areas (28–30). The physiological response of heavily calcified coccolithophores to ocean acidification is therefore of considerable biogeochemical significance. Growth and calcification rates in C. leptoporus and C. braarudii are sensitive to pH values predicted to prevail on a future decadal timescale (10, 15, 31, 32). However, a mechanistic understanding of the different sensitivity of coccolithophore species to changing ocean carbonate chemistry is lacking.The net H⁺ load in a cell is determined by the combination of metabolic processes that consume or produce H⁺. H⁺ fluxes in coccolithophores will be primarily determined by the balance of H⁺ consumed by photosynthesis and H⁺ generated by calcification, with uptake of different carbon sources a particularly important consideration (Fig. 1A). CO₂ uptake for photosynthesis results in no net production or consumption of H⁺, whereas uptake of HCO₃⁻ requires the equimolar consumption of H⁺ in order to generate CO₂. Growth at elevated CO₂ causes a switch from HCO₃⁻ uptake to predominately CO₂ uptake in E. huxleyi (33, 34). The associated net decrease in H⁺ consumption will therefore increase the H⁺ load in coccolithophores grown at elevated CO₂, which may exacerbate the potential for cytosolic acidosis caused by lower seawater pH.Open in a separate windowFig. 1.Physiology and H⁺ fluxes of C. braarudii cells grown at different seawater pH. (A) Schematic indicating H⁺ fluxes associated with photosynthesis and calcification in a coccolithophore cell. While many metabolic processes may contribute to the cellular H⁺ budget, these two processes are likely to be the major contributors. In a cell taking up HCO₃⁻, the overall H⁺ budget is determined by the relative rates of H⁺ consumed during photosynthesis and H⁺ generated during calcification. In a cell taking up CO₂, the H⁺ budget is determined primarily by calcification, as 2 H⁺ are produced for each molecule of CaCO₃ produced and H⁺ are no longer consumed during photosynthesis. In both scenarios, excess H⁺ may be removed from the cell by H⁺ transporters in the plasma membrane, such as voltage-gated H⁺ channels (H_v). Coccolithophores take up both HCO₃⁻ and CO₂ across the plasma membrane, with increasing proportions of DIC taken up as CO₂ as seawater CO₂ increases (34). (B) Growth rate of C. braarudii cells acclimated to different seawater pH. n = 3 replicates per treatment; line represents polynomial fit to mean. (C) Cellular production of POC through photosynthesis and PIC through calcification. The optima for both processes are close to the control conditions (pH 8.15). (D) As a consequence of the unequal changes in cellular POC and PIC production across the applied pH values, cellular PIC/POC ratios are minimal at pH 7.55 (∼1.0) and maximal at pH 8.45 (∼1.8). (E) Calculated net H⁺ budgets under the different pH regimes, based on rates of photosynthesis and calcification shown in C (see Materials and Methods). The concentration of CO₂ in seawater is also shown (dashed line). Estimates are shown for cells using taking up only HCO₃⁻ or only CO₂. As C. braarudii cells will likely take up a mixture of both DIC species, with a shift toward greater CO₂ usage at elevated CO₂, the shaded area represents the potential range of H⁺ production. Regardless of DIC species used C. braarudii produces excess H⁺ at all applied pH values, but H⁺ production is much lower at pH 7.55 due to the decrease in calcification.In this study we set out to better understand the cellular mechanisms underlying the sensitivity of coccolithophore calcification to lower pH. We subjected the heavily calcified species C. braarudii, which is commonly found in temperate upwelling regions (35, 36), to conditions that reflect the range of pH values it may experience in current and future oceans. We show that acclimation to low pH leads to loss of H⁺ channel function and disruption of cellular pH regulation in C. braarudii. These effects are coincident with very specific defects in coccolith morphology that can be reproduced by direct inhibition of H⁺ channels. We conclude that H⁺ efflux through H⁺ channels is essential for maintaining both calcification rate and coccolith morphology. By providing a mechanistic insight into pH regulation during the calcification process, our results indicate that disruption of coccolithophore calcification in a future acidified ocean is likely to be most severe in heavily calcified species.