Chemolithoheterotrophy in a metazoan tissue: thiosulfate production matches ATP demand in ciliated mussel gills.

The ribbed mussel Geukensia demissa inhabits sulfide-rich coastal sediments with a distribution that suggests a preference for exposure to sulfide. Although sulfide is a respiratory poison, it is also a potent reductant. Geukensia demissa gill mitochondria can use sulfide as a respiratory substrate for ATP production, and the gills of this species exhibit sulfide-supported oxygen consumption that matches the energy demand of ciliary beating. Here, we demonstrate (i) that the major product of G. demissa gill sulfide oxidation is thiosulfate and (ii) that the rate of sulfide oxidation also matches the cellular energy demand, resulting in a ratio near unity of oxygen consumed to sulfide oxidized at both low and high ciliary beat frequencies. A value for this ratio of unity is consistent with electrons from sulfide oxidation entering the mitochondrial electron transport chain. In the gills of the blue mussel Mytilus edulis from sulfide-free conditions, this ratio is 3-5 times higher, indicating an uncoupling of oxygen consumption from sulfide oxidation. Whereas M. edulis gills exhibit anaerobic metabolism during sulfide exposure, G. demissa gills do not, indicating a difference in sulfide tolerance between the two mussel species.

ATP production from the oxidation of sulfide in gill mitochondria of the ribbed mussel Geukensia demissa.

The ribbed mussel Geukensia demissa inhabits intertidal Spartina grass marshes characterized by sulfide-rich sediments. Sulfide poisons aerobic respiration, and G. demissa may cope in this seemingly inhospitable environment by oxidizing sulfide in gill mitochondria. Well-coupled mitochondria isolated from G. demissa gills were used to investigate sulfide oxidation and ATP synthesis. State 3 respiration, maximally stimulated by 5 micromol l(-)(1) sulfide with a P/O ratio of 0.89 and a respiratory control ratio (RCR) of 1.40, remained refractory to sulfide at higher concentrations except in the presence of salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidases. Sulfide-stimulated ATP production was 3-5 times greater than that stimulated by malate and succinate, respectively, giving an ATP/sulfide ratio of 0.63. The inhibition of sulfide-stimulated respiration and ATP production by the complex III inhibitors myxothiazol and antimycin A, respectively, suggests that electrons enter the electron transport chain before complex III. Combined with in vivo evidence for electron entry at cytochrome c, these data suggest that more than one type of sulfide-oxidizing enzyme may function in G. demissa gills. The SHAM-sensitive pathway of electron flux may be a critical component of a physiological strategy to tolerate sulfide. We conclude that G. demissa exploits the energy available from its reduced environment by using sulfide as a respiratory substrate for cellular ATP production.

An investigation of fibroblast mitochondria enzyme activity and respiration in response to metallic ions released from dental alloys.

Most cellular functions evaluated for biocompatibility are high-energy processes such as proliferation and therefore are not usually affected before a decrease in energy production is observed. Several studies have shown that metabolic functions are altered at much lower concentrations than several normally used biocompatibility tests such as viability. Therefore, the purpose of this study was to provide an in-depth evaluation of metallic ion effects on mitochondria function and thereby biocompatibility. These studies evaluated the mitochondrial function of human gingival fibroblasts exposed to the salt solutions of ions released from nickel-based dental alloys, particularly beryllium (Be(2+)), chromium (Cr(6+) and Cr(3+)), nickel (Ni(2+)), and molybdenum (Mo(6+)). Mitochondrial function was examined by NADH:CoQ reductase activity, succinate dehydrogenase activity, and oxygen consumption.

A High-Affinity Hemoglobin Is Expressed in the Notochord of Amphioxus, Branchiostoma californiense.

In the phylum Chordata, only members of the subphylum Vertebrata were thought to express hemoglobin (Hb). Here we document the existence of intracellular Hb expressed in members of the subphylum Cephalochordata. Hemoglobin is expressed in myotome tissue and in notochord cells within the body of amphioxus. Both notochord and myotome tissue Hbs have a molecular size consistent with a dimeric molecule made up of two non-covalently linked monomers each of approximately 19 kD. The notochord Hb has a relatively high oxygen-binding affinity, with an apparent P5O of 0.036 kPa (0.27mm Hg), and it does not bind oxygen cooperatively. The notochord Hb may be involved in facilitating oxygen delivery and providing a short-term oxygen store within the notochord cells in order to maintain a high level of aerobic metabolism in support of the sustained contraction necessary for notochord function.

Plasmodium falciparum: cyanide-resistant oxygen consumption.

It has been hypothesized that Plasmodium parasites utilize a branched chain respiratory pathway, consisting of a classical cyanide-sensitive branch and an alternative cyanide-resistant branch. To further explore this hypothesis, the effect of cyanide on Plasmodium falciparum was determined using a polarographic assay. The rate of oxygen consumption by saponin-freed parasites was approximately 5% that of control human white blood cells or of Toxoplasma gondii, consistent with an anabolic role for P. falciparum respiration. However, while all of the oxygen consumption of the control white blood cells and of T. gondii could be inhibited by cyanide, 25% of the oxygen consumption of the P. falciparum parasites was found to be insensitive to high concentrations of cyanide. The cyanide-resistant portion of the parasite oxygen consumption was completely inhibited by two inhibitors of alternative oxidase activities in other systems, propyl gallate and salicyclhydroxamic acid. These studies provide the first direct evidence for a branched chain respiratory pathway in P. falciparum. Furthermore, salicyclhydroxamic acid, propyl gallate, and related inhibitors of alternative oxidase activities were shown to inhibit the growth of P. falciparum in vitro. These results support the need for further investigation of alternative oxidase activity as an antimalarial chemotherapeutic target.

Sulfide-Stimulation of Oxygen Consumption Rate and Cytochrome Reduction in Gills of the Estuarine Mussel Geukensia demissa.

Organisms, such as the mussel Geukensia demissa, that inhabit high-sulfide sediments have mechanisms that impede sulfide poisoning of aerobic respiration. Oxygen consumption rates (nO2) of excised ciliated gills from freshly collected G. demissa were stimulated 3-fold at sulfide concentrations between 200 and 500 µM and remained stimulated at 1000 µM. Maintenance of mussels in sulfide-free conditions resulted in less stimulation of gill nO2 at <500 µM sulfide and inhibition between 500 and 1000 {mu}M sulfide. Gills of Mytilus galloprovincialis from a sulfide-free environment were inhibited by {ge}200 µM sulfide. These results indicate that sulfide stimulation of nO2 may be correlated to environmental exposure to sulfide. Serotonin, a neurohormonal stimulant of ciliary beating, further increased sulfide-stimulated nO2, possibly in support of energy demand. Sulfide-stimulated nO2 was negligible in boiled gills and was 61% inhibited by cyanide, implicating the participation of mitochondrial electron flux. Mitochondrial cytochromes c and oxidase oxidation/ reduction state changed little at <500 µM sulfide, but reduction occurred at 500-2000 µM sulfide, suggesting that although cytochrome oxidation/reduction state may be regulated in the face of increased electron flux, regulation may fail at inhibitory sulfide levels. Sulfide-stimulated nO2 may represent a detoxification mechanism in G. demissa.

Heat flux, oxygen flux, and mitochondrial redox state as a function of oxygen availability and ciliary activity in excised gills of Mytilus edulis.

The ciliated gill of bivalve molluscs is situated at an interface between animal and environment. Cilia propel water past the gills to deliver oxygen and nutrition to the animal. Ciliary activity is driven by dynein ATPases and requires a continual supply of ATP at a rate sufficient to match the rate of ATP hydrolysis. Control of the balance between ATP supply and demand in the ciliated gill, and how this balance may be altered by environmental stresses, is unknown. In this pilot study, metabolic flux of excised gills from the marine mussel Mytilus edulis was examined in response to oxygen availability and to serotonin-stimulated ciliary activity. Heat flux and oxygen flux were measured simultaneously with calorespirometry. In parallel experiments, the redox state of mitochondrial cytochromes was determined with in vivo spectrophotometry. Above 4 kPa pO2, heat flux was supported by aerobic metabolism. Anoxic heat flux was less than 5% of aerobic heat flux. Heat and oxygen fluxes nearly doubled in gills in the presence of 10 microM serotonin; however, half-maximal pO2 for heat and oxygen fluxes and for reduction of mitochondrial cytochromes remained unchanged from control levels. In gills having inactive cilia in half-strength seawater, half-maximal pO2 for heat and oxygen fluxes and for cytochrome reduction nearly doubled compared with valves in full-strength seawater. These data indicate that limitation to oxygen delivery imposed by boundary layers may be reduced when ciliary beat frequency is elevated, leading to enhanced oxygen flux to intracellular mitochondrial which matches the increased energy demand by the cilia.

Hydrogen Sulfide Reduction of Symbiont Cytochrome c552 in Gills of Solemya reidi (Mollusca).

The gill of the protobranch clam Solemya reidi houses a dense population of intracellular symbiotic chemoautotrophic sulfur-oxidizing bacteria that fix carbon dioxide into sugars and supply the carbon nutrition of the host. The gill is divided into a bacteriocyte (cells with intracellular symbionts) domain and a domain of mitochondria-rich, symbiont-free ciliated cells. Optical spectra, recorded separately from each domain, are dominated by hemoglobin. Only oxygenated and deoxygenated hemoglobin were detected in the gill. In sharp contrast to the gill of the congener Solemya velum, ferric hemoglobin sulfide was not detected, suggesting that this species, if formed, is short lived. The spectral contribution of hemoglobin may be cancelled or subtracted in difference spectra. Difference spectra of each gill domain in nitrogen minus the same tissue in air show a complement of reduced cytochromes, demonstrating that both symbiont and mitochondrial cytochromes are reduced by endogenous substrate. Difference spectra of the bacteriocyte domain exposed to hydrogen sulfide (air containing 1.4 torr hydrogen sulfide minus air) show only the contribution of reduced symbiont cytochrome c552. The extent of reduction increases monotonically with ambient pH2S, suggesting that, by analogy with some free-living sulfur-oxidizing bacteria, cytochrome c552 is near the point of entry of electrons into the symbiont electron transport chain. Difference spectra of muscle or of the ciliated domain under these same conditions show reduced cytochrome c550, cytochrome b and cytochrome oxidase, suggesting that host mitochondria may accept electrons from hydrogen sulfide.

Myoglobin function and energy metabolism of isolated cardiac myocytes: effect of sodium nitrite.

Inactivation of intracellular myoglobin by sodium nitrite or by carbon monoxide in isolated cardiac myocytes diminishes steady-state respiratory rate and phosphocreatine concentration (PCr) by approximately 25% at nonlimiting oxygen pressures; oxidative phosphorylation and glycolysis together are insufficient to maintain ATP, and PCr falls. At concentrations required to convert myoglobin to high-spin ferric myoglobin, nitrite does not affect the respiration of isolated aerobic heart mitochondria. The creatine phosphokinase-catalyzed equilibrium between PCr and ATP is not affected by nitrite. Myoglobin inactivation reduces PCr in cells in which glycolytic ATP production is blocked by iodoacetate. However, inhibition of electron transport by rotenone does block myoglobin-mediated oxygen uptake. These data suggest that functional myoglobin augments mitochondrial oxidative phosphorylation [myoglobin-mediated oxidative phosphorylation (30)]. Myoglobin itself does not cross mitochondrial membrane(s). At high oxygen pressures used here, myoglobin is everywhere saturated with oxygen, and facilitated oxygen diffusion vanishes. Oxidative phosphorylation must be augmented by some effector, such as NADH or a carrier of reducing or oxidizing equivalents that can transduce the effect of oxymyoglobin across the mitochondrial membrane(s).

Intracellular calcium and high-energy phosphates in isolated cardiac myocytes.

The relationship between intracellular calcium (Cai) and high-energy phosphates was studied in adult cardiac myocytes. Cai and high-energy phosphates were measured in the same population of cells. Cai, reported by the fluorescence of fura-2, was maintained at normal levels in the presence of increased transsarcolemmal calcium gradients, up to 5 mM extracellular calcium concentration. Cai was experimentally elevated by increasing calcium influx from the extracellular medium and/or by diminishing calcium efflux by Na-Ca exchange. Under these conditions, cells contracted and relengthened repetitively. The regulation of high-energy phosphates was challenged by increasing ATP utilization and by inhibiting ATP synthesis. Cai regulation was not affected by inhibition of glycolysis or NADH oxidation, so long as ATP concentration remained unchanged. High-energy phosphates were not depleted in beating cells with intact NADH oxidation, but inhibition of NADH oxidation caused a significant drop in phosphocreatine, demonstrating the increased rate of ATP consumption during beating. In beating cells, as in the working heart, ATP supply is increased to meet ATP demand, and steady-state ATP and phosphocreatine concentrations remain unchanged.

Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++.

Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.