O(2)-dependent K(+) fluxes in trout red blood cells: the nature of O(2) sensing revealed by the O(2) affinity, cooperativity and pH dependence of transport.

The effects of pH and O(2) tension on the isotonic ouabain-resistant K(+) (Rb+) flux pathway and on haemoglobin O2 binding were studied in trout red blood cells (RBCs) in order to test for a direct effect of haemoglobin O(2) saturation on K(+) transport across the RBC membrane. At pH values corresponding to in vivo control arterial plasma pH and higher, elevation of the O(2) partial pressure (PO(2)) from 7.8 to 157 mmHg increased unidirectional K(+) influx across the RBC membrane several-fold. At lower extracellular pH values, stimulation of K(+) influx by O(2) was depressed, exhibiting an apparent pK(a) (pK'(a)) for the process of 8.0. Under similar conditions the pK'(a) for acid-induced deoxygenation of haemoglobin (Hb) was 7.3. When trout RBCs were exposed to PO(2) values between 0 and 747 mmHg, O(2) equilibrium curves typical of Hb O(2) saturation were also obtained for K(+) influx and efflux. However, at pH 7.9, the PO(2) for half-maximal K(+) efflux and K(+) influx (P50) was about 8- to 12-fold higher than the P(50) for Hb-O(2) binding. While K(+) influx and efflux stimulation by O(2) was essentially non-cooperative, Hb-O(2) equilibrium curves were distinctly sigmoidal (Hill parameters close to 1 and 3, respectively). O(2)-stimulated K(+) influx and efflux were strongly pH dependent. When the definition of the Bohr factor for respiratory pigments (Phi = delta logP50 x delta pH(-1)) was extended to the effect of pH on O(2)-dependent K(+) influx and efflux, extracellular Bohr factors (Phi(o) of -2.00 and -2.06 were obtained, values much higher than that for Hb (Phi(o) = -0.49). The results of this study are consistent with an O(2) sensing mechanism differing markedly in affinity and cooperativity of O(2) binding, as well as in pH sensitivity, from bulk Hb.

Sulphaemoglobin formation in fish: a comparison between the haemoglobin of the sulphide-sensitive rainbow trout (Oncorhynchus Mykiss) and of the sulphide-tolerant common carp (Cyprinus Carpio).

A method for the quantitative determination of sulphaemoglobin (SHb) in a mixture of haemoglobin derivatives by spectral deconvolution is described. SHb formation was studied in haemolysates and in red blood cells of the sulphide-sensitive rainbow trout (Oncorhynchus mykiss) and of the sulphide-tolerant common carp (Cyprinus carpio). Addition of sulphide caused the formation of SHb in haemolysates of both animals. However, haemoglobin from common carp was much less sensitive to sulphide than was trout haemoglobin. The maximal obtainable SHb fraction was approximately 30 % in trout and 10 % in carp haemolysates. In both animals, the SHb fraction increased with increasing Hb and sulphide concentrations up to 100 micromol l(-)(1) and 1 mmol l(-)(1), respectively, and was favoured by a low pH. An increase of temperature between 5 and 25 degrees C strongly increased SHb formation in trout haemolysate. In contrast, temperature changes had almost no effect on SHb production in carp. Within trout red blood cells, approximately 7 % of total haemoglobin was converted to SHb during 60 min of incubation (with 2.5 mmol l(-)(1) sulphide), inducing a 20 % loss of haemoglobin oxygen-saturation. In carp red blood cells incubated under identical conditions, SHb formation was minimal and haemoglobin oxygen-saturation was not affected.

Animal adaptations for tolerance and exploitation of poisonous sulfide.

Many aquatic animal species can survive sulfide exposure to some extent through oxidation of the sulfide, which results mainly in thiosulfate. In several species, sulfide oxidation is localized in the mitochondria and is accompanied by ATP synthesis. In addition, blood-based and intracellular compounds can augment sulfide oxidation. The formation of thiosulfate requires oxygen, which results in an increase in oxygen consumption of some species. If not all sulfide is detoxified, cytochrome C oxidase is inhibited. Under these conditions, a sulfide-dependent anaerobic energy metabolism commences.

Mitochondrial sulfide oxidation in Arenicola marina. Evidence for alternative electron pathways.

Sulfide is oxidized in the mitochondria of the lugworm Arenicola marina. Mitochondrial sulfide oxidation is coupled with oxygen consumption and with an equimolar production of thiosulfate [Völkel, S. & Grieshaber, M. K. (1994) Mar. Biol. 118, 137-147]. Mitochondrial respiration in the presence of malate (or succinate) and ADP but without sulfide could be completely inhibited by rotenone, antimycin, cyanide, and sulfide. Only 40% inhibition was achieved by salicylhydroxamic acid. Sulfide oxidation (with sulfide as the only substrate) was fully inhibited by antimycin and by salicylhydroxamic acid but not by rotenone or sulfide. Moreover, sulfide oxidation was 3-4-fold less sensitive to cyanide as compared to normal respiration. The data indicate that sulfide oxidation in A. marina is linked to the respiratory electron transport chain. We suggest that electrons from sulfide enter the respiratory chain via ubiquinone or at the ubiquinol-cytochrome-c oxidoreductase. At sulfide concentrations higher than 10 microM, the cytochrome-c oxidase is blocked and electrons from sulfide are transferred to oxygen via an alternative terminal oxidase.

[Characterization of immunological responses to bone and cartilage xenografts]. / Zur Charakterisierung der immunologischen Abwehr bei xenogener Transplantation von Knochen und Knorpel.

The immunological responses to subcutaneous bone and cartilage grafts were investigated in rats using phenol oxidase, a leukocyte labelling enzyme. The resulting development of granulomas reflects a cell-mediated immunological process and is proof of the immunogenicity of bone and cartilage. This method is a new criterium for the assessment of the biocompatibility of graft and implant materials.

[Further studies on modification of phenoloxidase by effectors]. / Weitere Untersuchungen zur Beeinflussung der Phenoloxidase durch Effektoren.

With the aid of the histochemical phenoloxidase (PO) assay, 12 substances were tested. On the one side they can act as substrates or as inhibitors of phenoloxidase and on the other side as immunogenic or immunosuppressive compounds. In particular the following substances were investigated: o-cresol, Levamisol, prednisolonebisuccinate, Bleocin, siliciumdioxide, 4-methylbrenzcatechol, +-catechine, thiomersal, D-penicillamine, hydrochinonemonomethylether, hydrochinonemonoethylether, and hydrochinonemonobenzylether. No changes in the phenoloxidase activity and in the morphological behaviour of the cells could be detected using Levamisol, prednisolonebisuccinate, siliciumdioxide, and Bleomycin, indicating an immunogenic stimulation or immunosuppressive effect. 4-methylbrenzcatechol is probably transformed into a soluble reaction product by phenoloxidase. It could not clearly demonstrated by the histochemical method, if this substance is used as a true substrate. +-catechine, thiomersal, and D-penicillamine cause strong to complete enzyme inhibition of the phenoloxidase containing cells (POC), which are located in the spleen and gut of the white rat. Hydrochinonemonoethylether, hydrochinonemethylether, and hydrochinonemonobenzylether cause an increased pigment fermentation, but they are not used as substrates for the PO.