Thiol-dependent K∶Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

Summary The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO₃ or methane sulfonate (CH₃SO₃). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the KCl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983.J. Membrane Biol..73:237–246). Like NEM, diamide activated KCl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L₁, the allo-antibody known to inhibit OR K fluxes.     

Influence of medium osmolality on the in vitro growth ofPlasmodium falciparum: A morphologic and radioisotopic study

Summary The study of the growth rate and incorporation of [³H]hypoxanthine and [¹⁴C]isoleucine showed that in vitro variations ofPlasmodium falciparum parasitemia levels and incorporation rates of the two radiolabeled molecules have been correlated. In our experimental conditions,P. falciparum blood forms in vitro tolerate osmolalities ranging from 180 to 360 mOsm. A weak hypo-osmolality (241 mOsm) favored the development of the parasite. The highest sensitivity of the parasite to osmotic variations was observed during schizogony. The merozoite stage and reinvasion process seemed less affected by hypo-osmolalities than by hyperosmolalities. The minor alterations in morphology of the parasites in hypo- and hyperosmotic media suggested thatP. falciparum may have efficient osmoregulatory power.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

Kinetics of DIDS inhibition of swelling-activated K-Cl contrasport in low K sheep erythrocytes

Summary The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocy-anatostilbenes H₂DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC₅₀) of KCl cotransport was 60 m in the absence of external K and 3 m at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4 isothiocyanatostilbene-2,2-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4-dinitrostilbene-2,2-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mm. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).This work was supported by grants in aid from the American Heart Association.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

Nucleoside transport in mammalian cell membranes

Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK _m of 350 m and a maximal velocity (V) of 780 m·min^–1 (V/K _m =2.28 min^–1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK _m of 500 m and av of 1230 m·min^–1 (V/K _m =2.26 min^–1) and either a saturable component of highK _m or a nonsaturable component ofk=0.3 min^–1. For the saturable component, thev/K _m values were similar in both procedures.CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K _i <0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.     

Characteristics of the volume- and chloride-dependent K transport in human erythrocytes homozygous for hemoglobin C

Summary In human red cells homozygous for hemoglobin C (CC), cell swelling and acid pH increase K efflux and net K loss in the presence of ouabain (0.1mm) and bumetanide. We report herein, that K influx is also dependent on cell volume in CC cells: cell swelling induces a marked increase in the maximal rate (from 6 to 18 mmol/liter cell × hr) and in the affinity for external K (from 77±16mm to 28±3mm) of K influx. When the external K concentration is varied from 0 to 140mm, K efflux from CC and normal control cells is unaffected. Thus, K/K exchange is not a major component of this K movement. K transport through the pathway of CC cells is dependent on the presence of chloride or bromide; substitution with nitrate, acetate or thiocyanate inhibits the volume- and pH-dependent K efflux. When CC cells are separated according to density, a sizable volume-dependent component of K efflux can be identified in all the fractions and is the most active in the least dense fraction. N-ethylmaleimide (NEM) markedly stimulates K efflux from CC cells in chloride but not in nitrate media, and this effect is present in all the fractions of CC cells separated according to density. The persistence of this transport system in denser CC cells suggests that not only cell age, but also the presence of the positively charged C hemoglobin is an important determinant of the activity of this system. These data also indicate that the K transport pathway of CC cells is not an electrodiffusional process and is coupled to chloride.     

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mg _i ) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO₃ medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO₃) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations ?1 mm. The inhibitory effect of Q was manifested in Cl, but not in NO₃, whereas QD reduced K and Rb fluxes both in Cl and NO₃ media. The mean 50% inhibitory concentration (IC⁵⁰) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mm. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous. This work was supported by a grant from the National Institutes of Health (NIH DK5RO1 37,160).     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide

The ruthenium arene anticancer complex [(⁶-bip)Ru(en)Cl][PF₆] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺, and an N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), ¹⁵N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [¹H, ¹⁵N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .

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Passive potassium transport in LK sheep red cells. Modification by N- ethyl maleimide

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5- min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3- substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site.     

Properties of β-glucosidase purified fromAspergillus niger

Summary Extracellular -glucosidase fromAspergillus niger USDB 0355 was purified 120-fold. It gave a single band on PAGE and had an M_r of 325 000. It was optimally active at 60°C and pH 4.6. It had K_m values forp-nitrophenyl--glucoside and cellobiose of 0.82±0.10mm and 1.33±0.20mm, respectively. It was competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono--lactone.

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Resumen Se purificó la glucosidasa extracelular deAspergillus niger USDB 0355 120 veces. Su electroforesis en gel de poliacrilamida (PAGE) resultó en una única banda con peso molecular de 325000D. La actividad fue óptima a 60°C y pH 4.6. Los valores de la Km para elp-nitrofenol-glucosido y para la celobiosa fueron de 0.82±0.10mm y 1.33±0.20mm respectivamente. La glucosa inhibió competitivamente el enzima mientras que la glucono--lactona lo inhibió no competitivamente (inhibición mixta).

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Résumé La -glucosidase extracellulaire d'Aspergillus niger USDB 0355 a été purifiée 120 fois. L'enzyme a révélé une bande unique sur PAGE et avait un M_r de 325 kdaltons. Elle était optiquement active à 60°C et à pH 4.6. Elle présentait des valeur des Km pour lep-nitrophenyl--glucoside et la cellobiose, respectivement de 0.82±0.10mm et de 1.33±20mm. Elle était complètement inhibée par le glucose et présentait une inhibition non-compétitive mixte par la glucono--lactone.

     

Hämatologische Beobachtungen an Regenbogenforellen (Salmo gairdneri RICH.). V. Die Erythozytenverteilungskurve

Zusammenfassung Für die Fischpathologie wird als differentialdiagnostisches Verfahren die Erythozytenmessung vorgeschlagen. Zur Darstellung der Erythrozytenverteilungskurve wird wegen der elliptischen Form der Fisch-Erythrozyten die in der Humanmedizin übliche Technik modifiziert.An 102 Regenbogenforellen (Salmo gairdneri) beiderlei Geschlechts im Alter von 1 bis 3 Jahren wurde versucht, die Normalkurve für gesunde Tiere dieser Population zu ermitteln. Die Größenverteilung der Erythrozyten ergibt eine Gauss-Glockenkurve, deren Gipfel in die Größenklasse 30–35 µm²/ fällt, und deren Basis von 15–20 µm²/ bis 55–60 µm²/ reicht.Zwischen den Price-Jones-Kurven der Milchner (52 Individuen) und Rogner (50 Individuen) bestehen keine signifikanten Unterschiede. Ebenso konnte keine Beeinflussung dieser Normalkurve durch unterschiedliches Alter oder jahreszeitliche Einflüsse — untersucht wurden in dieser Hinsicht die F₂ — beobachtet werden.

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Summary As differential-diagnostic feature in the fish pathology it is suggested to measure the red blood cells. The normal technique used in human medicine for plotting the distribution curve of the red blood cells is modified, because the red blood cells of the fish are of elliptical shape.102 rainbow trouts (Salmo gairdneri), both, male and female, and 1–3 years of age, were examined to find the normal curve for healthy animals of this population. The size distribution of the red blood cells show a bell-shaped Gauss-curve, the peak of which is 30–35 µm²/, and the basis of which reaches from 15–20 µm²/ to 55–60 µm²/.The Price-Jones-curves show no significant differences between the milters (52 individuals) and the spawners (50 individuals). Besides, changes caused by different age or seasonal influences could not be observed on this normal curve. Analyzed were the F₂ individuals.

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Institut für Siedlungswasserbau und Wassergütewirtschaft der Universität Stuttgart Biologische Abteilung - Arbeitsgruppe Haider     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the basilar membrane, limbus spiralis and spiral ligament.These different parts continue one into the other and make up a single morphological and functional structure which may be called the supporting structure of Corti's organ (s.s.C.o.).It is formed by a tissue the components of which are the cells and an intercellular substance in which are arranged the capillary vessels.The cells can be classed in two groups, the first consisting of the cells proper (basilar membrane, limbus spiralis and spiral ligament cells) which present structural changes parallel with the growth mechanism of the intercellular substance; the second of the cochlear duct covering cells (Corti's organ cells, inner and outer spiral sulcus cells, interdental cells, stria vascularis cells).The intercellular substance is organised in laminae, fibrolaminae, bundles and microscopic fibers composed of filaments with an intervening ground substance.The filaments have a diameter ranging from 85 to 105 Å. Topochemical tests with polarised light microscope, enzymatic tests, diffractographic and chemical analyses suggest that these filaments unquestionably consist of protein material which have nothing to do with collagen or elastic fibers. Perhaps it may be classed in the K.E.M.F. group.The ground substance generally appear anhistous and transparent but in some parts of the basilar membrane it presents a cottony appearance.The possible different hypotheses about the classification of the s.s.C.o. tissue are discussed.The quantity and architecture of the cells and the intercellular substance vary appreciably in the basilar membrane, limbus spiralis and spiral ligament, which are examined in detail one by one.The demonstration that the s.s.C.o. is formed of a tissue possessing an intercellular substance containing filamentous scleroproteins clearly corroborates the theory that is performs supporting activity in respect of Corti's organ. The term supporting structure of Corti's organ is based on this interpretation.Research financed by C.N.R. grant.     

Behaviour and evolution in the genusEuplectes

Summary TheEuplectes species are polygynous weavers, showing marked sexual and seasonal dimorphism. The are territorial during the breeding season, and interspecific territorial behaviour is common. Courtship consists of a flight display followed by a perched display leading to copulation. The nest plays an important role in courtship. Throughout the year the birds feed and roost in large aggregations. The evolution of the genus is discussed; the two main behavioural changes have been an elaboration of the flight display, accompanied by a decline in nest-building by the .

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Zusammenfassung DieEuplectes-Arten sind polygame Webervögel mit einem ausgeprägten Sexualdimorphismus. Während der Brutzeit legen die das farbige Prachtkleid an und gründen Reviere, die häufig auch gegen artfremde verteidigt werden. Die Balz besteht aus zwei Elementen: einer Flugbalz, gefolgt von einer Stangenbalz, die zur Kopulation führt. Das Nest spielt bei der Balz eine wichtige Rolle. Das ganze Jahr hindurch versammeln sich die Vögel in großen Schwärmen zum Nahrungserwerb und am Schlafplatz. Der wahrscheinliche Verlauf der Evolution in dieser Gattung wird beschrieben. Zwei augenfällige Verhaltensänderungen sind eine Zunahme in der Dauer und Komplexität der Flugbalz, begleitet von einer Abnahme der Nestbauaktivität durch die .

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This research was supported by funds from the South African Council for Scientific and Industrial Research, and for one year by a stipendium from the Deutsche Akademische Austauschdienst. I am most grateful to Prof.G. L. Maclean and Prof. Dr.K. Immelmann for their help and advice.     

Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide

Summary The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent²²Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 85 m, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Ion transport by rabbit colon

Summary Descending rabbit colon, stripped ofmuscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO₃ secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit currentI _sc is determined solely by the rate of active Na transport and that the net movements of Cl and HCO₃ are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO₃ secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxesJ _sm ⁱ of Na, K and Cl suggest thatJ _sm ^Na ,J _sm ^K and one-third ofJ _sm ^Cl may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such thatP _KP _NaP _Cl=1.00.070.11. Electrolyte transport byin vitro rabbit colon closely resembles that reported fromin vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Catabolism of malate and related dicarboxylic acids in various Desulfovibrio strains and the involvement of an oxygen-labile NADPH dehydrogenase

Four out of five Desulfovibrio strains tested were able to oxidize l-malate to acetate in the presence of sulfate. Fumarate and succinate were also oxidized to acetate by these strains, but growth with the latter substrate was marginal. During growth on malate high NADP-dependent malic enzyme and NADPH DH activities were found in all strains. These activities were lower in lactate-or pyruvate-grown cells. An NADPH DH from D. gigas was partially purified. It was oxygen-labile, very sensitive to heavy metal ions and highly specific for NADPH. Growth yield studies indicated that energy conservation occurred during the transport of reducing equivalents from NADPH to the sulfate reduction pathway.Abbreviations DH dehydrogenase - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - HEPES 4-(2-hydroxyethyl-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - MES 2-(N-morpholino)-ethane sulfonic acid - DTT dithiothreitol     

[3H]Histamine uptake and release by astrocytes from rat brain: Effects of sodium deprivation,high potassium,and potassium channel blockers

Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [³H]-histamine uptake yielded a K_m of 0.19±0.03 M and a V_max of 3.12±0.75 pmol×mg protein^–1×min^–1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na⁺ with choline inhibited the initial rate of uptake showing a strong Na⁺-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K⁺-gradient (in>out) by high K⁺ or by K⁺-channel blockers, BaCl₂, (100 M), quinine (100 M) or Sparteine (20 M) produced also remarkable inhibitions in the uptake of [³H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 M with a K_i value of 2.8 M, indicating the specificity of the uptake. [³H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 M; and a V_max of 2.65±0.35 pmoles×mg protein^–1×min^–1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [³H]histamine efflux was sensitive to Na⁺ omission, ouabain treatment and high K⁺ or K⁺ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na⁺-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.Abbreviations used HA histamine - [³H]HA [2.5-³H]-histamine - dl--aAA dl-alpha-aminoadipic acid - (Na⁺+K⁺) ATP-ase sodium and potassium activated adenosine triphosphatase - SAH S-Adenosyl-d-Homocysteine - HNMT histamine-N-methyltransferase