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.     

Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes

A detailed kinetic study of K:Cl cotransport in hyposmotically swollen low K sheep red blood cells was carried out to characterize the nature of the outwardly poised carrier. The kinetic parameters were determined from the rate of K efflux and influx under zero-K-trans conditions in red cells with cellular K altered by the nystatin method and with different extracellular K or Rb concentrations. Although apparent affinities for efflux and influx were quite similar, the maximal velocity for K efflux was approximately two times greater than for influx. Furthermore, at thermodynamic equilibrium (i.e., when the ion product of K and Cl within the cell was equal to that outside) a temperature-dependent net K efflux was observed, approaching zero only when the external product reached approximately two times the internal product. The binding order of the ions to the transporter was asymmetric, being ordered outside (Cl binding first, followed by K) and random inside. K efflux but not influx was trans-inhibited by KCl. Trans inhibition of K efflux was used to verify the order of binding outside: trans inhibition by external Cl occurred in the absence of external K, but not vice versa. Thus K:Cl cotransport is kinetically asymmetric in hyposmotically swollen low K sheep red cells.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells

Summary Binding of highly purified¹²⁵I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75–100 and 500–750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3–6×10³ in HK (MM) and 1.5–4×10³ in LM (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2–1.8×10³ in LL and less in LM (LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na⁺K⁺ pumps measured previously in these cells by³H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na⁺K⁺ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and/or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na⁺K⁺ pump molecules is more complex than that in goat red cells previously reported by others.     

The effect of anti-L on ouabain binding to sheep erythrocytes.

Binding of 3H-ouabain was studied in high potassium (HK) and low potassium (LK) sheep red cells. In particular, we investigated the effect of anti-L, an antibody raised in HK sheep against L-positive LK sheep red cells, on 3H-oubain binding and its relation to K+ -pump flux inhibition in LK cells. HK cells were found to have about twice as many 3H-ouabain binding sites and a higher association rate for 3H-ouabain than homozygous LL-type LK cells. The number of 3H-ouabain molecules bound to heterozygous LM-type LK cells is lower than that on LL cells, but the rate of ouabain binding is between that of HK and LL red cells. A close correlation was observed between the rates of 3H-oubain binding and fraction K+-pump inhibition. Exposure of LM and LL cells to anti-L did not affect the number of 3H-ouabain molecules bound at saturation, but increased the rates of glycoside binding and K+ -pump inhibition proportionately, so that for LK cells in the presence of anti-L, the rates of the two processes approximate those of HK cells. These data exclude the possibility that anti-L generates entirely new pump sites in LK sheep cells, but suggest that the antibody increases the affinity of the existing -a+ -K+ pumps for the glycoside.     

Cation transport in different volume populations of genetically low K+ lamb red cells

During the first three months after birth lambs produce sequentially three erthryocyte populations of different mean volume as demonstrated by electric sizing methods (Valet, Franz, and Lauf, J. Cell. Physiol. 94 (1978) 215). We separated by centrifugal elutriation the small volume population (type II) red cells of a genotypically low K⁺ (LK) lamb from the population containing the larger volume type I and III cells, an admixture of fetal (I) and adult (III) erythrocytes. The cells were separated at various time intervals after birth and analyzed with respect to their volumes, cation contents, and cation flux properties by means of ⁸⁶Rb uptake. The effect of anti-L on K⁺ pump and leak fluxes was ascertained in unseparated and separated red cells. It was found that the small red cells of population II, transiently present for several weeks, were fully developed LK cells with K⁺ pumps responding characteristically to the stimulatory action of anti-L. In constrast, the larger cells of population I and III were of high K⁺ (HK) nature at early time points, the K⁺ pump activities approximately ten times higher than adult LK cells. These cells constitute an admixture of type I fetal HK cells, and type III reticulocytes which are precursors for the final type III adult LK cells, since anti-L had a small stimulatory effect. At later times, however, only adult type III LK cells predominated. The data directly support our earlier finding that the HK-LK transition in genotypically LK lambs is primarily governed by cellular replacement.     

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.     

Emission of gaseous nitrogen oxides from an extensively managed grassland in NE Bavaria, Germany.

We analysed the stable isotope composition of emitted N₂O in a one-year field experiment (June 1998 to April 1999) in unfertilized controls, and after adding nitrogen by applying slurry or mineral N (calcium ammonium nitrate). Emitted N₂O was analysed every 2–4 weeks, with additional daily sampling for 10 days after each fertilizer application. In supplementary soil incubations, the isotopic composition of N₂O was measured under defined conditions, favouring either denitrification or nitrification. Soil incubated for 48 h under conditions favouring nitrification emitted very little N₂O (0.024 mol g_dw ^–1) and still produced N₂O from denitrification. Under denitrifying incubation conditions, much more N₂O was formed (0.91 mol g_dw ^–1 after 48 h). The isotope ratios of N₂O emitted from denitrification stabilized at ¹⁵N = –40.8 ± 5.7 and ¹⁸O = 2.7 ± 6.3. In the field experiment, the N₂O isotope data showed no clear seasonal trends or treatment effects. Annual means weighted by time and emission rate were ¹⁵N = –8.6 and ¹⁸O = 34.7 after slurry application, ¹⁵N = –4.6 and ¹⁸O = 24.0 after mineral fertilizer application and ¹⁵N = –6.4 and ¹⁸O = 35.6 in the control plots, respectively. So, in all treatments the emitted N₂O was ¹⁵N-depleted compared to ambient air N₂O (¹⁵N = 11.4 ± 11.6, ¹⁸O = 36.9 ± 10.7). Isotope analyses of the emitted N₂O under field conditions per se allowed no unequivocal identification of the main N₂O producing process. However, additional data on soil conditions and from laboratory experiments point to denitrification as the predominant N₂O source. We concluded (1) that the isotope ratios of N₂O emitted from the field soil were not only influenced by the source processes, but also by microbial reduction of N₂O to N₂ and (2) that N₂O emission rates had to exceed 3.4 mol N₂O m^–2 h^–1 to obtain reliable N₂O isotope data.     

K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L) positive, than in high K (HK), M-antigen (M) positive, sheep red blood cells (SRBCs) and is inhibited by sheep allo-anti-L1 antibody. To answer the question of whether this difference in K-Cl co-transport activity resides at the level of the transporter or its regulation, a combined immunocytochemical and functional approach was taken. At least four isoforms of K-Cl COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclonal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino acids from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide from the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separate protein bands of 180 and 145 kDa molecular mass were detected in hemoglobin-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 and rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, the effect of the anti-L1 antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-L1 serum inhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approximately two-thirds of the transport activity is independent of the L1 antigen. That staurosporine altered the L1 antigen/antibody reaction is unlikely since the action of another antibody, anti-Lp, stimulating the Na/K pump flux, was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-L1 inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-KCC1 and anti-KCC3 antibodies.     

The inverted repeats of IS<Emphasis Type="Italic">1384</Emphasis>, a newly described insertion sequence from<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> strain H,represent the specific target for integration of IS<Emphasis Type="Italic">1383</Emphasis>

Analysis of a region on plasmid pPGH1 from Pseudomonas putida strain H that is flanked by two copies of IS1383 has revealed an additional element with the typical features of a bacterial insertion sequence. This new IS element, designated IS1384, contains a single ORF of 972 bp, and is flanked by 9-bp inverted repeats. Based on sequence homology and structural characteristics of the putative transposase it encodes, IS1384 belongs to the IS5 subgroup of the IS5 family. Two copies of IS1384 are present on plasmid pPGH1, whereas none could be detected on the chromosome of P. putida strain H. Sequence analysis revealed the presence of two truncated copies of IS1384 on the second plasmid in this strain, pPGH2. The inverted repeats of all IS1384 copies (including the truncated ones) are interrupted by the integration of an IS1383 element. All integrations were found to be site- and orientation-specific. PCR studies and sequence data indicate that IS1383 can form a circular intermediate on excision. In the circular form, the previously described 13-bp inverted repeats of IS1383 are separated by 10 bp that are identical to the 5-bp motif that flanks each side of the element when it is integrated in its target. We provide evidence that these additional nucleotides, although not of inverted symmetry, represent an essential part of the inverted repeats. Furthermore, the data indicate that IS1383 integrated into the inverted repeats of IS1384 by a site-specific recombination rather than a site-specific insertion event.