GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells

Thiol reagents activateK-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independentouabain-resistant K flux, in red blood cells (RBCs) of several species,upon depletion of cellular glutathione (GSH). K-Cl COT isphysiologically active in high potassium (HK), high GSH (HG) dog RBCs.In this unique model, we studied whether the same inverse relationshipexists between GSH levels and K-Cl COT activity found in other species.The effects of GSH depletion by three different chemical reactions[nitrite (NO₂)-mediated oxidation, diazene dicarboxylicacid bis-N,N-dimethylamide (diamide)-induceddithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD).After 85% GSH depletion, all three interventions stimulated K-Cl COThalf-maximally with the following order of potency: diamide > NO₂ > CDNB. Repletion of GSH reversed K-Cl COTstimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activationby NO₂ and diamide. K-Cl COT activation at concentrationratios of oxidant/GSH greater than unity was irreversible, suggestingeither nitrosothiolation, heterodithiol formation, or GST-mediateddinitrophenylation of protein thiols. The data support the hypothesisthat an intact redox system, rather than the absolute GSH levels,protects K-Cl COT activity and cell volume regulation from thiol modification.

     

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.     

Tolerability and efficacy of single dose albendazole,diethylcarbamazine citrate (DEC) or co-administration of albendazole with DEC in the clearance of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis>in asymptomatic microfilaraemic volunteers in Pondicherry,South India: a hospital-based study

Background  

The tolerability and efficacy of single dose albendazole (400 mg), diethylcarbamazine citrate (DEC) (6 mg/kg bodyweight) or co-administration of albendazole (400 mg) + DEC (6 mg/kg bodyweight) was studied in 54 asymptomatic Wuchereria bancrofti microfilaraemic volunteers in a double blind hospital-based clinical study.     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

A P Element Containing Suppressor of Hairy-Wing Binding Regions Has Novel Properties for Mutagenesis in Drosophila Melanogaster

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.     

Cloning and expression of sheep renal K-CI cotransporter-1.

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.     

K-Cl cotransport in red blood cells from patients with KCC3 isoform mutants.

Red blood cells (RBCs) possess the K-Cl cotransport (KCC) isoforms 1, 3, and 4. Mutations within a given isoform may affect overall KCC activity. In a double-blind study, we analyzed, with Rb as a K congener, K fluxes (total flux, ouabain-sensitive Na+/K+ pump, and bumetanide-sensitive Na-K-2Cl cotransport, Cl-dependent, and ouabain- and bumetanide-insensitive KCC with or without stimulation by N-ethylmaleimide (NEM) and staurosporine or Mg removal, and basal channel-mediated fluxes, osmotic fragility, and ions and water in the RBCs of 8 controls, and of 8 patients with hereditary motor and sensory neuropathy with agenesis of corpus callosum (HMSN-ACC) with defined KCC3 mutations (813FsX813 and Phe529FsX532) involving the truncations of 338 and 619 C-terminal amino acids, respectively. Water and ion content and, with one exception, mean osmotic fragility, as well as K fluxes without stimulating agents, were similar in controls and HMSN-ACC RBCs. However, the NEM-stimulated KCC was reduced 5-fold (p < 0.0005) in HMSN-ACC vs control RBCs, as a result of a lower Vmax (p < 0.05) rather than a lower Km (p = 0.109), accompanied by corresponding differences in Cl activation. Low intracellular Mg activated KCC in 6 out of 7 controls vs 1 out of 6 HMSN-ACC RBCs, suggesting that regulation is compromised. The lack of differences in staurosporine-activated KCC indicates different action mechanisms. Thus, in HMSN-ACC patients with KCC3 mutants, RBC KCC activity, although indistinguishable from that of the control group, responded differently to biochemical stressors, such as thiol alkylation or Mg removal, thereby indirectly indicating an important contribution of KCC3 to overall KCC function and regulation.