Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl^- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca²⁺-dependent Cl^- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca²⁺ transients via activation of a long-lived, store-operated Ca²⁺ entry pathway. The enhanced intracellular Ca²⁺ signal is associated with uncoupling of the Cl^- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca²⁺ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects. nelfinavir; clotrimazole; barium     

Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis

Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.     

Recent Advances in Alcoholic Liver Disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease

A significant body of evidence indicates that endotoxemia and endotoxin-mediated hepatocellular damage play a crucial role in the pathogenesis of alcoholic liver disease. A close correlation between endotoxemia and the severity of alcohol-induced liver injury is supported by a number of clinical and experimental studies. Elevated intestinal permeability appears to be the major factor involved in the mechanism of alcoholic endotoxemia and the pathogenesis of alcoholic liver disease. Ethanol and its metabolic derivatives, acetaldehyde in particular, alter intracellular signal-transduction pathways leading to the disruption of epithelial tight junctions and an increase in paracellular permeability to macromolecules. Studies addressing the mechanisms of such epithelial disruption and the protective factors that prevent ethanol and acetaldehyde-mediated disruption of epithelial tight junctions are critically important in the investigations toward the search of preventive and therapeutic strategies for alcoholic liver disease.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Aryl urea analogs with broad-spectrum antibacterial activity

The preparation and evaluation of novel aryl urea analogs as broad-spectrum antibacterial agents is described. Numerous compounds showed low micromolar minimum inhibitory concentrations (MIC) against both Gram-positive and Gram-negative bacteria. Selected analogs also exhibited in vivo efficacy in a lethal murine model of bacterial septicemia.     

Thioxanthene-derived analogs as sigma(1) receptor ligands

An investigation of the structure-affinity relationships for the binding of thioxanthene-related structures indicates that an intact thioxanthene ring is not required for binding at sigma(1) receptors, and that with the appropriate structural modifications, affinity can be enhanced to the subnanomolar level. Certain of the analogs displayed sigma(1)-fold selectivity for sigma(1) versus sigma(2) receptors.