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Biophysical characterisation of the transport properties of cultured renal inner medullary collecting duct cellsKoese, Hayrullah January 1999 (has links)
No description available.
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Search for the Basolateral Potassium Channel in the Shark Rectal Gland: Functional and Molecular Identification of a Task-1 Channel Coupled to Chloride SecretionTelles, Conner James 15 November 2006 (has links)
In the shark rectal gland (SRG), apical Cl[superscript]- secretion through CFTR channels is tightly coupled to a basolateral K[superscript]+ conductance. The identity of this K[superscript]+ conductive pathway is unknown. Studies were performed in the isolated perfused SRG with 16 K[superscript]+ channel inhibitors at their IC50 and with acidic perfusate. During maximal chloride secretion stimulated by forskolin and IBMX, secretion was inhibited >90% by barium chloride, a non-selective inhibitor of K[superscript]+ channels. Specific inhibitors of calcium sensitive, voltage sensitive, ATP sensitive, and inward rectifying K[superscript]+ channels had no effect on chloride secretion. The inhibitors quinidine, quinine, bupivicaine, anandamide, and low perfusate pH (6.0) abruptly and reversibly inhibited secretion by >90%, consistent with the presence of the Two-Pore-Domain (4TM 2P/KCNK/K2P) family of K+ channels. Degenerate primers were designed to regions of high amino acid homology in known mammal and teleost 4TM 2P K[superscript]+ channel subtypes: TWIK, THIK, TASK, TREK, and TRAAK. PCR with cDNA from several shark tissues identified a putative TASK-1 fragment (394 bp) in shark rectal gland, brain, gill, and kidney. 5and 3 RACE PCR was used to obtain the entire 3 sequence and partial 5 sequence of the shark gene. Genome walking was then used to obtain the remaining 5sequence, including 335 bp of untranslated region sequence upstream of the start codon. The full length clone (1282bp) had an open reading frame encoding a protein of 375 amino acids. This isoform was 80% identical at the amino acid level to the human TASK-1 protein (394 amino acids). Major structural features of the human protein were conserved in the shark ortholog, including the four transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2- and long COOH-termini, and an extended extracellular loop between M1 and P1. Shark and human TASK-1 full-length clones were expressed in Xenopus oocytes and studied with two electrode voltage clamp (TEVC) techniques. Both the shark and human TASK-1 channel showed identical current voltage relationships (outward rectifying) with a reversal potential near -90 mV compared to water injected controls. The responses to the inhibitor quinine, and the TASK-1 inhibitor bupivacaine, were identical in shark and human TASK-1. However, shark TASK-1 differed from the human ortholog in two critical responses: response to pH and the metal zinc. The pKa for shark TASK-1 was 7.75 vs. 7.37 for human TASK-1, values that are exceedingly close to the arterial pH for each species, suggesting that TASK-1 channels are regulated closely by the ambient pH. An antibody specific to shark TASK-1 was generated and expression of TASK-1 protein in the rectal gland was confirmed by confocal immuno-fluorescent microscopy which revealed localization to the basolateral membrane, with some apical staining. Shark rectal gland TASK-1 appears to be the major K[superscript]+ channel coupled to secretion in the SRG, is the oldest 4TM 2P family member identified to date, and is the first TASK-1 channel identified to play an essential role in chloride secreting epithelia.
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Wortmannin Inhibition of Forskolin-Stimulated Chloride Secretion by T84 CellsEcay, Tom W., Dickson, Jeffrey L., Conner, Tracy D. 31 July 2000 (has links)
The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (R(te)) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (I(SC)) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated I(SC). Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated I(SC). In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated I(SC) and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.
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