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Na+/Ca2+ exchanger in epithelial cells of the porcine cortical thick ascending limb. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F411-F418,1996.-Intracellular Ca2+ concentration ( [Ca2+]i) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+]; was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) had basal [Ca2+]. 10 f 86 IT 2 nM. Removal of extracellular Na (Na,f ) or voltage depolarization with KC1 (in the presence of Na,f) resulted in a rapid and reversible maximal elevation of [Ca2+]i (1,023 t 72 nM, n = ZS), which was dependent on the presence of external Ca 2+ (Caf’). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Naz or voltage depolarization were dependent on pretreatment with ouabain and increases in [Na+]i. The presence of a Na+/Ca2+ exchanger was confirmed with hybridization techniques, and the isoform was identified by sequencing the alternative splicing site within the intracellular loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tissue and single CTAL cells by reverse transcription-polymerase chain reaction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D, which is characteristic of one isoform (NACA3) of the renal Na+/Ca2+ exchanger. The results provide both functional and molecular evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.

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Abstract Modulation of Nat/ Ca2+ exchange in epithelial cells of porcine thick ascending limb. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F953-F959, 1996.-We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ( [Ca2+];) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) from 10 to 20 mM. These cells had normal basal [Ca2+]; (79 2 3 nM). Substitution of extracellular NaCl (50 mM) with KC1 resulted in the rapid elevation of [Ca2+]; to maximal levels of 795 t 60 nM (n = 17). The increments of [Ca2+]; were associated with [Na+]i. We next determined the modulation of Na+lCa2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca2+- calmodulin inhibitor, resulted in a shift of the [Na+]i dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in halfmaximal [Ca2+]; increase near normal [Na+]i of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+]i was not required to elicit increments in [Ca2+];. These data indicate that Na+/Ca2+ exchange is present in CTAL cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.

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