title: cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2).
abstract: NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal=100%; db-cGMP 100 microM=77+/-7%; 500 microM=54+/-10% and 1,000 microM=61+/-8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal=100%; 8-Br-cGMP=75+/-5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48+/-4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.
authors.affiliation: Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, and Department of Physiology, Wayne State University, 2799 West Grand Blvd., Detroit, MI 48202, USA.
authors.forename: Gustavo R
authors.initials: GR
authors.lastname: Ares
authors.forename: Paulo
authors.initials: P
authors.lastname: Caceres
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Alvarez-Leefmans
authors.forename: Pablo A
authors.initials: PA
authors.lastname: Ortiz
mesh_terms: D000818:Animals; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000378:metabolism*; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology*; D008138:Loop of Henle / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012965:Sodium Chloride / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1
chemical_list: D002712:Chlorides; D008565:Membrane Proteins; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; C016276:8-bromocyclic GMP; D003995:Dibutyryl Cyclic GMP; D012965:Sodium Chloride; D000242:Cyclic AMP; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517278:Pde2a protein, rat; D006152:Cyclic GMP