RGD Reference Report - cAMP stimulates apical exocytosis of the renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in the thick ascending limb: role of protein kinase A. - Rat Genome Database

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cAMP stimulates apical exocytosis of the renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in the thick ascending limb: role of protein kinase A.

Authors: Caceres, PS  Ares, GR  Ortiz, PA 
Citation: Caceres PS, etal., J Biol Chem. 2009 Sep 11;284(37):24965-71. doi: 10.1074/jbc.M109.037135. Epub 2009 Jul 10.
RGD ID: 7242914
Pubmed: PMID:19592485   (View Abstract at PubMed)
PMCID: PMC2757200   (View Article at PubMed Central)
DOI: DOI:10.1074/jbc.M109.037135   (Journal Full-text)

The apical renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 mediates NaCl absorption by the thick ascending limb (TAL) of Henle's loop. cAMP stimulates NKCC2 by enhancing steady-state apical membrane levels of this protein; however, the trafficking and signaling mechanisms by which this occurs have not been studied. Here, we report that stimulation of endogenous cAMP levels with either forskolin/3-isobutyl-1-methylxanthine (IBMX) or the V2 receptor agonist [deamino-Cys(1),d-Arg(8)]vasopressin increases steady-state surface NKCC2 and that the protein kinase A (PKA) inhibitor H-89 blocks this effect. Confocal imaging of apical surface NKCC2 in isolated perfused TALs confirmed a stimulatory effect of cAMP on apical trafficking that was blocked by PKA inhibition. Selective stimulation of PKA with the agonist N(6)-benzoyl-cAMP (500 microm) stimulated steady-state surface NKCC2, whereas the Epac-selective agonist 8-p-chlorophenylthio-2'-O-methyl-cAMP (100 and 250 microm) had no effect. To explore the trafficking mechanism by which cAMP increases apical NKCC2, we measured cumulative apical membrane exocytosis and NKCC2 exocytic insertion in TALs. By monitoring apical FM1-43 fluorescence, we observed rapid stimulation of apical exocytosis (2 min) by forskolin/IBMX. We also found constitutive exocytic insertion of NKCC2 in TALs over time, which was increased by 3-fold in the presence of forskolin/IBMX. PKA inhibition blunted cAMP-stimulated exocytic insertion but did not affect the rate of constitutive exocytosis. We conclude that cAMP stimulates steady-state apical surface NKCC2 by stimulating exocytic insertion and that this process is highly dependent on PKA but not Epac.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process

Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Slc12a1Ratditerpenoid metabolic process  IEP forskolinRGD 
Slc12a1Ratprotein insertion into membrane from inner side  IDA  RGD 

Cellular Component

Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Slc12a1Ratapical plasma membrane  IDA  RGD 
Slc12a1Ratcell surface  IDA  RGD 

Objects Annotated

Genes (Rattus norvegicus)
Slc12a1  (solute carrier family 12 member 1)

Additional Information