Protein kinase A-independent activation of ERK and H,K-ATPase by cAMP in native kidney cells: role of Epac I.
This study aimed at determining the signaling pathways underlying calcitonin- and isoproterenol-induced stimulation of H,K-ATPase in rat renal collecting duct. H,K-ATPase activity was determined in microdissected collecting ducts preincubated with or without either specific inhibitors or antibodies directed against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access of antibodies. The stimulation of H,K-ATPase by calcitonin and isoproterenol was mimicked by cAMP analogues and was abolished by adenylyl cyclase inhibition. Protein kinase A inhibition abolished isoproterenol but not calcitonin effect on H,K-ATPase. Calcitonin increased the phosphorylation of extracellular signal-regulated kinase (ERK) in a protein kinase A-independent manner, and the inhibition of the ERK phosphorylation prevented the stimulation of H,K-ATPase by calcitonin. Antibodies directed against either the cAMP-activated guanine-nucleotide exchange factor Epac I, the monomeric G protein Rap-1 or the kinase Raf-B, curtailed the stimulation of H,K-ATPase by calcitonin, whereas antibodies against the related monomeric G protein Ras or kinase Raf-1 had no effect. In conclusion, calcitonin stimulates H,K-ATPase through a cAMP/Epac I/Rap-1/Raf-B/ERK cascade.
Laroche-Joubert
Nicolas
N
Laboratoire de Biologie Intégrée des Cellules Rénales, Service de Biologie Cellulaire, Commissariat á l'Energie Atomique, Saclay, Unité de Recherche Associée 1859, CNRS, 91191 Gif-sur-Yvette Cedex, France.
Marsy
Sophie
S
Michelet
Stephanie
S
Imbert-Teboul
Martine
M
Doucet
Alain
A
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D017931:DNA Primers; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017506:H(+)-K(+)-Exchanging ATPase