Mechanism of activation of ERK and H-K-ATPase by isoproterenol in rat cortical collecting duct.
Isoproterenol stimulates H-K-ATPase activity in rat cortical collecting duct beta-intercalated cells through a PKA-dependent pathway. This study aimed at determining the signaling pathway underlying this effect. H-K-ATPase activity was determined in microdissected collecting ducts preincubated with or without specific inhibitors or antibodies against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access to antibodies. Isoproterenol increased phosphorylation of ERK in a PKA-dependent manner, and inhibition of the ERK phosphorylation prevented the stimulation of H-K-ATPase. Antibodies against the monomeric G protein Ras or the kinase Raf-1 curtailed the stimulation of H-K-ATPase by isoproterenol, whereas antibodies against the related proteins Rap-1 and B-Raf had no effect. Pertussis toxin and inhibition of tyrosine kinases with genistein also curtailed isoproterenol-induced stimulation of H-K-ATPase. It is proposed that activation of PKA by isoproterenol induces the phosphorylation of beta-adrenergic receptors and the switch from G(s) to G(i) coupling. In turn, betagamma-subunits released from G(i) would activate a tyrosine kinase-Ras-Raf-1 pathway, leading to the activation of ERK1/2 and of H-K-ATPase.
Laroche-Joubert
Nicolas
N
Laboratoire de Biologie Intégrée des Cellules Rénales, Service de Biologie Cellulaire, Commissariat a l'Energie Atomique, Saclay, France.
Marsy
Sophie
S
Luriau
Stéphanie
S
Imbert-Teboul
Martine
M
Doucet
Alain
A
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology*; D007672:Kidney Cortex; D007685:Kidney Tubules, Collecting / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D011943:Receptors, Adrenergic, beta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017506:H(+)-K(+)-Exchanging ATPase; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D007545:Isoproterenol