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.