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.

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.