No abstract

Rat collecting ducts exhibit type I or type III K(+)-ATPase activities when animals are fed a normal (NK) or a K(+)-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K(+)-ATPases. For this purpose, we searched for an effect on K(+)-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-D-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, alpha-intercalated cells, and beta-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and alpha-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K(+)-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K(+)-ATPase) and by dD-AVP in LK rats (type III K(+)-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K(+)-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K(+)-ATPase activity in alpha- and beta-intercalated CCD cells, whereas in LK rats it stimulates type III K(+)-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.

Isoproterenol stimulates H-K-ATPase activity in rat cortical collecting duct β-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 β-adrenergic receptors and the switch from Gs to Gi coupling. In turn, βγ-subunits released from Gi would activate a tyrosine kinase-Ras-Raf-1 pathway, leading to the activation of ERK1/2 and of H-K-ATPase.