Effect of cAMP on the activity and the phosphorylation of Na+,K+-ATPase inrat thick ascending limb of Henle

Background. In rat kidney medullary thick ascending limb of Henle's loop (M TAL), activation of protein kinase A (PKA) was previously reported to inhib it Na+,K+-ATPase activity. This is paradoxical with the known stimulatory e ffect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A(2) (PLA(2)) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K+-ATPas e in MTAL. Methods. Ouabain-sensitive Rb-86 uptake and Na+,K+-ATPase activity were mea sured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K+-ATPase were determined in suspensions of outer medullary tubules . Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. Results. cAMP analogues or forskolin associated with 3-isobutyl-1-methylxan thine (IBMX) inhibited ouabain-sensitive Rb-86 uptake in nonoxygenated MTAL s. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-s ensitive Rb-86 uptake and Na+,K+-ATPase activity. Improved oxygen supply wa s associated with increased intracellular ATP content. The phosphorylation level of the Na+,K+-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated t ubules. Under nonoxygenated conditions, the inhibition of Na+,K+-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygena ted conditions, the stimulatory effect of cAMP analogues on ouabain-sensiti ve 86Rb uptake was linearly related and cosaturated with the level of phosp horylation of the Naf,Ki-ATPase alpha subunit. Conclusion. In oxygenated MTALs, PKA-mediated stimulation of Na+,K+-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorptio n. Under nonoxygenated conditions, this stimulatory pathway is likely overr idden by the PLA(2)-mediated inhibitory pathway, a possible adaptation to p rotect the cells against hypoxic damage.