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

Effect of cAMP on the activity and the phosphorylation of Na + ,K + -ATPase in rat thick ascending limb of Henle. Background In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na + ,K + -ATPase activity. This is paradoxical with the known stimulatory effect 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 + -ATPase in MTAL. Methods Ouabain-sensitive 86 Rb uptake and Na + ,K + -ATPase activity were measured 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-methylxanthine (IBMX) inhibited ouabain-sensitive 86 Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86 Rb uptake and Na + ,K + -ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na + ,K + -ATPase α subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na + ,K + -ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86 Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na + ,K + -ATPase α subunit. Conclusion In oxygenated MTALs, PKA-mediated stimulation of Na + ,K + -ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA 2 -mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.