Regulation of Na+,K+-ATPase activity by protein phosphatase-1 and protein kinase C : studies in infant and adult renal tissue and on vascular smooth muscle cells

Abstract

Na+, K+-ATPase is short-term modulated by hormones, involving activation of signal transduction system. The present study was designed to elucidate the role of the signaling molecules PP-1, PKC and arachidonic acid (AA) metabolites in the regulation of Na+, K+-ATPase by reversible phosphorylation, the final step of the signal transduction, and to examine the ontogeny of the signaling system.

In situ hybridization and immunoblotting showed that PP-1 is present in the kidney. Inhibition of PP-1 with OA or CL-A phosphorylated Na+, K+-ATPase [alpha]1 subunit. Activation of PKC by PDBu phosphorylated Na+, K+-ATPase at Ser23 of the [alpha]1 subunit in vivo. Na+, K+-ATPase phosphorylation mediated by PP-1 inhibition or PKC activation was associated with inhibition of activity. PDBu and OA had synergistic effects on Na+, K+-ATPase phosphorylation and inhibition of Nat, K+-ATPase activity.

PKC is regulated by lipid second messengers. We found that in contrast to PLC pathway, the PLA2-AA pathway activates Ca2+-dependent conventional PKCs at low levels of [Ca2+]i. The AA metabolite 20-HETE synergized with phosphatidylserine and a DAG analog to activate PKC. 20-HETE phosphorylated Na+, K+-ATPase at the PKC consensus site, Ser23.

Atypical PKC is engaged in IGF-I signaling. We found that IGF-I stimulated VSMC Na+, K+-ATPase activity in a tyrosine kinase- and P13K-dependent manner. Atypical PKC was required for IGF-I action.

Regulation of renal Na+ excretion is known to change postnatally. We showed here that PLA2-AA intracellular signaling pathways modulating renal Na+, K+-ATPase are age-dependent. AA dose-dependently inhibited PCT Na+, K+-ATPase activity in both infant and adult rats via different AA metabolites; in infant rats the lipoxygenase pathway and in adult rats the CYP450 pathway. Lipoxygenase mRNA was only detected in infant rats. In contrast, renal isoforms of CYP450 mRNA were detected in both infant and adult cortex, but the expression was much more prominent in adult cortex.

In conclusion, the level of Na+, K+-ATPase phosphorylation is determined by the balance between protein kinases and phosphatases. Both PP-1 and PKC play important roles for the modulation of the level of phosphorylation and activity of Na+, K+-ATPase in rat renal tissue. Conventional PKCs, that modulate Na+, K+-ATPase, can be activated via lipid signaling pathway in a Ca2+-independent manner. Different isoforms of PKC may have different effects on Na+, K+-ATPase activity in different tissues. There are developmental changes in the intracellular signaling systems that regulate rat renal Na+, K+-ATPase. This may have an impact on the regulation of Na+ metabolism in infancy.