Phorbol diacetate potentiates Na⁺-K⁺ ATPase inhibition by a putative endogenous ligand, marinobufagenin

Several vasoconstrictor agents can regulate the phosphorylation status of the Na⁺-K⁺ ATPase (NKA). We have recently demonstrated that mammalian tissues contain an endogenous bufadienolide, digitalis-like α₁-NKA-selective ligand, marinobufagenin (MBG). Protein kinase C induces phosphorylation of the α₁-NKA isoform, the major isoform in vascular smooth muscle, kidney, and heart cells. We hypothesized that protein kinase C-induced phosphorylation of NKA can potentiate the effect of endogenous digitalis-like ligands, and that such potentiation can occur in an NKA isoform-specific fashion. A protein kinase C activator, phorbol 12,13-diacetate (PDA, 50 nmol/L), induced phosphorylation of the α₁-NKA from human mesenteric artery (HMA) sarcolemma and rat kidney but not that of the α₃-NKA from rat fetal brain. In HMA Sarcolemma, which predominantly contains α₁-NKA, PDA (50 nmol/L) potentiated the NKA-inhibitory effect of MBG at the level of high-affinity binding sites (0.05±0.03 nmol/L versus 4.0±1.7 nmol/L, P<0.05). In contrast, PDA did not affect the NKA inhibition by ouabain, an α₃-NKA ligand. In isolated endothelium-denuded HMA artery rings, 50 nmol/L PDA potentiated the MBG-induced vasoconstriction (EC₅₀, 17±6 nmol/L versus 150±40 nmol/L; P<0.01). Our results suggest that α₁-isoform-specific NKA inhibition by the endogenous digitalis-like ligand, MBG, is substantially enhanced via NKA phosphorylation by protein kinase C. Thus, an interaction of protein kinase C-dependent phosphorylation and MBG on NKA activity may underlie the synergistic vasoactive effects of MBG and other endogenous vasoconstrictors in hypertension.