Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Abstract-Several vasoconstrictor agents can regulate the phosphorylation status of the Na ϩ -K ϩ ATPase (NKA). We have recently demonstrated that mammalian tissues contain an endogenous bufadienolide, digitalis-like ␣ 1 -NKA-selective ligand, marinobufagenin (MBG). Protein kinase C induces phosphorylation of the ␣ 1 -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 ␣ 1 -NKA from human mesenteric artery (HMA) sarcolemma and rat kidney but not that of the ␣ 3 -NKA from rat fetal brain. In HMA sarcolemma, which predominantly contains ␣ 1 -NKA, PDA (50 nmol/L) potentiated the NKA-inhibitory effect of MBG at the level of high-affinity binding sites (0.05Ϯ0.03 nmol/L versus 4.0Ϯ1.7 nmol/L, PϽ0.05). In contrast, PDA did not affect the NKA inhibition by ouabain, an ␣ 3 -NKA ligand. In isolated endothelium-denuded HMA artery rings, 50 nmol/L PDA potentiated the MBG-induced vasoconstriction (EC 50 , 17Ϯ6 nmol/L versus 150Ϯ40 nmol/L; PϽ0.01). Our results suggest that ␣ 1 -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. Key Words: drug therapy Ⅲ ouabain Ⅲ Na ϩ -K ϩ -exchanging ATPase Ⅲ vasoconstriction Ⅲ protein kinases Ⅲ blood pressure Ⅲ bufanolides V arious vasorelaxants and vasoconstrictors can regulate vascular Na ϩ -K ϩ ATPase (NKA) activity via its phosphorylation/dephosphorylation by protein kinases and phosphatases. 1-3 Protein kinases phosphorylate the sodium pump in a tissue-and isoform-specific fashion. 4,5 Protein kinase C (PKC) directly phosphorylates the ␣ 1 -NKA isoform. 6,7 Several endogenous digitalis-like NKA inhibitors exist in mammalian plasma. 8 A cardenolide, a ouabain-like compound, was the first endogenous sodium pump inhibitor to be purified. 9 More recently, we have demonstrated that an endogenous bufadienolide NKA inhibitor, marinobufagenin (MBG), 10 exhibits greater affinity for the ␣ 1 -NKA isoform than for the ouabain-sensitive ␣ 3 -isoform. 11-13 The ␣ 1 -NKA is the major isoform in the kidney, vascular smooth muscle, and adult cardiomyocytes. 14,15 Although phosphorylation of NKA by PKC can affect the sensitivity of this enzyme to ouabain, 16 it is not known whether PKC phosphorylation of specific NKA isoforms is implicated in NKA inhibition by endogenous digitalis-like inhibitors, such as MBG. We hypothesized that protein kinase C-induced phosphorylation of the NKA can potentiate the effect of endogenous digitalis-like ligands, and that such potentiation can ...

Cicletanine antagonizes vasoconstriction induced by Na/K-ATPase inhibition via a PKC-dependent mechanism that does not involve inhibition of cyclic GMP phosphodiesterase (cGMP-PDE). This mechanism of action may be relevant to the greater potency of cicletanine in salt-sensitive hypertension in which plasma levels of endogenous digitalis-like cardiotonic steroids are elevated. Our findings also suggest that PKC is an important factor for cardiotonic steroid-Na/K-ATPase interactions on the vascular tone, and is therefore a potential target for therapeutic intervention in hypertension.

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