Phorbol Diacetate Potentiates Na ⁺ -K ⁺ ATPase Inhibition by a Putative Endogenous Ligand, Marinobufagenin

Abstract

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 ...

Endogenous sodium pump inhibitors and age-associated increases in salt sensitivity of blood pressure in normotensives

“…Specific antibodies against MBG prevent both the acute and sustained NaCl-induced increase in arterial pressure (10). A prohypertensive effect of SPI in response to salt loading in hypertensive prone rats has been repeatedly observed (9,10,12,13,29). Our results show that a subacute increase in dietary NaCl elicits a sustained increase in MBG that is directly related to an increase in renal FENa and inversely related to NaCl sensitivity of SBP in population of normotensive middle-age and older women.…”

Ouabain- and Marinobufagenin-Induced Proliferation of Human Umbilical Vein Smooth Muscle Cells and a Rat Vascular Smooth Muscle Cell Line, A7r5

“…Indeed, with higher concentrations of ouabain, cell proliferation was diminished. The similar pharmacological profile by which these compounds stimulate proliferation and inhibit Na ϩ ,K ϩ -ATPase activity in human and rat VSMCs as described in the literature 18,19 strongly suggests that the proliferative actions of these compounds are a result of their binding to the ␣-subunit of the sodium pump. The suggestion has been made that for the prostate cells, higher concentrations of ouabain activate apoptosis in these cells, and although there is some evidence that this may occur in rat VSMCs as well, this is still controversial.…”

“…This 10-fold difference in sensitivity in rat A7r5 cells is in general agreement with the 20-fold higher IC 50 values for ouabain that inhibit rat aortic Na ϩ /K ϩ -ATPase compared with MBG. 18,19 Thus, the similar rank order for ouabain-and MBG-induced proliferation and inhibition of Na ϩ /K ϩ -ATPase activity in human and rat VSMCs also suggests that the proliferative actions of ouabain and MBG are a result of their binding to the ␣-subunit of the sodium-pump.…”

ANP Differentially Modulates Marinobufagenin-Induced Sodium Pump Inhibition in Kidney and Aorta

“…36 Aortic rings were minced by scissors, processed with Polytron 20S homogenizer (Kinematica) in 250 mmol/L of sucrose and 5 mmol/L of histidine solution (4°C; pH 7.4), and centrifuged (6000g; 15 minutes; 4°C) in a Sorvall RC-5B centrifuge (DuPont Instruments). The pellet was homogenized in a glass-Teflon homogenizer and combined with the supernatant, which was respun at 20 000g for 30 minutes at 4°C, and the resultant supernatant centrifuged (Beckman L8-N; 148 000g; 90 minutes; 4°C).…”