Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na(+) pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14-16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (-8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta -60% and mesenteric artery -30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased ∼90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca(2+) entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).

The localization and density of Kir2.3 channels are influenced by the balance between PDZ protein interaction at the cell surface and routing into the endocytic pathway. Here, we explore mechanisms by which the Kir2.3 channel is directed into the endocytic pathway. We found that Kir2.3 channels are constitutively internalized from the cell surface in a dynamin-dependent manner, indicative of vesicle-mediated endocytosis. The rate of Kir2.3 endocytosis was dramatically attenuated following RNA interference-mediated knockdown of either ␣ adaptin (AP-2 clathrin adaptor) or clathrin heavy chain, revealing that Kir2.3 is internalized by an AP-2 clathrin-dependent mechanism. Structure-rationalized mutagenesis studies of a number of different potential AP-2 interaction motifs indicate that internalization of Kir2.3 is largely dependent on a non-canonical di-isoleucine motif (II413) embedded within the C terminus. Internalization assays using CD4-Kir2.3 chimeras demonstrate that the di-isoleucine signal acts in an autonomous and transplantable manner. Kir2.3 co-immunoprecipitates with ␣ adaptin, and disruption of the di-isoleucine motif decreased interaction of the channel with AP-2. Replacement of the di-isoleucine motif with a canonical di-leucine internalization signal actually blocked Kir2.3 endocytosis. Moreover, in yeast three-hybrid studies, the Kir2.3 di-isoleucine motif does not bind the AP-2 ␣C-2 hemicomplex in the way that has been recently observed for canonical di-leucine signals. Altogether, the results indicate that Kir2.3 channels are marked for clathrin-dependent internalization from the plasma membrane by a novel AP-2-dependent signal.

Endogenous ouabain (EO) is elevated and correlates with blood pressure (BP) in ~50% of patients with essential hypertension. “Ouabain‐like” inhibitors are increased in pre‐eclampsia (PE) and correlate with BP. EO and ouabain inhibit the α2‐Na+ pump isoform in arterial myocytes, enhance Ca2+ entry via NCX1.3, and raise BP. We explored whether EO per se is elevated in pregnancy and pseudopregnancy (PSP) and determined whether exogenous ouabain induces PE in otherwise normal pregnant rats. We implanted mini‐osmotic pumps containing ouabain in pregnant (E10), non‐pregnant, and PSP rats and measured BP by tail cuff. At E20 or equivalent, blood, adrenals, and aortas were collected to determine ouabain (RIA), ouabain‐like materials (RRA), NCX, and α2‐Na+ pump expression (Western blot). Plasma EO increased in pregnant (4.7 ± 1.9 nM plasma equiv) vs non‐pregnant (0.3 ± 0.02 nM plasma equiv, p<0.001) rats and was confirmed by HPLC‐RIA and mass spectrometry. Adrenal EO content was lower in pregnant (12.3 ± 5.0 nmoles/kg) vs non‐pregnant rats (32.5 ± 15.3 nmoles/kg, p<0.01). NCX and α2‐Na+ pump expression decreased in pregnancy. Ouabain infusion raised BP in non‐pregnant rats (+21.3 mmHg, p<0.01), but had no effect on BP in their pregnant or PSP counterparts. These data show: 1) that the pregnant and PSP conditions are physiologically high EO states; 2) both conditions are associated with the development of a physiological resistance to ouabain that prevents BP from increasing. 3) The mechanism of the ouabain resistance is exclusively maternal, and likely related to decreased expression of, and reduced Ca2+ entry by, vascular NCX. Failure of the maternal circulation to develop resistance to EO may underlie hypertension in PE.

Ouabain‐like inhibitors are elevated in ∼50% of patients with essential hypertension and patients with pre‐eclampsia (PE). Prolonged administration of ouabain induces hypertension in Sprague‐Dawley rats. Accordingly, we hypothesized that ouabain might induce PE in otherwise normal pregnant rats. We implanted ouabain pellets subcutaneously in pregnant (E10) and non‐pregnant rats and measured their blood pressures (BP) weekly by tail cuff. After delivery, trunk blood was collected to determine ouabain‐like activity by radioreceptor assay. Ouabain pellets raised BP significantly in non‐pregnant rats (+18.3 mmHg, p<0.05) compared to non‐pregnant controls (+5.4 mmHg). Surprisingly, there was no significant difference in BP between pregnant rats with ouabain and their pregnant controls. Moreover, there was a significant increase in ouabain‐like activity in all pregnant (57.0 ± 3.1 nmoles plasma equiv/L) verses non‐pregnant (12.4 ± 1.1 nmoles plasma equiv/L) rats, irrespective of BP. These data show 1) that normal pregnant rats become resistant to the hypertensive effect of ouabain; 2) the pregnant state is associated with elevated levels of polar ouabain‐like materials that compete with 3H‐ouabain for binding to Na/K‐ATPase. These ouabain‐like factors may act to prevent increases in BP, or oppose the hypotensive effects of pregnancy. Funded by NHLBI 078870/75584, NIGMS Initiative for Minority Student Development Grant (R25‐GM55036), and Procter and Gamble