High Affinity Ouabain Research Articles

Endogenous Ouabain: A Possible Link between High Salt Intake and Male Infertility

Male Infertility is an increasing global health issue, and one of the main findings in the semen of infertile men is spermatogenesis and sperm motility deficiencies. Despite the advances in male fertility, sperm abnormalities are complex and caused by multiple endogenous and exogenous factors. The Na, K ATPase α4 isoform is testis-specific, and its catalytic function is essential to maintain sperm quality, motility, and capacity. Exogenous ouabain is known for its inhibitory actions on the Na, K-ATPase, and α4 has a high affinity for ouabain. This heightened sensitivity is the basis for numerous experiments showing that Inhibition of the α4 by ouabain leads to sperm deterioration. It is interesting to note that endogenous ouabain is a cardiotonic steroid hormone found in animals and humans during normal conditions and increases in response to high salt intake. Also, infertile men with deficient sperm motility had increased levels of endogenous ouabain in the semen. Therefore, this article emphasizes the intersectionality between the following factors: 1) high salt intake and decreased sperm motility, 2) high salt intake and increased EO levels, 3) sperm Na, K-ATPase α4 isoform high affinity for ouabain, 4) effects of high ouabain on reduced sperm quality and motility, and 5) increased EO levels in infertile men with decreased sperm motility. Based on these observations, we hypothesize that endogenous ouabain is a possible link between high salt intake and male infertility.

The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter.

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na+,K+-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the α2 isoform of NKA (α2-NKA) that has a high affinity for ouabain, we hypothesized that α2-NKA mediates this effect. Knockdown of α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 μM), a Na+,Ca2+ exchanger (NCX) antagonist. α2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca2+ transients ([Ca2+]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca2+]t was observed at DIV5. At DIV6 almost no [Ca2+]t were observed either in control or in α2-siRNA-treated cultures. Immunocytochemical analyses showed that α2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of α2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that α2-NKA activity in OLCs affects NCX-mediated [Ca2+]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K+] changes, might locally influence NCX-mediated [Ca2+]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception.

Na,K-ATPase α4 is a testis-specific plasma membrane Na+ and K+ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

How does pressure overload cause cardiac hypertrophy and dysfunction? High-ouabain affinity cardiac Na+ pumps are crucial.

Left ventricular hypertrophy is frequently observed in hypertensive patients and is believed to be due to the pressure overload and cardiomyocyte stretch. Three recent reports on mice with genetically engineered Na+ pumps, however, have demonstrated that cardiac ouabain-sensitive α2-Na+ pumps play a key role in the pathogenesis of transaortic constriction-induced hypertrophy. Hypertrophy was delayed/attenuated in mice with mutant, ouabain-resistant α2-Na+ pumps and in mice with cardiac-selective knockout or transgenic overexpression of α2-Na+ pumps. The latter, seemingly paradoxical, findings can be explained by comparing the numbers of available (ouabain-free) high-affinity (α2) ouabain-binding sites in wild-type, knockout, and transgenic hearts. Conversely, hypertrophy was accelerated in α2-ouabain-resistant (R) mice in which the normally ouabain-resistant α1-Na+ pumps were mutated to an ouabain-sensitive (S) form (α1S/Sα2R/R or "SWAP" vs. wild-type or α1R/R α2S/S mice). Furthermore, transaortic constriction-induced hypertrophy in SWAP mice was prevented/reversed by immunoneutralizing circulating endogenous ouabain (EO). These findings show that EO and its receptor, ouabain-sensitive α2, are critical factors in pressure overload-induced cardiac hypertrophy. This complements reports linking elevated plasma EO to hypertension, cardiac hypertrophy, and failure in humans and elucidates the underappreciated role of the EO-Na+ pump pathway in cardiovascular disease.

Relationship Between Susceptibility to DMCM-Induced Generalized Motor Convulsions and Low-Affinity [3H]-Ouabain Binding in Membranes in Rat Brain.

Epilepsy is one of the most prevalent neurological disorders worldwide, but its underlying mechanisms have not yet been clarified. Among the possible molecular mechanisms that underlie its occurrence are those that are responsible for the neuronal ionic gradient, including the transmembrane enzyme Na+,K+;-adenosine triphosphatase (ATPase). Na+,K+-ATPase plays an important role in controlling neuronal excitability, and it is believed to be related to the pathophysiology of epilepsy. However, the specific isozymes that may be related to this neurological disorder remain to be determined. The α3 subunit-containing Na+,K+-ATPase isozyme has high affinity for ouabain and appears to play a major role in the pathogenesis of epilepsies. However, more studies are needed to evaluate the possible participation of Na+,K+- ATPase isozymes with lower affinity for ouabain (i.e., those that contain the α1 and α2 subunits). The present study investigated whether rats with high (HTR) and low (LTR) thresholds for clonic convulsions that are induced by a benzodiazepine inverse agonist differ in the binding of [3;H]- ouabain to Na+,K+-ATPase isozymes with lower affinity to ouabain in discrete brain regions. Compared with the HTR group, the LTR group exhibited lower binding of [3H]-ouabain in the brainstem and frontal cortex. This finding supports the hypothesis that epilepsy is associated with impairments in Na+,K+- ATPase activity. The results also suggest that Na+,K+;-ATPase isozymes that contain the α1/α2 subunits in these brain regions may underlie the susceptibility to methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate-induced convulsions.

Non-thermal microwave radiation-induced brain tissue dehydration as a potential factor for brain functional impairment

Based on our previous finding that metabolically controlled cell hydration is sensitive to the changes of physicochemical properties of cell aqua medium, which take place upon the effect of weak physical signals, including microwaves with non-thermal intensity (NT MW), it has been hypothesized that cell aqua medium serves as a primary target for NT MW effect on brain tissue. To elucidate the nature of the metabolic pathway through which the effect of NT MW-induced changes of physicochemical properties of cell aqua medium modulate cell hydration, the effects of intraperitoneal (IP) injection of PS treated by NT MW on brain tissue hydration, 45Ca2+ uptake, [3H]-ouabain binding with cell membrane and intracellular cyclic nucleotides have been studied. The obtained data indicate that PS treated by NT MW through the activation of high affinity ouabain receptors in membrane stimulates cAMP-dependent Na+/Ca2+ exchange in reverse mode (R), which leads to brain tissue hydration in healthy (young) and dehydration in non-healthy (old) rats.As NT MW radiation-induced activation of R Na+/Ca2+ exchange leads to the increase of intracellular Ca2+ ([Ca2+]i), it is considered as a potential factor leading to the brain functional impairment, especially when brain metabolic activity is depressed (e.g. during aging).

Pivotal role of α2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease.

Reduced smooth muscle (SM)-specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensinII (AngII) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases AngII/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain-resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific α2 knockout, cardio-specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by AngII and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain-sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+ /Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction.

Arterial α2-Na+ pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific α2 mice.

Arterial myocytes express α1-catalytic subunit isoform Na(+) pumps (75-80% of total), which are ouabain resistant in rodents, and high ouabain affinity α2-Na(+) pumps. Mice with globally reduced α2-pumps (but not α1-pumps), mice with mutant ouabain-resistant α2-pumps, and mice with a smooth muscle (SM)-specific α2-transgene (α2 (SM-Tg)) that induces overexpression all have altered blood pressure (BP) phenotypes. We generated α2 (SM-DN) mice with SM-specific α2 (not α1) reduction (>50%) using nonfunctional dominant negative (DN) α2. We compared α2 (SM-DN) and α2 (SM-Tg) mice to controls to determine how arterial SM α2-pumps affect vasoconstriction and BP. α2 (SM-DN) mice had elevated basal mean BP (mean BP by telemetry: 117 ± 4 vs. 106 ± 1 mmHg, n = 7/7, P < 0.01) and enhanced BP responses to chronic ANG II infusion (240 ng·kg(-1)·min(-1)) and high (6%) NaCl. Several arterial Ca(2+) transporters, including Na(+)/Ca(2+) exchanger 1 (NCX1) and sarcoplasmic reticulum and plasma membrane Ca(2+) pumps [sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and plasma membrane Ca(2+)-ATPase 1 (PMCA1)], were also reduced (>50%). α2 (SM-DN) mouse isolated small arteries had reduced myogenic reactivity, perhaps because of reduced Ca(2+) transporter expression. In contrast, α2 (SM-Tg) mouse aortas overexpressed α2 (>2-fold), NCX1, SERCA2, and PMCA1 (43). α2 (SM-Tg) mice had reduced basal mean BP (104 ± 1 vs. 109 ± 2 mmHg, n = 15/9, P < 0.02) and attenuated BP responses to chronic ANG II (300-400 ng·kg(-1)·min(-1)) with or without 2% NaCl but normal myogenic reactivity. NCX1 expression was inversely related to basal BP in SM-α2 engineered mice but was directly related in SM-NCX1 engineered mice. NCX1, which usually mediates arterial Ca(2+) entry, and α2-Na(+) pumps colocalize at plasma membrane-sarcoplasmic reticulum junctions and functionally couple via the local Na(+) gradient to help regulate cell Ca(2+). Altered Ca(2+) transporter expression in SM-α2 engineered mice apparently compensates to minimize Ca(2+) overload (α2 (SM-DN)) or depletion (α2 (SM-Tg)) and attenuate BP changes. In contrast, Ca(2+) transporter upregulation, observed in many rodent hypertension models, should enhance Ca(2+) entry and signaling and contribute significantly to BP elevation.

Ligand orientation in a membrane-embedded receptor site revealed by solid-state NMR with paramagnetic relaxation enhancement.

NMR relaxation enhancement by paramagnetic metals provides powerful restraints on the three-dimensional structures of proteins in solution, and this approach has recently been utilized in several NMR structural investigations of proteins in the solid-state. Here we utilize paramagnetic relaxation enhancement (PRE) by Mn(2+) with cross-polarization magic-angle spinning (CP-MAS) solid-state NMR to investigate the interaction of a membrane-embedded protein the Na,K-ATPase (NKA) with a cardiotonic steroid inhibitor. The inhibitor, a diacetonide derivate of the cardiac glycoside ouabain, with (13)C labelled acetonide groups in the rhamnose sugar and steroid moieties ([(13)C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the (13)C CP-MAS solid-state NMR spectra of the NKA-[(13)C2]ODA complex exhibit distinct signals for the two (13)C labels of the inhibitor when bound to the ouabain site of membrane-embedded NKA. Recent crystal structures of NKA indicate that the catalytic α-subunit binds a single Mn(2+) in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn(2+) broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn(2+) site than is the steroid group. These observations agree with computational molecular docking simulations and are consistent with ODA adopting an inverted orientation compared to ouabain in the cardiac glycoside site, with the modified rhamnose group drawn toward the transmembrane centre of the protein. This work demonstrates that PRE can provide unique information on the positions and orientations of ligands within their binding pockets of transmembrane proteins.

Abstract 19: Blood Pressure Correlates Directly with the Expression Level of High Ouabain Affinity a2 Na+ Pumps in Arterial Smooth Muscle

High ouabain affinity α2 Na + pumps are expressed in arterial smooth muscle (ASM). They help regulate blood pressure (BP) in several forms of hypertension, but their influence on basal BP is controversial. We determined basal BP (telemetry), and the effects of subcutaneous (sc) angiotensin II (Ang II) and dietary salt on BP in: 1) α2 SM/Tg mice, which express an α2 transgene controlled by an α-actin smooth muscle (SM) promoter; 2) α2 SM/DN mice, which express a truncated α2 gene, dominant negative for native α2 Na + pumps, controlled by the SM myosin heavy chain promoter. Expression (immunoblots) of α2 was significantly up- and down-regulated in SM from α2 SM/Tg and α2 SM/DN mice, respectively. Basal 24 hr mean BP (MBP) was reduced in α2 SM/Tg mice: 103±1 ( n =15) vs 110±2 mm Hg ( n =9) in WT controls; P &lt;0.01. Ang II, 400 ng/kg/min sc x 2 wks, increased MBP in both WT and transgenic mice ( n =5 each; P &lt;0.01), but the increase (Δ) was smaller in α2 SM/Tg , Δ=20.3±3.3, than in WT mice, Δ=30.9±6.4 mm Hg . Also, a high (2% x 2 wks) NaCl diet, along with a sc 300 ng/kg/min (low dose) Ang II infusion, elevated systolic BP (SBP) in the WT mice (Δ=12.6±1.8 mm Hg; n =4; P &lt;0.05), but not in the α2 SM/Tg mice (Δ=7.2±2.0 mm Hg; n =9; NS=not significant). In contrast, in α2 SM/DN mice, basal SBP was elevated: 128±4 mm Hg ( n =11) vs 109±4 mm Hg ( n =10) in WT; P &lt;0.01. Furthermore, both high NaCl (HS, 6% x 10 days), and low dose sc Ang II infusion (240 ng/kg/min x 8 days), individually induced significantly greater increases in MBP in α2 SM/DN than in WT mice. In α2 SM/DN mice, the HS diet raised MBP by Δ=6.8±0.7 mm Hg ( n =5; P &lt;0.01); Ang II elevated MBP by Δ=17.1±3.7 mm Hg ( n =5; P &lt;0.05). In WT mice, however, HS did not elevate MBP (Δ=2.6±1.4; n =5; NS), and Ang II elevated MBP by only Δ=6.9±4.0 mm Hg; n =5; P &lt;0.05). Conclusion: ASM α2 Na + pumps help regulate basal BP and vascular reactivity. Reduced α2 expression raises basal BP and augments arterial responses to salt and Ang II; increased ASM α2 expression has the opposite effects. The α2 Na + pumps co-localize with Na/Ca exchangers in plasma membrane (PM) microdomains at MP-sarcoplasmic reticulum junctions; they are a crucial link between circulating endogenous ouabain and the control of ASM Ca 2+ homeostasis, arterial contraction and tone.

Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling.

BackgroundInsulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions.Results[125I]Insulin binding (Kd = 54 ± 6 nM) at the oocyte plasma membrane activates membrane serine protease(s), followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi). Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft) membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi.ConclusionsUnder physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

Age‐dependent magnetosensitivity of heart muscle ouabain receptors

In our previous work we have shown that the age-dependent decrease in the magnetosensitivity of heart muscle hydration is accompanied by a dysfunction of the Na(+) /K(+) pump. The reciprocal relation between the Na(+/) K(+) pump and Na(+) /Ca(2+) exchange in development was suggested as a possible pathway for the age-dependent decrease in the magnetosensitivity of heart muscle hydration (water content). Because high and low affinity ouabain receptors in cell membranes are involved in Na(+) /Ca(2+) exchange and Na(+) /K(+) pump functions, respectively, the effect of a 0.2 T static magnetic field (SMF) on dose-dependent, ouabain-induced hydration and [(3) H]-ouabain binding with heart muscle tissues in young, adult and older rats was studied. Three populations of receptors in membranes with high (10(-11) -10(-9) M), middle (10(-9) -10(-7) M) and low (10(-7) -10(-4) M) affinity to [(3) H]-ouabain were distinguished, which had specific dose-dependent [(3) H]-ouabain binding kinetics and effects on muscle hydration. The magnetosensitivity of [(3) H]-ouabain binding kinetics with high affinity receptors was prominent in all the three age groups of animals, while with low affinity receptors it was more expressed only in the young group of animals. All three types of receptors that caused modulations of muscle hydration were age dependent and magnetosensitive. Based on the obtained data we came to the conclusion that heart muscle hydration in young animals is more magnetosensitive due to the intense expression of high affinity ouabain receptors, which declines with aging.

Abstract 525: Nanomolar Ouabain Increases Na/Ca Exchanger-1 Expression and Enhances Ca2+ Signaling in Human Arterial Myocytes: A Mechanism That Links Salt to Increased Vascular Resistance?

The mechanisms by which excess dietary NaCl raises blood pressure (BP) in hypertension are unresolved. Much evidence indicates that endogenous ouabain (EO, an adrenocortical hormone and Na + pump blocker) plays a role. In rodents, arterial smooth muscle cell (ASMC) Na + pumps with an α2 catalytic subunit (ouabain EC 50 ≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na + pumps have an α1 subunit (ouabain EC 50 ≈5 μM). Human α1 Na + pumps, however, have high ouabain affinity (EC 50 ≈ 20 nM). Therefore, we studied the expression, distribution and function of Na + pump α subunit isoforms in ASMCs from surgically-recovered human artery segments. Immunoblots and immunocytochemistry reveal that human ASMCs (hASMCs) express α1 and α2 Na + pumps. As in rodents, α2, but not α1, pumps are confined to plasma membrane (PM) microdomains adjacent to sarcoplasmic reticulum, SR (identified with ER Tracker, an SR stain, and anti-SR Ca 2+ pump-2, SERCA2, antibodies). Na/Ca exchanger-1 (NCX1) and TRPC6 (component of receptor-operated channels, ROCs) co-localize with α2 in the PM microdomains. Incubation (72 hr) with 100 nM ouabain (blocks nearly all α1 and α2 pumps) was toxic to most cultured hASMCs, but 10 nM ouabain (blocks 90-95% of α2 and &lt;&lt;50% of α1 pumps) increased NCX1 and SERCA2 expression by 45±10% and 31±6%, respectively ( P &lt;0.05; n =5 patients). Ca 2+ transients were measured with fura 2: The 72 hr, 10 nM ouabain pre-treatment increased 10 μM ATP-induced SR Ca 2+ release in 0Ca 2+ media by 27±8% ( P &lt;0.05; n =3), and increased Ca 2+ influx when external Ca 2+ was restored (ATP still present) by 96±15% ( P &lt;0.01; n =3 patients). This Ca 2+ influx was likely mediated primarily by ROCs and store-operated Ca 2+ channels. Thus, α2 Na + pumps, NCX1, ROCs, and the SR are structurally and functionally linked. Freshly-isolated myocytes from the arteries of several rat hypertension models display comparable protein expression and Ca 2+ signaling changes. We conclude that the same molecular mechanisms regulate long-term Ca 2+ homeostasis and signaling in human and rodent ASMCs. These ouabain/EO-modulated mechanisms underlie the ‘whole body autoregulation’ associated with increased vascular resistance and elevation of BP in human hypertension.

Nanomolar ouabain increases NCX1 expression and enhances Ca2+signaling in human arterial myocytes: a mechanism that links salt to increased vascular resistance?

The mechanisms by which NaCl raises blood pressure (BP) in hypertension are unresolved, but much evidence indicates that endogenous ouabain is involved. In rodents, arterial smooth muscle cell (ASMC) Na(+) pumps with an α(2)-catalytic subunit (ouabain EC(50) ≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na(+) pumps have an α(1)-subunit (ouabain EC(50) ≈ 5 μM). Human α(1)-Na(+) pumps, however, have high ouabain affinity (EC(50) ≈ 10-20 nM). We used immunoblotting, immunocytochemistry, and Ca(2+) imaging (fura-2) to examine the expression, distribution, and function of Na(+) pump α-subunit isoforms in human arteries and primary cultured human ASMCs (hASMCs). hASMCs express α(1)- and α(2)-Na(+) pumps. Further, α(2)-, but not α(1)-, pumps are confined to plasma membrane microdomains adjacent to sarcoplasmic reticulum (SR), where they colocalize with Na/Ca exchanger-1 (NCX1) and C-type transient receptor potential-6 (receptor-operated channels, ROCs). Prolonged inhibition (72 h) with 100 nM ouabain (blocks nearly all α(1)- and α(2)-pumps) was toxic to most cultured hASMCs. Treatment with 10 nM ouabain (72 h), however, increased NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and augmented ATP (10 μM)-induced SR Ca(2+) release in 0 Ca(2+), ouabain-free media, and Ca(2+) influx after external Ca(2+) restoration. The latter was likely mediated primarily by ROCs and store-operated Ca(2+) channels. These hASMC protein expression and Ca(2+) signaling changes are comparable with previous observations on myocytes isolated from arteries of many rat hypertension models. We conclude that the same structurally and functionally coupled mechanisms (α(2)-Na(+) pumps, NCX1, ROCs, and the SR) regulate Ca(2+) homeostasis and signaling in hASMCs and rodent ASMCs. These ouabain/endogenous ouabain-modulated mechanisms underlie the whole body autoregulation associated with increased vascular resistance and elevation of BP in human, salt-sensitive hypertension.

Age dependency of high-affinity ouabain receptors and their magnetosensitivity

Age dependency of [3H]-ouabain binding, 45Ca2+ eflux and its magnetosensitivity in rats’ brain cortex and heart muscle tissues were studied. Curves of dose-dependent [3H]-ouabain binding consisted of three components with different affinities (10−7–10−4 M (α1); 10−9–10−7 M (α2); and 10−11–10−9 M (α3)). These curves were also characterized by different dose-dependent kinetics. [3H]-ouabain binding with α3 receptors in brain cortex and heart muscle tissues of young and adult animals had a dose-dependent character, while that in old ones had a dose-independent character. A 0.2 T static magnetic field (SMF) exposure had modulation effect on ouabain binding with α1, α2 and α3 receptors in young rats, while in adult ones, only α3 receptors were magnetosensitive. In old animals, SMF exposure had no significant effect on ouabain binding with α3 receptors in brain cortex, while in heart muscle, it had inhibitory effect on it. Age-dependent effect of ouabain impact on 45Ca2+ efflux showed that all concentrations of ouabain lead to inhibitory effect in young animals’ brain cortex and heart muscle (with the exception of 10−10 and 10−6 M), while in old ones, it had activation effect as compared with data received without ouabain. SMF exposure in young animals had activation effect on 45Ca2+ efflux from brain cortex and heart muscle in data without ouabain, and in old rats, 45Ca2+ efflux from brain cortex was magnetic insensitive. In old animals, SMF increased 45Ca2+ efflux even after extra low concentration of ouabain. It is suggested that α3 receptors having a crucial role in the regulation of Na+/Ca2+ exchange serve as age-dependent magnetosensors of excitable cells.

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1 <or= alpha3 < alpha2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K(D) alpha1 = alpha3 = alpha2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K(+) ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately alpha2-selective. This supports a major role of alpha2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Progesterone binding to the α1-subunit of the Na/K-ATPase on the cell surface: Insights from computational modeling

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [ 3H]ouabain and [ 3H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112 kDa α1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding α1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar β and hydrophobic α surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg 118, Asp 129 and possibly Glu 122–124 in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys 111 near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.

Analysis of the gastric H,K ATPase for ion pathways and inhibitor binding sites.

New models of the gastric H,K ATPase in the E1K and E2P states are presented as the first structures of a K+ counter-transport P2-type ATPase exhibiting ion entry and exit paths. Homology modeling was first used to generate a starting conformation from the srCa ATPase E2P form (PDB code 1wpg) that contains bound MgADP. Energy minimization of the model showed a conserved adenosine site but nonconserved polyphosphate contacts compared to the srCa ATPase. Molecular dynamics was then employed to expand the luminal entry sufficiently to allow access of the rigid K+ competitive naphthyridine inhibitor, Byk99, to its binding site within the membrane domain. The new E2P model had increased separation between transmembrane segments M3 through M8, and addition of water in this space showed not only an inhibitor entry path to the luminal vestibule but also a channel leading to the ion binding site. Addition of K+ to the hydrated channel with molecular dynamics modeling of ion movement identified a pathway for K+ from the lumen to the ion binding site to give E2K. A K+ exit path to the cytoplasm operating during the normal catalytic cycle is also proposed on the basis of an E1K homology model derived from the E12Ca2+ form of the srCa ATPase (PDB code 1su4). Autodock analyses of the new E2P model now correctly discriminate between high- and low-affinity K+ competitive inhibitors. Finally, the expanded luminal vestibule of the E2P model explains high-affinity ouabain binding in a mutant of the H,K ATPase [Qiu et al. (2005) J. Biol. Chem. 280, 32349-32355].

Conversion of the Low Affinity Ouabain-binding Site of Non-gastric H,K-ATPase into a High Affinity Binding Site by Substitution of Only Five Amino Acids

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.