Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and ß subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

Whither digitalis? What we can still learn from cardiotonic steroids about heart failure and hypertension.

Cloning of the "Na+ pump" (Na+,K+-ATPase or NKA) and identification of a circulating ligand, endogenous ouabain (EO), a cardiotonic steroid (CTS), triggered seminal discoveries regarding EO and its NKA receptor in cardiovascular function and the pathophysiology of heart failure (HF) and hypertension. Cardiotonic digitalis preparations were a preferred treatment for HF for two centuries, but digoxin was only marginally effective in a large clinical trial (1997). This led to diminished digoxin use. Missing from the trial, however, was any consideration that endogenous CTS might influence digitalis' efficacy. Digoxin, at therapeutic concentrations, acutely inhibits NKA but, remarkably, antagonizes ouabain's action. Prolonged treatment with ouabain, but not digoxin, causes hypertension in rodents; in this model, digoxin lowers blood pressure (BP). Furthermore, NKA-bound ouabain and digoxin modulate different protein kinase signaling pathways and have disparate long-term cardiovascular effects. Reports of "brain ouabain" led to the elucidation of a new, slow neuromodulatory pathway in the brain; locally generated EO and the α2 NKA isoform help regulate sympathetic drive to the heart and vasculature. The roles of EO and α2 NKA have been studied by EO assay, ouabain-resistant mutation of α2 NKA, and immunoneutralization of EO with ouabain-binding Fab fragments. The NKA α2 CTS binding site and its endogenous ligand are required for BP elevation in many common hypertension models and full expression of cardiac remodeling and dysfunction following pressure overload or myocardial infarction. Understanding how endogenous CTS impact hypertension and HF pathophysiology and therapy should foster reconsideration of digoxin's therapeutic utility.

NO-induced vasodilation correlates directly with BP in smooth muscle-Na/Ca exchanger-1-engineered mice: elevated BP does not attenuate endothelial function.

Arterial smooth muscle Na+/Ca2+ exchanger-1 (SM-NCX1) promotes vasoconstriction or vasodilation by mediating, respectively, Ca2+ influx or efflux. In vivo, SM-NCX1 mediates net Ca2+ influx to help maintain myogenic tone (MT) and neuronally activated constriction. SM-NCX1-TG (overexpressing transgenic) mice have increased MT and mean blood pressure (MBP; +13.5 mmHg); SM-NCX1-KO (knockout) mice have reduced MT and MBP (-11.1 mmHg). Endothelium-dependent vasodilation (EDV) is often impaired in hypertension. We tested whether genetically engineered SM-NCX1 expression and consequent BP changes similarly alter EDV. Isolated, pressurized mesenteric resistance arteries with MT from SM-NCX1-TG and conditional SM-NCX1-KO mice, and femoral arteries in vivo from TG mice were studied. Acetylcholine (ACh)-dilated TG arteries with MT slightly more than control or KO arteries, implying that SM-NCX1 overexpression does not impair EDV. In preconstricted KO, but not TG mouse arteries, however, ACh- and bradykinin-triggered vasodilation was markedly attenuated. To circumvent the endothelium, phenylephrine-constricted resistance arteries were tested with Na-nitroprusside [SNP; nitric oxide (NO) donor] and cGMP. This endothelium-independent vasodilation was augmented in TG but attenuated in KO arteries that lack NCX1-mediated Ca2+ clearance. Baseline cytosolic Ca2+ ([Ca2+]cyt) was elevated in TG femoral arteries in vivo, supporting the high BP; furthermore, SNP-triggered [Ca2+]cyt decline and vasodilation were augmented as NO and cGMP promote myocyte polarization thereby enhancing NCX1-mediated Ca2+ efflux. The TG mouse data indicate that BP elevation does not attenuate endothelium-dependent vasodilation. Thus, in essential hypertension and many models the endothelial impairment that supports the hypertension apparently is not triggered by BP elevation but by extravascular mechanisms.NEW & NOTEWORTHY Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na+/Ca2+ exchangers (NCX1) are upregulated in many forms of hypertension. Surprisingly, mildly hypertensive smooth muscle-specific (SM)-NCX1 transgenic mice exhibited modestly enhanced EDV and augmented endothelium-independent vasodilation (EIV). Conversely, mildly hypotensive SM-NCX1-knockout mice had greatly attenuated EIV. These adaptations help compensate for NCX1 expression-induced alterations in cytosolic Ca2+ and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.

Na+/Ca2+ exchanger overexpression in smooth muscle augments cytosolic Ca2+ in femoral arteries of living mice.

Plasma membrane Na+/Ca2+ exchanger-1 (NCX1) helps regulate the cytosolic Ca2+ concentration ([Ca2+]CYT) in arterial myocytes. NCX1 mediates both Ca2+ entry and exit and tends to promote net Ca2+ entry in partially constricted arteries. Mean blood pressure (telemetry) is elevated by ≈10 mmHg in transgenic (TG) mice that overexpress NCX1 specifically in smooth muscle. We tested the hypothesis that NCX1 overexpression mediates Ca2+ gain and elevated [Ca2+]CYT in exposed femoral arteries that also express the Ca2+ biosensor exogenous myosin light chain kinase. [Ca2+]CYT and the NCX1-dependent (SEA0400-sensitive) component, ≈15% of total basal constriction in controls, were increased in TG arteries, but constrictions to phenylephrine and ANG II were comparable in TG and control arteries. Normalized phenylephrine dose-response curves and constriction to 30 and 300 ng/kg iv ANG II were virtually identical in control and TG arteries. ANG II-evoked constrictions, superimposed on elevated basal tone, accounted for the larger blood pressure responses to ANG II in TG arteries. TG and control mouse arteries fit the same pCa-constriction relationship over a wide range of pCa (≈125-500 nM). Vasodilation to acetylcholine, normalized to passive diameter, was also comparable in TG and control arteries, implying normal endothelial function. TG artery Na+ nitroprusside (nitric oxide donor)-induced dilations were, however, shifted to lower Na+ nitroprusside concentrations, indicating that TG myocyte vasodilator mechanisms were augmented. Maximum arterial dilation was comparable in TG and control mice, although passive diameter was ≈6-7% smaller in TG mice. The changes in TG arteries were apparently largely functional rather than structural, despite the congenital hypertension. NEW & NOTEWORTHY Smooth muscle Na+/Ca2+ exchanger-1 transgene overexpression (TG mice) increases femoral artery basal cytosolic Ca2+ concentration ([Ca2+]CYT) and tone in vivo and raises blood pressure. Arterial constriction to phenylephrine and angiotensin II are normal but superimposed on the augmented basal [Ca2+]CYT and tone (constriction) in TG mouse arteries. Similar effects in resistance arteries would explain the elevated blood pressure. Acetylcholine-induced vasodilation is unimpaired, implying a normal endothelium, but TG arteries are hypersensitive to sodium nitroprusside.

The pump, the exchanger, and the holy spirit: origins and 40-year evolution of ideas about the ouabain-Na+ pump endocrine system.

Two prescient 1953 publications set the stage for the elucidation of a novel endocrine system: Schatzmann's report that cardiotonic steroids (CTSs) are all Na+ pump inhibitors, and Szent-Gyorgi's suggestion that there is an endogenous "missing screw" in heart failure that CTSs like digoxin may replace. In 1977 I postulated that an endogenous Na+ pump inhibitor acts as a natriuretic hormone and simultaneously elevates blood pressure (BP) in salt-dependent hypertension. This hypothesis was based on the idea that excess renal salt retention promoted the secretion of a CTS-like hormone that inhibits renal Na+ pumps and salt reabsorption. The hormone also inhibits arterial Na+ pumps, elevates myocyte Na+ and promotes Na/Ca exchanger-mediated Ca2+ gain. This enhances vasoconstriction and arterial tone-the hallmark of hypertension. Here I describe how those ideas led to the discovery that the CTS-like hormone is endogenous ouabain (EO), a key factor in the pathogenesis of hypertension and heart failure. Seminal observations that underlie the still-emerging picture of the EO-Na+ pump endocrine system in the physiology and pathophysiology of multiple organ systems are summarized. Milestones include: 1) cloning the Na+ pump isoforms and physiological studies of mutated pumps in mice; 2) discovery that Na+ pumps are also EO-triggered signaling molecules; 3) demonstration that ouabain, but not digoxin, is hypertensinogenic; 4) elucidation of EO's roles in kidney development and cardiovascular and renal physiology and pathophysiology; 5) discovery of "brain ouabain", a component of a novel hypothalamic neuromodulatory pathway; and 6) finding that EO and its brain receptors modulate behavior and learning.

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.

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 angiotensin II (Ang II) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases Ang II/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 Ang II 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.

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.

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.

Ouabain-digoxin antagonism in rat arteries and neurones.

'Classic' cardiotonic steroids (CTSs) such as digoxin and ouabain selectively inhibit Na+, K+ -ATPase (the Na+ pump) and, via Na+ / Ca2+ exchange (NCX), exert cardiotonic and vasotonic effects. CTS action is more complex than previously thought: prolonged subcutaneous administration of ouabain, but not digoxin, induces hypertension, and digoxin antagonizes ouabain's hypertensinogenic effect. We studied the acute interactions between CTSs in two indirect assays of Na+ pump function: myogenic tone (MT) in isolated, pressurized rat mesenteric small arteries, and Ca2+ signalling in primary cultured rat hippocampal neurones. The 'classic' CTSs (0.3-10 nm) behaved as 'agonists': all increased MT70 (MT at 70 mmHg) and augmented glutamate-evoked Ca2+ (Fura-2) signals. We then tested one CTS in the presence of another. Most CTSs could be divided into ouabain-like (ouabagenin, dihydroouabain (DHO), strophanthidin) or digoxin-like CTS (digoxigenin, digitoxin, bufalin). Within each group, the CTSs were synergistic, but ouabain-like and digoxin-like CTSs antagonized one another in both assays: For example, the ouabain-evoked (3 nm) increases in MT70 and neuronal Ca2+ signals were both greatly attenuated by the addition of 10 nm digoxin or 10 nm bufalin, and vice versa. Rostafuroxin (PST2238), a digoxigenin derivative that displaces 3H-ouabain from Na+, K+ -ATPase, and attenuates some forms of hypertension, antagonized the effects of ouabain, but not digoxin. SEA0400, a Na+ / Ca2+ exchanger (NCX) blocker, antagonized the effects of both ouabain and digoxin. CTSs bind to the α subunit of pump αß protomers. Analysis of potential models suggests that, in vivo, Na+ pumps function as tetraprotomers ((αß)4) in which the binding of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxin-like CTSs to reactivate the ouabain-inhibited complex can be explained by de-oligomerization of the tetrameric state. The interactions between these common CTSs may be of considerable therapeutic relevance.

Nanomolar ouabain augments Ca2+ signalling in rat hippocampal neurones and glia.

Linkage of certain neurological diseases to Na(+) pump mutations and some mood disorders to altered Na(+) pump function has renewed interest in brain Na(+) pumps. We tested nanomolar ouabain on Ca(2+) signalling (fura-2) in rat hippocampal neurone-astrocyte co-cultures. The neurones and astrocytes express Na(+) pumps with a high-ouabain-affinity catalytic subunit (α3 and α2, respectively); both also express pumps with a ouabain-resistant α1 subunit. Neurones and astrocytes were identified by immunocytochemistry and by stimulation; 3-4 µM L-glutamate (Glu) and 3 µM carbachol (CCh) evoked rapid Ca(2+) transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.5-1 µM ATP evoked Ca(2+) transients only in astrocytes. Both cell types responded to 5-10 µM Glu or ATP. The signals evoked by 3-4 µM Glu in neurones were markedly inhibited by 3-10 µm MPEP (blocks metabotropic glutamate receptor mGluR5) and 10 µm LY341495 (non-selective mGluR blocker), but not by 80 µm AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre-incubation (0.5-10 min) with 1-10 nm ouabain (EC50 < 1 nm) augmented Glu- and CCh-evoked signals in neurones. This augmentation was abolished by a blocker of the Na(+)-Ca(2+) exchanger, SEA0400 (300 nm). Ouabain (3 nm) pre-incubation also augmented 10 µM cyclopiazonic acid plus 10 mm caffeine-evoked release of Ca(2+) from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits α3 Na(+) pumps, increases (local) intracellular Na(+), and promotes Na(+)-Ca(2+) exchanger-mediated Ca(2+) gain and increased storage in the adjacent ER. Ouabain (3 nm) also increased ER Ca(2+) release and enhanced 0.5 µM ATP-evoked transients in astrocytes; these effects were mediated by α2 Na(+) pumps. Thus, nanomolar ouabain may strongly influence synaptic transmission in the brain as a result of its actions on the high-ouabain-affinity Na(+) pumps in both neurones and astrocytes. The significance of these effects is heightened by the evidence that ouabain is endogenous in mammals.

Salt sensitivity, endogenous ouabain and hypertension.

PURPOSE OF REVIEW: Endogenous cardiotonic steroids (CTS) exert long-term effects on salt and blood pressure homeostasis. Here we discuss recent observations on mechanisms of salt sensitivity that involve endogenous ouabain and novel pathways in the brain and discuss their possible relationship to arterial and renal function in hypertension. RECENT FINDINGS: Chronic elevation of brain sodium promotes sustained hypertension mediated by central endogenous ouabain and the Na(+) pump α-2 catalytic subunit. The intermediary pressor mechanism in the brain involves aldosterone biosynthesis, activation of mineralocorticoid receptors and increased epithelial sodium channel activity. In the periphery, elevated plasma CTS raise contractility and blood pressure by augmentation of sympathetic nerve responses, increasing arterial Ca(2+) signaling and blunting nitric oxide production in the renal medulla and collecting ducts. SUMMARY: Endogenous ouabain in the brain appears to play a critical role in salt sensitivity and hypertension. In the periphery, the J-shaped relationship of plasma endogenous ouabain in response to short-term changes in salt balance in humans raises the possibility that endogenous ouabain contributes to the increased risk of adverse cardiovascular events associated with both low and high salt intakes.

Livin' with NCX and lovin' it: a 45 year romance.

This conference commemorates, almost to the day, the 45th anniversary of the discovery of the Na(+)/Ca(2+) exchanger (NCX). The discovery was serendipitous, as is so often the case with scientific breakthroughs. Indeed, that is what is so fascinating and romantic about scientific research. I will describe the discovery of NCX, but will begin by explaining how I got there, and will then discuss how the discovery influenced my career path.

Cross talk between plasma membrane Na(+)/Ca (2+) exchanger-1 and TRPC/Orai-containing channels: key players in arterial hypertension.

Arterial smooth muscle (ASM) Na(+)/Ca(2+) exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na(+) pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na(+) metabolism, Ca(2+) signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention.

Cation flux through SUR1-TRPM4 and NCX1 in astrocyte endfeet induces water influx through AQP4 and brain swelling after ischemic stroke.

Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na+/Ca2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na+ through SUR1-TRPM4 induced Ca2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca2+. This increase in Ca2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.

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.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension.

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.