Ouabain-induced Hypertension Research Articles

Chronic cyclooxygenase-2 inhibition prevents the worsening of hypertension and endothelial dysfunction induced by ouabain in resistance arteries of spontaneously hypertensive rats

AimPrevious studies raise cyclooxygenase (COX) activation as a possible mechanism involved in the pathophysiology of ouabain-induced hypertension. We hypothesized that inhibition of COX-2 activity might prevent ouabain-induced vascular dysfunction and worsening of hypertension in spontaneously hypertensive rats (SHR). MethodsSHR were exposed to ouabain or vehicle and treated or not with the selective COX-2 inhibitor nimesulide for 5 weeks. Systolic blood pressure was measured by plethysmography. Vascular reactivity by wire myograph and protein expression by Western-blot were assessed in mesenteric resistance arteries (MRA) of groups. Thromboxane A2 (TXA2) production by ELISA was evaluated in MRA supernatants of groups. ResultsNoradrenaline-induced maximal contraction (Emax) was greater in MRA from SHR receiving ouabain than those of vehicle group. In situ inhibition of COX-2, TXA2 synthase, or TP receptor reduced the Emax to noradrenaline in MRA of ouabain to vehicle levels. TXA2 production was higher in ouabain than in vehicle group. Ouabain enhanced expression of cytoplasmic tyrosine kinase Src (c-Src)/ERK1/2/COX-2/TXA2 synthase/TP receptor in SHR MRA, but did not change NFkB/iKB ratio. Anticontractile effect of nitric oxide (NO) was smaller in MRA from ouabain- than vehicle-treated SHR, as well as eNOS and nNOS expression. Nimesulide co-treatment prevented the ouabain-induced worsening of hypertension and noradrenaline MRA hypercontractility in SHR. ConclusionOuabain worsen hypertension and induce MRA hypercontractility in SHR associated with upregulated c-Src/ERK1/2/COX-2/TXA2 synthase/TXA2/TP receptor axis. These effects were prevented by COX-2 inhibition.

Flaxseeds (L. Usitatissimum) attenuates blood pressure, acetylcholinesterase activity and oxidative stress in ouabain-induced hypertension in normal Wistar rats

PurposeThe purpose of this study is to evaluate the effects of flaxseed (L. usitatissimum [Linn]) on blood pressure, redox status markers and acetylcholinesterase enzyme activity in ouabain-induced hypertension in normal Wistar rats.Design/methodology/approachMale Wistar rats weighing 250 ± 7 g (n = 24) fed with an experimental diet containing 20 per cent casein were divided into three groups (n = 8) and received a daily subcutaneous injection of either 0.9 per cent saline solution (T group) or 10 µg/kg/day of ouabain diluted in saline solution-treated Oub-Lu or not with 1 per cent of flaxseed (L. usitatissimum) mixed in the diet for 21 days.FindingsThe results showed that treatment with flaxseed had a significant effect (p < 0.05) in decreasing systolic, diastolic blood pressure, mean arterial pressure and the heart rate in hypertensive rats. Total and non-HDL cholesterol levels were reduced by –26 and –35 per cent; p = 0.04, respectively. Concentrations of thiobarbituric acid-reactive substances were significantly decreased by –85 and –42 per cent (p = 0.001 and p = 0.04), respectively in the plasma and heart. Nitric oxide levels were increased in the aorta (+ 63 per cent; p = 0.001). Moreover, in the heart and aorta, a significant increase was noted in the thiol contents (+81 and +69 per cent; p = 0.001, respectively), glutathione peroxidase (+50 per cent; p = 0.03 and p = 0.01, respectively) and acetylcholinesterase activities (75 and +19 per cent, respectively; p = 0.001 and p = 0.04).Originality/valueThese results suggest hypotensive, cardiomoderating and antioxidant effects of flaxseed in ouabain-induced hypertension in the rat. In addition, it promotes a significant increase of the acetylcholinesterase activity in tissues.

Role of GSPE in improving early cerebral vascular damage by inhibition of Profilin-1 expression in a ouabain-induced hypertension model.

Grape seed proanthocyanidin extract (GSPE), as one of the most popular natural drug extracted from the grape, has been reported to improve endothelial function and arteriosclerosis. However, little is known about the influence of GSPE on hypertension and vascular remodeling. Profilin-1, an Actin-binding protein, is closely involved in the remodeling of large vessels in ouabain-induced hypertension. To date, there is no effective prevention or treatment in place for the high incidence of ischemic stroke associated with hypertension. In this study, we aimed to determine the role of GSPE via inhibition Profilin-1 in ischemic cerebral cortices of ouabain-hypertension rats and potentially provide a new target to prevent stroke associated with hypertension. The blood pressure of male Sprague-Dawley (SD) rats was measured during a period of ouabain-induced hypertension. The expression of Profilin-1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the cerebral cortex were determined by quantitative Real Time-PCR (qRT-PCR) and Western blot. Histopathological and behavioral tests were also conducted. Blood pressure elevation started at week 5 and reached clinical standards for hypertension at week 8. GSPE was proved to suppress Profilin-1 and VEGF levels through inhibition of Profilin-1-protein kinase B (AKT)-hypoxia inducible factor-1α (HIF-1α) signal pathway and promote eNOS expression. Moreover, the histopathological and ethiological improvement was observed in GSPE over-expression and Profilin-1 inhibition groups. We detected that GSPE could improve cerebral vascular damage through inhibiting Profilin-1 in an ouabain-induced hypertension model.

Chronic ouabain treatment induces Rho kinase activation.

Ouabain is an endogenous Na(+)/K(+)-ATPase inhibitor whose chronic administration induces hypertension. Endogenous ouabain levels increase in human essential hypertension. On the other hand, Rho/Rho kinase (ROCK) pathway has been implicated in various animal models of hypertension. In the current work, we evaluated the possible involvement of Rho kinase in ouabain-induced hypertension. Ouabain was administered daily (20 µg/kg, i.p.) to Wistar rats for 6 weeks. After the ouabain treatment, we evaluated the possible changes in vascular responses to KCl and phenylephrine alone and in the presence of Rho kinase inhibitor Y27632. We also determined the expressions of ROCKs, Rho A and phosphorylation of myosin binding subunit of myosin light chain phosphatase (pMYPT) and activation of Rho A. Agonist-induced contractions in the presence of Y27632 are significantly decreased and Y27632-induced relaxations in aortas precontracted with phenylephrine are significantly enhanced with the chronic treatment of ouabain. Although the expressions of ROCK I and ROCK II remained unchanged, pMYPT expression was significantly increased in ouabain-treated group. Moreover, Rho A expression and activation were decreased after treatment with ouabain. Although Rho kinase expression did not change in aortas, increased basal Rho kinase activation may contribute to the development of ouabain-induced hypertension. Our current data present the first evidence that Rho kinase is involved in the development of ouabain-induced hypertension in rats.

Why isn't endogenous ouabain more widely accepted?

A new idea is the most quickly acting antigen known to science . —Wilfred Trotter During the past few years, several distinguished senior colleagues who visited our department and inquired about the status of endogenous ouabain (EO) have asked, “How come I didn't know about these [published]

Grape seed proanthocyanidin extract alleviates ouabain-induced vascular remodeling through regulation of endothelial function

Recent studies indicate that chronic ouabain treatment leads to hypertension and hypertensive vascular remodeling. Grape seed proanthocyanidin extract (GSPE) has been reported to be effective in treating arteriosclerosis, while little is known about its effect on systolic blood pressure and vascular remodeling. In this study, the effects of GSPE on systolic blood pressure and vascular remodeling were analyzed by treating ouabain-induced hypertensive rats with GSPE (250 mg/kg·d). The expression of nitric oxide (NO) and endothelin-1 (ET-1) in thoracic aorta was examined by ELISA; the mRNA and protein levels of TGF-β1 were detected using real-time PCR and western blotting, respectively. The results showed that the systolic blood pressure was significantly decreased following treatment with GSPE, with blocked vascular remodeling. The ET-1 content was reduced while NO production was increased in the GSPE group, which showed improved vascular endothelial function. Moreover, GSPE also reduced TGF-β1 expression in the thoracic aorta, which is a determinant in vascular remodeling. In conclusion, GSPE antagonized ouabain-induced hypertension and vascular remodeling and is recommended as a potential anti-hypertensive agent for patients with hypertensive vascular diseases.

Dopamine‐mediated inhibition of renal Na+/K+‐ATPase in HK‐2 cells is reduced by ouabain

1. Abnormal renal sodium handling is considered a major contributing factor in hypertension associated with chronic ouabain treatment. However, the molecular mechanisms involved in abnormal renal sodium handling have not been elucidated. Therefore, we investigated whether chronic ouabain treatment perturbs dopamine D(1) receptor function. 2. The expression and phosphorylation levels of the D(1) receptor in cells of the human proximal tubule cell line (HK-2) were determined using western blot analysis and reverse transcription polymerase chain reaction. The activity of the renal sodium/potassium pump (Na(+)/K(+)-ATPase) was measured using a colourimetric assay, and cyclic adenosine monophosphate accumulation was determined by performing a radioimmunoassay. 3. We showed that chronic ouabain treatment decreased the protein and mRNA expression levels of the D(1) receptor and increased the basal phosphorylation of the D(1) receptor in HK-2 cells. We also showed that in the presence of ouabain, HK-2 cells did not reveal the cyclic adenosine monophosphate accumulation and Na(+)/K(+)-ATPase inhibition induced by the D(1) receptor agonist fenoldopam. 4. We hypothesize that the ouabain-induced decrease in renal D(1) receptor function is responsible for the increase in renal sodium reabsorption, which eventually leads to ouabain-induced hypertension.

Low-dose ouabain constricts small arteries from ouabain-hypertensive rats: implications for sustained elevation of vascular resistance

Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries (approximately 320 microm internal diameter) as a result of collagen deposition in the artery media. Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 +/- 3 versus 124 +/- 4 mmHg in controls (P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had approximately 165-microm internal diameters and approximately 20-microm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 microM) and high K(+)-induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 microM phenylephrine and by 10 mM caffeine in Ca(2+)-free media indicated that releasable sarcoplasmic reticulum Ca(2+) stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by approximately 26 microm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a approximately 90% increase in resistance to flow in these small arteries; thus ouabain at EC(50) of approximately 0.66 nM should raise resistance by approximately 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.

Ouabain treatment changes the role of endothelial factors in rat resistance arteries

This study investigates the participation of the endothelial factors in the α-adrenoceptor contractile responses in mesenteric resistance arteries from 15 days ouabain-treated (25 µg/kg/day) and untreated rats. Ouabain treatment increased blood pressure and heart rate without changing the contractile response to phenylephrine (3 nM–30 µM). Endothelium removal or N G-nitro- l-arginine methyl ester ( l-NAME, 100 µM), increased the responses to phenylephrine. The endothelial modulation was similar in both rat groups, but the l-NAME effects were bigger in arteries from ouabain-treated rats. However, the endothelial NOS expression and the relaxation to acetylcholine (0.1 nM–10 µM) remained unaltered after ouabain treatment. The coincubation with l-NAME and indomethacin (100 µM) leftward shifted the concentration–response curves to phenylephrine in arteries from untreated rats similarly to the displacement after incubation only with l-NAME. However, in mesenteric arteries from treated rats, the co-incubation with indomethacin and l-NAME did not alter the response to phenylephrine. The addition of the inhibitor of calcium activated potassium channels tetraethylammonium (2 mM) further leftward shifted the phenylephrine curves only in arteries from untreated rats. Cyclooxygenase-2 (COX-2) expression was greater in vessels from ouabain-treated rats. In conclusion, the chronic ouabain treatment for 15 days modified the participation of endothelial factors in response to phenylephrine in mesenteric resistance arteries, by increasing the release of NO and prostanoids and impairment the endothelium-derived hyperpolarizing factor (EDHF) release. This was accompanied by an increased COX-2 expression. Although this balance avoids changes in the phenylephrine concentration–response curves, these vascular changes might contribute to maintain the ouabain-induced hypertension.

CHRONIC OUABAIN TREATMENT ENHANCES CARDIAC MYOSIN ATPase ACTIVITY IN RATS

1. Chronic ouabain administration increases blood pressure and produces a positive inotropic effect. However, the temporal changes capable of affecting both arterial and ventricular pressures and myosin ATPase activity during the induced hypertension have not been determined. 2. The aim of the present study was to investigate the time-course of the induction of hypertension to define when changes occur in Wistar rats treated with 25 mg/kg per day, s.c., ouabain for 3, 7, 15 or 30 days. 3. In anaesthetized rats, diastolic blood pressure increased after 7 days treatment with ouabain and after 15 and 30 days treatment, increases were observed in systolic blood pressure, left ventricular systolic pressure and myosin ATPase activity. After 15 days treatment, heart rate (HR) also increased, but after 30 days treatment HR returned to control levels. However, only after 30 days treatment did the left ventricular positive and negative first derivatives of intraventricular pressure (dP/dt(max) and dP/dt(min), respectively) increase. Increased arterial and left ventricular systolic pressures and myosin ATPase activity observed after 15 days treatment maintained similar levels as those after 30 days treatment. 4. The results suggest that changes in arterial and left ventricular pressures, HR and myosin ATPase activity induced by chronic ouabain treatment are time dependent, increasing after 15 days treatment. After 30 days treatment, the increase in systolic and diastolic arterial and ventricular pressures remained stable, as did inotropism. Normalization of HR after 30 days treatment suggests that during the period from Day 16 to Day 30 ouabain-induced hypertension is dependent, at least in part, on increased sympathetic activity.

Ouabain‐induced hypertension and rho kinase

Rho/Rho kinase pathway has been implicated various models of hypertension. However, there is no report whether this pathway involves in ouabain‐induced hypertension. In our current work, we evaluated possible involvement of rho kinase in ouabain‐induced hypertension in rats. Ouabain was injected to rats 20μg/kg per day for six weeks. After ouabain treatment, Rho kinase‐1 and Rho kinase‐2 expressions of aortas were evaluated by Western Blotting. It was shown that expressions of neither 1 nor 2 isoform of Rho kinase were altered in ouabain group. Dose dependent relaxations Y27632 were determined in KCl and Phenylephrine precontracted aortas of both control and ouabain treated groups. Although there was no statistically significant difference between KCl precontracted tissues of control and hypertensive groups, it was shown that Y27632 relaxations were enhanced in Phenylephrine precontracted aortas of ouabain treated group compared to control. As a conclusion, the results of this study indicate that although Rho kinase expressions did not contribute to ouabain induced hypertension, Rho kinase activation may have a role in this hypertension model.

Sodium pumps: ouabain, ion transport, and signaling in hypertension

TO THE EDITOR: A recent Editorial Focus in this journal (7) addresses the newly recognized role of the sodium pump as both an ion transport protein and a hormone receptor with its own signaling cascade. The relative importance of the cardiotonic steroids (CTS) as endogenous signaling hormones versus their ability to inhibit the sodium pump is of great interest. Indeed, the article noted that “although all digitalislike compounds inhibit Na-K-ATPase-mediated transport, they differ considerably in terms of their hypertensive effects.” The author concludes that “these findings do not support the prevailing hypothesis that the pathophysiological effect of digitalis results from inhibition of . . . transport activity.” This implies that the hypertensive effect of CTS may be due to the action of these agents as regulators of signal transduction, entirely unrelated “to its (Na-K-ATPase’s) role as an ion pump.” The discoveries related to Src kinase-mediated signal transduction by Na-K-ATPase add a new dimension to the view that the endogenous cardiotonic steroids comprise a novel hormone system. Nevertheless, the view that CTS induced hypertension is unrelated to inhibition of ion transport is inconsistent with published reports. Regrettably, the Editorial Focus does not cite key articles that support the conclusion that CTS hypertension is secondary to inhibition of sodium pumps. It is true that the tendency of certain CTS to induce hypertension does not correlate with their relative potency as Na-KATPase inhibitors (8, 9). The most obvious comparison is that ouabain induces hypertension while digoxin (1, 9) and a digitoxigenin derivative, PST-2238 (4), are antihypertensive. This need not imply that ouabain’s effect on blood pressure is unrelated to inhibition of Na transport. Instead, these data are consistent with the interpretation that some CTS have mixed agonist/antagonist properties. Viability of the hypothesis that enhancement of cytoplasmic Ca 2 responses by ouabain is related to inhibition of Na-KATPase relies on the secondary effect of Na on Na/Ca exchange. There is now direct evidence that elevation of blood pressure is a consequence of reduced transport activity of the ouabain-sensitive Na-K-ATPase 2-isoform (2, 3, 10) and the resultant Ca 2 gain mediated by Na/Ca exchange (3, 5, 10). Moreover, reduced 2 expression leads to murine hypertension (10). The vascular myogenic effect of that model is prevented by the Na/Ca exchange inhibitor SEA0400 (10). A key role for Na/Ca exchange is revealed by the observation that SEA0400 lowers blood pressure in ouabain-induced hypertension and other forms of salt-dependent hypertension, but not salt-independent hypertension (5). These findings demonstrate that ouabain’s hypertensive effect depends on its inhibition of the Na-K-ATPase and Ca 2 gain via Na/Ca exchange. Thus Src signaling, alone, appears to be insufficient to raise blood pressure in ouabain-induced hypertension although an accessory role is not excluded. Comparison of the effect of reduced expression of the 2 Na-K-ATPase (6) with that of low-dose ouabain on cell signaling may be a useful way to determine the roles of altered Na and Ca 2 in mediating the effects of ouabain on Src kinase signaling.

Reply to Blaustein et al.

reply: Blaustein et al. ([2][1]) have responded to the Editorial Focus ([9][2]) by stating that it “did not cite key articles that support the conclusion that CTS (cardiotonic steroids) hypertension is secondary to inhibition of sodium pumps.” To support their view, they cited several articles

Ouabain-induced hypertension enhances left ventricular contractility in rats

Chronic ouabain treatment produces hypertension acting on the central nervous system and at vascular levels. However, cardiac effects in this model of hypertension are still poorly understood. Hence, the effects of hypertension induced by chronic ouabain administration (∼ 8 μg day − 1 , s.c.) for 5 weeks on the cardiac function were studied in Wistar rats. Ouabain induces hypertension but not myocardial hypertrophy. Awake ouabain-treated rats present an increment of the left ventricular systolic pressure and of the maximum positive and negative d P/d t. Isolated papillary muscles from ouabain-treated rats present an increment in isometric force, and this effect was present even when inotropic interventions (external Ca 2+ increment and increased heart rate) were performed. However, the sarcoplasmic reticulum activity and the SERCA-2 protein expression did not change. On the other hand, the activity of myosin ATPase increased without changes in myosin heavy chain protein expression. In addition, the expression of α 1 and α 2 isoforms of Na +, K +-ATPase also increased in the left ventricle from ouabain-hypertensive rats. The present results showed positive inotropic and lusitropic effects in hearts from awake ouabain-treated rats, which are associated with an increment of the isometric force development and of the activity of myosin ATPase and expression of catalytic subunits of the Na +, K +-ATPase.

Central and peripheral renin-angiotensin systems in ouabain-induced hypertension

Chronic subcutaneous infusion of ouabain causes hypertension via central pathways involving angiotensin type 1 (AT(1)) receptor stimulation. The present study assessed plasma and tissue ANG I and II levels as well as AT1 receptor and angiotensin-converting enzyme (ACE) mRNA levels and binding densities by real-time PCR and in vitro autoradiography in relevant brain nuclei and peripheral tissues (heart and kidney) in rats at 1 and/or 2 wk after start of ouabain infusion at 50 microg/day. After 2 wk (but not after 1 wk), blood pressures significantly increased (+15 mmHg). At 2 wk, plasma ANG I and II levels were markedly suppressed by ouabain. In contrast, in the heart and kidneys, ANG I levels were not affected, and ANG II levels tended to decrease, whereas in the hypothalamus ANG II content clearly increased. At 1 wk, no changes in ACE and AT1 receptor densities were seen. After 2 wk, there were significant decreases in AT(1) receptor mRNA and densities in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and paraventricular nucleus (PVN). ACE densities decreased only in the OVLT and SFO, but ACE mRNA showed more variable responses (decrease in OVLT vs. increase in PVN). In the kidneys, at 2 wk both AT1 receptor and ACE densities were decreased, but mRNA abundance did not change. The heart showed no significant changes. The increase in hypothalamic ANG II content and associated decreases in central AT1 receptor and ACE densities support the involvement of the brain renin-angiotensin system in the central hypertensive mechanism of action of ouabain.

How does salt retention raise blood pressure?

A critical question in hypertension research is: How is long-term blood pressure controlled? Excessive NaCl ingestion or NaCl retention by the kidneys and the consequent tendency toward plasma volume expansion lead to hypertension. Nevertheless, the precise mechanisms linking salt to high blood pressure are unresolved. The discovery of endogenous ouabain, an adrenocortical hormone, provided an important clue. Ouabain, a selective Na+ pump inhibitor, has cardiotonic and vasotonic effects. Plasma endogenous ouabain levels are significantly elevated in approximately 40% of patients with essential hypertension and in animals with several forms of salt-dependent hypertension. Also, prolonged ouabain administration induces hypertension in rodents. Mice with mutant Na+ pumps or Na/Ca exchangers (NCX) and studies with a ouabain antagonist and an NCX blocker are revealing the missing molecular mechanisms. These data demonstrate that alpha2 Na+ pumps and NCX1 participate in long-term regulation of vascular tone and blood pressure. Pharmacological agents or mutations in the alpha2 Na+ pump that interfere with the action of ouabain on the pump, and reduced NCX1 expression or agents that block NCX all impede the development of salt-dependent or ouabain-induced hypertension. Conversely, nanomolar ouabain, reduced alpha2 Na+ pump expression, and smooth muscle-specific overexpression of NCX1 all induce hypertension. Furthermore, ouabain and reduced alpha2 Na+ pump expression increase myogenic tone in isolated mesenteric small arteries in vitro, thereby tying these effects directly to the elevation of blood pressure. Thus, endogenous ouabain, and vascular alpha2 Na+ pumps and NCX1, are critical links between salt and hypertension. New pharmacological agents that act on these molecular links have potential in the clinical management of hypertension.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the alpha2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in alpha2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the alpha2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

Brain Na +,K +-ATPase isozyme activity and protein expression in ouabain-induced hypertension

In normotensive rats, chronic infusion of exogenous ouabain causes hypertension involving central mechanisms. To determine whether ouabain-induced hypertension is associated with specific changes in brain Na +,K +-ATPase activity and expression, we assessed brain Na +,K +-ATPase isozyme activity and protein expression in rats treated with ouabain (50 μg/day s.c. or 10 μg/day i.c.v. for 14 days). Resting mean arterial pressure (MAP) was higher in s.c.- and i.c.v.-ouabain-treated animals vs. control (124±2 vs. 105±2 and 130±2 vs. 109±2, respectively, p<0.01). Ouabain infused s.c. or i.c.v. for 14 days had no effect on Na +,K +-ATPase isozyme activity in hypothalamic, pontine/medullary or cortical microsomes. However, the percent increase in total Na +,K +-ATPase activity produced in vitro by antibody Fab fragments that bind ouabain with high affinity (Digibind) was two-fold greater in s.c.- and i.c.v.-ouabain-treated rats vs. control, but only in hypothalamic microsomes. Thus, ouabain infused s.c. or i.c.v. does appear to directly inhibit Na +,K +-ATPase activity in the hypothalamus. On the other hand, in the hypothalamus, s.c.- and i.c.v.-ouabain infusions tended to increase α 3 (by 30–44%), but had no effect on α 1 or α 2 Na +,K +-ATPase isozyme protein expression. In addition, ouabain was found to partially dissociate from the Na +,K +-ATPase enzyme following sample processing. Thus, the inability to detect a decrease in enzyme activity in the hypothalamus in response to ouabain may be due, in part, to an increase in enzyme expression and the dissociation of ouabain during sample processing.

Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors.

The involvement of nitric oxide (NO), prostaglandins, and calcium-dependent potassium channel (K(Ca)) activators on the negative modulation of phenylephrine-induced contractions was evaluated on the isolated aorta and caudal (CAU) artery obtained from rats treated with ouabain for 5 wk to induce hypertension. In ouabain-treated rats, the reactivity to phenylephrine was reduced in the endothelium-intact aorta but not the CAU segments. Endothelial modulation of phenylephrine contraction, as demonstrated by endothelium removal, NO synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester and aminoguanidine, as well as K(Ca) inhibition with tetraethylammonium, was more pronounced in segments from ouabain-treated animals, and here greater effects were seen in the aorta than in CAU. An increased expression of endothelial NOS and neuronal NOS was seen in the aorta after ouabain treatment. In CAU, only endothelial NOS was detected and ouabain treatment did not alter its expression. These results suggest that ouabain-induced hypertension is accompanied by increased NO release derived from endothelial NOS and neuronal NOS and increased release of an endothelial hyperpolarizing factor that presumably opens K(Ca), all of which contribute to the increased negative modulation of the phenylephrine contraction.