K-ATPase Inhibitor Research Articles

Ouabain, a Na–K-ATPase inhibitor, enhances wakefulness in rats

Sleep disturbances are common findings in bipolar affective disorder (BD), and they may be related to the pathophysiology of the disease. REM sleep deprivation, which has an antidepressant effect, increases Na–K-ATPase (NKA) activity. NKA might be involved in the induction of rebound sleep. Decrements in NKA are thought to be implicated in the pathophysiology of BD. A single intracerebroventricular (ICV) microinjection of ouabain (OUA), an NKA inhibitor, results in long-lasting suppression of NKA activity. It has been proposed to be used as a model of BD. To elucidate the role of NKA, we studied the effects of ICV administered OUA on the sleep–wake activity of rats. Rats were ICV injected with saline on the baseline day and 2.5 μL 20 μM (n = 8) or 2 mM (n = 6) OUA on the subsequent OUA day. Saline was administered 1, 4, and 8 days after OUA injection. Sleep–wake activity was recorded for 12 h or 24 h post-injection. OUA enhanced wakefulness, but there was only little similarity between the changes in sleep and in BD after OUA treatment. These findings give only limited support for the NKA hypothesis of BD. Not the decrease in NKA activity itself that may be the important factor in the pathophysiology of BD, but rather the dysfunction of those regulatory processes that influence NKA.

Effect of photobiomodulation therapy on neuronal injuries by ouabain: the regulation of Na, K-ATPase; Src; and mitogen-activated protein kinase signaling pathway

BackgroundTo determine whether photobiomodulation (PBM) rescued the disruption of Na+/Ca2+ homeostasis and mitochondrial membrane potential by ouabain; the Na, K-ATPase inhibitor. For PBM in this study, a 660 nm LED array was used at energy densities of 0.78, 1.56, 3.12, 6.24, and 9.36 J/cm2.ResultsHCN-2 neuronal cells treated with ouabain showed loss of cell polarity, disrupted cell morphology, and decreased cell viability, which were improved after PBM treatment. We found that ouabain-induced Na, K-ATPase inhibition promoted activation of downstream signaling through Src, Ras, and mitogen-activated protein kinase (MAPK), which were suppressed after PBM treatment. This provided evidence of Na, K-ATPase α-subunit inactivation and intracellular Ca2+ increase. In response to ouabain, we observed activation of Src and MAPK by Na, K-ATPase, decreased mitochondrial membrane potential, and Na+-dependent Ca2+ increases, which were restored by PBM treatment.ConclusionsThis study demonstrated that Na+/K+ imbalance could be regulated by PBM treatment in neuronal cells, and we suggest that PBM is a potential therapeutic tool for Na, K-ATPase targeted neuronal diseases.

Increased Reabsorption of Alveolar Edema Fluid in the Obese Zucker Rat

Diabetic patients have a decreased incidence of acute respiratory distress syndrome, but the mechanism responsible for the decreased incidence is uncertain. Reabsorption of alveolar edema fluid (alveolar fluid clearance) has been considered to play an important role in resolution of acute respiratory distress syndrome. However, little is known regarding alveolar fluid clearance in diabetes mellitus. Since the obese Zucker rat has been used as an experimental model for diabetes mellitus, we determined if alveolar fluid clearance increased in the obese Zucker rat. First, we compared alveolar fluid clearance in obese Zucker rats with that in lean Zucker rats and Sprague-Dawley (SD) rats. Then, we determined the role of sodium channel, Na,K-ATPase, and beta(2)-adrenoceptor, which drives alveolar fluid clearance, in obese Zucker rats. Alveolar fluid clearance was estimated by the progressive increase in alveolar albumin concentrations in the isolated lungs. We found that basal alveolar fluid clearance in obese Zucker rats was two-fold greater than that in lean Zucker rats and SD rats. The mRNA expression of alpha(1)-, beta(1)-Na, K-ATPase and beta(2)-adrenoceptor, but not mRNA expression of sodium channel, increased in obese Zucker rats. A selective beta(2)-agrenergic antagonist, but not a Na, K-ATPase inhibitor, specifically inhibited the increase in alveolar fluid clearance in obese Zucker rats. These results indicate that overexpression of beta(2)-adrenoceptor primarily increases basal alveolar fluid clearance in the obese Zucker rat. We speculate that the stimulation of alveolar fluid clearance ameliorates acute respiratory distress syndrome in patients with diabetes mellitus.

Endogenous brain Na pumps, brain ouabain-like substance and the α2 isoform in salt-dependent hypertension

An endogenous ouabain-like substance (OLS) plays a critical role in the etiology of experimental models of human hypertension induced by a high salt diet. Early on, evidence for a role of this Na, K-ATPase inhibitor in blood pressure regulation was provided mainly by correlations of blood pressure with the levels of circulating Na, K-ATPase inhibitor. However, over the past decade, numerous studies have shown that endogenous Na pump inhibitors in the brain mediate salt-dependent hypertension in a variety of experimental models, including Dahl salt-sensitive (Dahl-S) and spontaneously hypertensive (SHR) rats on a high-salt diet. Other forms of hypertension that are known to be mediated by endogenous ouabain-like substances include steroid/salt- (e.g., DOCA-salt) and ACTH-induced hypertension. Even when exogenous ouabain is peripherally administered and/or the plasma ouabain/OLS level is increased in rats, the resulting hypertension is of CNS origin. After peripheral ouabain administration, ouabain levels increase in the plasma and the inhibitor subsequently accumulates in the brain. The ensuing hypertension is abolished by the intracerebroventricular (icv) administration of an anti-ouabain antibody (but not by the same antibody dose given iv), by discrete excitotoxic lesions in the brain or by ganglionic blockade, demonstrating that the response is neurally mediated. The pressor response to stimuli that increase the brain OLS (high salt diet, icv sodium) or to icv ouabain is abolished by icv losartan, demonstrating that the brain OLS activates the brain renin-angiotensin system (RAS) downstream. There are three isoforms of the catalytic α subunit of the Na, K-ATPase in the brain and cardiovascular system (α1, α2 and α3), but it is not known which brain isoform(s) mediate the hypertensive effects of circulating/CNS ouabain. Preliminary studies in gene-targeted mice suggest that the α2 isoform plays a critical role.

MULTIFOCAL GASTRIC CARCINOID TUMOR IN A PATIENT WITH PERNICIOUS ANEMIA RECEIVING LANSOPRAZOLE

In an international consensus statement, determination of serum gastrin levels in patients receiving H, K-ATPase inhibitors was not advised since gastric carcinoid tumor development is associated with serum gastrin levels greater than 1000 pg/ml. This is a discussion of a patient with type I diabetes mellitus being treated with once daily, oral lansoprazole, 30 mg. This patient underwent upper endoscopy for evaluation of nausea and was found to have multifocal gastric carcinoid tumor involving the cardia and fundus of the stomach, with no evidence for Helicobacter pylori on gastric biopsies. Subsequent investigation then revealed the presence of parietal cell antibodies (1:320 titer), elevation of serum chromogranin A level (53.5 ng/ml), and hypergastrinemia (1111 pg/ml). Computerized tomography of the abdomen showed gastric wall thickening with no lymphadenopathy, ascites, or liver abnormality. [In-111]-Octreotide scan was unrevealing. Off of lansoprazole, the patient's gastric p H was 2.0, and there was a marked reduction in her hypergastrinemia (to 483 pg/ml). These findings support the notion that achlorhydria is not a prerequisite for hypergastrinemia with subsequent formation of multifocal gastric carcinoid tumor in patients with pernicious anemia. Treatment of non-specific symptoms with a H, K-ATPase inhibitor could increase the risk of developing gastric carcinoid tumor by facilitating hypergastrinemia. In select patients, consideration should be given to obtaining a fasting serum gastrin level prior to initiation of treatment with a H, K-ATPase inhibitor.

Aplysiallene, a new bromoallene as an Na, K-ATPase inhibitor from the sea hare, Aplysia kurodai.

A new bromoallene metabolite, named aplysiallene, was isolated from the Japanese sea hare, Aplysia kurodai, as an Na, K-ATPase inhibitor. Its structure was elucidated by spectroscopic methods. The known metabolites, laurinterol and debromolaurinterol, isolated from this animal were also evaluated for their Na, K-ATPase inhibitory activity.

Inotropic properties of a novel Na, K-ATPase inhibitor, PST 2107

Inotropic properties of a novel Na, K-ATPase inhibitor, PST 2107

F013 Ouabain-like factors in human urine: Identification of a NaK-ATPase inhibitor as vanadium-diascorbate adduct

We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of > 30g NaCVday 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Se hadex G-10, one of three OLFs eluted in the early subfractions FIV/1-2 close to H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with M, around 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF-1 (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 crossreacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-KATPase with ICso of around 1.5 x 10-5M and 1.5 x 104M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance ('H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium 07) diascorbates (M, 403) with similar elution times from RP-HPLC I:

Ouabain as an amplifier of mineralocorticoid-induced hypertension.

Ouabain has recently been reported to be an endogenous Na, K-ATPase inhibitor. To evaluate whether it exerts hypertensive action itself or amplifies the hypertensive action of small doses of mineralocorticoids, 5 mg deoxycorticosterone acetate (DOCA), 1 mg ouabain, or a combination of both were injected into mononephrectomized rats weekly for 6 weeks, and changes in blood pressure were evaluated. The blood pressures of control, DOCA-treated, ouabain-treated, and the combination treatment group at the sixth week were 138 +/- 3 (SE), 160 +/- 6, 144 +/- 6, and 201 +/- 14 mmHg, respectively. The blood pressure of rats given DOCA or ouabain alone was not significantly different from that of controls. In contrast, the blood pressure of rats given the combination of DOCA and ouabain was significantly higher than that of control rats and those given DOCA or ouabain separately. Cardionephromegaly and histopathological changes found in rats given the combination of DOCA and ouabain were consistent with the effects of an elevation of blood pressure. Further evaluation revealed that the amplification effect of ouabain on the hypertensive action of DOCA was dose dependent, with the minimum dose that caused the amplification effect being 0.25 mg/week. These results indicate that ouabain, although devoid of hypertensive action itself, amplifies the hypertensive action of small doses of DOCA and can cause a hypertensive state similar to that induced by larger doses of DOCA. It is inferred that the amplification effect of ouabain on mineralocorticoids is important in the genesis of hypertension.

Higher salt consumption, digoxin-like factor, and nifedipine response are associated with salt sensitivity in essential hypertension.

In addition to demonstrating evidences of increased sympathetic nervous system activity and marked left ventricular hypertrophy in salt-sensitive hypertensives, our group has also reported increased weight gain with salt overload in these patients. The increased weight gain suggests volume expansion, a situation already shown to increase plasma levels of a Na, K-ATPase inhibitor. Therefore, in the present study, digoxin-like factor (DLF) serum levels, spontaneous salt ingestion, nifedipine hypotensive effect, and plasma renin activity were evaluated in essential hypertensive subjects. Thirteen essential hypertensive outpatients were studied sequentially on an ad lib diet, a low salt diet (LSD = 30 mEq Na/day), and a high salt diet (HSD = LSD + 171 mmol/L NaCl/day), 1 week each. On the seventh day of LSD and HSD, DLF levels, mean blood pressure (MBP) response to nifedipine (10 mg sublingual), and plasma renin activity were measured. The MBP percent change from the seventh day of LSD to the seventh day of HSD (salt sensitivity) ranged from -13.7 to 20.9%. A positive correlation (r = 0.64, P < .01) was observed between salt sensitivity and 24-h urinary sodium excretion with an ad lib diet. The DLF serum levels correlated with the salt sensitivity both on LSD (r = 0.50, P < .05) and on HSD (r = 0.53, P < .05). Salt sensitivity was positively correlated with the difference of response to nifedipine between HSD and LSD (r = 0.78, P < .001). Plasma renin activity correlated inversely with DLF on LSD (r = -0.51, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1–34 (hPTH (1–34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.

The role of the Na, K-ATPase inhibitor in renal sodium handling in patients with essential hypertension

The present study aimed to elucidate the role of Na, K-ATPase inhibitor in renal sodium metabolism in essential hypertension. Mean arterial pressure (MAP), heart rate(HR), urine volume (UV), urinary excretion of sodium (UNaV), endogenous creatinine clearance (Ccr), fractional excretion of sodium (FENa), plasma renin activity(PRA) plasma aldosterone concentration(PAC), plasma noradrenaline concentration (PNA) and urinary excretion of noradrenaline(UNA) were measured before and after intravenous injection of ouabain (0.1 mg/m2.BSA) in 12 normotensive(NT) and 22 mild-to-moderate essential hypertensive subjects(EHT). Following ouabain injection, UV, UNaV FENa significantly increased, but PRA decreased, in both NT and EHT. MAP, HR, Ccr, PNA, and UNA did not change significantly in either group. On the other hand, a significant decrease in PAC was observed in NT, but not in EHT. The changes of UNaV and FENa were significantly attenuated in EHT as compared to NT. No significant difference in change of MAP, HR, UV, Ccr, PNA, UNA, or PRA was demonstrated between NT and EHT. A significantly positive correlation was found between delta UNaV and delta FENa in both NT and EHT, while no significant correlation was observed between delta UNaV and delta MAP, delta UV, delta Ccr, delta PRA, delta PAC, delta PNA and delta UNA in either group. These results suggest that 1) Na, K-ATPase inhibitor clearly augments natriuresis by suppression of sodium reabsorption in renal tubules, 2) since this augmentation was attenuated, there is an elevation of endogenous Na, K-ATPase inhibitor(s) should be considered in EHT, and 3) an increase of the inhibitor might participate to the hypertensive mechanism in EHT.

Isolation of a hexaprenylhydroquinone sulfate from the marine spongeDysidea sp. as an H, K-ATPase inhibitor

A hexaprenylhydroquinone sulfate has been isolated as an H,K-ATPase inhibitor from a marine sponge Dysidea sp. It also inhibited phospholipase A2 as well as secretion of gastric acid in rats.

Specific endogenous Na, K-ATPase inhibitor purified from bovine adrenal

The endogenous inhibitor of Na, K-ATPase has been implicated in the pathogenesis of salt-induced hypertension. In spite of an intensive search the inhibitor has long remained elusive. We have been able to purify such an inhibitor from methanol extract of bovine adrenal glands by multiple steps of high-performance liquid chromatography (HPLC). This compound with a molecular mass of 336 showed striking similarity to the cardiac glycoside ouabain in its dose dependency in the inhibition of Na, K-ATPase and Na-pump activity, competitive binding to the ouabain-binding site, and dependence of these effects on K + concentration. These inhibitory activities were potentiated with reduced K + concentration in parallel with ouabain. These results indicates that vertebrate animals possess a regulator of Na, K-ATPase and suggests the possibility that it may be the mediator of salt-induced high blood pressure.

A new in vivo method for repeatedly studying gastric acid secretion and other secretory parameters in awake guinea pig

A new model for measuring gastric secretory parameters in awake guinea pigs is described. A chronic cannula was surgically implanted in the stomach of each guinea pig. The rates of gastric secretion and changes in intragastric volume were measured using a dye dilution technique. In contrast to previous techniques in small laboratory animals, there was no collection of gastric juice via drainage, no oral intubation for aspiration was involved, no special or sophisticated equipment was used, no anesthesia was employed, and there was no stress associated with acute surgery. This method offers a valuable advantage by combining the chronic gastric cannula with a dye dilution technique in that the same animal can be used several times and finally, several gastric secretory parameters can be measured simultaneously. The animals were used from 3 weeks to 10 months after surgery and as many as 15 studies were performed on the same guinea pig. Samples were collected at 10-min intervals and analyzed for acid and dye concentration from which the onset and kinetics of gastric secretion were followed. Basal gastric secretion (11.8 ± 1.6 μeq/kg/min; all mean ± 1 SEM) was increased within 20 min after subcutaneous infusion of histamine (30 μg/kg/hr) and peaked by 40–60 min at a mean acid output rate of 41 ± 3 μeq/kg/min. Histamine also increased the intragastric volume from 6.3 to 13.4 ml as it increased fluid output from 1.6 ± 0.1 ml/10 min to 3.4 ± 0.2 ml/10 min. The increase in acid output caused by histamine was inhibited by the H 2-antagonists cimetidine (3 μmole/kg) and ranitidine at 0.5 μmole/kg. Omeprazole (1.2 μmole/kg), an HK-ATPase inhibitor, almost abolished acid output under both basal and histamine-stimulated conditions. Thus, the present method is simple and suitable to study the physiology and pharmacology of gastric secretion in the guinea pig with a particular emphasis on the action of histamine. Furthermore, because of the species involved, there is also a significant economical advantage and the guinea pig can also be used as a potential model for studying experimental ulcer.

Influence of 16β formylation on Na, K-ATPase inhibition by cardiac glycosides

The inhibitory effect of formylated cardiac steroids (gitaloxin and its derivatives) on guinea-pig heart Na, K-ATPase was compared to that of other cardiac steroids with various hydroxy substituents. The decreasing order of potency of aglycones at equilibrium was as follows: gitaloxigenin greater than digitoxigenin greater than ouabagenin greater than digoxigenin greater than gitoxigenin greater than diginatigenin. This sequence was different to the sequence of drugs hydrophobic character. The compounds with hydroxy groups in the vicinity of the lactone ring (gitoxigenin, diginatigenin) were less potent than the hydrophilic compound ouabagenin. We propose that intramolecular bounding between 16 beta-OH and the lactone ring contributes to the relatively low potency of gitoxigenin and diginatigenin. The formylation of 16 beta-OH increased the potency of gitoxigenin by a factor of 41. The formylated compound (gitaloxigenin) was 5-fold more potent than digitoxigenin. The 3 beta-glycosylation of digoxigenin lead to pseudo-irreversible inhibitors of Na, K-ATPase. The half-time to achieve the equilibrium (for 5 mumol/l) was equal to 54 s, 90 s and 108 s respectively for digoxigenin monodigitoxoside, digoxin and desacetyllanatoside C. However, at equilibrium the three glycosides were equipotent, suggesting the existence of steric effects at the sugar site of the receptor. The sequence of potency observed for monodigitoxosides, monodigitalosides and tridigitoxosides after 60 min incubation was similar to that observed for the corresponding aglycones. These results suggest that the strongly negative inductive group 16 beta-OCHO is tightly bound to Na, K-ATPase, possibly to the same receptor site than that which is thought forming hydrogen and ionic bonds with the lactone ring. They show that the high toxicity of gitaloxin in guinea-pig heart is likely due to its high potency as Na, K-ATPase inhibitor.