Inhibition Of Na-K ATPase Research Articles

Systemic Hypertension

Essential hypertension has been shown to be significantly associated with an increased risk for cardiovascular disease and is not well controlled in many patients. In a large portion of people with essential hypertension, sodium intake has been shown to play a significant role in the production of their hypertension. The mechanism through which increased sodium intake manifests hypertension is unresolved and likely multifactorial. Endogenous cardiac glycosides such as endogenous ouabain (EO) and marinobufagenin have been proposed to play a role in salt-sensitive essential hypertension through their inhibition of Na/K ATPase (NKA). The normal function of the NKA pump is to extrude Na from the intracellular environment and import K. Blocking the NKA disrupts its normal maintenance function. EO is proposed to produce alteration in smooth muscle cell contractility by inhibiting the α2-isoform of NKA, altering Na in a microdomain of the cell. In this region of the plasma membrane the α2-isoform of the NKA colocalizes with another transmembrane protein, the Na/Ca exchanger (NCX). The normal function of NCX is to extrude Ca and import Na. Inhibition of NKA produces an increase in Na within the microdomain, which in turn alters the function of the NCX so that less Ca is extruded, leading to increased intracellular Ca and increased vascular contraction. EO has been shown to be synthesized and secreted by the adrenal cortex in response to chronically elevated sodium intake. The levels of EO have been shown to be significantly elevated in 40% of all untreated hypertensive patients. Marinobufagenin, another cardiac glycoside, has also been implicated as a possible cause of essential hypertension through its preferential inhibition of the α1-isoform of NKA. Antagonism of the endogenous inhibitors of NKA is currently a target of clinical research for the development of innovative antihypertensive treatments.

RNG105 Deficiency Impairs the Dendritic Localization of mRNAs for Na+/K+ATPase Subunit Isoforms and Leads to the Degeneration of Neuronal Networks

mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.e., α3, FXYD1, FXYD6, and FXYD7). The loss of dendritic mRNA localization is accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, we show that RNG105 deficiency affects the formation and maintenance of synapses and neuronal networks. These phenotypes are partly explained by an inhibition of NKA, which is known to influence synaptic functions as well as susceptibility to neurotoxicity. The present study first demonstrates the in vivo role of RNG105 in the dendritic localization of mRNAs and uncovers a novel link between dendritic mRNA localization and the development and maintenance of functional networks.

MARINOBUFAGENIN-INDUCED SODIUM PUMP INHIBITION ASSOCIATES WITH PRESSOR RESPONSE TO MILD SALT LOADING IN PATIENTS WITH RESISTANT HYPERTENSION: 4B.07

Objective: Marinobufagenin (MBG), an endogenous natriuretic and vasoconstrictor cardiotonic steroid, is implicated in natriuretic and pressor responses to salt-loading (SL) in normotensive human subjects. We hypothesized that MBG-induced Na/K-ATPase (NKA) inhibition is associated with salt-sensitivity of blood pressure (BP) in hypertension (HTN). Design and Methods: We studied 22 patients (11 males, 11 females; 56Â ± 6 yrs) with resistant HTN on a combined therapy (lisnopril/amlodipine/hydrochlorothiazide) and 16 healthy normotensive subjects (7 males, 9 females; 44Â ± 2 yrs). SL was performed via i.v. infusion of 1000 mL saline for 1 hour. Beat-to-beat BP was registered using Finometer Pro device. Activity of NKA in erythrocytes measured in the absence and in the presence of a monoclonal anti-MBG antibody (mAb) at baseline and following SL was correlated with SL-induced BP changes and with central BP assessed by applanation tonometry. Results: Within 1 hour of SL patients with HTN exhibited elevation of systolic and diastolic BP (188Â ± 6 vs. 161Â ± 4 and 104Â ± 2 vs. 89Â ± 2 mm Hg, both P < 0.01 vs. baseline). In one hour following SL, BP returned to baseline (158Â ± 4 and 86Â ± 2 mm Hg, respectively). At baseline, activity of the NKA in HTN was suppressed vs. that in controls (1.97Â ± 0.08 vs. 2.84Â ± 0.10 umol Pi/ml/hr; P < 0.01) and was negatively associated with the magnitude of NaCl-induced response of systolic (r = −0.48; P = 0.02) and diastolic (r = −0.44; P = 0.04) BP. Within 60 min of SL, NKA activity was inhibited (1.26Â ± 0.08 umol Pi/ml/hr; P < 0.01), and magnitude of NKA inhibition was positively related to NaCl-induced increase in systolic BP (r = 0.54;P = 0.02) and to central diastolic BP (r = 0.54; P = 0.02). Ex vivo, anti-MBG mAb restored NKA activity (2.66Â ± 0.12 umol Pi/ml/hr; P < 0.01), and magnitude of mAb effect was positively associated with systolic BP following SL (r = 0.47; P = 0.02) and with central diastolic BP (r = 0.67; P = 0.003). Conclusion: In patients with resistant HTN, NKA activity is suppressed and is negatively associated with the magnitude of pressor response to SL. MBG-dependent NKA inhibition associates with NaCl-induced BP elevation.

Na/K-ATpase α2-Subunit Preferentially Modulates Ca Transients and SR Ca Release in Cardiac Myocytes

Na/K-ATPase (NKA) is essential in regulating [Na]i, and thus cardiac myocyte Ca and contractility via Na/Ca exchange. Different NKAα-subunit isoforms are present in heart and may differ functionally, depending on differential membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with endo(sarco)plasmic reticulum, where they could regulate local Na, and indirectly junctional cleft [Ca]. In contrast, NKA-α1 is ubiquitously distributed and may regulate bulk [Na]i. It is controversial whether this model holds for cardiac myocytes. We measured the effect of selective NKA-α2/NKA-α1 inhibition with low concentrations of ouabain on [Na]i, Ca transients and fractional SR Ca release in cardiac myocytes from wild-type (WT) mice and transgenic mice in which NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant (SWAP mice), respectively. Different [ouabain] (5 and 0.1 μM, respectively) were used to attain a similar level of total NKA inhibition in WT and SWAP mice. Ouabain increased Ca transients in WT (F/F0=1.84±0.13 vs. 1.54±0.12 under control conditions) but not in SWAP mice (1.49±0.15 vs. 1.46±0.12), despite a similar and modest increase in [Na]i (≤2 mM). Ca transient increase in WT mice was mediated specifically by NKA-α2 inhibition, because 5 μM ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain-resistant. Ouabain also significantly increased the fractional SR Ca release in WT mice (from 23±2% to 28±2%) but not SWAP mice (23±3% with and without ouabain). Dual-color immunofluorescence measurements coupled with spatial cross-correlation analysis revealed that NKA-α2 is co-localized to a significant extent with both Na/Ca exchanger (43% co-localization) and ryanodine receptors (43% co-localization) in cardiac myocytes. In conclusion, our data indicate that NKA-α2 has a more prominent role (vs. NKA-α1) in modulating Ca transients and SR Ca release in cardiac myocytes.

Endogenous ouabain-like compounds in locus coeruleus modulate rapid eye movement sleep in rats

Although the detailed mechanism of spontaneous generation and regulation of rapid eye movement sleep (REMS) is yet unknown, it has been reported that noradrenergic REM-OFF neurons in the locus coeruleus (LC) cease firing during REMS and, if they are kept active, REMS is significantly reduced. On the other hand, the activity as well as expression of Na-K ATPase has been shown to increase in the LC following REMS deprivation. Ouabain is a specific inhibitor of Na-K ATPase, and endogenous ouabain-like compounds are present in the brain. These findings led us to propose that a decrease in the level of ouabain-like compounds spontaneously available in and around the LC would stimulate and increase the REM-OFF neuronal activities in this region and thus would reduce REMS. To test this hypothesis, we generated anti-ouabain antibodies and then microinjected it bilaterally into the LC in freely moving chronically prepared rats and recorded electrophysiological signals for evaluation of sleep-wakefulness states; suitable control experiments were also conducted. Injection of anti-ouabain antibodies into the LC, but not into adjacent brain areas, significantly reduced percent REMS (mean +/- SEM) from 7.12 (+/-0.74) to 3.63 (+/-0.65). The decrease in REMS was due to reduction in the mean frequency of REMS episode, which is likely due to increased excitation of the LC REM-OFF neurons. Control microinjections of normal IgG did not elicit this effect. These results support our hypothesis that interactions of naturally available endogenous ouabain-like compounds with the Na-K ATPase in the LC modulate spontaneous REMS.

Exercise as an antioxidant decreases age‐associated oxidative stress and protein kinase C activity and restores D1 receptor function in renal proximal tubules of old rats

Age‐associated oxidative stress (OS) via protein kinase C (PKC) activation causes D1 receptor G‐protein (D1R/G) uncoupling resulting in inability of dopamine to inhibit Na,K‐ATPase (NKA) in proximal tubules (PT) of old compared to adult Fisher 344 rats. Our hypothesis is that 3‐month exercise in old rats reduces OS, normalizes PKC activity and restores D1R/G coupling in PT. Biochemical, pharmacological and immunological studies were undertaken in PT of exercised and sedentary adult and old rats. We found that OS markers malondialdehyde and protein carbonyls in PT decreased in exercised rats. This was accompanied by an increase in antioxidant enzymes (AEs), superoxide dismutase and hemeoxygenase. Moreover, Nrf‐2 and NFκB in PT nuclei, transcription factors involved in AEs genes transcription, increased in exercised rats. Interestingly, PKC activity decreased and D1R agonist SKF38393‐ mediated 35S‐GTPγS binding and inhibition of NKA increased in PT of exercised rats. Also, D1R numbers and D1R proteins increased in PT of exercised rats. Furthermore, exercise decreased proteinuria and increased phosphaturia in rats. These results suggest beneficial effects of exercise in increasing anti‐oxidant defenses and improving kidney function in general and D1R function in particular in aging. Both Nrf‐2 and NFκB seem to be involved in this phenomenon. [Funding: NIH/NIA AG25056, AG29904].

Istaroxime, a first in class new chemical entity exhibiting SERCA-2 activation and Na–K-ATPase inhibition: a new promising treatment for acute heart failure syndromes?

Acute heart failure syndromes (AHFS) are associated with the rapid onset of heart failure (HF) signs and symptoms. Hospitalizations for AHFS continue to rise and are associated with significant mortality and morbidity. Several pharmacological agents are currently approved for the treatment of AHFS, but their use is associated with an increase in short-term mortality. There is a need for new agents that can be given in the acute setting with increased efficacy and safety. Istaroxime is a unique agent with both inotropic and lusitropic properties which is currently being studied for the treatment of AHFS. Istaroxime inhibits the sodium-potassium adenosine triphosphatase (ATPase) and stimulates the sarcoplasmic reticulum calcium ATPase isoform 2 (SERCA-2) thereby improving contractility and diastolic relaxation. Early data from human studies reveal that istaroxime decreases pulmonary capillary wedge pressure (PCWP) and possibly improves diastolic function without causing a significant change in heart rate (HR), blood pressure, ischemic or arrhythmic events. Most commonly reported side effects were related to gastrointestinal intolerance and were dose related. In conclusion, istaroxime is a novel agent being investigated for the treatment of AHFS whose mechanism of action and cellular targets make it a promising therapy. Further studies with longer infusion times in patients with hypotension are required to confirm its efficacy and safety.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension

Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

Endogenous sodium pump inhibitors, diabetes mellitus and preeclampsia: Preliminary observations and a hypothesis

Endogenous inhibitors of the Na/K-ATPase (NKA) and diabetes mellitus (DM) are both risk factors for preeclampsia and NaCl sensitive hypertension. Our goal was to test the hypothesis that NaCl supplementation, induces preeclampsia-like symptoms in pregnant rats with DM via stimulation of marinobufagenin (MBG), a natriuretic and vasoconstrictor inhibitor of the NKA. Type 2 DM in female Sprague–Dawley rats was induced by administration of 65 mg/kg streptozotocin at day 4 post-partum. In intact rats, pregnancy was associated with a twofold increase in MBG levels and a mild impairment in glucose tolerance. Pregnant rats with DM exhibited fetal macrosomia, greater impairment of glucose tolerance, and higher levels of MBG as compared to that in normal pregnant rats. As compared to intact pregnant rats, NaCl supplementation of diabetic pregnant rats (drinking 1.8% NaCl during days 12–19 of pregnancy) was associated with an increase in systolic blood pressure, decreased fetal and placental weight, fivefold elevation of MBG excretion, and 42% inhibition of NKA in erythrocytes. In nonpregnant rats, in vivo pretreatment with anti-MBG antibody produced an exaggerated response of plasma levels of glucose and insulin in oral glucose tolerance test. These results suggest that MBG is a common factor in the pathogenesis of DM and preeclampsia, and that regulation of glucose tolerance may be one of the physiological functions of endogenous cardiotonic steroids.

Sex differences in heat shock protein 72 expression and localization in rats following renal ischemia-reperfusion injury

Previously, we demonstrated gender differences in Na-K-ATPase (NKA) expression and function after renal ischemia-reperfusion (I/R) injury (Sex differences in the alterations of Na(+), K(+)-ATPase following ischemia-reperfusion injury in the rat kidney. J Physiol 555: 471-480, 2004). Postischemic membrane destruction causes inhibition of NKA, whereas heat shock protein (HSP) 72 helps to preserve it. We tested the sex differences in postischemic expression of HSP72 and colocalization with NKA. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 (T2), 16 (T16), and 24 h (T24) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls. Postischemic blood urea nitrogen (BUN), serum creatinine, and renal histology were analyzed. HSP72 mRNA expression was detected by RT-PCR, protein levels by Western blot analysis. Fluorescent immunohistochemistry was performed to evaluate the localization of HSP72 and NKA alpha(1)-subunit. Postischemic BUN and creatinine were higher, and renal histology showed more rapid progression in M vs. F (P < 0.05). HSP72 mRNA expression was higher in F vs. M in control and in all I/R groups (P < 0.05). Similar changes were observed in HSP72 protein levels (F vs. M, P < 0.05, control, T2, T16, T24, respectively). Immunohistochemical localization of HSP72 and NKA alpha(1) was similar in control F and M. In postischemic F kidneys, the majority of NKA alpha(1) and HSP72 was colocalized on the basolateral membrane of tubular cells, whereas in M prominent staining was observed in the cytosol and apical domain. This study indicates that in female kidneys the higher basal and postischemic levels of HSP72 and different colocalization with NKA might contribute to the gender differences in renal I/R injury.

Endogenous digitalis-like ligands and Na/K-ATPase inhibition in experimental diabetes mellitus

Dysregulation of the Na/K-ATPase (NKA) in the kidney, cardiovascular system, and peripheral nervous system is believed to contribute to pathogenesis of diabetes mellitus (DM) and its complications. Recently we demonstrated that, in addition to endogenous ouabain (EO), mammalian tissues contain another NKA inhibitor, a bufadienolide marinobufagenin (MBG). In vitro MBG, a natriuretic and a vasoconstrictor, acts as a selective inhibitor of alpha-1 NKA, the main isoform of the sodium pump in renal tubules and vascular smooth muscle. To determine whether digitalis-like NKA inhibitors are linked to NKA dysregulation in DM, we studied changes in renal excretion and plasma levels of MBG and EO and the activity of erythrocyte NKA in male Wistar rats with type 1 DM and type 2 DM. Rats with type 1 DM were studied four weeks following a single intraperitoneal injection of 65 mg/kg streptozotocin (STZ)(n = 12), and rats with type 2 DM were studied 10 weeks after intraperitoneal injection of STZ during the neonatal period (n = 12). Renal excretion and plasma levels of EO did not change in rats with both types of DM as compared to that in the control groups. Renal excretion (57.5 +/- 9.4 pmol/kg/ 3 hours vs. 12.6 +/- 2.1 pmol/kg/ 3 hours; P less than 0.01) and plasma levels (2.23 +/- 0.82 nmol/L vs. 0.29 +/- 0.07 nmol/L; P less than 0.01) of MBG increased, and NKA activity in erythrocytes was inhibited by 50% in rats with type 1 DM as compared to controls. In rats with type 2 DM, plasma levels (1.48 +/- 0.09 nmol/L vs. 0.46 +/- 0.02 nmol/L; P less than 0.01) and renal excretion (21.3 +/- 3.2 pmol/kg/ 3 hours vs. 13.1 +/- 2.1 pmol/kg/ 3 hours) of MBG also became elevated, but less than in the animals with type 1 DM. Accordingly, activity of NKA in erythrocytes from rats with type 2 DM was inhibited by 35%. In vitro treatment of erythrocytes from rats with type 1 and type 2 DM with anti-MBG antibody reversed the DM induced inhibition of the NKA. These results suggest that digitalis-like factors are involved in the pathogenesis of DM and that MBG, rather than EO, is responsible for DM-induced NKA inhibition.

Renal dopamine D1receptor dysfunction is acquired and not inherited in obese Zucker rats

In essential hypertension, the defect in renal dopamine (DA) D(1) receptor function is intrinsic to proximal tubules as this phenomenon is also seen in primary proximal tubule cultures from spontaneously hypertensive rats (SHR) and essential hypertensive patients. Previously, a defect was reported in renal D(1) receptor function in obese Zucker rats. In the present study, we sought to determine whether this D(1) receptor dysfunction is intrinsic in these animals. In primary proximal tubular epithelial cells (PTECs) from lean and obese rats, DA inhibited Na-K-ATPase (NKA) activity in PTECs from both groups of rats. Basal NKA activity, D(1) receptor protein expression, and their coupling to G proteins were similar in cells from both groups. However, when PTECs from lean and obese rats were cultured in 20% serum from obese rats, DA failed to inhibit NKA activity, which was accompanied by a reduction in D(1) receptor expression and a defect in D(1) receptor-G protein coupling. No such defects in the inhibitory effect of DA on NKA activity, D(1) receptor numbers, or coupling were seen when PTECs from both lean and obese rats were grown in 20% serum from lean or rosiglitazone-treated obese (RTO) rats. RTO rat serum had normal blood glucose and reduced plasma levels of insulin compared with serum from obese rats. Furthermore, chronic insulin treatment of PTECs from lean and obese rats caused an attenuation in DA-induced NKA inhibition, a decrease in D(1) receptor expression, and D(1) receptor-G protein uncoupling. These results suggest that defective D(1) receptor function in obese Zucker rats is not inherited but contributed to by hyperinsulinemia and/or other circulating factors associated with obesity.

PTH and DA regulate Na-K ATPase through divergent pathways.

Parathyroid hormone (PTH) and dopamine (DA) inhibit Na-K ATPase activity and sodium-phosphate cotransport in proximal tubular cells. We previously showed that PTH and DA inhibit phosphate transport in opossum kidney (OK) cells through different signaling pathways. Therefore, we hypothesized that PTH and DA also inhibit Na-K ATPase through divergent pathways. We measured PTH and DA inhibition of Na-K ATPase activity in the presence of inhibitors of signaling pathways. PTH and DA inhibited Na-K ATPase in a biphasic manner, the early inhibition through protein kinase C (PKC)- and phospholipase A(2) (PLA(2))-dependent pathways and the late inhibition through protein kinase A- and PLA(2)-dependent pathways. Inhibition of extracellular signal-regulated kinase (ERK) activation blocked early and late inhibition of Na-K ATPase by PTH but not by DA. Pertussis toxin blocked early and late inhibition by DA but not by PTH. Treatment with DA, but not PTH, resulted in an early downregulation of basolateral membrane expression of the alpha-subunit, whereas total cellular expression remained constant for both agonists. We conclude that PTH and DA regulate Na-K ATPase by different mechanisms through activation of divergent pathways.

Role of endothelial nitric oxide and smooth muscle potassium channels in cerebral arteriolar dilation in response to acidosis.

Potassium channels or nitric oxide or both are major mediators of acidosis-induced dilation in the cerebral circulation. However, these contributions depend on a variety of factors such as species and vessel location. The present study was designed to clarify whether potassium channels and endothelial nitric oxide are involved in acidosis-induced dilation of isolated rat cerebral arterioles. Cerebral arterioles were cannulated and monitored with an inverted microscope. Acidosis (pH 6.8 to 7.4) produced by adding hydrogen ions mediated dilation of the cerebral arterioles in a concentration-dependent manner. The role of nitric oxide and potassium channels in response to acidosis was examined with several specific inhibitors and endothelial damage. The dilation was significantly inhibited by potassium chloride (30 mmol/L) and glibenclamide (3 micromol/L; ATP-sensitive potassium channel inhibitor). We found that 30 micromol/L BaCl2 (concentration-dependent potassium channel inhibitor) also affected the dilation; however, an additional treatment of 3 micromol/L glibenclamide did not produce further inhibition. Tetraethylammonium ion (1 mmol/L; calcium-activated potassium channel inhibitor) and 4-aminopyridine (100 micromol/L; voltage-dependent potassium channel inhibitor) as well as ouabain (10 micromol/L; Na-K ATPase inhibitor) and N-methylsulphonyl-6-(2-proparglyloxyphenyl) hexanamide (1 micromol/L; cytochrome P450 epoxygenase inhibitor) did not alter acidotic dilation. N(omega)-Monomethyl-L-arginine (10 micromol/L) and N(omega)-nitro-L-arginine (10 micromol/L) as nitric oxide synthase inhibitor blunted the dilation. Furthermore, the dilation was significantly attenuated after the endothelial impairment. Additional treatment with glibenclamide (3 micromol/L) further reduced the dilation in response to acidosis. Endothelial nitric oxide and smooth muscle ATP-sensitive potassium channels contribute to acidosis-induced dilation of rat cerebral arterioles. Endothelial damage caused by pathological conditions such as subarachnoid hemorrhage or traumatic brain injury may contribute to reduced blood flow despite injury-induced cerebral acidosis.

Phorbol diacetate potentiates na(+)-k(+) ATPase inhibition by a putative endogenous ligand, marinobufagenin.

Several vasoconstrictor agents can regulate the phosphorylation status of the Na(+)-K(+) ATPase (NKA). We have recently demonstrated that mammalian tissues contain an endogenous bufadienolide, digitalis-like alpha(1)-NKA-selective ligand, marinobufagenin (MBG). Protein kinase C induces phosphorylation of the alpha(1)-NKA isoform, the major isoform in vascular smooth muscle, kidney, and heart cells. We hypothesized that protein kinase C-induced phosphorylation of NKA can potentiate the effect of endogenous digitalis-like ligands, and that such potentiation can occur in an NKA isoform-specific fashion. A protein kinase C activator, phorbol 12,13-diacetate (PDA, 50 nmol/L), induced phosphorylation of the alpha1-NKA from human mesenteric artery (HMA) sarcolemma and rat kidney but not that of the alpha(3)-NKA from rat fetal brain. In HMA sarcolemma, which predominantly contains alpha(1)-NKA, PDA (50 nmol/L) potentiated the NKA-inhibitory effect of MBG at the level of high-affinity binding sites (0.05 +/- 0.03 nmol/L versus 4.0 +/- 1.7 nmol/L, P<0.05). In contrast, PDA did not affect the NKA inhibition by ouabain, an alpha(3)-NKA ligand. In isolated endothelium-denuded HMA artery rings, 50 nmol/L PDA potentiated the MBG-induced vasoconstriction (EC(50), 17 +/- 6 nmol/L versus 150 +/- 40 nmol/L; P<0.01). Our results suggest that alpha(1)-isoform-specific NKA inhibition by the endogenous digitalis-like ligand, MBG, is substantially enhanced via NKA phosphorylation by protein kinase C. Thus, an interaction of protein kinase C-dependent phosphorylation and MBG on NKA activity may underlie the synergistic vasoactive effects of MBG and other endogenous vasoconstrictors in hypertension.

An Endogenous Ligand of α-1 Sodium Pump in Hypertensive Dahl Salt Sensitive Rats

P97 Dahl salt sensitive rats (DS), due to a mutation of α-1 subunit of Na/K ATPase (NKA), exhibit impaired pressure natriuresis and on a high NaCl diet retain Na and increase their blood pressure (BP). Recently, we demonstrated that mammalian tissues contain marinobufagenin (MBG), a bufodienolide with a high affinity to α-1 subunit of NKA. The present study investigated possible roles of MBG and another endogenous NKA inhibitor, ouabain (Ou) in hypertension which develops in DS on a high NaCl intake. Dahl salt resistant rats (DR; n=12) and DS (n=12) were put on an 8% NaCl diet for 4 weeks. In DS the excretion of Ou peaked at week 1 of NaCl loading (78±20 pmol vs. 6.4±1.1 pmol in baseline; P Bufo marinus toad for its ability of inhibit the NKA from rat kidney (which expresses only α-1 NKA) an the level of high- and low-affinity binding sites (Table). MBG acts as a selective inhibitor of ouabain-resistant α-1 NKA subunit, as one could expect from a putative natriuretic hormone. Our results sugest that in hypertensive DS, the α-1 ligand, MBG, is elaborated to promote natriuresis and contributes to the sustained BP elevation.

An Endogenous Ligand of α-1 Sodium Pump in Hypertensive Dahl Salt Sensitive Rats

P97 Dahl salt sensitive rats (DS), due to a mutation of α-1 subunit of Na/K ATPase (NKA), exhibit impaired pressure natriuresis and on a high NaCl diet retain Na and increase their blood pressure (BP). Recently, we demonstrated that mammalian tissues contain marinobufagenin (MBG), a bufodienolide with a high affinity to α-1 subunit of NKA. The present study investigated possible roles of MBG and another endogenous NKA inhibitor, ouabain (Ou) in hypertension which develops in DS on a high NaCl intake. Dahl salt resistant rats (DR; n=12) and DS (n=12) were put on an 8% NaCl diet for 4 weeks. In DS the excretion of Ou peaked at week 1 of NaCl loading (78±20 pmol vs. 6.4±1.1 pmol in baseline; P&lt;0.01)and then returned to baseline. In contrast, MBG exhibited a sustained increase (66±13 pmol at week 4 vs. 11±1 pmol in baseline; p&lt;0.01) which paralled the sustained increases in systolic BP (174±10 mm Hg at week 4 vs. 110±2 mm Hg in baseline; p&lt;0.01). No such effects occured in DR on an 8% NaCl intake. MBG-immunoreactive (MBG-ir) material was purified from urine of hypertensive DS via three steps of reverse phase HPLC and compared with ouabain and MBG from Bufo marinus toad for its ability of inhibit the NKA from rat kidney (which expresses only α-1 NKA) an the level of high- and low-affinity binding sites (Table). MBG acts as a selective inhibitor of ouabain-resistant α-1 NKA subunit, as one could expect from a putative natriuretic hormone. Our results sugest that in hypertensive DS, the α-1 ligand, MBG, is elaborated to promote natriuresis and contributes to the sustained BP elevation.

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:

Role of The Ouabain-Like Factor and Na-K Pump in Rat and Human Genetic Hypertension

Endogenous ouabain-like factor (OLF) is present in mammai tissues and after standardized extraction procedure can be similarly quantified by two independent assays: RIA and Na-KATPase inhibition. OLF was quantified both from plasma and tissues obtained from MHS hypertensive and MNS normotensive rats, maintained under the same enviromental and dietary conditions, and from plasma of healthy volunteers and essential hypertensive patients. OLF biochemical characterization shows that it behaves like ouabain except for a 1000-fold higher affinity for the ouabain low-affinity Na-KATPase isoforms than ouabainTissue and plasma levels of OLF are higher in MHS than in MNS rats and are not influenced by exogenous OLF sources. Plasma OLF is also increased in a subgroup of hypertensive patients. Both in rats and humans a primary cell membrane alteration affecting ion transports seems to be linked to the increased levels of OLF. An antihypertensive compound which selectively antagonizes the pressor effect of OLF and corrects the ion transport defect is under development and can represent a new pharmacological approach to the treatment of hypertension

Regulation of Sodium Transport by Endogenous Dopamine Production in Proximal Tubular and OK Cells

Inhibition of AADC for several hours increases the activity and mass of NaKATPase in proximal tubular basolateral membranes and reduces phosphate (Pi) and citrate excretion, but has only a small effect on Na excretion. The reduction in citrate excretion is consistent with the observed increase in brush border Na+/H+ exchange. Thus, in Na replete rats, endogenous D inhibits Na entry into proximal tubular cells, through cotransport with Pi and exchange with H+ and inhibits exit through the Na pump. Tonic D inhibition of NaKATPase and Na+/H+ antiporter activity is not found in the SH rats’ kidneys, which have defective linkage of proximal tubular D receptors to adenylate cyclase. The inhibitory action of endogenous D on Pi reabsorption is retained in SHR kidneys. This suggests that different signaling systems are responsible for the effects of D on NaPi transport and Na+/H+ exchange. In OK cells D inhibits NaPi cotransport (Ki 0.2 μM). The D effect is not blocked by cAMP, adenylate cyclase, PKA or PKC inhibitors. Thus it appears that D regulates NaPi transport by a non-cAMP, non-PKC mechanism and is a homeostatic regulator of phosphate reabsorption by SHR.