Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Khundmiri, Syed Jalal; Salyer, Sarah A.; Farmer, Brandon C.; Qipshidze-Kelm, Natia; Murray, Rebecca; Clark, Barbara J.; Xie, Zijian; Pressley, Thomas A.; Lederer, Eleanor

doi:10.1016/j.bbamcr.2014.02.005

Cited by 22 publications

(21 citation statements)

References 31 publications

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“…Interestingly, total internal reflection fluorescence microscopy (TIRFM) showed that deletion of the N-terminal CBD of Na C /K C ATPase resulted in the failure of trafficking of Na C /K C ATPase a-subunit and NHE1 to the basolateral membranes. Mutations in the scaffold domain or the activity domain of NHE1 prevented the induction of the increase in Na C /K C ATPase activity by ouabain (Khundmiri et al 2014). These data indicate that an association between Na C /K C ATPase and NHE1 triggered by low-dose ouabain is essential for Src activation and the increase in sodium reabsorption in the proximal tubular cells (Fig.…”

Section: Atpase Activitymentioning

confidence: 50%

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Khundmiri¹

2014

Journal of Endocrinology

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Cardiotonic steroids have been used for the past 200 years in the treatment of congestive heart failure. As specific inhibitors of membrane-bound Na C /K C ATPase, they enhance cardiac contractility through increasing myocardial cell calcium concentration in response to the resulting increase in intracellular Na concentration. The half-minimal concentrations of cardiotonic steroids required to inhibit Na C /K C ATPase range from nanomolar to micromolar concentrations. In contrast, the circulating levels of cardiotonic steroids under physiological conditions are in the low picomolar concentration range in healthy subjects, increasing to high picomolar levels under pathophysiological conditions including chronic kidney disease and heart failure. Little is known about the physiological function of low picomolar concentrations of cardiotonic steroids. Recent studies have indicated that physiological concentrations of cardiotonic steroids acutely stimulate the activity of Na C /K C ATPase and activate an intracellular signaling pathway that regulates a variety of intracellular functions including cell growth and hypertrophy. The effects of circulating cardiotonic steroids on renal salt handling and total body sodium homeostasis are unknown. This review will focus on the role of low picomolar concentrations of cardiotonic steroids in renal Na C /K C ATPase activity, cell signaling, and blood pressure regulation.

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Section: Atpase Activitymentioning

confidence: 50%

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Khundmiri¹

2014

Journal of Endocrinology

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“…Cells were washed three times with serum-free low-phosphate (0.1 mM phosphate) MEM without phenol red and incubated in 2 ml low-phosphate MEM. Total internal reflection fluorescence (TIRF) microscopy was performed in a humidified incubation chamber maintained at 37°C and 5% CO 2 as previously described (17).…”

Section: Methodsmentioning

confidence: 99%

Role of Na⁺/H⁺exchanger regulatory factor 1 in forward trafficking of the type IIa Na⁺-P_icotransporter

Ketchem

Khundmiri

Gawęda

et al. 2015

American Journal of Physiology-Renal Physiology

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Ketchem CJ, Khundmiri SJ, Gaweda AE, Murray R, Clark BJ, Weinman EJ, Lederer ED. Role of Na ϩ /H ϩ exchanger regulatory factor 1 in forward trafficking of the type IIa Na ϩ -Pi cotransporter. Am J Physiol Renal Physiol 309: F109 -F119, 2015. First published May 20, 2015 doi:10.1152 doi:10. /ajprenal.00133.2015 ϩ /H ϩ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na ϩ -Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-P i medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-P i medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-P i medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFPNpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZbinding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi. endoplasmic reticulum/Golgi; Na ϩ /H ϩ exchanger regulatory factor 1; sodium phosphate cotransporter 2a; plasma membrane; trafficking DESPITE THE IMPORTANCE of Na ϩ -P i cotransporter (NpT2a) in the regulation of total body phosphate homeostasis, our understanding of the factors regulating the apical membrane expression of this transporter remains incomplete. Major physiological regulators of NpT2a expression include parathyroid hormone (PTH), dopamine, fibroblast growth factor-23, and high-phosphate diet, which decrease apical membrane expression by stimulation of endocytosis and degradation of NpT2a as well as decreased mRNA expression (40). A low-phosphate diet has the opposite effect, stimulating insertion of NpT2a into the apical membrane and inhibiting endocytosis (22, 31). Thus, the regulation of the protein is dynamic and responsive to multiple physiological regulatory mechanisms. NpT2a is an intrinsic membrane protein for which hydropathy analysis predi...

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“…The vehicle group received PBS intraperitoneally and the ouabain group received 1 μg/kg body weight/day ouabain for 9 days. Blood was collected from carotid artery and serum was separated after measuring blood pressure (blood pressure data was reported previously)[3,6]. …”

Section: Materials and Experimental Methodsmentioning

confidence: 99%

“…We and others have demonstrated that cardioglycosides like ouabain exert a biphasic effect on NKA activity, low concentrations stimulating and higher concentrations inhibiting the activity of NKA [2–5]. Our laboratory has demonstrated that ouabain-mediated stimulation of NKA involves an NHE1 dependent activation of a signaling complex that includes EGFR, Src kinase, ERK, and Akt [3,6]. We have also demonstrated that low concentrations of ouabain increase blood pressure in Sprague Dawley rats in an NHE1 dependent manner [3].…”

Section: Introductionmentioning

confidence: 99%

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Low dose ouabain stimulates Na K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

Ketchem

Conner

Murray

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of Na-K ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of Na-K ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1 μg/kg body wt./day) for 9 days and increased angiotensin II levels in cell culture media after 24 h treatment with ouabain in human (HKC11), mouse (MRPT), canine (MDCK) kidney cells, and human adrenal cells. Ouabain 10 pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

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Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Cited by 22 publications

References 31 publications

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Role of Na⁺/H⁺exchanger regulatory factor 1 in forward trafficking of the type IIa Na⁺-P_icotransporter

Low dose ouabain stimulates Na K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

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Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Cited by 22 publications

References 31 publications

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

Role of Na+/H+exchanger regulatory factor 1 in forward trafficking of the type IIa Na+-Picotransporter

Low dose ouabain stimulates Na K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

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Role of Na⁺/H⁺exchanger regulatory factor 1 in forward trafficking of the type IIa Na⁺-P_icotransporter