Background: It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Results: Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. Conclusion: A420P mutant has normal pumping but not signaling function. Significance: Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase.

Pase is a fundamental component of ion transport. Four ␣ isoforms of the Na-K-ATPase catalytic ␣ subunit are expressed in human cells. The ubiquitous Na-K-ATPase ␣1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, ␣2 expression is limited to a few cell types including myocytes, where it is coupled to the Na ϩ /Ca 2ϩ exchanger. To test whether rat Na-KATPase ␣2 is capable of cellular signaling like its ␣1 counterpart in a recipient mammalian system, we used an ␣1 knockdown pig renal epithelial cell (PY-17) to create an ␣2-expressing cell line with no detectable level of ␣1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to ␣1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in ␣2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, ␣2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase ␣1, which downregulates basal Src activity, the corresponding ␣2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in ␣2 cells. These findings indicate that Na-K-ATPase ␣2 does not regulate Src and, therefore, may not serve the same role in signal transduction as ␣1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where ␣1 and ␣2 isoforms play distinct roles in mediating contraction and signaling in myocytes.rat Na-K-ATPase isoforms; Src tyrosine kinase; membrane transporters; ouabain; signal transduction THE NA-K-ATPASE, discovered in 1957 by Jens Skou, is a member of the P-type ATPase family and an integral membrane protein maintaining cellular ion homeostasis by pumping Na ϩ and K ϩ across the cell membrane (32). The protein consists of two noncovalently linked subunits, ␣ and ␤. The ␣-subunit contains the binding sites for substrates (e.g., ions and ATP) and ligands (e.g., ouabain) as it undergoes E1 and E2 conformational changes during a transport cycle. Four isoforms of the Na-K-ATPase ␣-subunit are expressed in humans. While ␣1 is expressed ubiquitously, ␣2 and ␣3 are primarily found in myocytes and neurons, respectively, and ␣4 is detected in sperm (3,4,31,45,50). Recent studies have revealed major differences in the physiological functions of each isoform. For example, the ␣2 isoform appears to possess the unique ability to regulate intracellular Ca 2ϩ levels in myocytes and coresides with the Na ϩ /Ca 2ϩ exchanger (5, 24). In addition, recent experiments using SWAP mice (ouabainsensitive ␣1 Na-K-ATPase mutant and ouabain-resistant ␣2 Na-K-ATPase mutant) suggest that the ␣2 isoform plays a more prominent role in calcium release in cardiac and smooth muscle myocytes than ␣1 (12).Over the last decade, we have also come to realize that the Na-K-ATPase may have many regulatory functions other than pumping ions across cell membranes. Studies fr...

Purpose: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. Experimental Design: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to −2 and P < 0.05. Results: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair–related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. Conclusions: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.

The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1.

Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Like ischemic preconditioning (IPC) induced by brief episodes of ischemia and reperfusion, ouabain preconditioning (OPC) mediated by Na/K-ATPase signaling protects the heart against IR injury. Class I PI3K activation is required for IPC, but its role in OPC has not been investigated. While PI3K-IB is critical to IPC, studies have suggested that ouabain signaling is PI3K-IA-specific. Hence, a pharmacological approach was used to test the hypothesis that OPC and IPC rely on distinct PI3K-I isoforms. In Langendorff-perfused mouse hearts, OPC was initiated by 4 min of ouabain 10 μM and IPC was triggered by 4 cycles of 5 min ischemia and reperfusion prior to 40 min of global ischemia and 30 min of reperfusion. Without affecting PI3K-IB, ouabain doubled PI3K-IA activity and Akt phosphorylation at Ser473. IPC and OPC significantly preserved cardiac contractile function and tissue viability as evidenced by left ventricular developed pressure and end-diastolic pressure recovery, reduced lactate dehydrogenase release, and decreased infarct size. OPC protection was blunted by the PI3K-IA inhibitor PI-103, but not by the PI3K-IB inhibitor AS-604850. In contrast, IPC-mediated protection was not affected by PI-103 but was blocked by AS-604850, suggesting that PI3K-IA activation is required for OPC while PI3K-IB activation is needed for IPC. Mechanistically, PI3K-IA activity is required for ouabain-induced Akt activation but not PKCε translocation. However, in contrast to PKCε translocation which is critical to protection, Akt activity was not required for OPC. Further studies shall reveal the identity of the downstream targets of this new PI3K IA-dependent branch of OPC. These findings may be of clinical relevance in patients at risk for myocardial infarction with underlying diseases and/or medication that could differentially affect the integrity of cardiac PI3K-IA and IB pathways.