| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR METABOLISM
-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae
Department of Physiology, Pharmacology, Metabolism, and Cardiovascular Sciences, Medical University of Ohio, Toledo, Ohio
Submitted 4 January 2006 ; accepted in final form 16 April 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na+-K+-ATPase with its signaling partners. Because both - and 
-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na+-K+-ATPase, we have examined the relative distribution of two major subunit isoforms (1 and 1) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na2CO3 or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of 1-subunits and 80–90% of 1-subunits. Use of Na2CO3 was shown to inactivate Na+-K+-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained 
75% of total myocyte Na+-K+-ATPase activity. Sealed isolated caveolae exhibited active Na+ transport. Confocal microscopy supported the presence of ,-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na+-K+ fluxes, and the sites where the pumping and signaling functions of Na+-K+-ATPase are integrated. Preferential concentration of 1-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of 1 and 1 in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na+-K+-ATPase in cardiac caveolae. cardiac myocyte; caveolin; oligomer; ouabain; sodium pump
and ) are essential for ATPase and ion pumping function, and some preparations also contain a member of the FXYD group of proteins that regulate function (6, 16, 39). In recent years, it has been realized that, in addition to pumping ions, Na+-K+-ATPase also acts as a signal transducer, i.e., the enzyme interacts with neighboring membrane proteins, such as Src and EGFR, to relay messages to intracellular signaling complexes and the nucleus (44). Our recent studies on the signal transducing function of Na+-K+-ATPase in cardiac myocytes (23) showed the presence of a significant fraction of the enzyme's -subunits in the caveolae microdomains of the plasma membrane and suggested that the caveolar pool of the enzyme may be responsible for its signaling function because of its proximity to the signaling partners within the restricted space of the caveolae microdomain. This raised the question of whether or not the caveolar Na+-K+-ATPase also has ATPase activity and ion pumping functions. The present studies were initiated to address this question. Unexpectedly, our findings show that, because of the almost exclusive localization of the -subunit of the cardiac enzyme in caveolae, the catalytic/pumping function of the enzyme is largely restricted to these sarcolemmal microdomains of the cardiac myocytes. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
1-, 2-, 1-, and 3-subunits of Na+-K+-ATPase, EEA1, and Src (clone GD11) from Upstate Biotechnology (Lake Placid, NY); Rab5A (S-19), Rab7 (H50), sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) 2 (N-19), extracellular signal-regulated kinase (ERK) 1/2, goat anti-rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP from Santa Cruz Biotechnology (Santa Cruz, CA); caveolin-1 (clone C060), caveolin-2 (clone 65), caveolin-3 (clone 26), and clathrin heavy chain from BD Transduction Laboratories (Lexington, KY); Na+-K+-ATPase 1 (6F) from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA); vimentin, sarcomeric tropomyosin, and smooth muscle heavy chain from Sigma; and von Willebrand factor from DAKOPATTS (Copenhagen, Denmark). Customer-designed antibody against residues 58–72 of phospholemman (26) was produced by Sigma-Genosys (Woodlands, TX). Protein A/G plus agarose was purchased from Santa Cruz Biotechnology. 22Na+ was purchased from Perkin-Elmer Life Sciences (Boston, MA). Kidney medullas were prepared by dissection from kidneys. Pig kidneys were obtained from a slaughterhouse. All research on rats was done according to procedures and guidelines approved by the Institutional Animal Care and Use Committee. Cell preparation and culture. Calcium-tolerant adult rat cardiac myocytes were prepared from isolated hearts as described before (45). Freshly made suspensions were used for fractionation and caveolae isolation. Cardiac fibroblasts were obtained from the same hearts used for myocyte preparation. After collagenase treatment of the hearts, the disaggregated myocytes and nonmyocytes were separated by gravity (4), and fibroblasts were further purified and cultured as described previously (5). Only passage 2–3 cells that were positive for vimentin but negative for von Willebrand factor, smooth muscle myosin heavy chain, and sarcomeric tropomyosin were used. Neonatal rat cardiac myocytes were prepared from ventricles of 1- to 2-day-old rats and cultured as previously described (30). A7r5 cells (American Type Culture Collection) were cultured as noted before (22).
Fractionation of cell lysates for the preparation of caveolar and noncaveolar membranes. The carbonate-based and detergent-free procedure of Song et al. (38) was used as described before (23). Briefly, cells were lysed at 4°C in a solution containing 0.5 M Na2CO3 (pH 11), 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 10 nM okadaic acid, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. The samples were then homogenized, sonicated, and centrifuged by flotation through sucrose gradients at 4°C as indicated (23). After collection of twelve 1-ml fractions, each fraction was diluted in 10 vol of MBS [25 mM 2-(N-morpholino)ethanesulfonic acid, 150 mM NaCl, pH 6.5] and centrifuged at 100,000 g for 1 h at 4°C. The membrane pellet from each fraction was resuspended in MBS and used or frozen. A modification of the above procedure designed to avoid the denaturing effect of carbonate (see RESULTS) involved the replacement of 0.5 M Na2CO3 with 0.5 M KCl, prepared in MBS, in both the lysis and the gradient solutions. When ATPase activity was to be assayed in the fractionated membranes, Na3VO4 and NaF were omitted from the lysis solution.
Preparation of endosomes. Membrane fractions enriched in early and late endosomes were prepared from neonatal rat cardiac myocytes by the procedures of Gorvel et al. (11) as described by Liu et al. (20).
Other membrane preparations. Microsomes from rat and pig kidney medullas were prepared by previously described procedures (31). A crude membrane preparation was obtained from adult cardiac myocytes by suspending Ca2+-tolerant myocytes isolated from rat hearts in 15 ml of a solution containing 0.25 M sucrose, 30 mM histidine, and 1 mM EDTA. The suspension was homogenized by a Teflon-glass homogenizer, further disrupted by a Polytron homogenizer, filtered through gauze, and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was then centrifuged for 1 h at 115,000 g at 4°C. The sediment was suspended in the above sucrose solution and used or stored frozen. To obtain a crude adult rat heart membrane preparation, one ventricle was minced and then processed as described above.
Immunofluorescence microscopy.
Immunofluorescent staining of myocytes attached to coverslips was conducted as described before (22, 23) with minor modifications. Briefly, cells were fixed with 2% paraformaldehyde for 10 min at room temperature and then permeabilized in 0.0025% saponin for 10 min. Cells were blocked by Image-iT FX signal enhancer (Molecular Probes) for 30 min. Incubations involving both monoclonal and polyclonal primary antibodies were done simultaneously overnight at 4°C. The following pairs of antibodies were used: anti-Na+-K+-ATPase 1-monoclonal antibody (1:50; Upstate Biotechnology) and anti-caveolin-3 polyclonal antibody (1:200; Affinity BioReagents); anti-Na+-K+-ATPase 1-monoclonal antibody (1:50; Upstate Biotechnology) and anti-Na+-K+-ATPase 1-polyclonal antibody (1:100: Upstate Biotechnology). Antigen-antibody complexes were visualized with Alexa Fluor 488- or 546-conjugated secondary antibodies. The cover slips were mounted with ProLong Gold antifade reagent (Molecular Probes). Confocal images were detected by a Leica TCS SP2 spectral confocal scanner and a Leica DMIRE2 microscope (Leica, Mannheim, Germany) equipped with a x63 oil-immersion objective. To avoid cross talk between the two fluorescence dyes, we used the sequential method in which the two laser lines 488 and 546 nm were applied to the cells alternatively. Visualization and analysis were performed using Leica Confocal Microscope Systems software.
Immunoblot analysis and immunoprecipitation.
Membrane samples were subjected to SDS-PAGE and probed with appropriate antibodies as described before (23). For quantitative comparisons, different dilutions of the samples were used to ensure that quantitations were made within the linear range of the assays (e.g., see 
Fig. 2A).
|
|
Na+-K+-ATPase activity. The various membrane preparations were assayed at 37°C by measuring the initial rate of release of Pi from ATP in a medium containing (in mM) 100 NaCl, 25 KCl, 3 MgCl2, 1 EGTA, 2 ATP, and 20 Tris·HCl (pH 7.4). Each assay was done in the presence and absence of 1 mM ouabain, and the ouabain-sensitive component was considered as Na+-K+-ATPase activity. When indicated, alamethacin was included in the assay as described before (46). Pi was measured with Malachite Green (17), using the commercially available Biomol Green reagent (BIOMOL Research Laboratories, Plymouth Meeting, PA).
Na+ transport assay. ATP-dependent Na+ uptake by inside-out membrane vesicles was measured by modification of a procedure described before (47). Caveolar membranes of adult cardiac myocytes prepared by the KCl procedure (see above) were suspended in a solution containing 160 mM choline chloride, 2 mM KCl, and 20 mM MOPS (pH 7.2) at a protein concentration of 2–4 mg/ml. A sample (20 µl) was added to 80 µl of a solution containing (in mM) 150 choline chloride, 5 22NaCl, 0.2 EGTA, 3 MgCl2, 2 ATP, and 20 MOPS (pH 7.2). ATP was omitted from the control to measure ATP-independent uptake. After incubation at 24°C for 15 min, 3 ml of ice-cold stop solution containing 160 mM KCl and 20 mM MOPS (pH 7.2) was added, and the mixture was passed through a 0.45-µm Millipore filter. The filter was washed three times with the stop solution and counted. Membranes pretreated with 0.1 mM Na3VO3 or 0.1 mM digitoxigenin (47) were used to test the sensitivity of the ATP-dependent component of the Na+ transport to these established inhibitors of Na+-K+-ATPase (37).
Other assays. Cholesterol contents of various membrane fractions were assayed by the fluorescent Amplex red cholesterol assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions, using a plate reader. Protein was measured using DC colorimetric assay (Bio-Rad, Hercules, CA).
Analysis of data. Unless stated otherwise, data are means ± SE of the results of a minimum of three experiments. Student's t-test was used, and significance was accepted at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
30% of the cellular contents of the -subunit isoforms of Na+-K+-ATPase are retained in these plasma membrane microdomains (23). In our previous studies, although we also demonstrated the presence of the -subunit of the enzyme in caveolae (23), we did not compare the relative distributions of the two subunits in caveolar and noncaveolar membranes. To do this, we extended our previous work, focusing on isolated adult rat cardiac myocytes.
The most widely used detergent-free procedures for the biochemical isolation of cardiac caveolar membranes are variations of the carbonate-based procedure of Song et al. (38). We used this procedure to fractionate the whole cell lysates of isolated adult cardiac myocytes. However, because our primary interest was on membrane proteins, we sedimented the membrane component of each fraction, assayed the membrane fractions for protein and cholesterol, and used specific antibodies to detect the distribution of the Na+-K+-ATPase subunits and caveolins-2 and -3 in these membrane fractions (Fig. 1). The low buoyant density membranes (fractions 4 and 5) that were rich in cholesterol and contained most of the cellular content of caveolin-3 and -2 also contained significant quantities of the 1- and 2-subunits of Na+-K+-ATPase (Fig. 1), in agreement with our prior findings (23). Surprisingly, however, the 1-subunit seemed to be quite restricted to caveolar membranes (Fig. 1). That the immunoreactive band designated as 1 is indeed this subunit was confirmed as indicated before (23) by its complete conversion to a band of higher mobility (35 kDa) upon deglycosylation (data not shown). In agreement with previous observations of others (25), we did not detect the 2-isoform of Na+-K+-ATPase in rat cardiac myocytes. The 3-isoform was detected in these myocytes as expected from the previous report of the presence of small quantities of 3 in the rat heart (1); however, this minor isoform was also restricted to caveolae (Fig. 1). In the adult rat cardiac myocytes, 1 and 2 are the only isoforms present, with the former constituting 80% of the total -subunit (24, 45).
The immunoblots of Fig. 1 also show the distribution of some signaling partners of Na+-K+-ATPase (Src and ERK1/2) and a number of marker proteins in the membranes fractionated by the carbonate-based procedure. The results indicate that the noncaveolar membranes (fractions 6–12) obtained by this procedure consist of mixtures of sarcoplasmic reticulum (SERCA2), endosomes (Rab5, Rab7), and noncaveolar plasma membrane (clathrin). Certainly, they must also contain other internal membranes for which markers were not used, e.g., the mitochondria. Of interest is the distribution pattern of the sarcolemmal protein phospholemman (FXYD1), which is known to regulate the cardiac Na+-K+-ATPase (39); its distribution seems to be more similar to that of the -subunit.
To quantitate the relative distributions of the muscle-specific caveolin-3 and the two major subunits of Na+-K+-ATPase (1 and 1) in caveolar and noncaveolar membranes, blots such as those shown in Fig. 1 were obtained from five fractionated myocyte samples and quantitated as described in MATERIALS AND METHODS. The results (Fig. 2) show the uneven distribution of the ATPase subunits in caveolar and noncaveolar pools. Interestingly, these data suggest that, in myocytes, the 1-subunit may be a better marker of the caveolar membrane than caveolin-3. That in adult rat cardiac myocytes a significant fraction of caveolin-3 is in heavy noncaveolar membranes has also been noted recently by others (12).
In our previous studies on adult cardiac myocytes (23), we used immunostaining and confocal microscopy to provide support for the partial colocalization of the 1- and 2-subunits of Na+-K+-ATPase with caveolin-3 at peripheral sarcolemma and T tubules. In similar experiments (Fig. 3), we explored the subcellular locations of the 1-subunits and their proximities to caveolin-3 and the 1-subunits. The sample images clearly indicate colocalization of the 1 with caveolin-3 and 1, particularly in T tubules (Fig. 3). Considering the well-established existence of , association, these findings are consistent with the fractionation data (Figs. 1–2), indicating the presence of ,-subunits in some, if not all, of the cardiac caveolae. That in adult rat cardiac myocytes there are distinct populations of caveolae containing different occupants has been suggested (36).
|
-subunit in the caveolar membranes, and because the -subunit is required for catalytic function, we wished to assay Na+-K+-ATPase activities of the different membrane pools obtained by the carbonate-based fractionation procedure. Cell lysates that are subjected to this fractionation procedure often contain vanadate and fluoride to inhibit tyrosine phosphatases. These agents, however, are also high-affinity or irreversible inhibitors of Na+-K+-ATPase (34, 37). When these inhibitors were left out of the lysis buffer (MATERIALS AND METHODS), the relative distributions of Na+-K+-ATPase subunits were the same as those presented in Fig. 1; however, we still could not detect significant ouabain-sensitive Na+-K+-ATPase activity in any of the fractions. This suggested that the use of Na2CO3 (pH 11) in the fractionation procedure may cause inactivation of the enzyme. To test this directly, we compared Na+-K+-ATPase activities of a crude rat heart membrane preparation and kidney microsomes (preparations with vastly different specific activities) before and after exposure to Na2CO3. The results (Fig. 4) showed that such exposure indeed causes significant inactivation of the enzyme. We were prompted, therefore, to modify the carbonate-based fractionation procedure to avoid the denaturation of Na+-K+-ATPase.
|
1-subunit, and 1-subunit (Fig. 5) were similar to those obtained in the carbonate-based procedure (Figs. 1 and 2). The distributions of several marker proteins in the KCl fractions (not shown) were also the same as those shown in Fig. 1.
|
|
|
/.
Although the data of Figs. 1 and 2 clearly show that the ratio of / is lower in caveolar than in noncaveolar membranes, they do not provide information on the molar ratio of the two subunits in either pool. To address this issue, we took advantage of the fact that the molar ratio of 1/1 in rat kidney microsomes has been determined to be close to one (18). By procedures similar to those of the experiments of Fig. 2, we determined the relative 1 and 1 contents of different dilutions of rat kidney microsomes and crude cellular membranes of myocytes (MATERIALS AND METHODS) prepared from five different hearts. Assuming the molar ratio of one for kidney microsomes, we found the molar 1/1 ratio for cardiac membranes to be 2.02 ± 0.28 (mean ± SE; n = 5). Based on this value and the data of Fig. 2B, it may easily be estimated that the molar 1/1 ratio is about one in caveolae and about five in noncaveolar membranes. Evidently, in the latter, there is a large molar excess of 1 over 1.
Interactions of caveolins with Na+-K+-ATPase.
Caveolin-3 oligomerization is responsible for the formation of flask-shaped caveolar invaginations (33, 41), and heterooligomerization of caveolin-3 and caveolin-2 has also been demonstrated in skeletal muscle cells and neonatal cardiac myocytes (2, 35). To assess the possibility of interactions of the ,-oligomers of Na+-K+-ATPase with the caveolin oligomers, coimmunoprecipitation studies were done. When neonatal myocytes were solubilized in a detergent mixture (octylglucoside and Triton X-100) that is known to preserve caveolin oligomers (2, 35, 41), and subjected to immunoprecipitation with an antibody specific to 1-subunit, the Na+-K+-ATPase subunits and caveolin-3 were detected in the precipitate (Fig. 8A). With the use of neonatal or adult myocyte lysates, or caveolar fractions isolated from adult myocytes, immunoprecipitation with an antibody specific for the caveolin-3 also resulted in the isolation of detergent-solubilized oligomers containing the 1-subunit, caveolin-3, and caveolin-2 (Fig. 8B).
|
- and -subunits in caveolar and noncaveolar membranes of cells other than adult cardiac myocytes.
In neonatal rat cardiac myocytes, the relative distributions of the subunits in the membrane fractions (Fig. 9) were similar to those of the adult cardiac myocytes (Fig. 1). However, in two other tested cell types (A7r5 cells and adult rat cardiac fibroblasts), the 1-subunit was not as restricted to caveolae as it is in cardiac myocytes (Fig. 9).
|
- and -subunits in cardiac myocyte endosomes.
Because different internalization pathways of the subunits of Na+-K+-ATPase may be responsible for their uneven distributions in the caveolar and noncaveolar membranes (see DISCUSSION), it was of interest to examine the relative levels of the subunits in endosomes. Using the same preparation of neonatal cardiac myocytes, we prepared caveolar membranes by the carbonate-based procedure and endosomal membranes by a procedure that is commonly used for the preparation of two membrane fractions that are enriched with early and late endosomes (11), and we subjected the three membrane fractions to Western analysis with antibodies against caveolin-3, the endosomal marker EEA1, and the - and -subunits (Fig. 10). The relative levels, per unit of protein, of caveolin-3 and EEA1 in the three membrane fractions were as expected (Fig. 10A). Importantly, the / ratio was clearly higher in late endosomes than in early endosomes or caveolae (Fig. 10B). These findings suggest different internalization paths of the two subunits.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-subunits) of Na+-K+-ATPase but also demonstrate that the major cardiac -subunit of the enzyme resides almost exclusively in caveolar membranes. In agreement with the established dependence of the catalytic activity on ,-association (9, 16), we also find that most of the Na+-K+-ATPase activity of these cardiac myocytes is also concentrated in the caveolar membranes. Evidently, the caveolar inpocketings that constitute 20% of cardiac myocyte plasma membrane surface (7, 19) are the major sites where the linked active transport of Na+ and K+ occurs. Uneven distribution of the active enzyme among caveolar and noncaveolar membranes. To obtain functional Na+-K+-ATPase in caveolar and noncaveolar membranes, it was necessary to modify the widely used carbonate-based fractionation procedure by replacing Na2CO3 with KCl because of the carbonate-induced inactivation of the enzyme (Fig. 4). This finding is consistent with previous observations on the denaturation of Na+-K+-ATPase at highly alkaline pH (32). Replacement of Na2CO3 with NaCl has also been found useful for the detergent-free cell fractionation and preparation of caveolae where inactivation of a membrane receptor by carbonate has been observed (10). It is appropriate to note, however, that not all intrinsic membrane proteins are inactivated by high alkalinity (e.g., see Ref. 32).
What kind of cardiac membranes contain the enzyme subunits that are not in caveolae? Although the carbonate-based fractionation procedure, or its KCl modification, is suitable for the separation of low buoyant-density caveolar membranes from the heavier noncaveolar membranes, the latter fractions are clearly the unresolved mixtures of several cellular membranes (Fig. 1). In spite of this heterogeneity, because Na+-K+-ATPase activity is detected in noncaveolar membranes that are clearly separated from caveolae (Fig. 6), properly folded and active ,-oligomers must also exist in noncaveolar membranes. It is reasonable to assume that these active units are in the bulk plasma membrane and perhaps in Golgi and sarcoplasmic reticulum/endoplasmic reticulum where the ,-assembly and maturation are known to occur (9, 16). That active enzyme is detectable in endoplasmic reticulum and Golgi has been shown in a heterologous expression system (8).
The molar ratio of / seems to be close to one in caveolar membranes, but there is a large excess of in noncaveolar membranes (see RESULTS). Because of the heterogeneity of the noncaveolar membrane fractions, it is not possible to say whether or not the excess coexists with the active ,-oligomers in the same membrane compartment. There are several prior observations indicating that, in different cell types, or different membrane pools, excess of one subunit or the other may exist (18 and references therein).
Origin of the uneven distributions of Na+-K+-ATPase subunits.
If it is true, as most evidence for the assembly of oligomeric proteins indicates (13), that and associate early in the endoplasmic reticulum upon synthesis and membrane integration, and remain associated in the course of delivery to the plasma membrane (8, 9), how may the clear uneven occurrence of the two subunits in various membrane fractions be explained? A possibility that has been considered before is the independent synthesis and maturation of the unaccompanied subunit (18). An alternative is that the two subunits part company on the return trip from the plasma membrane and its microdomains and move at different rates on their respective degradation pathways. It was for this reason that, in the exploratory experiments of Fig. 10, we compared the relative levels of - and -subunits in membrane fractions enriched with early and late endosomes. The apparent absence of from the late but not the early endosomes and the presence of in both suggest that the two subunits remain together in early recycling endosomes but not so in the late endosomes that lead to lysosomal degradation. Although this may not fit the classical pathway of clathrin-mediated endocytosis, we should note that there is now ample evidence for caveolae/raft-mediated endocytosis and that the two alternative pathways may merge by undetermined mechanisms, perhaps through caveosomes (27, 29, 40). That epithelial Na+-K+-ATPase is internalized through the clathrin-dependent route has been known for a long time (e.g., see Ref. 3), and recent evidence also suggests its internalization by a caveolin-dependent route (21). Clearly, further comparative studies on the degradation pathways and rates of the subunits of cardiac Na+-K+-ATPase are required. It is important to note, however, that mechanisms responsible for the uneven distribution of the subunits in various membranes must be highly cell specific, as evident by the present (Fig. 9) and previous findings (18).
Oligomeric state of the caveolar enzyme.
The present findings suggest interactions between the ,-subunits of cardiac Na+-K+-ATPase, caveolin-3, and caveolin-2 (Fig. 8). Whether these interactions are direct or through other proteins within a detergent-resistant assembly cannot be determined by the immunoprecipitation experiments. There is extensive previous evidence indicating that, in caveolae microdomains, caveolin-3 and caveolin-2 (or caveolin-1 and caveolin-2) exist as large oligomers in association with other proteins (33, 41). In epithelial caveolae, interaction of the -subunit of Na+-K+-ATPase with caveolin-1 and other caveolar-signaling proteins has been shown recently (43, 48). There is also a large body of evidence, accumulated over decades, to indicate that the minimum quaternary structure of Na+-K+-ATPase is a tetramer of the ,-dimer (31, 42). Taking these facts together, we consider it most likely that cardiac caveolar Na+-K+-ATPase exists as a large aggregate of Na+-K+-ATPase subunits, caveolins, and related signaling proteins. In spite of the complexity of this assembly, it is evident that at least some of its Na+-K+-ATPase subunits are capable of performing the essential ion-pumping function of the enzyme. Studies are in progress to determine whether or not there are significant differences between the catalytic and transport functions of caveolar and noncaveolar pools of Na+-K+-ATPase.
Functional implications.
The preponderance of the pumping ,-oligomers in cardiac caveolae, relative to other cardiac membranes, strengthens our previous suggestion that these microdomains are the sites of initiation of digitalis-induced signaling, leading to transcriptional regulation of growth-related genes, stimulation of protein synthesis, and myocyte hypertrophy (23, 44). Although the initial proposal (23) was based on the mere caveolar presence of Na+-K+-ATPase subunits, and their resulting proximities to a host of caveolar signaling partners, the present data indicating that cardiac caveolae are the major portals of active Na+-K+ fluxes lend more credibility to the hypothesis that these inpocketings of the sarcolemma/T tubules are indeed responsible for the spacial integration of the pumping and signaling functions of Na+-K+-ATPase. Considering this, and the well-established fact that changes in the active Na+-K+ fluxes of cardiac myocytes are linked to altered intracellular Ca2+ concentration and contractility (30, 44), the question arises as to whether cardiac caveolae are also the sites for the integration of the multiple function of Na+-K+-ATPase with those of the cardiac Ca2+-transporting proteins. This is likely because of a larger body of experimental evidence pointing to the role of caveolae of different cell types, including myocytes, in the regulation of Ca2+ entry in cells and Ca2+-dependent signaling (15). However, because of the apparent existence of different cargo-specific populations of cardiac caveolae (36), it remains to be determined if Na+-K+-ATPase and related Ca2+-regulatory proteins reside in the same cardiac caveolae.
Our limited experiments on different cell types (Fig. 9) suggest that the heavy concentration of the ion pumping Na+-K+-ATPase in caveolar membranes may be specific to cardiac myocytes. Although the cause(s) of the cell specificity of this phenomenon remains to be established, it is appropriate to comment on its functional implications. Studies of the recent past on the signal-transducing function of Na+-K+-ATPase have clearly shown that, although there are similarities in some early stimulus-induced signaling events detected in different cells (e.g., transactivations of Src and EGFR), there is also significant diversity in the nature of activated signaling pathways and the downstream consequences (e.g., inhibition or activation of growth) in various cell types (22, 44). Some of the potential causes of this diversity have been discussed before (44). An additional possibility is that caveolar and noncaveolar pools of the plasma membrane Na+-K+-ATPase may both be involved in stimulus-induced signaling via transactivations of different signaling partners and that the relative distribution of Na+-K+-ATPase in the two pools contributes to the diversity of the downstream events.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
3 subunit. J Biol Chem 272: 22405–22408, 1997.2. Capozza F, Cohen AW, Cheung MWC, Sotgia F, Schubert W, Mattista M, Lee H, Frank PG, and Lisanti MP. Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells Am J Physiol Cell Physiol 288: C677–C691, 2005.
3. Chibalin AV, Katz AI, Berggren PO, and Bertorello AM. Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its - and -subunits. Am J Physiol Cell Physiol 273: C1458–C1465, 1997.
4. Communal C, Singh K, Pimental DR, and Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the -adrenergic pathway. Circulation 98: 1329–1334, 1998.
5. Crabos M, Roth M, Hahn AWA, and Erne P. Characterization of angiotensin II receptors in cultured adult rat cardiac fibroblasts. J Clin Invest 93: 2372–2378, 1994.[Web of Science][Medline]
6. Crambert G, Füzesi M, Garty H, Karlish S, and Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci USA 99: 11476–11481, 2002.
7. Gabella G. Inpocketings of the cell membrane (caveolae) in the rat myocardium. J Ultrastruc Res 54: 135–147, 1978.
8. Gatto C, McLoud SM, and Kaplan JH. Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits. Am J Physiol Cell Physiol 281: C982–C992, 2001.
9. Geering K. The functional role of subunits in oligomeric P-type ATPases. J Bioenerg Biomembr 33: 425–438, 2001.[CrossRef][Web of Science][Medline]
10. Gimpl G and Fahrenholz F. Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane. Eur J Biochem 267: 2483–2497, 2000.[Web of Science][Medline]
11. Gorvel JP, Chavrier P, Zerial M, and Gruenberg J. Rab5 controls early endosome fusion in vitro. Cell 64: 915–925, 1991.[CrossRef][Web of Science][Medline]
12. Head BP, Patel HH, Roth DV, Lai NC, Niesman IR, Farquhar MG, and Insel PA. G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3 associated microdomains in adult cardiac myocytes. J Biol Chem 280: 31036–31044, 2005.
13. Helenius A, Marquardt T, and Braakman I. The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol 2: 227–231, 1992.[CrossRef][Medline]
14. Huang W, Rhee HM, Chiu TH, and Askari A. Re-evaluation of the relationship between the positive inotropic effect of ouabain and its inhibitory effect on (Na++K+)-dependent adenosine triphosphatase in rabbit and dog hearts. J Pharmacol Exp Ther 211: 571–582, 1979.
15. Isshiki M and Anderson RGW. Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction. Traffic 4: 717–723, 2003.[CrossRef][Web of Science][Medline]
16. Kaplan JH. Biochemistry of Na,K-ATPase. Annu Rev Biochem 71: 511–535, 2002.[CrossRef][Web of Science][Medline]
17. Lanzetta PA, Alvarez LJ, Reinach PS, and Candia OA. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100: 95–97, 1979.[CrossRef][Web of Science][Medline]
18. Lavoie L, Levenson R, Martin-Vasallo P, and Klip A. The molar ratios of and subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle. Biochemistry 36: 7726–7732, 1997.[CrossRef][Medline]
19. Levin KR and Page E. Quantitative studies on plasmalemmal folds and caveolae of rabbit ventricular myocardial cells. Circ Res 46: 244–255, 1980.
20. Liu J, Kesiry R, Periyasamy SM, Malhotra D, Xie Z, and Shapiro JI. Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Kidney Int 66: 227–241, 2004.[CrossRef][Web of Science][Medline]
21. Liu J, Liang M, Liu L, Malhotra D, Xie Z, and Shapiro JI. Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1. Kidney Int 67: 1844–1854, 2005.[CrossRef][Web of Science][Medline]
22. Liu L, Abramowitz J, Askari A, and Allen JC. Role of caveolae in ouabain-induced proliferation of cultured vascular smooth muscle cells of the synthetic phenotype. Am J Physiol Heart Circ Physiol 287: H2173–H2182, 2004.
23. Liu L, Mohammadi K, Aynafshar B, Wang H, Li D, Liu J, Ivanov AV, Xie Z, and Askari A. Role of caveolae in signal-transducing function of cardiac Na+-K+-ATPase. Am J Physiol Cell Physiol 284: C1550–C1560, 2003.
24. Lucchesi PA and Sweadner KJ. Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. J Biol Chem 266: 9327–9331, 1991.
25. McDonough AA, Zhang Y, Shin V, and Frank JS. Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes. Am J Physiol Cell Physiol 270: C1221–C1227, 1996.
26. Moorman JR, Palmer CJ, John JE III, Durieux ME, and Jones LR. Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes. J Biol Chem 267: 14551–14554, 1992.
27. Nichols B. Caveosomes and endocytosis of lipid rafts. J Cell Sci 116: 4707–4714, 2003.
28. Page E, Winterfield J, Goings G, Bastawrous A, Upshaw-Earley J, and Doyle D. Water channel proteins in rat cardiac myocyte caveolae: osmolarity-dependent reversible internalization. Am J Physiol Heart Circ Physiol 274: H1988–H2000, 1998.
29. Parton RG and Richards AA. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4: 724–738, 2003.[CrossRef][Web of Science][Medline]
30. Peng M, Huang L, Xie Z, Huang WH, and Askari A. Partial inhibition of Na+/K+-ATPase by ouabain induces the Ca2+-dependent expressions of early-response genes in cardiac myocytes. J Biol Chem 271: 10372–10378, 1996.
31. Periyasamy SM, Huang WH, and Askari A. Subunit associations of (Na++K+)-dependent adenosine triphosphatase. J Biol Chem 258: 9878–9885, 1983.
32. Philipson KD, McDonough AA, Frank JS, and Ward R. Enrichment of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by alkaline extraction. Biochim Biophys Acta 899: 59–66, 1987.[Medline]
33. Razani B, Woodman SE, and Lisanti MP. Caveolae: From cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002.
34. Robinson JD, Davis RL, and Steinberg M. Fluoride and beryllium interact with the (Na+K)-dependent ATPase as analogs of phosphate. J Bioenerg Biomembr 18: 521–531, 1986.[CrossRef][Web of Science][Medline]
35. Rybin VO, Grabham PW, Elouardighi H, and Steinberg SF. Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3. Am J Physiol Heart Circ Physiol 285: H325–H332, 2003.
36. Scriven DRL, Klimek A, Asghari P, Bellve K, and Moore EDW. Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors. Biophys J 89: 1893–1901, 2005.[CrossRef][Web of Science][Medline]
37. Skou JC and Esmann M. The Na,K-ATPase. J Bioenerg Biomembr 24: 249–261, 1992.[Web of Science][Medline]
38. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, and Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. J Biol Chem 271: 9690–9697, 1996.
39. Sweadner KJ. Phospholemman: a new force in cardiac contractility. Circ Res 97: 510–511, 2005.
40. Tagawa A, Mezzacasa A, Hayer A, Longatti A, Pelkmans L, and Helenius A. Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters. J Cell Biol 170: 769–779, 2005.
41. Tang Z, Scherer PE, Okamoto T, Song K, Chu C, Kohtz DS, Nishimoto I, Lodish HF, and Lisanti MP. Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol Chem 271: 2255–2261, 1996.
42. Taniguchi K, Kaya S, Abe K, and Mårdh S. The oligomeric nature of Na/K-transport ATPase. J Biochem 129: 335–342, 2001.
43. Wang H, Haas M, Liang M, Cai T, Tian J, Li S, and Xie Z. Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase. J Biol Chem 279: 17250–17259, 2004.
44. Xie Z and Askari A. Na+/K+-ATPase as a signal transducer. Eur J Biochem 269: 2434–2439, 2002.[Web of Science][Medline]
45. Xie Z, Wang Y, Askari A, Huang WH, Klaunig JE, and Askari A. Studies on the specificity of the effects of oxygen metabolites on cardiac sodium pump. J Mol Cell Cardiol 22: 911–920, 1990.[CrossRef][Web of Science][Medline]
46. Xie Z, Wang Y, Ganjeizadeh M, McGee R Jr, and Askari A. Determination of total (Na++K+)-ATPase activity of isolated or cultured cells. Anal Biochem 183: 215–219, 1989.[CrossRef][Web of Science][Medline]
47. Xie Z, Wang Y, Liu G, Zolotarjova N, Periyasamy SM, and Askari A. Similarities and differences between the properties of native and recombinant Na+/K+-ATPases. Arch Biochem Biophys 330: 153–162, 1996.[CrossRef][Web of Science][Medline]
48. Yuan Z, Cai T, Tian J, Ivanov AV, Giovannucci DR, and Xie Z. Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex. Mol Biol Cell 16: 4034–4045, 2005.
This article has been cited by other articles:
|
|
 |
|
  C. N. White, G. A. Figtree, C.-C. Liu, A. Garcia, E. J. Hamilton, K. K. M. Chia, and H. H. Rasmussen Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase Am J Physiol Cell Physiol, April 1, 2009; 296(4): C693 - C700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Garg, J. Jiao, and K. Hu Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes Cardiovasc Res, April 1, 2009; 82(1): 51 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Zadeh, H. Xu, M. E. Loewen, G. P. Noble, D. F. Steele, and D. Fedida Internalized Kv1.5 traffics via Rab-dependent pathways J. Physiol., October 15, 2008; 586(20): 4793 - 4813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Cai, H. Wang, Y. Chen, L. Liu, W. T Gunning, L. E. M. Quintas, and Z.-J. Xie Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase J. Cell Biol., September 22, 2008; 182(6): 1153 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. William, E. J. Hamilton, A. Garcia, H. Bundgaard, K. K. M. Chia, G. A. Figtree, and H. H. Rasmussen Natriuretic peptides stimulate the cardiac sodium pump via NPR-C-coupled NOS activation Am J Physiol Cell Physiol, April 1, 2008; 294(4): C1067 - C1073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu, X. Zhao, S. V. Pierre, and A. Askari Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1489 - C1497. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
