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(Clinical Chemistry. 2000;46:234-241.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Sodium Pump Isoforms in Xenotransplantation: Importance of Biochemical Compatibility

Andrea M. Rose1,1, Hassan M. Qazzaz1, Nina Zolotarjova1, Brenda J. Mellett1, Alvin W. Martin1 and Roland Valdes Jr1,2,a

Departments of
1 Pathology and Laboratory Medicine and
2 Biochemistry and Molecular Biology, University of Louisville, School of Medicine, Louisville, KY 40292.
a Address correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292. Fax 502-852-1177; e-mail rvaldes{at}louisville.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Xenotransplantation of pig hearts to humans could be hampered by the reportedly reduced affinity for digoxin of pig heart. We examined the hypothesis that expression of the individual {alpha}-subunit isoforms of the sodium pump [Na+,K+-ATPase (NKA)], the receptor for the plant-derived cardiac glycosides, may be responsible for this difference.

Methods: We used a NKA-inhibition assay in combination with Western analysis, immunohistochemistry, and phosphorylation of the NKA {alpha} subunit to identify the distribution and expression of {alpha} isoforms in four chambers of porcine and human hearts.

Results: We confirmed that tissue from porcine heart is less sensitive to digitalis (IC50 = 1740 nmol/L) when compared with human heart (IC50 = 840 nmol/L), whereas porcine cerebral cortex-mix had an affinity comparable to that of human heart (IC50 = 910 nmol/L). Our data show that porcine cerebral cortex-mix and human heart contain all three {alpha} isoforms, whereas porcine heart expresses only the {alpha}1 isoform.

Conclusions: The different expressions of sodium pump isoforms in human vs porcine cardiac tissues suggests that porcine hearts may not be pharmacologically or endocrinologically compatible when used in humans. Studies of both pharmacologic and endocrinologic tissue compatibility are needed prior to selection of organs for xenotransplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heart failure currently affects >2 million people in the United States. Many severely affected individuals, identified as potential heart transplant recipients, die while waiting for donor organs. Porcine heart has been proposed and is actively under investigation as a replacement organ for failing human hearts (1). Although nonhuman primates are physiologically and genetically closest to humans, social and ethical concerns make more widely divergent species such as pig attractive. In assessing organ-transfer viability between species, structural and immunological compatibility seem to be key hurdles. However, compatibility of pharmacologic and endocrinologic function must also be characterized. The immunohisto-incompatibility between more divergent species leads to hyperacute rejection within minutes of transplantation. Immunologic tolerance to discordant xenografts currently is being addressed (2)(3)(4), and clinical trials using transgenic pigs that express human proteins are slated to begin (5). With the expectation that porcine-to-human heart xenotransplantation may be a viable alternative, porcine heart tissue should also be characterized biochemically with respect to its pharmacologic response to commonly used cardioactive drugs as well as to endogenous hormones specific to regulating cardiac function.

The sodium pump [Na+,K+-ATPase (NKA);2 EC 3.6.1.37] is an integral membrane-anchored protein that couples the energy released in the enzymatic hydrolysis of ATP to the translocation of sodium and potassium ions across the cell membrane. In heart muscle, NKA plays a key role in regulating strength of contraction (inotropy) and rhythmicity (chronicity). NKA is the receptor for the cardiac glycosides (e.g., digitalis) (6). The NKA holoenzyme is composed of two subunits ({alpha} and ß), each of which has at least three genetically distinct isoforms (7)(8)(9). Selective pressure to maintain different NKA isoforms in mammalian and non-mammalian species supports the hypothesis that the isoforms differ functionally (10) and that isoform expression is linked to function. Pharmacologic treatment with digitalis is based on the interaction of these drugs with the sodium pump (i.e., inhibition of pump ion-transport activity). We previously have suggested that pig heart has a reduced affinity for digoxin compared with human heart (11). The basis for this is not known, but one explanation is that a difference in expression of individual NKA isoforms may be responsible for the reduced sensitivity to cardiac glycosides.

Biochemical compatibility in response to pharmacologic agents as well as to endogenous hormones must be characterized as part of establishing the compatibility of organs used in xenotransplantation. The case of digitalis is particularly important in view of its high prevalence of usage as a prophylactic cardiac drug as well as the recent evidence indicating existence of endogenous hormone-like counterparts [e.g., ouabain-like factor (OLF) and digoxin-like immunoreactive factor (DLIF)] (12). In this study, we confirmed the difference in digitalis sensitivity for heart from these two species and used three lines of evidence (Western analysis, immunohistochemistry, and a specific ouabain-stimulated phosphorylation) to demonstrate a different expression of NKA {alpha} isoforms in human and porcine heart tissues. Whereas human heart expresses the {alpha}1, {alpha}2, and {alpha}3 isoforms of NKA, porcine heart expresses only {alpha}1. Our results may have general implications in demonstrating biochemical incompatibility for the use of pig hearts for transplantation into humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and reagents
All chemicals used were reagent grade. Ouabain, digoxin, all reagents for catalytic inhibition of NKA (ATP, ammonium molybdate, Tween-80, and bovine serum albumin), and all reagents for gel electrophoresis [acrylamide, bis-acrylamide, N,N,N',N'-tetraethylethylenediamine, Tris, sodium dodecyl sulfate (SDS), glycine, ß-mercaptoethanol, and bromphenol dye] were purchased from Sigma. Ammonium persulfate was obtained from Amresco. Prestained protein markers were obtained from Bio-Rad. Phosphorus-32-phosphate (32Pi; 1 mCi in 100 µL) was purchased from NEN, Life Science Products. Acetonitrile (chromatographic grade) was obtained from Aldrich.

Serial dilutions (10-6 to 10-3) of both ouabain and digoxin were used in catalytic activity inhibition of NKA and ouabain-stimulated phosphorylation studies.

equipment and materials
To verify the purity of the cardiac glycosides, we used HPLC with a C18 reversed-phase µBondapak column (3.9 x 300 mm; 10-µm particle size) connected to a Waters 600E system controller and a Waters 996 photodiode array detector.

organ procurement
Human brain samples (13) were obtained from the Neuropathology Section, Clinical Brain Disorders Branch, National Institutes of Mental Health Neuroscience Center. Porcine cerebral cortex-mix was obtained from Sigma. Human heart and kidney were purchased from the International Institute for the Advancement of Medicine, or provided by the Kentucky Organ Donor Association, Louisville, KY. Dog heart was purchased from Pel-Freez Biologicals, and pig heart was purchased from Pel-Freez or provided by a local abattoir. Human red blood cells were obtained locally from healthy volunteers. Approval for the use of human tissue was obtained from the human studies Internal Review Board of the University of Louisville School of Medicine.

tissue preparation and fractionation
Heart and kidney were prepared and fractionated as described previously (14). All preparative steps were performed at 4 °C. If previously frozen, tissue samples were thawed on ice in homogenization buffer (0.32 mol/L sucrose, 50 mmol/L Tris, 50 mmol/L EDTA, pH 7.4) plus the protease inhibitors (1 mg/L each of aprotinin, pepstatin, and antipain). Samples were minced with scissors and homogenized with a polytron at two-thirds speed for 1 min. After filtration through cheesecloth, the samples were rehomogenized with a glass homogenizer and Teflon pestle (five strokes; 20 s per stroke) and centrifuged at 20 000g for 40 min. Pellets were rehomogenized three times in sucrose buffer and centrifuged at 4000g for 15 min after each rehomogenization. All supernates were combined and centrifuged at 40 000g for 30 min. Pellets were washed twice by resuspension in ice-cold Tris buffer (50 mmol/L Tris, pH 7.4, 0.5 mmol/L EDTA, 80 mmol/L NaCl, 4 mmol/L MgSO4 · 7 H2O) followed by centrifugation at 40 000g for 90 min. Final pellets were resuspended in 200 mmol/L Tris, 200 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L MgCl2, 2 mmol/L EGTA, pH 7.4.

Human brain tissue samples were sonicated on ice in 50 mmol/L Tris-HCl buffer, pH 7.5, using a Heat Systems MicrosonTM Ultrasonic Cell Disrupter, and then were resuspended in loading buffer for Western analysis.

Total protein concentration was determined by the Bio-Rad microassay procedure for microtiter plates and adjusted to 1 g/L.

nka inhibition assay
The NKA assay used to measure the effect of digoxin on the release of phosphate during hydrolysis of ATP was based on the method of Chan and Swaminathan (15) with minor modifications to increase the sensitivity. We performed the inhibition assay by pipetting 20 µL of sample containing the desired concentration of glycoside (Tris buffer was used for the no-inhibitor control) into a well of a disposable nonsterile 96-well flat-bottomed polystyrene microtiter plate (Corning) preincubated at 37 °C in a water bath for 10 min. Porcine cerebral cortical NKA solution (20 µL of a 1 kU/L solution containing three isoforms of the {alpha} subunit) in Tris buffer, pH 7.8, was added for an additional 20-min incubation. ATP (20 µL of a 10 mmol/L solution in Tris buffer, pH 7.8) was added and allowed to react for an additional 15 min. The final concentrations in the mixture were as follows: 3.3 mmol/L potassium, 133.3 mmol/L sodium, 3.3 mmol/L magnesium, and 3.3 mmol/L ATP in Tris-HCl buffer, pH 7.8.

After the incubation, we added 150 µL of molybdate solution [1.0 mmol/L molybdate, 11 mmol/L sulfuric acid, and 142 mL/L Tween-80:methanol solution (12:88, by volume)]. Color development was allowed to proceed for a maximum of 30 min, after which the color intensity was measured immediately in triplicate at 340 nm on a DuPont Microplate Reader II, Multiskan MCC/340. The color intensity is proportional to the release of phosphate ions, which is a direct indicator of ATP hydrolysis and, therefore, NKA activity. For statistical purposes, all samples were assayed three to five times each with duplicates that were corrected for background (assay buffer only), averaged, and normalized to ouabain-sensitive NKA activity (100% inhibition at 1 mmol/L ouabain). The percentage of inhibition of NKA activity by digoxin represents the percentage of total ouabain-specific inhibition. Similar studies were performed with porcine heart and human heart.

western analysis
Samples were diluted fivefold in loading buffer (250 mmol/L Tris-HCl, pH 6.8, 100 mL/L SDS, 30 mL/L ß-mercaptoethanol, 500 mL/L glycerol, 0.1 g/L bromphenol blue) and heated at 65 °C for 5 min (10) before loading on an 8% polyacrylamide gel. Gel electrophoresis to resolve the proteins of interest was performed on both minigel and vertical slab gel electrophoresis units from Hoefer Scientific Instruments (Models SE 245, SE 260, and SE400). Gels were dried on a slab gel dryer (SDG 4050) connected to a Savant gel pump (GP 100). Filters were incubated for 1 h with NKA isoform-specific antibodies: monoclonal mouse anti-rabbit NKA isoform-specific antibodies ({alpha}1 monoclonal antibodies) from Upstate Biotechnology, {alpha}2 polyclonal antibody (PAb), or {alpha}3 PAb (10), followed by incubation for 1 h with goat anti-mouse or goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad). Signal was detected on autoradiograms by chemiluminescence per the manufacturer’s instructions (Amersham Life Science).

ouabain-specific 32PiPHOSPHORYLATION OF NKA ISOFORMS
Ouabain-specific phosphorylation of NKA by Pi was performed as described elsewhere (16) with minor modifications. To evaluate the extent of phosphorylation, the radioactively labeled acid-stable phosphoenzyme/intermediate was resolved by gel electrophoresis. Membrane preparations (100–200 µg for porcine and human heart and 40–60 µg for porcine cerebral cortex-mix) were preincubated at room temperature for 30 min with or without 1 mmol/L ouabain in the reaction medium (2 mmol/L MgCl2, 50 mmol/L Tris-HCl, pH 7.2). A negative control was preincubated with 20 µL of the reaction buffer only. Carrier-free H332PO4 (New England Nuclear) was diluted with Tris-MgCl2 buffer and filtered through a Millipore Filter (0.22 µm) to remove polyphosphates. The reaction was started by the addition of the 32Pi (final concentration, 30 µmol/L containing 8 µCi of 32Pi). After a 15-min incubation at room temperature, the reaction was terminated by the addition of 1 mL of 80 mL/L HClO4. The sample was resuspended in 5x Laemmli sample buffer containing at a final concentration 5 mL/L HClO4, 25 g/L SDS, 100 mL/L glycerol, and 1 g/L pynomin Y dye. Samples (100 µL) were loaded onto a 7.5% SDS-polyacrylamide gel and run for 4–5 h at 30 mA in the coldroom at 4 °C. For autoradiography, the gel was fixed in a mixture of 400 mL/L methanol and 100 mL/L acetic acid, dried, and exposed to x-ray film. Radioactivity was quantified by soft laser-scanning densitometry. Before phosphorylation, the gel electrophoretic mobility of the three {alpha} isoforms of NKA from porcine cerebral cortex were confirmed by Western blot analysis with isoform-specific antibodies to the three {alpha} isoforms. Each isoform on the blot was visualized with a horseradish peroxidase-conjugated goat anti-mouse antibody, and the signal was detected on an autoradiogram by chemiluminescence as described above.

immunohistochemical analysis
Human and porcine heart tissue were excised and dissected, and contiguous sections were either fixed in neutral-buffered formalin for paraffin embedding or snap frozen in isopentane cooled in liquid nitrogen. Frozen sections were cut into 6-µm sections, mounted onto silanized slides, fixed in acetone, and immunostained along with the paraffin-embedded tissue. Paraffin-embedded tissues were cut into 3-µm sections, floated onto a protein-free water bath, and picked up on silanized glass slides. After removal of the paraffin and hydration to distilled water, the slides were steamed for 20 min in citrate buffer, pH 5.7. Tissue sections were incubated for 25 min with {alpha} isoform-specific PAbs (described above) and visualized with a standard three-step immunohistochemistry procedure, using a commercially available avidin-biotin-labeled detection system including buffers (ChemMateTM Detection System; Ventana Bio Tek Medical Systems). Diaminobenzene was used as the chromogen and was counterstained with hematoxylin. The specificity of each reaction was further checked by replacing the PAb with the diluting buffer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We confirmed the relative tissue sensitivities to digoxin for pig and human hearts. We used both immunochemical and phosphorylation approaches to identify the presence of {alpha}-subunit isoforms of NKA in all four chambers of hearts from porcine and humans. Brain tissue was used to control for possible genetic epitope differences in immunochemical detection. All techniques applied in the course of this study (Western analysis, immunohistochemical staining, phosphorylation, and catalytic activity of the {alpha} isoforms of NKA) converged to consistent results.

tissue sensitivities to digoxin
The relative inhibition (IC50, in µmol/L) of NKA catalytic activity by digoxin, using porcine cerebral cortex-mix, porcine heart, and human heart, is shown in Table 1 . These data confirm that porcine cerebral cortex-mix and human heart have comparable IC50 values, whereas the sensitivity of NKA to inhibition by digoxin in porcine heart is approximately twofold lower.


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  		Table 1. Inhibition of NKA catalytic activity (IC50, µmol/L) by digoxin in porcine cerebral cortex, porcine heart, and human heart tissues.

immunochemical identification of {alpha} isoforms in human and porcine heart tissue
Differential centrifugation was used to obtain membrane fractions from human and porcine atrial and ventricular outer free-wall tissues. Tissues used for Western analysis were isolated in our laboratory with the exception of a commercially acquired porcine cerebral cortex-mix NKA. The {alpha} isoforms present and the specificity of the NKA isoform-specific antibodies [Ref. (10) and Upstate Biotechnology] were consistent with previously published reports for human heart, dog heart, human brain, pig brain, rat brain, human kidney, and human red blood cells (12). Fig. 1 shows the distribution pattern of NKA {alpha} isoforms observed with seven tissue specimens taken from four human hearts (cause of death was head trauma or cerebral aneurysm) and four specimens taken from one porcine heart. Western analysis of outer free wall from four porcine left ventricles with antibodies specific for the individual isoforms (data not shown) confirmed that unlike human heart, which showed three {alpha} isoforms, porcine heart showed only {alpha}1.



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  		Figure 1. Western analysis of porcine cerebral cortex-mix (PCC) and porcine (P) and human (H) heart tissues.

Heart tissue samples from outer free wall. Chamber designations: R, right; L, left; A, atrium; V, ventricle. Human and porcine heart samples loaded at 5.0 µg of total protein in 13 µL; PCC loaded at 0.5, 1.0, 2.0, and 5.0 µg of total protein in 13 µL. We used 1-h incubations with primary (anti-{alpha}1, -{alpha}2, or -{alpha}3; diluted 1:1000) and secondary (goat anti-rabbit IgG conjugated to horseradish peroxidase; diluted 1:5000) antibodies, respectively.

Immunohistochemical staining of all four chambers of human heart (Fig. 2 ) detected all three {alpha} isoforms ({alpha}3 not shown), whereas tissue from comparably treated pig heart stained only for {alpha}1. These results are consistent with those obtained with Western analysis, demonstrating the presence of all three {alpha} isoforms in human tissue but only {alpha}1 in tissue in porcine heart.



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  		Figure 2. Immunohistochemical staining of porcine and human heart tissue with antibodies specific for the {alpha}1 and {alpha}2 isoforms of NKA.

Staining for {alpha}2 (data for {alpha}3 not shown) is absent in tissue from porcine heart, but {alpha}1 is detected. Arrows indicate staining.

ouabain-specific phosphorylation of {alpha} isoforms by 32Pi
NKA can be phosphorylated by Pi (16). The phosphate is incorporated covalently on the same aspartic acid residue (amino acid residue 369), and a phospho-intermediate is formed that is identical to the intermediate formed during phosphorylation by ATP (17)(18). Phosphorylation of crude membrane preparations by Pi in the presence of Mg2+ and ouabain allows detection of the phosphorylated {alpha} polypeptide in the absence of contributions from other non-ouabain-dependent ATPases (19). Similar techniques have been used previously to distinguish (16) and quantify (20) different isoforms of NKA. The uniqueness of this technique is that phosphorylation of NKA is enhanced and stabilized in the presence of ouabain (21).

We phosphorylated crude membrane preparations from porcine cerebral cortex-mix, porcine heart, and human heart with the addition of 30 µmol/L 32Pi in the presence or absence of 1 mmol/L ouabain. Phosphorylation in the presence of 1 mmol/L ouabain followed by gel electrophoresis demonstrated one {alpha}-subunit band in pig heart and two distinct {alpha}-subunit bands in porcine cerebral cortex-mix and human heart (Fig. 3 ). Western analysis confirmed that the higher molecular weight band in human heart and porcine cerebral cortex-mix represents {alpha}2 and {alpha}3, which comigrate and move more slowly than does {alpha}1. No higher molecular weight band was observed with porcine heart tissue. These data are consistent with the immunochemical staining results discussed above that show only the {alpha}1 isoform in porcine heart.



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  		Figure 3. Ouabain-stimulated phosphorylation of {alpha} isoforms of NKA.

The {alpha} isoforms of NKA from porcine cerebral cortex-mix (PCC), porcine heart (PH), and human heart (HH) were separated electrophoretically after incubation with ouabain and 32Pi. Note that in porcine heart, only the {alpha}1 is detected. The {alpha}2 and {alpha}3 isoforms comigrate as indicated and are detected in both porcine cerebral cortex-mix and human heart.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A growing shortage and increasing demand for human organs have stimulated research to determine whether interspecies organ transplants (xenotransplantation) are a viable alternative. Porcine heart, which is physiologically similar to human heart, is under investigation as a replacement organ for failing human hearts (5). We confirmed that both porcine cerebral cortex-mix (IC50 = 900 nmol/L) and human heart (IC50 = 840 nmol/L) have higher affinity for digoxin compared with porcine heart (IC50 = 1740 nmol/L), and we hypothesize that this difference is attributable to different distributions of {alpha}-subunit isoforms. In addition, Western analysis, immunohistochemistry, and specific ouabain-stimulated phosphorylation demonstrated that human heart expresses three {alpha} isoforms of NKA ({alpha}1, {alpha}2, {alpha}3), as does porcine brain. However, only the {alpha}1 isoform was detected in porcine heart by all techniques used in this study. Our results with porcine heart are identical to previous observations for sheep heart, which also expresses only {alpha}1 (22), but are different from the results obtained in studies with dog heart, which expresses {alpha}1 and {alpha}3 (23). In the rat, it appears that the distribution of NKA isoforms is specific to the type of heart cell, where {alpha}2 and {alpha}3 are preferentially expressed in specialized cardiac conduction tissue as opposed to adjacent working myocytes (24). A similar distribution pattern to that in rat may be present in porcine heart. Using immunohistochemistry, Spinale et al. (25) reported detection of {alpha}2 but not {alpha}3 in porcine left ventricular sections from the subendocardial region. It is possible that, analogous to rat heart, expression of {alpha}2 and/or {alpha}3 may be cell specific and confined to specialized cardiac conduction tissue (e.g., Purkinje fibers) in the pig. Our membrane preparations likely contained Purkinje fibers in addition to myocytes. If {alpha}2 and/or {alpha}3 expression are confined to cardiac conduction tissue in the pig, the low ratio of Purkinje fibers to myocytes might explain our inability to detect either isoform by Western analysis. In contrast to Spinale et al. (25), we were unable to detect {alpha}2 or {alpha}3 expression, using immunohistochemistry or phosphorylation. It is unlikely that our inability to detect expression of the {alpha}2 and {alpha}3 isoforms in porcine tissue is attributable to preferential degradation of {alpha}2 and {alpha}3 during tissue preparation and cell fractionation. Porcine samples were prepared for immunohistochemistry within 2 h of sacrifice, and protease inhibitors were routinely included in our cell fractionations. In addition, human heart, dog heart, porcine brain, and rat brain, all isolated in our laboratory, gave isoform expression patterns consistent with previously published reports. Analysis of human membrane preparations from these same regions of the heart showed expression of {alpha}1, {alpha}2, and {alpha}3; thus, clear differences in isoform expression in heart tissue exist between the two species, humans and pigs.

Some studies have suggested that porcine heart has the appropriate hemodynamic characteristics needed to function in humans (26). However, considerable differences in tissue response to digitalis drugs between and within various animal species have been demonstrated, as have differences in response to deglycosylated and chemically reduced digitalis metabolites (27). Evidence now suggests that these observations are attributable to differences in NKA isoform response to the cardiac glycosides (28) and their related endogenous mammalian factors (29). Interspecies differences in NKA isoform expression and affinity for digitalis-related compounds likely reflect differences in physiological requirements and probably mimic tissue responses to endogenous NKA modulators such as the putative endogenous DLIF and OLF (29)(30).

Differences in activity and response to cardiac glycosides may have important implications regarding the effect of standard drug therapy or the response of porcine hearts to the human hormone milieu after transplantation into human recipients. The expression of NKA {alpha}1, {alpha}2, and {alpha}3 isoforms is differentially regulated by hormones (31). This regulation has been studied in experiments examining the 5'-flanking sequences of the human {alpha}-isoform genes, and data suggest that each contains several potential transacting and hormone-binding sites that are not conserved among the three {alpha}-isoform genes (31)(32). Those findings strongly support the concept of differential regulation of the sodium pump genes. Thus, an altered presence of these isoforms (e.g., as the result of reduced expression or a lack of expression of {alpha}2 and {alpha}3) could increase the concentration of endogenous digitalis-like regulators (OLF or DLIF) needed to effect proper cardiac function or to compensate for cardiac dysfunction during disease. Although the absence of the {alpha}2 and {alpha}3 isoforms in porcine heart likely explains the decreased affinity for ouabain compared with human heart in our study, previous reports of species-specific differences in binding affinities for individual isoforms should be considered (33). However, it is very difficult to measure the ouabain binding affinity for the individual isoforms from any species.

The issue of therapy with digoxin, for example, may be very important in transplantation of porcine hearts into humans. If digitalis-type drugs were to be required posttransplantation, the concentrations needed to affect the transplanted porcine heart may be much higher than those tolerated by other human organs, thus precipitating complications or preventing the use of this drug altogether. Whereas a pharmacologic incompatibility may well be an important consideration, the lack of an alternative organ source in an emergent situation might take precedence. However, from an endocrinologic perspective, the presence of the endogenous mammalian cardenolides as hormonal-axis may require immediate compatibility independent of the above considerations of pharmacologic interest. Our present study suggests that differences exist between human and porcine cardiac tissues relative to this important receptor. Defining the physiological and biochemical differences between pig and human hearts is essential if the feasibility of xenotransplantation using porcine organs is to be assessed rigorously. For example, it may be necessary to consider incorporating expression of the "missing" NKA isoforms as part of the transgenic strategy for porcine hearts.


   Acknowledgments
 
Support for this work was provided in part by Grants HL R01-36172 and HL R01-59404 from NIH and Grant EPSCoR-9452895 from the National Science Foundation (all to R.V.) and the Department of Pathology’s Special Procedures Laboratory (to A.W.M.). We thank Dr. Thomas A. Pressley, Department of Physiology, Texas Tech University, Health Science Center, Lubbock, TX for antibodies used in this study. We also acknowledge the expert technical assistance of Sharon C. Lear in performing the immunohistochemical analyses.


   Footnotes
 
1 Present address: Roche Diagnostics Corporation, Roche Laboratory Systems, Indianapolis, IN 46250-0457.

2 Nonstandard abbreviations: NKA, Na+,K+-ATPase; OLF, ouabain-like factor; DLIF, digoxin-like immunoreactive factor; SDS, sodium dodecyl sulfate; and PAb, polyclonal antibody.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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