Anti-CD3 Antibody Treatment Reduces Scar Formation in a Rat Model of Myocardial Infarction.

: Introduction: Antibody treatment with anti-thymocyte globulin (ATG) has been shown to be cardioprotective. We aimed to evaluate which single anti-T-cell epitope antibody alters chemokine expression at a level similar to ATG and identified CD3, which is a T-cell co-receptor mediating T-cell activation. Based on these results, the effects of anti-CD3 antibody treatment on angiogenesis and cardioprotection were tested in vitro and in vivo. METHODS: Concentrations of IL-8 and MCP-1 in supernatants of human peripheral blood mononuclear cell (PBMC) cultures following distinct antibody treatments were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). In vivo, anti-CD3 antibodies or vehicle were injected intravenously in rats subjected to acute myocardial infarction (AMI). Chemotaxis and angiogenesis were evaluated using tube and migration assays. Intracellular pathways were assessed using Western blot. Extracellular vesicles (EVs) were quantitatively evaluated using fluorescence-activated cell scanning, exoELISA, and nanoparticle tracking analysis. Also, microRNA profiles were determined by next-generation sequencing. RESULTS: Only PBMC stimulation with anti-CD3 antibody led to IL-8 and MCP-1 changes in secretion, similar to ATG. In a rat model of AMI, systemic treatment with an anti-CD3 antibody markedly reduced infarct scar size (27.8% (Inter-quartile range; IQR 16.2-34.9) vs. 12.6% (IQR 8.3-27.2); p < 0.01). The secretomes of anti-CD3 treated PBMC neither induced cardioprotective pathways in cardiomyocytes nor pro-angiogenic mechanisms in human umbilical vein endothelial cell (HUVECs) in vitro. While EVs quantities remained unchanged, PBMC incubation with an anti-CD3 antibody led to alterations in EVs miRNA expression. CONCLUSION: Treatment with an anti-CD3 antibody led to decreased scar size in a rat model of AMI. Whereas cardioprotective and pro-angiogenetic pathways were unaltered by anti-CD3 treatment, qualitative changes in the EVs miRNA expression could be observed, which might be causal for the observed cardioprotective phenotype. We provide evidence that EVs are a potential cardioprotective treatment target. Our findings will also provide the basis for a more detailed analysis of putatively relevant miRNA candidates.

Klotho improves diabetic cardiomyopathy by suppressing the NLRP3 inflammasome pathway.

AIMS: NLRP3 inflammasome activation is essential for the development and prognosis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho is suggested to modulate tissue inflammatory responses. The aim of the present study was to examine the protective effects of Klotho on DCM. MAIN METHODS: A streptozotocin-induced diabetes mouse model was established to assess the effects of Klotho in vivo, which was administered for 12 weeks. The characteristics of type 1 DCM were evaluated by general status, echocardiography, and histopathology. The expression of associated factors was determined by RT-qPCR and western blotting. Parallel experiments to determine the molecular mechanism through which Klotho prevents DCM were performed using H9C2 cells exposed to high glucose (35 mM). KEY FINDINGS: Diabetes-induced increases in serum creatine kinase-muscle/brain and lactate dehydrogenase levels, cardiac fibrosis, cardiomyocyte apoptosis, and cardiac dysfunction were ameliorated by Klotho. Additionally, Klotho suppressed TXNIP expression, NLRP3 inflammasome activation, and expression of the inflammatory cytokines tumor necrosis factor ɑ, interleukin-1ß, and interleukin-18 in vivo. In high glucose-cultured cardiomyocytes, Klotho and N-acetylcysteine significantly downregulated intracellular reactive oxygen species generation and TXNIP/NLRP3 inflammasome activation. Pretreatment of H9C2 cells with NLRP3 siRNA or Klotho prevented high glucose-induced inflammation and apoptosis in H9C2 cells. SIGNIFICANCE: Our results demonstrate that the protective effect of Klotho on diabetes-induced cardiac injury is associated with inhibition of the NLRP3 inflammasome pathway, suggesting its therapeutic potential for DCM.

Mechanisms of cardioprotection via modulation of the immune response.

Both morbidity and mortality as a result of cardiovascular disease remain significant worldwide and account for approximately 31% of annual deaths in the US. Current research is focused on novel therapeutic strategies to protect the heart during and after ischemic events and from subsequent adverse myocardial remodeling. After cardiac insult, the immune system is activated and plays an essential role in the beginning, development, and resolution of the healing cascade. Uncontrolled inflammatory responses can cause chronic disease and exacerbate progression to heart failure and therefore, constitute a major area of focus of cardiac therapies. In the present overview, we share novel insights and promising therapeutic cardioprotective strategies that target the immune response.

Effects of antidigoxin antiserum on endoxin levels, apoptosis and the expression of Bax and Bcl-2 protein in ischaemia-reperfusion myocardium.

The aim of the present study was to investigate the effects of antidigoxin antiserum (ADA), an endoxin special antagonist, on endoxin levels, apoptosis and the expression of the apoptosis-related protein bcl-2 and bax in myocardial ischaemia-reperfusion (MIR). The left anterior descending coronary artery was subjected to 30 min ischaemia followed by 45 min reperfusion in open-chest anaesthetized rats. The rats were divided randomly into seven groups: a sham-operated group, an MIR group, a vehicle control (normal saline) group, and groups receiving verapamil (5 mg/kg) or ADA (9, 18 and 36 mg/kg). The drugs were injected into rats via the femoral vein before reperfusion was commenced. Myocardial endoxin levels were measured by radioimmunoassay. Apoptotic cells was detected using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling method. The expression of the apoptosis-related proteins bcl-2 and bax was detected by immunohistochemistry and their semiquantification scores were recorded by a computer image analysis system. Myocardial endoxin levels, the number of apoptotic cells and bax protein expression were increased in the MIR group compared with the sham group. Although bcl-2 protein expression was elevated in the MIR group, there was no significant difference between the MIR and sham groups. However, the ratio of bcl-2/bax was significantly decreased in the MIR group. In the group receiving 36 mg/kg ADA, myocardial endoxin levels, the number of apoptotic cells and bax protein expression were significantly decreased; bcl-2 protein expression was enhanced. The bcl-2/bax ratio was increased. The results suggest that ADA inhibited myocardial apoptosis induced by MIR in rats. The mechanisms involved require further investigation, but the present study may suggest that ADA prevents bax upregulation and enhances bcl-2 upregulation by antagonizing the effects of endoxin.

Neutralization of free digoxin-like immunoreactive components of oriental medicines Dan Shen and Lu-Shen-Wan by the Fab fragment of antidigoxin antibody (Digibind).

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.

Measurement of beta-methyldigoxin level in serum from patients by enzyme immunoassay using novel specific antiserum with a phenyl boric acid column.

The authors compared serum beta-methyldigoxin (MDx) levels in digitalized patients by enzyme immunoassay (EIA) using anti-MDx 3'-hemisuccinate BSA antiserum (antiserum-I) with commercial antidigoxin antiserum (antiserum-II). The usefulness of a phenyl boric acid (PBA) column for pretreatment of the serum samples was also investigated. The assay using antiserum-I demonstrated good accuracy and precision in the concentration range of 0.5 to 5 ng/mL. When the specificities of antiserum-I and antiserum-II were assessed by cross-reactivity studies with various related compounds, antiserum-I was much more specific for MDx antiserum-II. Using a phenyl boric acid (PBA) column, MDx, and digoxigenin, which exhibits a negligible cross-reactivity, were separated from serum, including MDx and its metabolites. The recovery tests of MDx using antiserum-I with a PBA column in human serum were satisfactory and no interference of metabolites of MDx was observed. Mean MDx concentrations in serum samples (n = 30) from digitalized patients by EIA using antiserum-I with PBA column, antiserum-I, and antiserum-II were 1.06, 1.30, and 1.74 ng/mL, respectively. These results indicate that our EIA system using antiserum-I with a PBA column for pretreatment of serum samples is useful to more precisely measure the unchanged type of MDx in patients.

Immediate control of life-threatening digoxin intoxication in a child by use of digoxin-specific antibody fragments (Fab).

Digoxin-immune antibody fragments (Fab) for treatment of digitalis intoxication was introduced in 1976. Many reports have been published concerning this therapy for children, but few have focused on its immediate reversal of cardiac as well as extracardiac life-threatening manifestations of digoxin toxicity. We present a case of life-threatening digitalis intoxication in a child with postoperative renal insufficiency, after a Sennings procedure for transposition of the great arteries. Digoxin administration according to the nationally recommended dosage and intervals unexpectedly resulted in serum levels in the toxic range. Severe cardiac arrhythmias, haemodynamic instability and a rapid-increasing serum potassium level resulted. This report demonstrates how administration of Fab according to the manufacturer's dosage recommendation reversed the tachyarrhythmia immediately and re-established a normal level of serum potassium within minutes.

Effect of the traditional Chinese medicines Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng on serum digoxin measurement by Tina-quant (Roche) and Synchron LX system (Beckman) digoxin immunoassays.

Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng are traditionally used to treat a number of conditions, including cardiovascular disease. All of these traditional Chinese medicines exhibit cardioactive properties. Digoxin is a cardioactive drug with a narrow therapeutic range (0.8-1.9 ng/mL). A patient taking digoxin may also take these Chinese medicines for their cardiotonic effects. Moreover, the active components of these medicines that are responsible for cardiotonic effects bear structural similarities to digoxin. Therefore, we studied the potential interference of these Chinese medicines with two digoxin immunoassays--the Tina-quant (Roche Diagnostics) and the Beckman (Synchron LX system)--and compared the values with the fluorescence polarization immunoassay (FPIA; Abbott Laboratories). When very small amounts (2-5 microL) of aqueous extract of Chan Su or Lu-Shen-Wan were added to drug-free serum, we observed high digoxin-like immunoreactivity with the FPIA. In contrast, when ethyl acetate extract of Dan Shen or microliter amounts of ginseng extract were added to drug-free serum, we observed modest digoxin-like immunoreactivity with the FPIA, but no apparent digoxin activity with the Roche and Beckman digoxin immunoassays. When aliquots of a digoxin pool prepared from patients receiving digoxin were supplemented with these Chinese medicines, we observed the most significant interference with the FPIA. The presence of endogenous digoxin-like immunoreactive substances can have additive effects with these Chinese medicines and falsely increase apparent digoxin levels by the FPIA. On the other hand, the Roche and Beckman assays were free from interference from DLIS but showed significant interference from Chan Su and Lu-Shen-Wan. We conclude that the FPIA showed the most significant interference from all four of the Chinese medicines we studied. However, the Roche and Beckman assays showed no interference from two (Dan Shen and Asian ginseng) of the four Chinese medicines we studied.

Cysteine-free mutant of aequorin as a photolabel in immunoassay development.

The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.

Measurement of serum digitoxin in patients by radioimmunoassay using specific antiserum.

INTRODUCTION: Digitoxin is used to treat patients with heart failure. METHODS: A radioimmunoassay procedure for the specific determination for digitoxin in serum was developed using the antiserum (antiserum (A)) raised against digitoxin 3'-hemisuccinate-BSA conjugate. RESULTS: The intra- and interassay variability were <10% in the range of 5-100 ng/ml. The specificities of antiserum (A) and the commercial anti-digitoxin antiserum (antiserum (B)) were assessed by cross-reactivity studies with various related compounds. Antiserum (A) was highly specific for digitoxin. Mean digitoxin concentrations in serum samples (n=34) from digitalized patients by RIA using these antisera were 10.0 and 12.4 ng/ml, respectively. CONCLUSION: This RIA using antiserum (A) measure unmetabolized digitoxin and may be applicable for pharmacokinetic studies.

A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs.

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

Binding of ouabain and human ouabainlike substance to different Na+, K+-ATPase isoforms.

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+,K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%). In this system, two distinct ouabain immunoreactive peaks were resolved. Peak I demonstrated a polarity identical with that of authentic ouabain. In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS. Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y=0.945x-2.532, r2=0.977) and porcine (y=0.428x-1.685; r2=0.993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine. In conclusion, these data suggest that like authentic ouabain, peak I OLS is a-isoform and species selective.

Falsely elevated serum digitoxin concentrations due to cross-reactivity of water-extractable digitoxin-like immunoreactivity of Chinese medicine Chan SU: elimination of interference by use of a chemiluminescent assay.

Chinese medicines are available without prescription in health food stores. One such Chinese preparation, Chan SU, is used as a cardiotonic agent. Digoxin-like immunoreactivity of Chan SU has been reported in the past. In this report we demonstrated significant digitoxin-like immunoreactivity of Chan SU. For example, when a 20-microl aliquot of an aqueous extract of Chan SU (2 mg/ml) was added to drug-free serum, the observed digitoxin-like immunoreactivity was 51.40 ng/ml by the fluorescence polarization assay. In contrast, a new chemiluminescent assay for digitoxin did not show any immunoreactivity. When very small amount of aqueous extract of Chan SU was added into serum containing digitoxin, the observed digitoxin concentrations were falsely elevated when measured by the fluorescence polarization immunoassay (FPIA), but did not change significantly when measured by the chemiluminescent immunoassay (CLIA). Significant digitoxin-like immunoreactivity was also observed (FPIA) in mice after feeding with Chan SU. Because bufalin, cinobufotalin and cinobufagin are major components of Chan SU, digitoxin-like immunoreactivity of these purified compounds was also studied. Bufalin was identified as the major digitoxin-like immunoreactive compound responsible for most of the interference in serum digitoxin measurement using the FPIA.

Effect of digoxin fab antibody on the measurement of total and free digitoxin by fluorescence polarization and a new chemiluminescent immunoassay.

Digoxin fab antibody (Digibind; Burroughs Wellcome, Research Triangle Park, NC, USA) is used in the treatment of digoxin overdose. The effect of digibind on the measurement of total and free digoxin has been extensively studied. However, the effect of digibind on digitoxin measurements has not been studied thoroughly. The authors studied the effect of digibind on the measurement of total and free digitoxin in vitro using the fluorescence polarization immunoassay and a new chemiluminescent immunoassay. We also studied the capability of digibind to bind digitoxigenin, the major aglycon metabolite of digitoxin. Digibind neutralized both digitoxin and digitoxigenin in vitro, as evidenced by significant reductions in free digitoxin and digitoxigenin (measured as digitoxin equivalent) concentrations. Digibind caused negative interference in the measurement of total digitoxin concentrations by both fluorescence polarization and chemiluminescent assays. However, the magnitude of negative interference was significantly higher with the chemiluminescent assay. For example, in a serum pool supplemented with 80 ng/mL of digitoxin, the concentrations of total and free digitoxin measured by the fluorescence polarization immunoassay were 82.1 ng/mL and 3.3 ng/mL respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 73.9 ng/mL and none detected, respectively. In another serum pool supplemented with 70 ng/mL of digitoxin, the concentrations of total and free digitoxin as measured by the chemiluminescent assay were 69.1 ng/mL and 3.8 ng/mL, respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 29.0 ng/mL and none detected, respectively. Because this effect may also occur in vivo, the progress of Digibind therapy in treating a patient with digitoxin overdose may be monitored by measuring the free digitoxin concentrations.

Comparison of microparticle enzyme and fluorescence polarization immunoassays in pediatric patients not receiving digoxin.

Previously, investigators have measured endogenous digoxin-like immunoreactive substances (DLIS) in pediatric patients. Digoxin-like immunoreactive substances may cross-react with digoxin assays to produce false-positive digoxin concentrations; hence, the validity of digoxin concentrations in pediatric patients is questionable. The authors compared the presence and magnitude of apparent DLIS using the microparticle enzyme immunoassay (MEIA) AxSYM Digoxin II and the fluorescence polarization immunoassay (FPIA) TDx Digoxin II, in the serum of 80 pediatric patients who were hospitalized with normal serum creatinine but had not been administered digoxin. Patients ranged in age from 1 day to 16 years (mean age, 4.96 +/- 5.17 years). Serum creatinine and total bilirubin were 0.5 +/- 0.18 mg/dl and 1.3 +/- 0.17 mg/dl, respectively. Forty-eight percent of MEIA samples and 79% of FPIA samples had measurable DLIS values. Digoxin-like immunoreactive substance concentrations for the MEIA (0.07 +/- 0.09 ng/ml) and FPIA assays (0.1 +/- 0.1 ng/ml) were statistically different (p = 0.01); however, no sample had a DLIS value >0.38 ng/ml. A poor correlation was noted between patient age, serum creatinine, total bilirubin, and DLIS concentration. The MEIA and FPIA assays effectively minimized DLIS cross-reactivity making both technologies clinically acceptable for serum digoxin measurement in pediatric patients with normal serum creatinine and total bilirubin.

Bidirectional (positive/negative) interference in a digoxin immunoassay: importance of antibody specificity.

The importance of high specificity in immunoassays used in therapeutic monitoring is highlighted by a case study in which therapeutic-to-toxic borderline digoxin levels were measured by a digoxin immunoassay in the serum sample from a patient administered digitoxin rather than digoxin. The sample, mistakenly sent to the laboratory for digoxin analysis, gave discordant results in three digoxin immunoassays: 1.99 and 0.79 ng/ml in assays using polyclonal antibodies (fluorescence-polarization immunoassay and microparticle enzyme immunoassay, respectively), and <0.1 ng/ml in a chemiluminescent immunoassay using more specific monoclonal antibody. The presence of digitoxin (approximately 40 ng/ml) in the sample was confirmed by three different digitoxin immunoassays. Based on these results, the interference of different levels of digitoxin was studied in the presence of 0, 0.85, 1.9, and 4.7 ng/ml digoxin in all three digoxin assays. The chemiluminescent assay showed no significant interference. The fluorescence-polarization immunoassay showed positive interference in all cases; however, the microparticle enzyme immunoassay showed a bidirectional interference: a positive interference observed at digoxin level <1.8 ng/ml, changing to a negative interference at higher digoxin concentrations. The authors conclude that in countries such as Germany, where both digoxin and digitoxin may be prescribed, caution should be used to interpret digoxin immunoassay results. Digoxin assays, with cross-reactivity to digitoxin <0.1% should be used.

Exogenous ouabain is accumulated in the adrenals and mimics the kinetics of endogenous digitalis-like factor in rats.

Ouabain has been isolated as an endogenous pathogenetic factor in salt-induced hypertension and has been shown to be rich in the adrenals. In this study, organ accumulation of orally administered [3H]ouabain was examined in rats. Exogenous [3H]ouabain was accumulated in high levels in the adrenals, especially in the zona intermedia, and was not metabolized in the rat. Accumulated [3H]ouabain mimicked the movement of "endogenous" digitalis-like factor, since 1) the plasma [3H]ouabain level decreased in bilaterally adrenalectomized rats, 2) the plasma [3H]ouabain level increased accompanied by a decrease in [3H]ouabain content in the adrenals in reduced renal mass hypertensive rats, and 3) [3H]ouabain levels in plasma and in the adrenals increased in spontaneously hypertensive rats, as compared with those in respective control animals. Moreover, the rat diet contained a relatively high amount of ouabain-like immunoreactivity (OLI), and the ratio of the [3H]ouabain content to OLI in each organ was comparable to that of the daily intake of dietary [3H]ouabain to OLI. Furthermore, high 3H-radioactivities were also observed in the adrenals of rats that ingested [3H]digoxin and [3H]digitoxin. These data suggest that exogenous ouabain, related cardiotonic glycosides of plant origin, or both accumulate in the adrenals and, at least in part, act as "endogenous" digitalis-like factor(s).

Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.