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3H-Ouabain binding to human mononuclear leucocytes
Insights
Human mononuclear leucocytes bind 3H-ouabain similarly to heart muscle, but large variations in binding sites make them unsuitable for drug studies.
Area of Science:
- Pharmacology
- Cell Biology
- Biochemistry
Background:
- Cardiac glycosides are crucial drugs affecting heart muscle function.
- Understanding cardiac glycoside binding is vital for therapeutic applications and disease monitoring.
- Human blood cells offer a potential model for studying these binding dynamics.
Purpose of the Study:
- To investigate 3H-ouabain binding to intact human mononuclear leucocytes.
- To compare the binding characteristics with those of human heart muscle.
- To assess the suitability of leucocytes as a model for physiological or disease-induced changes in cardiac glycoside binding.
Main Methods:
- Incubation of leucocyte suspensions from normal subjects with 3H-ouabain.
- Quantification of 3H-ouabain binding using radioligand assay.
- Analysis of binding affinity (KD), association (k+1), and dissociation (k-1) rates.
- Determination of the number of ouabain binding sites per leucocyte.
Main Results:
- 3H-ouabain exhibited specific binding to a single type of site on mononuclear leucocytes.
- The affinity (KD) of binding was comparable to that observed in human heart muscle.
- Binding association and dissociation rates were slow.
- Significant inter- and intra-individual variations in the number of binding sites were observed.
Conclusions:
- The ouabain binding site on human heart muscle is likely identical to that on intact mononuclear leucocytes.
- Despite similar binding affinity, the substantial variability in binding site number within leucocyte mixtures limits their use for quantifying drug- or disease-induced changes.
Abstract:
Specific binding of cardiac glycosides to intact human blood cells may be a suitable model for physiological or disease-induced changes in cardiac glycoside binding to human heart muscle. Since the erythrocyte contains no nucleus and has relatively few binding sites compared with heart muscle, intact mononuclear leucocytes were investigated in the present study. Using leucocyte suspensions from 34 normal subjects, 133 measurements of 3H-ouabain binding-were obtained. 3H-Ouabain bound to one type of binding site with an affinity (KD) of 2.8 +/- 1.2 X 10(-9) M, similar to that of human heart muscle. Association and dissociation were slow processes (k+1, 3.9 X 10(4) M-1 sec-1; k-1, 8.1 X 10(-5) sec-1, n = 2). The number of ouabain binding sites/leucocyte varied from 18,000 to 60,000 (mean +/- SD, 34,600 +/- 9,700), with no correlation with the proportion of monocytes present or with the serum K+-level of the donors. Large inter- and intra-individual differences in binding site number were measured which are probably a result of the heterogeneity of the cell suspension used. Thus, the ouabain binding site on human heart muscle and intact mononuclear leucocytes is probably identical. However, the number of binding sites in mixtures of mononuclear leucocytes shows large and inconsistent intraindividual variations, making these studies unsuitable for quantifying drug- or disease-induced changes in ouabain binding site number.