Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter |
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Authors: | R James Turner Janet N George |
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Institution: | (1) Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, 20892 Bethesda, Maryland |
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Abstract: | Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK
D
=3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent22Na transport and tracer 3H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK
0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol.
94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both Na] and K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 1 1 andK
0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and22Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site. |
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Keywords: | loop diuretics exocrine gland fluid secretion parotid acinar cell ion transport |
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