Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil.
1. The inhibition of short circuit current in frog skin by benzamil (N-benzyl-amidino-3,5-diamino-6-chloropyrazine carboxamide) was investigated. When skins were bathed on both sides by Ringer solution (pH 7.6) the affinity was 5 x 10(7) M-1. When the sodium concentration was reduced to 1.1 mM and the pH adjusted to 6.5 the affinity increased to 8.5 x 10(8) M-1. 2. A method is described for measuring uptake of [3H]benzamil into the mucosal (outer) surface of pieces of isolated epithelium, 0.95 cm2 in area, under open circuit conditions. 3. The relation of [3H]benzamil uptake at the mucosal surface to its concentration was measured in solutions containing 1.1 mM-sodium and adjusted to pH 6.5. Uptake could be resolved into a linear component (10.2 f-mole nM-1) and a saturable component (21.5 f-mole cm-2) with a half saturating concentration of 1 nM. 4. In the presence of amiloride (1 microM) or unlabelled benzamil (1 microM) uptake was linear with concentration, and was, respectively, 9.2 f-mole nM-1 and 8.8 f-mole nM-1. When the pH was reduced to 3.5 uptake was again linear but reduced to 3.3 f-mole nM-1. 5. The identity of the saturable component of [3H]benzamil uptake to sodium entry sites is discussed. The results suggest a sodium entry site density of around 130 micron-2 of mucosal surface.
D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001001:Anura; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D003864:Depression, Chemical; D004848:Epithelium / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008722:Methods; D009092:Mucous Membrane / Q000378:metabolism; D009994:Osmolar Concentration; D011719:Pyrazines / Q000378:metabolism*; D011896:Rana temporaria; D012867:Skin / Q000378:metabolism*; D012879:Skin Physiological Phenomena; D012964:Sodium / Q000378:metabolism*
D011719:Pyrazines; C013407:benzamil; D000584:Amiloride; D012964:Sodium