Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy

Abstract

Apical cell membranes from Na+-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spin-labeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na+ transport. Frog skin short-circuit current was inhibited by 50% in the presence of 7 X 10(-8) M ASp, whereas 4 X 10(-7) M amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 micrograms of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 X g fraction. All of the ASp binding in frog skin is found in the 10,000 X g fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.

Intracellular binding of spin‐labeled amiloride: An alternative explanation for amiloride's effects at high concentration

“…Further, it is interesting to note that in this system benzamil, an amiloride derivative, is approximately 27 times less effective than amiloride, based on K0.5 values, in blocking Na+ movement. When the efficacy of these two compounds to inhibit Na+ transport in frog skin are compared, the results are reversed: benzamil half inhibits Na+ transport at a concentration about 8 times lower than amiloride (Costa et al, 1984). Heikkila et al (1983) also report that amiloride has a direct effect on DNA synthesis as well as its usual inhibition of ion fluxes.…”

“…Liver was chosen as a source of tissue because, as we have reported, the plasma membranes isolated from trout liver display little Asp binding (Costa et al, 1984). Therefore, any contamination of the organelle preparations by plasma membrane will not contribute to the Asp binding of the nuclei or mitochondria.…”

“…The demonstration of both high and low affinity amiloride binding to these organelles and to the nucleic acids, especially DNA, adds further evidence for the argument that amiloride has direct effects at high concentration and may alert workers in this area that data from amiloridetreated cell systems require cautious interpretation. amino] -2,2,6,6-tetramethyl-l-piperidinyloxy free radical hemihydrate, inhibits Na+ transport across frog skin in a manner similar to the parent compound and can be quantitatively detected at micromolar concentrations (Costa et al, 1984). The nitroxide derivative produces a signal that is proportional to the concentration of the molecules free in solution.…”

“…The nitroxide derivative produces a signal that is proportional to the concentration of the molecules free in solution. When the spin-label is immobilized, as when bound to a large molecule, the signal is reduced in size (Costa et al, 1984; c.f. also Berliner and Wong, 1975).…”

“…Using a spin-labeled derivative of amiloride (Asp) with transport inhibiting properties that are similar to the parent compound, (Costa et al, 1984) we have examined the notion that amiloride binds to isolated mitochondria and nuclei. Further, we have measured the binding of ASp to commercially prepared DNA and RNA.…”