AJP - Renal Physiology, Vol 261, Issue 3 437-F442, Copyright © 1991 by American Physiological Society
Polyclonal antibodies in study of ADH-induced water channels in frog urinary bladder
G. Valenti, G. Calamita and M. Svelto
Istituto di Fisiologia Generale, Universita di Bari, Italy.
It is now generally accepted that changes in water permeability in
anti-diuretic hormone (ADH)-responsive target epithelial cells result from
the insertion in the plasma apical membrane of new components that contain
channels for water. The specificity of these channels suggests that they
are formed by intrinsic proteins having access to both facies and spanning
the whole membrane. We have previously shown that Triton X-100 apical
extracts from ADH-stimulated frog urinary bladder contain some proteins
inserted under hormonal stimulation. In the present study we have developed
polyclonal antibodies using Triton X-100 extract as an immunogen. After
considering the inhibitory effect exerted by the whole immune serum on the
osmotic water flow, we used different adsorption steps to select, from the
immune serum, antibodies to apical membrane proteins inserted in response
to the hormone. Immunoblot analysis of these selected antibodies shows that
they recognize seven to eight proteins, of which 55-, 35-, 26-, and 17-kDa
proteins are always present. Antibodies to these four proteins, affinity
purified on nitrocellulose sheets, inhibited ADH-induced osmotic water
flow. Altogether these results strongly suggest that proteins of 55, 35,
26, and 17 kDa (or at least one of them) are likely to be involved in the
mechanism of water transport.
