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Journal of Experimental Biology 104,217-230 (1983)
Published by Company of Biologists 1983

³H-Ouabain Binding Autoradiography in the Abdominal Nerve Cord of the Hawk Moth, Manduca Sexta

ANDREW L. RUBIN ¹, CHARLES E. STIRLING ², and WILLIAM L. STAHL ²

¹ Departments of Physiology and Biophysics, and Medicine (Neurology), University of Washington School of Medicine, and the Neurochemistry Laboratory, Veterans Administration Medical Center, Seattle, Washington, U.S.A; Joint Program in Neonatology, Harvard Medical School, 75 Francis Street, Boston, MA 02115
² Departments of Physiology and Biophysics, and Medicine (Neurology), University of Washington School of Medicine, and the Neurochemistry Laboratory, Veterans Administration Medical Center, Seattle, Washington, U.S.A

Ouabain, a specific inhibitor of the Na⁺,K⁺-ATPa9e, was used to study localization of this enzyme in the abdominal nerve cord of the hawk moth, Manduca sexta. Treatment of nerve cords with urea was necessary to permit access of ouabain to binding sites within the nerve cord, probably due to opening of the perineurial barrier. Specific, saturable ³H-ouabain binding below 10 µM was observed in the urea-treated nerve cord, and the total number of specific binding sites was 17.8 pmol/mg protein and those sites were half-occupied (K_D) at 1.8 µM-ouabain. The urea treatment did not alter the total number of ³H-ouabain binding sites in this tissue. Localization of the ³H-ouabain binding sites was studied by light microscopic autoradiography. Potassium, an established inhibitor of ouabain binding, at 50 mM blocked ³H-ouabain binding in abdominal ganglia by 59%. The majority of ouabain binding sites in the ganglion are in the neuropil, consistent with the high transport requirement in active nerve processes of small diameter. Significant, but lower binding than found in the neuropil was present in the ganglionic perineurium and this structure may be involved in the transport of sodium from the haemolymph into the neural extracellular space.

Key words: Ouabain binding, abdominal nerve cord, Manduca sexta

Submitted on November 16, 1982
Accepted on October 26, 1982