To investigate the participation of the pineal gland and its hormone melatonin on Na(+)-K(+)-ATPase (the sodium pump) in rat brain, we used Scatchard plots to analyze the changes in rat cerebral cortex of [3H]ouabain high-affinity binding in groups of intact, pinealectomized (PX), and sham-PX rats. Only one type of binding site, with a dissociation constant of approximately 3 nM and site number (Bmax) of approximately 250 fmol/mg protein, was apparent with our assay conditions. PX or sham-PX rats (subjected to surgery 15 days earlier) were killed at six different time intervals during the 24-h cycle. Intact and sham-PX animals showed a similar biphasic pattern in diurnal rhythm of ouabain binding, with a minimal concentration of binding sites at 1600 h and a maximal concentration at 0400 h. Pinealectomy induced a significant increase in Bmax at all time intervals studied, with the largest rise appearing at night and coinciding with the nocturnal peak, whereas the daytime minimum was blunted. Time-dependent experiments indicated that the Bmax of ouabain high-affinity binding in PX rats attained maximal values at 7 days after surgery and decreased somewhat 7 days later, while sham-PX animals showed only a small transient increase in Bmax up to 7 days after surgery, with values returning to normal by the 15th day. Melatonin administration at a single subcutaneous dose of 25 micrograms/kg body wt given 3 h before death was enough to counteract the PX-induced increase of ouabain high-affinity binding. Melatonin was able to enhance the binding of [3H]ouabain to its receptor site, increasing binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of Membrane-Bound and Solubilized High-Affinity Binding Sites for 5'-N-Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex
