MODULATION BY PINEAL-GLAND OF OUABAIN HIGH-AFFINITY BINDING-SITES IN RAT CEREBRAL-CORTEX

To investigate the participation of the pineal gland and its hormone melationin on Na+-K+-ATPase (the sodium pump) in rat brain, we used Scatchard plots to analyze the changes in rat cerebral cortex of [H-3]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 (B(max)) 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 B(max) 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 B(max) 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 B(max) up to 7 days after surgery, with values returning to normal by the 15th day. Melatonin administration at a single subcutaneous dose of 25-mu-g/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 [H-3]ouabain to its receptor site, increasing binding affinity. This effect was also seen in red blood cell preparations with an assay used to measure sodium pump activity, in which melatonin potentiated the effect of ouabain on Na+-K+-ATPase pump activity.