Characterization of the alpha +-like Na+,K+-ATPase which mediates ouabain inhibition of adrenergic induction of N-acetyltransferase (EC 2.3.1.87) activity: studies with isolated pinealocytes.

Ouabain inhibits (IC50 congruent to 200 nM) the congruent to 100-fold adrenergic cyclic AMP stimulation of rat pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87, serotonin N-acetyltransferase, NAT) activity in intact pineal glands. In the present study, ouabain binding sites in pineal membranes were characterized in detail and compared to sites in isolated pinealocytes, which mediate the inhibition of Na+,K+-ATPase, as indicated by 86Rb uptake and norepinephrine (NE) stimulation of NAT activity. High affinity ouabain-binding sites were identified in crude preparations of pineal membranes (Kd congruent to 14 nM; Bmax congruent to 4 pmol/mg of protein) and similar sites were also found in ovine and bovine pineal tissue. The ouabain Kd value for the rat pineal binding sites was similar to the estimated ouabain IC50 values for 86Rb uptake and the NE stimulation of NAT activity in intact rat pinealocytes. In addition, the relative orders of potency of four cardiac glycosides in displacing [3H]ouabain from high affinity binding sites and inhibiting both 86Rb uptake and NE stimulation of NAT activity were the same (acetyldigitoxin greater than ouabain greater than digitoxin greater than strophanthidin). The similarities in the characteristics of the high affinity [3H]ouabain-binding sites and the sites involved in the inhibition of 86Rb uptake and stimulation of NAT activity indicate that an alpha +-like Na+,K+-ATPase mediates the inhibitory effects of ouabain on the adrenergic induction of pineal NAT activity.