Molecular Pharmacology, Vol 11, 241-255, Copyright © 1975 by the American Society for Pharmacology and Experimental Therapeutics

Blockade by Ouabain or Elevated Potassium Ion Concentration of the Adrenergic and Adenosine Cyclic 3',5'-Monophosphate-Induced Stimulation of Pineal Serotonin N-Acetyltransferase Activity

¹ Section on Physiological Controls, Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014.
² Department of Pharmacology, Ohio State University College of Medicine, Columbus, Ohio 43210

Rat pineal glands were cultured for 48 hr and then treated with 10 µM norepinephrine. This resulted in the predictable 50-100-fold increase in acetyl-CoA:serotonin N-acetyltransferase) (EC 2.3.1.5) activity at 6 hr. This increase failed to occur in glands simultaneously treated with 1 µM ouabain or 80 mM K⁺. Prior treatment of pineal glands with ouabain or K⁺ did not, however, depress the 100-200-fold norepinephrine-stimulated increase in the pineal content of adenosine cylic 3',5'-monophosphate (cAMP) observed at 15-20 min; neither was there any alteration in the rate of disappearance of cAMP after this time. Ouabain or K⁺ alone failed to depress the conversion of L-[³H]tryptophan to either [³H]5-hydroxytryptophol or [³H]5-hydroxyindoleacetic acid. The conversion of L-[³H]tryptophan to [³H]serotonin was increased in the presence of 80 mM K⁺ but was not affected by the presence of 1 µM ouabain. This suggests that ouabain or K⁺ does not depress either the activity of the process responsible for the uptake and hydroxylation of L-[³H]tryptophan or the general indole metabolism in the pineal gland. Treatment with N6, O²'-dibutyryladenosine cyclic 3',5'-monophosphate (DBcAMP) or theophylline, compounds which are known to mimic the effects of norepinephrine on pineal N-acetyltransferase activity, did not stimulate N-acetyltransferase activity in the presence of 80 mM K⁺ or 1 µM ouabain. Ouabain given in vivo also reduced by 55-80% the effect of an injection of 20 mg/kg of isoproterenol on pineal N-acetyltransferase activity. Intracellular microelectrode recordings from acutely explanted pineal glands indicated that both 40 mM and 80 mM K⁺ caused a modest depolarization of the pineal cell membrane. In contrast, treatment with 1 µM ouabain for several minutes did not significantly change the resting membrane potential. However, both 80 mM K⁺ and 1 µM ouabain attenuated or reversed the hyperpolarization associated with exposure to norepinephrine. A small but significant hyperpolarizing effect was also elicited by treating the pineal gland with cAMP or DBcAMP. These findings indicate that ouabain or K⁺ blocks the effect of norepinephrine on N-acetyltransferase activity; it appears that the blockade is at some point in the sequence of events leading to the increase in enzyme activity which follows the generation of cAMP.

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