Stimulation of 32Pi Incorporation into Phosphatidylinositol and Phosphatidylglycerol by Catecholamines and β-Adrenergic Receptor Blocking Agents in Rat Pineal Organ Cultures

Abstract

Abstract The effect of norepinephrine, other catecholamines, phenylethylamines, and β-adrenergic receptor blocking drugs on the incorporation of 32Pi into phospholipids of intact rat pineal glands was studied in organ culture. Individual pineal phospholipids were assayed for radioactivity after separation by two-dimensional thin layer chromatography. Improved chromatographic separations were obtained (Getz, G. S., Jakovcic, S., Heywood, J., Frank, J., and Rabinowitz, M. (1970) Biochim. Biophys. Acta 218, 441–452), if thin layer plates were washed with acetone instead of ether after development in the first solvent system. Addition of l-norepinephrine (0.3 mm) significantly stimulated the incorporation of isotope into phosphatidylinositol within 5 min and into phosphatidylglycerol after 15 min. After 3 hours' incubation, l-norepinephrine and other phenylethylamine derivatives, including d-norepinephrine, epinephrine, dopamine, tyramine, and octopamine stimulated incorporation into phosphatidylglycerol (100 to 500%) and phosphatidylinositol (100 to 200%). Incorporated radioactivity continued to increase for up to 16 hours, but norepinephrine-induced stimulation steadily declined. The stimulating effects of l-norepinephrine were essentially linear from 0.3 mm to 3 µm. No stimulation was observed in the presence of N6, O2'-dibutyryl adenosine 3',5'-monophosphate at a concentration as high as 10 mm. Several substances with widely differing potencies as β-adrenergic receptor blocking agents, including dl,-propranolol, d-propranolol, dichloroisoproterenol, and alprenolol, but not sotalol, increased 32Pi incorporation into pineal acidic phospholipids. However these agents gave a labeling pattern different from that produced by norepinephrine: labeling of phosphatidylglycerol was elevated 1000 to 1500%, of phosphatidylinositol up to 100%, and of phosphatidic acid, 50 to 150%. Neither sotalol nor propranolol prevented the stimulation of phospholipid metabolism caused by norepinephrine. These findings indicate that the increase in phospholipid radioactivity is not related to the formation of melatonin which in the cultured pineal is stimulated by either norepinephrine or N6, O2'-dibutyryl adenosine 3',5'-monophosphate. This conclusion is also supported by the fact that propranolol fails to block enhanced phospholipid labeling whereas it blocks enhanced melatonin formation. The data further indicate that the stimulation of phospholipid metabolism caused by drugs with β-adrenergic receptor blocking activity is not brought about through interaction with β-adrenergic receptors.