Protein Phosphorylation in Rat Pineal Gland and Its Regulation in Supersensitive and Subsensitive States

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Abstract
The phosphorylation of specific proteins in pineal homogenate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cyclic AMP had the capacity to stimulate in a dose-dependent manner the incorporation of 32P in protein bands of apparent molecular weights 59K, 56K, and 35K with a maximal effect at 1 .mu.M. On the other hand, calcium alone did not induce a marked increase in 32P incorporation with the exception of a dose-dependent phosphorylation of a 46K protein with a peak effect at 0.2 mM calcium concentration. The addition of exogenous calmodulin enhanced 32P incorporation in proteins migrating in the 62K and 52K regions, an effect that was antagonized by the calmodulin inhibitor trifluoperazine. However, also under these conditions, the stimulation of pineal protein phosphorylation was rather weak compared to that observed in other brain areas. In an attempt to investigate the functional changes of these biochemical processes during environmental lighting and adrenergic stimulation, it was found that the administration of (-)-isoproterenol (5 mg/kg, s.c.), a .beta.-receptor agonist, induced a clear-cut enhancement of 32P incorporation into the cyclic AMP-sensitive 59K and 56K proteins only in animals exposed for 18 h to the light, whereas it was almost ineffective in those kept in the dark for the same period. This effect was antagonized by (-)-propranolol pretreatment (20 mg/kg), suggesting that the changes in cyclic AMP-dependent protein phosphorylation observed in supersensitive pineals may represent a .beta.-receptor mediated process. Thus the cyclic AMP-dependent protein kinase activity seems to be correlated with the sensitivity state of the .beta.-adrenergic receptor system and may take part in the development of pineal supersensitivity. On the other hand, the phosphorylation of the 46K calcium-sensitive protein was completely unaffected, confirming the action specificity of the adrenergic system on cyclic AMP-induced pineal protein phosphorylation.