Evidence for the involvement of Ca2+-calmodulin and cyclic AMP in the regulation of the tyrosine hydroxylase system in rat striatal tissue slices.

To determine if both the Ca2+-calmodulin system and the cyclic AMP system may regulate tyrosine hydroxylase (TH) activity in situ, rat striatal tissue slices that contain all of the components of the TH, cyclic AMP and Ca2+-calmodulin systems were subjected to experimental manipulations. Incubation of striatal tissue slices in a medium containing W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], a specific inhibitor of calmodulin, resulted in a dose-dependent decrease of DOPA formation. The concentration of W-7 producing 50% inhibition (3 X 10(-5)M) of DOPA formation was in good agreement with the binding affinity of W-7 to calmodulin. W-7 did not affect TH activity in vitro or cellular cyclic AMP level. A structurally unrelated calmodulin antagonist, trifluoroperazine, also inhibited DOPA formation. On the other hand, incubation of striatal tissue slices in a medium containing dibutyryl cyclic AMP (DBcAMP) increased DOPA formation dose dependently. Kinetic analysis revealed that the enzyme in homogenates of control tissue slices had two different Km values for a cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH4), indicating the presence of two forms in striatal tissue slices: a less active form with a relatively low affinity for the pterin cofactor and a more active form with a relatively high affinity. W-7 produced an increase of the high Km form and a decrease of the low Km form respectively. In contrast, incubation of tissue slices in the presence of DBcAMP resulted in almost complete activation of the enzyme to the low Km form. These results suggest that both the Ca2+-calmodulin-dependent system and the cyclic AMP-dependent system may regulate TH activity in the rat striatum in vivo. The different types of kinetic change produced by DBcAMP and W-7 indicate that the two processes may act in different fashions and that the basal level of catechol formation in striatal tissue slices may be dependent upon an activated form of TH.