Cyclic AMP-dependent modulation of vesicular monoamine transport in pheochromocytoma cells

Abstract: Cyclic AMP (cAMP) is well known to enhance tyrosine hydroxylase activity in PC12 cells. We were able to demonstrate, however, that the cellular dopamine level in PC12 was lowered by dibutyryl cAMP. Furthermore, the decrease in the cellular level of dopamine was accompanied by about a 10-fold increase in the medium. The aim of this work was to elucidate the effect of cAMP on catecholamine transport. Dibutyryl cAMP did not induce exocytotic release of norepinephrine but rather inhibited its uptake. As with forskolin and cholera toxin, physiological signaling molecules such as vasoactive intestinal polypeptide (VIP) and AMP, for which PC12 cells are known to have receptors linked to activation of adenylate cyclase, also inhibited norepinephrine uptake. The inhibitory effects of dibutyryl cAMP, VIP, and AMP were dose dependent, and EC50 values were estimated to be 100 µM, 10 nM, and 1.0 µM, respectively. The inhibition profile of dibutyryl cAMP over the time course of norepinephrine uptake was biphasic: Inhibition became clearly detectable after the cytosolic pool of norepinephrine had been saturated. This profile is similar to that of reserpine. Nomifensine, however, inhibited uptake at a rather constant rate throughout the entire time course. The ATP-dependent serotonin uptake by digitonin-permeabilized cells was lowered to ∼50% that of the control by dibutyryl cAMP treatment before permeabilization, indicating inhibition of vesicular monoamine transport. This effect was also dependent on a dibutyryl cAMP concentration with an EC50 of ≤100 µM. These results suggest that cAMP may be capable of elevating extracellular dopamine levels in the nervous system by inhibiting its translocation into storage vesicles while enhancing its synthesis in the cytosol. Moreover, endogenous neurotransmitters such as VIP, AMP, and adenosine may act as intrinsic antidepressants via the cAMP pathway.