Presence of 5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type H⁺-ATPase in neurosecretory granules from bovine posterior pituitary

Electrogenic H⁺-ATPase was found in neurosecretory granules from bovine posterior pituitary. This enzyme was sensitive to bafilomycin, a specific inhibitor of vacuolar H⁺-ATPase, and was inactivated completely by cold treatment in the presence of MgATP and NaNO₃. Immunoblot analysis showed the presence of immunologically identical polypeptides (72, 57, and 34 kDa) in the ATPases of the neurosecretory granules and chromaffin granules. The granules showed MgATP-dependent activity for 5-hydroxytryptamine (serotonin) uptake. This uptake was temperature-dependent and showed saturation kinetics (apparent K(m) for 5-hydroxytryptamine, 2 μM) and counter-flow. Reserpine and tetrabenazine at 1 μM inhibited the uptake, whereas imipramine at 2 μM had no effect. Dopamine, epinephrine and norepinephrine were also inhibitory. The uptake was abolished by various treatments that dissipated the electrochemical H⁺ gradient or inhibited the H⁺-ATPase. These results indicate that a vacuolar type H⁺-ATPase in the neurosecretory granules forms an electrochemical H⁺ gradient that drives 5-hydroxytryptamine uptake by a specific transport system. A similar granular fraction from the anterior pituitary had no ATP-dependent activity for 5-hydroxytryptamine uptake.