Does the carrier of chromaffin granules transport the protonated or the uncharged species of catecholamines?

Abstract

By osmotic lysis in the presence of urea ghosts (60–100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4–6 μmol catecholamine/mg prot.) of the bovine adrenal medulla. In the presence of 1–300 μmol/l3H-catecholamine and ATP-Mg2+, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (Δp). The same uptake was found by3H-counting or by fluorimetric measurements. At various pH-values (pH 6.2–82.) theK m andV max of the ATP-stimulated rate of uptake of3H-catecholamine into ghosts was determined (at 30°C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (ΔpH=pHout-pHin) and the electrical potential difference (ΔΨ) were determined to calculate Δp under conditions of3H-catecholamine uptake. When the pHout was increased (pH 6.2, 7.4, 8.2), the apparentK m of uptake decreased (50, 5, 1–2 μmol/l), showing a linear relation between pH and logarithm ofK m. TheK m was calculated for the uncharged catecholamine (with pK1=8.8 and pK2=10.0); it was nearly pH-independent and amounted to about 0.2 μmol/l. TheV max declined only in the extreme pH-range. Between pH 6.6 and 7.8V max and Δp showed a slight increase from 16 to 20 nmoles/(mg prot.·min) and from 110 to 140 mV, resp. In the same pH-range the pHin inside ghosts increased from pH 5.2 to 5.7, whereas ΔΨ was constant (30 mV). At constant pHout (=7.3) ammonium (0–30 mmol/l) caused an increase of pHin from 5.5 to 6.6. The increase of pHin was accompanied by an increase ofK m from 5 to 20 μmol/l3H-catecholamine and by a decrease of bothV max and Δp from 20 to 5 nmoles/(mg prot.·min) and from 123 to 85mV, respectively. From the dependence of theK m of uptake on pHout is concluded that the uncharged species of catecholamine is transported, whereas the dependence ofK m on pHin suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.