Abstract
Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca(2+) evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their K(i) values not only could increase cytoplasmic catecholamines but also could displace Ca(2+) from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca(2+), as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca(2+)-free solutions, supporting an internal Ca(2+) source. Depletion of endoplasmic reticulum and mitochondrial Ca(2+) pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca(2+) can play an important role in exocytosis and under some conditions may be involved in initiating this process.
Highlights
We have shown that vesicular Ca2ϩ in bovine chromaffin cells can be mobilized by exposure to agents that cause an alkalinization of the vesicle interior (9)
The results indicate that VMAT1 inhibitors are capable of mobilizing vesicular Ca2ϩ in addition to catecholamine, that the effects of this mobilization are reflected in altered characteristics of stimulated secretion, and that inhibitors with high affinity for VMAT can trigger exocytosis in the absence of any other secretagogue
The vesicles of chromaffin cells isolated from the adrenal medulla contain nearly 60% of the total Ca2ϩ contained within the cell (5)
Summary
Cultured Chromaffin Cells—Primary cultures of bovine adrenal chromaffin cells were prepared as described previously (15). Chemical stimulations and incubations of the cells were performed with the desired agents dissolved in this solution for the Ca2ϩ-containing experiments. Cytoplasmic catecholamine levels were determined by incubating the cells with the indicated drug for 15 min and permeabilizing it via the pressure ejection of 10 M digitonin (17). In Ca2ϩ-free buffer, pressure ejection of 10 mM caffeine was used to test the exocytotic viability of the cells (18). Cells were rinsed with a Ca2ϩ-free buffer and maintained in that solution while agents were pressure-ejected onto the cells. The cells were rinsed, and release was tested in Ca2ϩ-free buffer (the procedure was modified from Ref. 20). Chromaffin cells were incubated in a buffer solution containing 1 M Fura-2 AM and 0.1% bovine albumin for 20 min at 22 °C. All solutions were prepared in distilled, deionized water (Mega Pure System MP-3A, Corning Glass)
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