ABSTRACT
Bovine chromaffin cells cultured for 5 days in the presence of depolarizing concentrations of K+ ions show a decreased number of secretory (chromaffin) granules per cell. These cells were still capable of exocytosis. Their contents in catecholamine and chromogranin A, components of the granule matrix, and cytochrome b561, a major protein of the granule membrane, were decreased to 35, 30, and 50% of control cells, respectively. However, in the same cells, the number of [3H]dihydrotetrabenazine binding sites, a specific ligand of the vesicular monoamine transporter, was increased to 180% of controls. In situ uptake of noradrenaline in permeabilized cells indicated that [3H]dihydrotetrabenazine binding sites were associated with a functional vesicular monoamine transporter. When analyzed by isopycnic centrifugation, these sites cosedimented with catecholamine, chromogranin A, and cytochrome b561, in a peak with a density lighter than that from controls. The composition of this peak suggests that it contains incompletely matured secretory granules, with a 3-5-fold increase in the vesicular monoamine transporter content of this membrane. This increase might indicate that an adaptative process occurs which allows a faster filling of the granules in continuously secreting cells.
Subject(s)
Chromaffin Granules/metabolism , Glycoproteins/metabolism , Intracellular Membranes/metabolism , Membrane Glycoproteins , Membrane Transport Proteins , Neuropeptides , Animals , Biological Transport , Catecholamines/metabolism , Cattle , Cell Fractionation , Cell Membrane Permeability , Cells, Cultured , Chromaffin System/cytology , Chromaffin System/ultrastructure , Chromogranin A , Chromogranins/isolation & purification , Cytochrome b Group/analysis , Exocytosis , Fluorescent Antibody Technique , Norepinephrine/metabolism , Potassium/pharmacology , Stimulation, Chemical , Tetrabenazine/analogs & derivatives , Tetrabenazine/metabolism , Tyrosine 3-Monooxygenase/isolation & purification , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport ProteinsABSTRACT
The transsynaptic induction of the monoamine transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
