Co-localization of vesicles and P/Q Ca2+-channels explains the preferential distribution of exocytotic active zones in neurites emitted by bovine chromaffin cells.
We have taken advantage of the differences between the preferential localization of secretion in the terminals of neurite-emitting bovine chromaffin cells in contrast with the random distribution secretion in spherical cells to study the possible molecular factors determining such localization by using immunofluorescence and confocal microscopy techniques. By analyzing the distribution of dopamine beta-hydroxylase present in the membrane of chromaffin granules, we found that vesicles migrate and accumulate in dense packages in the terminals of neurite processes. Neither members of the fusion core complex such as SNAP-25, nor nicotinic receptors are preferentially located in the terminals as would be expected from elements defining sites of release, thereby suggesting the presence of additional factors. Interestingly, we observed a preferential distribution of the P/Q subtype of Ca2+ channels in these neurite terminals and co-localization with vesicles present in these structures, in sharp contrast with the overall distribution of the L subtype channels. Using the same immunofluorescence techniques we were unable to detect N-type calcium channels. In addition, omega-agatoxin IVA was able to block 70% of the exocytotic release occurring into the neurites, whereas L-type blockers had a weak effect. Taken together our results strongly indicate that the co-localization of vesicles and clusters of P/Q Ca2+ channels may explain the precise localization of exocytotic sites in the terminals of neurite-emitting chromaffin cells, whereas the distribution of secretory sites in round cells may arise from the random presence of these factors as indicated by their partial co-localization.
Gil
A
A
Instituto de Neurociencias, Centro Mixto, CSIC-Universidad Miguel Hernández, Alicante/Spain.
Viniegra
S
S
Neco
P
P
Gutiérrez
L M
LM
D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000032:analysis*; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000737:chemistry*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis; D016501:Neurites / Q000737:chemistry / Q000502:physiology*; D017729:Presynaptic Terminals / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000032:analysis; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000737:chemistry; D033921:Vesicular Transport Proteins; D020867:omega-Agatoxin IVA / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D002395:Catecholamines; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D020867:omega-Agatoxin IVA