PUNCTATE APPEARANCE OF DOPAMINE-BETA-HYDROXYLASE ON THE CHROMAFFIN CELL-SURFACE REFLECTS THE FUSION OF INDIVIDUAL CHROMAFFIN GRANULES UPON EXOCYTOSIS

A secretion from cultured bovine chromaffin cells was stimulated to ex amine the pattern of exocytotic fusion on the plasma membrane. Confoca l microscopy revealed that dopamine-beta-hydroxylase immunofluorescenc e in intact cells stimulated for 20s with the nicotinic agonist 1,1-di methyl-4-phenylpiperazinium was almost entirely punctate and evenly di stributed on the cell surface. The basis for the fine, punctate appear ance of dopamine-beta-hydroxylase was investigated. Dopamine-beta-hydr oxylase presentation on the surface of permeabilized cells stimulated with 1-30 mu M Ca2+ was punctate and similar to that on the plasma mem brane of intact cells. The fluorescence intensities of both surface do pamine-beta-hydroxylase sites and internal chromaffin granules were es timated by computerized digital image analysis. The surface area of pu nctate surface dopamine-beta-hydroxylase (0.218+/-0.013 mu m(2), mean/-S.E.M.) is similar to the surface area of a 0.28 mu m diameter chrom affin granule (0.25 mu m(2)). The average fluorescence intensity integ rated over the area of the surface spots was 25-30% of the average chr omaffin granule intensity, a fraction that is similar to the published values of 40-50% of the dopamine-beta-hydroxylase in the chromaffin g ranule being membrane bound. The surface density of the spots is consi stent with the number of granules undergoing exocytosis. The spots do not tend to be clumped. The key conclusions from this work are that ea ch individual punctate site of dopamine-beta-hydroxylase represents th e fusion of a single chromaffin granule and that the distribution of d opamine-beta-hydroxylase spots over the cell surface is extensive and random, suggesting that each individual granule associates with its ow n release site.