Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs

We used fluorescence imaging of individual exocytic events together with el ectron microscopy to study the regulation of dense core granule-to-plasma m embrane fusion and granule-to-granule fusion events that occur during secre tion from rat pituitary lactotrophs, Stimulating secretion with elevated ex tracellular potassium, with the calcium ionophore ionomycin, or with thyrot ropin releasing hormone or vasoactive intestinal polypeptide resulted in ab undant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulati on conditions there appeared to be a finite number of plasma membrane fusio n sites, similar to 11 sites around each cellular equator. However, a granu le could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granu le-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the num ber of granule-to-granule fusion events without altering the number of gran ule-to-plasma membrane fusion events. In contrast, the phorbol eater PMA. w hich activates protein kinase C increased both granule-to-granule and granu le-to-plasma membrane fusion events. These results provide a cellular mecha nism that can account for the previously demonstrated potentiation of secre tion from lactotrophs by cAMP- and PKC-dependent pathways.