Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Abstract

1. We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry.2. Stimulation of exocytosis through voltage-dependent Ca 2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 ± 0.9 % and the fluorescence by 6.6 ± 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner.3. Intracellular dialysis (0.5-4.5 min) with elevated Ca 2+ (1.5-100 µM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 ± 10 %; 21 cells). Interestingly during Ca 2+ dialysis the capacitance did not significantly change (2 ± 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane.4. Sustained endocytosis was not blocked when the intracellular GTP (300 µM) was replaced with GTPyS. Replacing intracellular Ca 2+ (100 µM) with Ba 2+ (300 µM) or Sr 2+ (200 µM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. 5. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

SUMMARY1. The ionic basis of tetanic and post-tetanic potentiation (TP and PTP) was studied at the rat soleus neuromuscular junction (NMJ), using the miniature endplate potential (MEPP) frequency as an index for transmitter release. Conventional intracellular recording and computer-assisted data analysis were employed.2. The experimental results in this study indicate that contrary to previous suggestions, there is a substantial similarity in the ionic basis of TP and PTP at the mammalian and amphibian motor nerve terminals which can be subdivided into [Ca2+]0-dependent and [Ca2+]0-independent parts.3. Tetanic and post-tetanic increase in MEPP frequency at the rat soleus NMJ is similar to that at the frog NMJ in the following aspects: (i) Tetanic potentiation is substantially larger in calcium-containing solutions than in calcium-deficient solutions. About 90 % of tetanic potentiation is contributed by extracellular calcium. 6. Tetanic potentiation is manifested earlier in calcium-containing media than in calcium-deficient media. This difference may indicate that sodium entry into the terminal during tetanic stimulation is at locations remote from the releasing sites. Alternatively, this time difference may be due to the delay between intracellular sodium accumulation and the increase in transmitter release.

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