THE SEPARATION OF EXOCYTOSIS FROM ENDOCYTOSIS IN RAF MELANOTROPH MEMBRANE CAPACITANCE RECORDS

1. Using the patch-clamp technique, we have monitored the secretory ac tivity of single rat melanotrophs. Changes in membrane capacitance (C- m) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measu re the amplitude of discrete steps in C-m. Records of C-m were convert ed into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in C-m was easily distinguish ed from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer-based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dC(m)/dt versus C-m, from which the m agnitude and direction of transients were determined. Transients due t o 2 fF steps (equivalent to a signal-to-noise ratio of 1) were detecte d with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detecti ons of 0.075 s(-1) was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs wit h an anti-adrenocorticotrophin (ACTH) antibody. Secretory organelles i n cultured and non-cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organell es of similar diameter stained positively with the anti-ACTH antibodie s. 4. Small discrete steps in C-m, recorded with the whole-cell config uration and loosely buffered cytosolic calcium, were similar to the es timated C-m of secretory organelles from morphological data. Thus, mea sured discrete steps in C-m reflect interactions between single organe lle size and plasma membrane. Exocytotic and endocytotic steps were fo und to be of similar size. 5. To separate exocytosis from endocytosis in C-m records, we assumed that the rates of exocytosis and endocytosi s were related to the respective frequencies of discrete steps in C-m. A relationship between the frequency of exocytotic, but not endocytot ic events, and the rate of change in C-m was observed. Thus, under our experimental conditions, an increase in C-m could be explained by an increased rate of exocytosis in rat melanotrophs.