Thirty-seventh Annual Meeting February 14-18, 1993 Washington Convention Center Washington, D.C. Tuesday Symposia and Posters, Part IV

1993 Biophysical Journal  
Recently carbon-fiber microelectrodes have been used for electrochemical detection of catecholamine release from single vesicles of adrenal chromaffin cells (Wightman et al, 1991, PNAS 88:10754-10758; Chow et al, 1992, Nature 356:60-63). Using Monte Carlo simulations incorporating realistic geometry, we have analysed some of the factors Influencing the shape of the oxidation current transient: the diffusion constant D, the distance x from the release site to the detecting surface, and the time
more » ... ourse of release. The main part of experimental traces were readily fit assuming Instantaneous release of molecules from the cell surface, but required either that x > 1 pm (larger than expected) or that D >> 5.5 x 10-6 cm2/s (D of freely diffusing catecholamine). This observation points to either 1) Irregular carbon fiber sufaces or cell infoldings, 2) retarded diffusion, perhaps due to a slowly diffusing complex, or 3) non-instanteous release, perhaps reflecting the kinetics of dilation of the fusion pore or escape from a matrix. When D was adjusted to 3 x 10-7 cm2/s to fit the main part of the data traces, the simulations still did not show the siow initial onset seen in many of the experimental trces. This slow "foot' signal was previously suggested to be due to the siow leak of molecules out of the narrow fusion pore that initialy connects the vesicle lumen with the outside, and that later dilates to complete exocytosis. Simulatins of altered time courses for release of molecules confirmed that details of the release time course are best appreciated near the detector, with the shape of the rising phase being most Informative. inciting Ca2i current. Also, the depression of AC., responses seen during a short train of voltage clamp pulses (0.2 Hz) is reduced. (2) Using conventional whole cell recording with a free Ca2+ concentration of -1.5 M in the pipette, and the cell clamped at -70 mV to prevent depolarization dependent Ca2+ entry, the rate of rise of C" after cell "pop-in" was enhanced by the presence of 150 AM cAMP in the pipette. These results suggest that maneuvers which raise cytosolic cAMP may contribute to the enhancement of depolarization induced secretion by mobilization of granules into an available pool, as well as by enhancing Ca2+ entry. (Support: NIH DK37380). A12 Because calcium uptake into intular organeles is dependent on free calcium ion concention, mot stdies ave ed calcium chelators lile EA toA ldiree caldum constant We examined whether EGTA had any effedt on Ca, uptae. Perneabilizd synaptosomes (.25mg/ml saponin) were incubated in an itracelular ionic media at pH 7.4 (20mM Heps), 370C, and 3mM ATP for either 1, 10, 30, or60 secondin the presence of four different EGTA c trations (0, 10, 100, or 1000pM). In all cases, free calcium was held at -0.7#M whic was checked with calcium sensitive mini-electrodes. We found that SCa + uptake increased with time up to 30 seconds, and that this increase could be abolished by the addition pftth calcium ionophore A23187. With no EGTA, ionopore sensitive 4Ca'+ upte incrased 0.02+ 0.003 smol/g protein. In presence of 10pM EGTA, uptake increased 0.1i 0.014 pmol/g protin; in 100pM EGTA, uptake increased 0.25± 0.05 pmol/g protein; and in 1000pM EGTA, ionophore sensitive calcium up increased 1.0 0.25 pmol/g protein in 30 s. This uptake was ATP d t and not affected by KCN plus oligomycin-B. Our interp tion is that a mobile buffer lie EGTA may enhance calcium uptakoe by overcoming diffusion limitations in the space surrounding the uptakce sites. Supped by NIH NS-19194. INTERCELLULAR COMMUNICATION A198
doi:10.1016/s0006-3495(93)81407-3 fatcat:e2el5ck7qneidoaqcbrj4lmbs4