Different Secretory Vesicles Can Be Involved in Depolarization-Evoked Exocytosis Elena A. Lukyanetz 1 Bogomoletz Institute of Physiology, Bogomoletz Str. 4, 01024 Kiev-24, Ukraine Received October 8, 2001 The relationship between Ca 2 influx through volt- age-activated Ca 2 channels, resting intracellular Ca 2 level (Ca i ) and Ca 2 -dependent exocytosis was studied in bovine adrenal chromaffin cells by using patch- clamp, capacitance, and fluorescent measurements. It was established that depolarization-induced exocyto- sis passed over two steps, both of which linearly de- pend on Ca i . At Ca i lying below critical point (200 –300 nM) the slope of the relationship was 4.43 and at Ca i exceeding the critical point the slope was equal to 31.63. The vesicular mechanism describing experimen- tal two-step dependence of exocytosis on intracellular Ca 2 (Ca i ) is proposed. According to the model at Ca i below critical point only small-sized vesicles fuse with plasma membrane, whereas at higher Ca i , larger ves- icles started to fuse. © 2001 Academic Press Key Words: calcium; exocytosis; chromaffin cells; se- cretory vesicles; calcium channels. Exocytosis is one of the main cellular processes, which enables the cell to influence its environment. This mechanism underlies the intercellular communi- cation in the nervous system via neurotransmitter re- lease in synapses and thus is a basis for integrative function of the brain. The involvement of numerous intracellular molecules in secretory pathway has been already shown. However, a major intracellular stimu- lus for exocytotic proceeding in excitable cells is a rise of intracellular Ca 2+ concentration (Ca i ). It has been previously shown that Ca 2+ -dependence of secretion has a two-step character which can be explained by several models, viz. ‘pools’ model in chromaffin cells (1, 2) or ‘prepared stage’ model of secretion in nerve ter- minals (3). Recently the involvement of a protein ki- nase C mechanism into biphasic phenomenon has also been proposed (4). The ‘pools’ model assumes the exis- tence of three vesicular pools and vesicle “migration” from large reserve to a release-ready and then to se- creted pools. The existence of large reserve and release- ready pools determines a two-step secretion with the third-power Ca 2+ -dependence of the final secretory response. The ‘prepared stage’ model describes two steps—a threshold phase being a preparative stage for secretion and a secretory phase itself. The preparative stage serves as Ca 2+ -dependent “priming” step of se- cretion. ‘PKC model’ explains the two phases of secre- tion by PKC-dependent and -independent processes depending on Ca i . In this paper we propose ‘vesicular’ mechanism that describes experimental data and im- plies significance of this phenomenon in the function of secretory cells. MATERIALS AND METHODS Cell culture. Chromaffin cells were prepared by enzymatic disso- ciation of bovine adrenal glands and maintained in tissue culture for 2–5 days. Then the cells were plated on poly-L-lysine-coated glass coverslips and maintained in Medium 199 supplemented with 10% fetal calf serum, 1% bovine serum albumin and 2 mM glutamine for cell culture, see (5) for more details. We did not separate adrenaline- and noradrenaline-secreted cells. Electrophysiological measurements. Cells were voltage clamped using the whole-cell patch-clamp technique (6). Whole-cell Ca 2+ cur- rents were recorded at high time resolution with a computer-controlled patch-clamp amplifier EPC-9 and ‘Pulse’ software (HEKA Electronic, Lambrecht, Germany). Standard bath solution contained in mM: NaCl, 140; KCl, 2.8; CaCl 2 , 2; MgCl 2 , 2, Hepes, 10; TEACl, 10; tetrodotoxin, 0.01; pH 7.2; bath solutions with high Ca 2+ concentration contained: NaCl, 40; CaCl 2 , 60; MgCl 2 , 2, Hepes, 10; TEACl, 40; tetrodotoxin, 0.01; pH 7.2. Protocol of experiments was as described previously (5). The intracellular solution contained in mM: CsCl, 64; Cs 2 SO 4 , 28; ATP, 2; MgCl 2 , 2; EGTA, 0.5; Hepes, 10; N-methyl-D-glucamin, 10; GTP, 0.3; pH 7.4. The osmolarity of the solutions was adjusted to 320 mOsM with glucose. All compounds were obtained from Sigma. All experiments were performed at room temperature (21–24°C). Capacitance measurements. To measure the secretion, we used a high resolution measurement of membrane capacitance ( C m ) (7). The measurements were realized in whole-cell experiments by applying a sine wave stimulus of about a DC holding potential -70 mV with a software “Lock-in.” A 1-kHz, 10 mV peak-to peak sine wave was generated by an ITC-16 multichannel interface (Instrutech, Inc., NY) controlled by Mac computer. The sine generation as well as phase sensitive detection of C m were fulfilled with the software ‘Lock-in amplifier’ controlled by ‘Pulse’ software and EPC-9 amplifier (HEKA Electronics). Differences in C m ( C m ) before and after a depolarizing 1 Fax: (380)-44-2536458. E-mail: elena@serv.biph.kiev.ua. Biochemical and Biophysical Research Communications 288, 844 – 848 (2001) doi:10.1006/bbrc.2001.5844, available online at http://www.idealibrary.com on 844 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.