Name: monitoring the effect of osmotic stress on secretory vesicles and exocytosis
Uploaded: 2018-02-19T13:30:00
Description: Watch this Scientific Journal Video about Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis at JoVE.com

The authors would like to thank the Swedish Research Council (349-2007-8680) for funding and Dalsj&#246;fors K&#246;tt AB (Dalsj&#246;fors, Sweden) for donation of bovine adrenal glands.

1. Cell Culture of Bovine Chromaffin Cells Isolated by Enzymatic Digestion from Adrenal Glands
<ol>
	<li>To isolate chromaffin cells collected from bovine adrenal glands, sterilize cells with 70% ethanol solution. Trim away fat and connective tissue using a scalpel. To remove blood, rinse the adrenal veins using Locke&#39;s solution (NaCl 154 mM, KCl 5.6 mM, NaHCO3 3.6 mM, hydroxyethyl piperazineethanesulfonic acid (HEPES) 5.6 mM at pH 7.4) that has been thermostated to 37 &#176;C.</li>
	<li>To digest the tissue, infla...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Mg2++ion-activated+ATPase+and+a+pH+gradient+in+the+storage+of+catecholamines+in+synaptic+vesicles.">Role of Mg2+ ion-activated ATPase and a pH gradient in the storage of catecholamines in synaptic vesicles.</a> Biochemistry. 17 (13), 2517-2523 (1978).</li><li>Terland, O., Flatmark, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+as+a+natural+constituent+of+chromaffin+granules+from+the+bovine+adrenal+medulla.">Ascorbate as a natural constituent of chromaffin granules from the bovine adrenal medulla.</a> FEBS letters. 59 (1), 52-56 (1975).</li><li>Camacho, M., Machado, J. D., Montesinos, M. S., Criado, M., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intragranular+pH+rapidly+modulates+exocytosis+in+adrenal+chromaffin+cells.">Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells.</a> J Neurochem. 96 (2), 324-334 (2006).</li><li>Jankowski, J. A., Schroeder, T. J., Ciolkowski, E. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+characteristics+of+quantal+secretion+of+catecholamines+from+adrenal+medullary+cells.">Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells.</a> J Biol Chem. 268 (20), 14694-14700 (1993).</li><li>Daniels, A., Williams, R., Wright, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+character+of+the+stored+molecules+in+chromaffin+granules+of+the+adrenal+medulla:+a+nuclear+magnetic+resonance+study.">The character of the stored molecules in chromaffin granules of the adrenal medulla: a nuclear magnetic resonance study.</a> Neurosci. 3 (6), 573-585 (1978).</li><li>Borges, R., Travis, E. R., Hochstetler, S. E., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+external+osmotic+pressure+on+vesicular+secretion+from+bovine+adrenal+medullary+cells.">Effects of external osmotic pressure on vesicular secretion from bovine adrenal medullary cells.</a> J Biol Chem. 272 (13), 8325-8331 (1997).</li><li>Troyer, K. P., Mundorf, M. L., Fries, H. E., Wightman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+vesicle+fusion+and+exocytosis+in+hypertonic+conditions.">Separating vesicle fusion and exocytosis in hypertonic conditions.</a> Ann NY Acad Sci. 971 (1), 251-253 (2002).</li><li>Brodwick, M. S., Curran, M., Edwards, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+osmotic+stress+on+mast+cell+vesicles+of+the+beige+mouse.">Effects of osmotic stress on mast cell vesicles of the beige mouse.</a> J Membrane Biol. 126 (2), 159-169 (1992).</li><li>Amatore, C., Arbault, S., Bonifas, I., Lemaitre, F., Verchier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+exocytosis+under+hypotonic+conditions+shows+two+distinct+populations+of+dense+core+vesicles+in+bovine+chromaffin+cells.">Vesicular exocytosis under hypotonic conditions shows two distinct populations of dense core vesicles in bovine chromaffin cells.</a> Chem Phys Chem. 8 (4), 578-585 (2007).</li><li>Hampton, R. Y., Holz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+changes+in+osmolality+on+the+stability+and+function+of+cultured+chromaffin+cells+and+the+possible+role+of+osmotic+forces+in+exocytosis.">Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis.</a> J Cell Biol. 96 (4), 1082-1088 (1983).</li><li>Troyer, K. P., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+separation+of+vesicle+release+from+vesicle+fusion+during+exocytosis.">Temporal separation of vesicle release from vesicle fusion during exocytosis.</a> J Biol Chem. 277 (32), 29101-29107 (2002).</li><li>Pihel, K., Travis, E. R., Borges, R., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytotic+release+from+individual+granules+exhibits+similar+properties+at+mast+and+chromaffin+cells.">Exocytotic release from individual granules exhibits similar properties at mast and chromaffin cells.</a> Biophys J. 71 (3), 1633 (1996).</li><li>Jankowski, J. A., Finnegan, J. M., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ionic+composition+alters+kinetics+of+vesicular+release+of+catecholamines+and+quantal+size+during+exocytosis+at+adrenal+medullary+cells.">Extracellular ionic composition alters kinetics of vesicular release of catecholamines and quantal size during exocytosis at adrenal medullary cells.</a> J Neurochem. 63 (5), 1739-1747 (1994).</li><li>Leszczyszyn, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+receptor-mediated+catecholamine+secretion+from+individual+chromaffin+cells.+Chemical+evidence+for+exocytosis.">Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis.</a> J Biol Chem. 265 (25), 14736-14737 (1990).</li><li>Wightman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+resolved+catecholamine+spikes+correspond+to+single+vesicle+release+from+individual+chromaffin+cells.">Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.</a> P Natl Acad Sci. 88 (23), 10754-10758 (1991).</li><li>Fathali, H., Dunevall, J., Majdi, S., Cans, A. -. 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We here present a protocol and the advantages of combining three complementary analytical methods to analyze secretory vesicles and the exocytosis process to gain a better understanding of how a physical force such as osmotic pressure can affect secretory vesicles and the exocytosis process in secretory cells. These methods include carbon fiber microelectrode amperometry, which is an established method for recording exocytosis activity, intracellular electrochemical cytometry, which is used to determine quantal size of v...

Chromaffin cells in adrenal glands are neuroendocrine cells that release neurotransmitter molecules into the blood stream. This occurs through a cellular process that involves the docking and fusion of neurotransmitter-filled vesicles, resulting in content release from vesicles to the extracellular space in a process called exocytosis. The neurotransmitters (adrenaline and noradrenaline) in chromaffin cells are actively transported by membrane proteins into large dense core vesicles (LDCVs) and stored at high concentrations (~0.5-1 M)1,2. Accumulation of neurotransmitters inside the LDCVs is accomplished by the affinity of catecholamine molecules to the intravesicular dense core protein matrix comprised of chromogranin proteins (~169 mg/mL)3,4,5,6, and an intravesicular cocktail solution containing key components for catecholamine loading and storage into the vesicle such as ATP (125-300 mM)7, Ca2+ (50-100 &#181;M in the solution and ~40 mM bound to the protein matrix)8, Mg2+ (5 mM)9, ascorbate (10-30 mM)10, and a pH of ~5.511,12. The LDCVs maintain an iso-osmotic condition with the cell cytoplasm (310 mOsm/kg)13, even though the theoretical solute concentration inside the vesicles sum up to more than 750 mM. The composition of the intravesicular components is not only essential for the loading and storage of catecholamines, but also for aggregation of solutes to the dense core protein matrix. This significantly reduces the osmolarity of the vesicles is significantly reduced and can affect the amount catecholamine that is released during exocytosis5,6.
Studies on the effect of extracellular osmotic pressure on the exocytosis process by amperometric recording have reported that high extracellular osmotic pressure inhibits exocytosis activity and reduces the number of neurotransmitters secreted from single vesicle compartments4,14,15,16,17,18,19. The explanations of these observations have speculated on the possible enhancement of macromolecular crowding in the cell cytoplasm inhibiting vesicle fusion events, and an alteration in membrane biophysical properties affecting the number of neurotransmitters released during exocytosis. These thoughts assumed that high extracellular osmotic stress does not affect the vesicle quantal size, which defines the number of neurotransmitter molecules stored in a vesicle compartment at prior stage of triggered exocytosis14,15,17,19,20,21. In amperometric measurements of exocytosis release in single cells, a carbon fiber disc microelectrode is placed in close contact with the cell surface, creating an experimental set up mimicking the synapse configuration, where the amperometric electrode serves as a postsynaptic detector (Figure 1)22,23. By stimulating a cell to exocytosis, one can induce neurotransmitter-filled vesicles to fuse with the cell plasma membrane and release part or the full vesicle content into the extracellular space. These neurotransmitter molecules released at the surface of the electrode can be detected electrochemically if the neurotransmitters are electroactive (e.g., catecholamines) by applying a redox potential of +700 mV vs a Ag/AgCl reference electrode. Consequently, a series of current spikes mark the detection of individual exocytosis events. From the current versus time trace in an amperometric recording, the area under each single amperometric spike represents the charge detected per exocytosis event and can be converted to the mole of neurotransmitters released, using the Faraday equation. Hence, the amperometric recordings provide quantitative information on the amount neurotransmitters expelled from single exocytosis events and report on the frequency of exocytosis events, but do not present quantitative information on the secretory vesicles content before vesicle fusion and neurotransmitter release has been initiated.
Therefore, to get a better understanding of how secretory vesicles in the cell cytoplasm respond to extracellular osmotic stress before the cell is triggered to undergo exocytosis, other complementary analytical methods can be used to enrich this information. For instance, to investigate if osmotic stress alters vesicle volume, transmission electron microscopy (TEM) imaging analysis can be used to measure the vesicle size of cells after chemical fixation. To examine if osmotic stress affects vesicle quantal size, a recently developed amperometric technique called intracellular electrochemical cytometry, can be applied for quantification of vesicle neurotransmitter content at secretory vesicles in their native state when still residing in the cytoplasm of live cells26. In the intracellular electrochemical cytometry technique, a nanotip cylindrical carbon fiber electrode is gently inserted into the cytoplasm of live cells and, by applying a +700 mV potential to this electrode (vs a Ag/AgCl reference electrode), the catecholamine content in vesicles can be quantified by the detection of a redox current spike from single vesicles colliding, adsorbing, and subsequently stochastically rupturing at the electrode surface and thereby releasing their contents against the electrode surface26. Hence, in the amperometric current versus time trace, each single vesicle rupturing event can result in a current transient and, by integrating the area of each current spike, the vesicle quantal size can be calculated using Faraday&#180;s law.
Therefore, by linking the quantitative information gained from vesicle size measurements using TEM imaging together with vesicle quantal size analysis, as recorded by intracellular electrochemical cytometry, vesicle neurotransmitter concentration can also be determined. This allows vesicle characterization when cells are exposed to different osmotic conditions and provides a better view on how vesicles may respond to extracellular osmotic stress at the stage prior to exocytosis. The results from combining these methods have shown that in the presence of extracellular high osmotic pressure, vesicles shrink and adjust their quantal size and comparing the quantitative information on the relative changes from these measurements shows that while shrinking, vesicles adjust their contents and size to maintain a constant neurotransmitter concentration24. Thus, this understanding is valuable in connecting to the observations made on neurotransmitter release in cells exposed to osmotic stress. In these protocols, we describe the use of these three complementary methodologies that allow the characterization of how secretory vesicles in their native environment respond to extracellular osmolality and the effects of this response on the exocytosis process. In addition to our previous observations regarding the effect of high osmotic pressure on exocytosis24, we present additional experiments that describe cell recovery after osmotic shock and the effect of multiple barium stimulations in chromaffin cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:631px;" width="631">
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		<col />
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	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">NaCl</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">KCl</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">NaHCO3</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">HEPES</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">MgCl2</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">M-2670</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glucose</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Collagenase P</td>
			<td style="width:129px;">Roche, Sweden</td>
			<td style="width:119px;">11 213 857 001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">100-&#181;M Nylon mesh</td>
			<td style="width:129px;">Fisher Sientific</td>
			<td style="width:119px;">08-771-19</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Percoll</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td style="width:119px;">P1677</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Collagen IV coated 60 mm plastic dish</td>
			<td style="width:129px;">VWR</td>
			<td style="width:119px;">354416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:129px;">Avanti J-20XP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Borosilicate glass capillary</td>
			<td style="width:129px;">Sutter instrument Co., Novato, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Micropipette puller</td>
			<td style="width:129px;">Narishing Inc., Japan</td>
			<td style="width:119px;">PE-21</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Epoxy solutions (A and B)</td>
			<td style="width:129px;">Epoxy technology, Billerica, MA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Beveller</td>
			<td style="width:129px;">Narishing Inc.</td>
			<td style="width:119px;">EG-400</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Microscope</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:119px;">IX81</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Patch clamp Instrument</td>
			<td style="width:129px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:119px;">Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Micromanipulator</td>
			<td style="width:129px;">Burleigh Instrument Inc., USA</td>
			<td style="width:119px;">PCS-5000</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Butane Flame</td>
			<td style="width:129px;">Multiflame AB, H&#228;sselholm, Sweden</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission electron microscopy</td>
			<td style="width:129px;">Omega</td>
			<td style="width:119px;">Leo 912 AB</td>
			<td></td>
		</tr>
	</tbody>
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monitoring the effect of osmotic stress on secretory vesicles and exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

We here describe the protocol for how combining TEM imaging together with two electrochemical methodologies, carbon fiber amperometry and intracellular electrochemical cytometry, can provide information that gains a broader view alluding to the effect of extracellular osmotic pressure on secretory vesicles and the exocytosis process. By comparing representative amperometric recordings of exocytosis release at single chromaffin cells using the experimental set up (shown in Figure 1), a signif...

Watch this Scientific Journal Video about Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis at JoVE.com

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

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