Extracellular Osmotic Stress Reduces the Vesicle Size while

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Extracellular osmotic stress reduces the vesicle size while keeping a constant neurotransmitter concentration Hoda Fathali, Johan Dunevall, Soodabeh Majdi, and Ann-Sofie Cans ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00350 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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ACS Chemical Neuroscience

Extracellular osmotic stress reduces the vesicle size while keeping a constant neurotransmitter concentration

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†

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Hoda Fathali, Johan Duneval, Soodabeh Majdi , and Ann-Sofie Cans

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Department of Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden ‡ Department of Chemistry and Molecular Biology, University of Gothenburg, 41296 Gothenburg, Sweden Abstract Secretory cells respond to hypertonic stress by cell shrinking, which causes a reduction in exocytosis activity and the amount of signaling molecules released from single exocytosis events. These changes in exocytosis have been suggested to result from alterations in biophysical properties of cell cytoplasm and plasma membrane, and were based on the assumption that osmotic stress does not affect the secretory vesicles content and size prior to exocytosis. To further investigate whether vesicles in secretory cells are affected by the osmolality of the extracellular environment, we used intracellular electrochemical cytometry together with transmission electron microscopy imaging to quantify and determine the catecholamine concentration of dense core vesicles in situ before and after cell exposure to osmotic stress. In addition, single cell amperometry recordings of exocytosis at chromaffin cells were used to monitor the effect on exocytosis activity and quantal release when cells were exposed to osmotic stress. Here we show that hypertonic stress hampers exocytosis secretion after the first pool of readily releasable vesicles have been fused and that extracellular osmotic stress causes catecholamine filled vesicles to shrink, mainly by reducing the volume of the halo solution surrounding the protein matrix in dense core vesicles. In addition, the vesicles demonstrate the ability to perform adjustment in neurotransmitter content during shrinking and intracellular amperometry measurements in situ suggest that vesicles reduce the catecholamine content to maintain a constant concentration within the vesicle compartment. Hence, the secretory vesicles in the cell cytoplasm are highly affected and respond to

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extracellular osmotic stress, which gives a new perspective to the cause of reduction in quantal size by these vesicles when undergoing exocytosis.

Keywords: osmotic stress, dense core vesicle, chromaffin cell, exocytosis, catecholamine concentration, vesicle size, amperometry, and transmission electron microscopy.

Introduction

Vesicles in secretory cells are involved in storage of signaling molecules and release of these molecules during the exocytosis process. In chromaffin cells from the adrenal medulla, large dense core vesicles contain remarkable concentrations of catecholamines (adrenaline and noradrenaline), with an average concentration estimated to 0.5-1 M.1,

2

This high concentration of

neurotransmitters is accomplished by association of the catecholamines with the acidic proteins from the chromogranin family, which makes up the dense core protein matrix with a protein density of ≈169 mg/mL.3-6 This protein core is often surrounded by a solution referred to as the halo, in which a remainder of the non-bound part of the catecholamine content is dissolved. The storage of catecholamine occurs in the presence of other components such as ATP (125300 mM),7 Ca2+ (free calcium in halo solution ≈50-100 μM and calcium bound to the dense core ≈40 mM),8 Mg2+ (5 mM),9 ascorbate (10-30 mM)10 and an intravesicular pH of ≈5.5.11, 12 Hence, the total soluble concentration of components stored inside large dense core vesicles sums up to more than 750 mM. Yet, the association of all these molecules occurs at an isoosmotic condition of 310 mOsm/kg in the cytoplasm in order to maintain vesicle stability.13 This is explained by the aggregation of components to the dense core protein matrix at the acidifying intravesicular conditions, and is maintained by the V-ATPase activity that is responsible for controlling the pH gradient against the cytosol. The intravesicular composition is thus maintained constant until an exocytosis event is initiated. During exocytosis events, the vesicles dock to the plasma membrane and upon vesicle fusion, a nanometer fusion pore is formed. The pore formation allows ion


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exchange of vesicle content with the extracellular medium. Then fusion pore expansion might occur, prompted in part, by swelling of the dense core matrix inside the vesicle and placing hydrodynamic pressure on the vesicle membrane.14-17 The exposure to the extracellular media causes dissociation of intravesicular components from the dense core matrix, which then can be released. The presence of certain extracellular ions,18,

19

and changes in

temperature,18 pH,12 or osmolality20-27 have been reported to affect the exocytosis process. However, whether these parameters affect the vesicle catecholamine content during storage in the cytoplasm or the amount catecholamine released during the exocytosis process is still not clear and needs further investigation.

The effect of external osmotic pressure on exocytosis was reported previously, showing that high osmotic pressure inhibits exocytosis, and significantly reduces the amount of catecholamine released per exocytosis event .20, 24, 26 These reports suggested that hypertonic external media changes the properties of the cell membrane and reduces the catecholamine released by inhibiting swelling of the dense core matrix. However, these studies were limited to amperometric quantification of the amount catecholamine released during the exocytosis process, and conclusions were based on the assumption that extracellular osmotic stress does not significantly affect the catecholamine content of vesicles in the cell cytoplasm that are ready to be released by exocytosis. To answer the question whether extracellular osmotic pressure affect the catecholamine content of secretory vesicles during storage in cytoplasm, we examined the effects of high osmolality on the vesicular catecholamine content and size prior to exocytosis and compared this to the amount of catecholamine released when cells were stimulated to undergo secretion. To quantify the vesicle catecholamine content in situ, a recently developed technique, called intracellular vesicle electrochemical cytometry28 was used. This technique employs a flame etched carbon microelectrode with a conical shaped nanotip that is placed directly into the cell cytoplasm to carry out intracellular amperometry in live cells. In this approach, intracellular vesicles collide, adsorb, and subsequently rupture at the electrode surface. The vesicle catecholamine


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content is oxidized at the electrode surface upon release and is quantified. In addition, to determine how the fraction of the total vesicle catecholamine content released is affected by extracellular osmotic stress during exocytosis, single cell amperometry was performed by placing a carbon fiber microelectrode in close proximity to the cell plasma membrane to quantify the amount of catecholamine released by cells during secretion. To further relate the change in vesicle neurotransmitter quantal size to a change in vesicle catecholamine concentration during osmotic stress, the vesicle size in cells exposed to external isotonic and hypertonic buffers was determined by transmission electron microscopy (TEM) imaging. By comparing the quantitative change of vesicle catecholamine content to changes in vesicle size, we here show that native vesicles in situ reduce the catecholamine content as the volume shrinks due to the cell exposure to hypertonic conditions. Furthermore, the vesicles appear to adjust the catecholamine content to maintain a constant vesicle catecholamine concentration.

Results and Discussion

Extracellular osmotic pressure inhibits exocytosis activity and decreases the amount catecholamine released per exocytosis event To investigate the effect of osmotic stress on the exocytosis process and the neurotransmitter content of secretory vesicles, analytical methods were used to characterize secretory vesicles and their content release during exocytosis from cultured chromaffin cells exposed to either an isotonic or a hypertonic environment. An extracellular hypertonic solution was prepared by adjusting an isotonic buffer (310 mOsm/kg) with NaCl to an osmolality of 730 mOsm/kg All buffers were prepared to be calcium-free to eliminate the effect of external Ca2+ in these measurements. In these experiments a 5-s injection pulse of 5-mM Ba2+ solution was used to trigger exocytotic release at single chromaffin cells,29 and amperometric exocytosis recording was carried out by placing a 5-μm in diameter carbon fiber disc microelectrode in close contact to the cell surface, as illustrated in Figure 1a. To determine the alteration in quantal content release


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due to extracellular osmotic stress, chromaffin cells were first incubated in an isotonic buffer prior to a barium stimulation and amperometric recording of exocytosis release, followed by a 10 min exposure to either a hypertonic solution or an isotonic buffer serving as control and amperometric recording of exocytosis release were performed within the 10 min incubation time . During the amperometric recordings, a series of amperometric current spikes marked the detection of single vesicle exocytosis release events. The size of each current spike corresponds to the collective amount of catecholamine released from a single vesicle during an exocytosis event. The integrated current or area under each amperometric current spike can be converted to the number of catecholamine molecules released using Faraday’s law, N=Q/nF. Here, Q is the integrated charge under each spike, n is the number of electrons transferred in the oxidation reaction of catecholamine (n=2), F is Faraday’s constant (96485 Cequivalent-1), and N is the number of moles of catecholamine. Hence, using this equation, the number of molecules released from each particular exocytosis event was calculated and was used to compare the size of quantal release from cells exposed to either isotonic or hypertonic conditions We found as shown in Figure 1b, that similar to previous work on chromaffin cells,20, 24 a smaller number of molecules are released from each exocytosis event when cells are subjected to osmotic stress compared to cells in isotonic condition. The magnitude of this reduction in the number of molecules released when cells are osmotically stressed corresponds to about 56% of the quantal size released from cells at isotonic condition. The kinetic information on single vesicle exocytosis release from these recordings is presented in the supporting information as summarized in Table S1 and S2. Several reports suggest that during exocytosis from cells in isosmotic condition, extracellular solution enters through the vesicular fusion pore and induces swelling of the vesicular dense core protein matrix.24, 26, 30-33 The swelling caused by hydration allows further penetration of solvated ions, which eventually leads to dissolution of the dense core and release of the intravesicular catecholamine contents. It has therefore been suggested that upon vesicle fusion pore formation a hypertonic environment can interrupt the process of swelling and inhibits dissociation of


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catecholamine molecules bound to the dense core matrix.20, 21, 34 This is one possible mechanism for the observed decrease in catecholamine release when cells are exposed to a hypertonic environment. However, this only considers the reduction in the amount catecholamine that is released during secretion and does not consider potential changes in vesicle content prior to exocytosis release when cells are exposed to osmotic stress. When the average frequency of exocytosis events triggered by the barium stimulation is plotted versus time, as shown in Figure 1c, the initial first group of readily releasable vesicles display the same level of exocytosis activity in both osmotic conditions. Then a dramatic decrease in frequency of exocytosis events when cells are exposed to hypertonic media is observed. This observation might be related to that high osmolality affects the biophysical properties of the cell plasma membrane from the induced cell shrinking, which causes a decrease in cell membrane tension that have been shown to result in a reduced probability of successful vesicle fusion events.20,

24, 35

In addition, potentially vesicle

transport to the plasma membrane for docking and priming is affected by the increased molecular crowding in the cytoplasm during osmotic shock.36-39 Hence this might serve as the explanation to the subsequent drop in exocytosis activity when cells are exposed to osmotic stress; the pool of vesicles that is already placed in close proximity or docked to the plasma membrane are still able to fuse with the same frequency as vesicles in cells at isotonic conditions, but as the transport of new vesicles to the plasma membrane is obstructed by the increase in molecular crowding of the cytoplasm, the exocytosis activity readily declines. Amperometric traces from recordings of exocytosis release at cells in isotonic and hypertonic conditions are presented in the supporting information, as displayed in Figure S2. External hypertonic condition induces vesicles to shrink and reduces the vesicle catecholamine content We used intracellular electrochemical cytometry28 to quantify and compare the vesicular catecholamine content in cells exposed to isotonic and hypertonic buffers. In these experiments, a nanotip conical carbon micro-electrode (Figure


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2b) was inserted into the cell cytoplasm by gently pushing the electrode using mechanical force from a micromanipulator as illustrated in Figure 2a to perform amperometric measurement of catecholamine content release from single vesicles stochastically rupturing at the surface of the electrode. In the supporting information amperometric spikes from intracellular cytometry measurements at cells in isotonic and hypertonic conditions are presented, as shown in Figure S3. The intracellular electrochemical cytometry measurements were carried out at cells exposed to the same buffer solutions as used in the amperometric exocytosis recording experiments to maintain identical osmolality conditions and the same incubation times. As demonstrated in Figure 2c, when the cells are exposed to an external hypertonic environment, the average number of catecholamine molecules in vesicles decreases to 59% of the vesicles catecholamine content in cells exposed to isotonic condition. Hence, it appears that extracellular osmotic stress affects the retention of catecholamine in vesicles prior to the exocytosis process. A comparison shows that the relative decline in vesicle catecholamine content (59% of isotonic conditions as determined by intracellular cytometry) is consistent to the relative reduction in the amount catecholamine released during exocytosis (56% of isotonic conditions as determined by amperometric recording of exocytosis). Therefore, this suggests that the effect of osmotic stress on the quantal amount of catecholamine released during secretion is due to a regulation of the total catecholamine content in the vesicle prior to exocytosis. It seems from these measurements performed at live chromaffin cells that these alterations in vesicle content occur within the 10-min time scale for the extracellular incubations used in these experiments. Cells behave as osmometers and can rapidly equilibrate the cytoplasmic volume to the osmolality of extracellular solution.25 The efflux of water from the cytoplasm during cell exposure to a hypertonic condition, which shrinks the cell and results in an increase in molecular crowding of the cytoplasm, and lowers the tension of the plasma membrane and might influence the secretory vesicle membrane as well. These alterations can directly affect the dynamics of the rupturing process at the electrode surface during intracellular amperometry


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measurements. This can be monitored by the effect on recorded amperometric spike kinetics reflecting the rate of catecholamine release for single vesicles rupture at the electrode surface. Table 1 summarizes the kinetic information of amperometric current spikes recorded during intracellular amperometry at cells exposed to isotonic and hypertonic conditions. Here, the peak rise time (trise) is considered to indicate the kinetic of catecholamine release through the initial pore formed when vesicle ruptures at the electrode surface is initiated. The peak half width (t½) corresponds to the duration of catecholamine release in the rupturing process. The values for the kinetic parameters listed in Table 1 show that catecholamine is released significantly faster by vesicle rupture at the intracellular electrode when cells are subjected to hypertonic buffer compared to cells in an isotonic environment. An explanation for faster kinetics of catecholamine release could be either due to changes in physical properties of the vesicle membrane or might correspond to changes in vesicle size, which affects the rate of vesicle content release. It has been observed previously in isolated large dense core vesicles that osmotic pressure can alter vesicle size and morphology and that isolated vesicles can behave as osmometers in vitro. 4, 23, 40, 41

Therefore, in order to investigate if the size of vesicles in situ is affected by the

extracellular osmotic pressure, transmission electron microscopy (TEM) imaging was carried out on fixed chromaffin cells after incubation in hypertonic and isotonic solutions. In all TEM images of chromaffin cells that were examined, dense core vesicles could be resolved with moderately high electron dense content and clearly distinguished from all the other cellular compartments in the cytoplasm. Additionally, in all images that was used for vesicles analysis, the vesicle membrane could be discerned clearly from the dense core protein. Representative TEM images of chromaffin cells in isotonic and hypertonic buffers are presented in the supporting information, as shown in Figure S1. The average vesicle radius was determined to be 168 ± 10 nm in cells (n=12) subjected to isotonic buffer and 147±4 nm in cells (n=9) exposed to a hypertonic solution (error bars are SEM). The vesicle size was calculated as the average vesicle diameter in x- and y-direction, and was presented as the average vesicle radius. Size measurements were collected from 31±1 vesicles per single


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chromaffin cell. The vesicle radius has been adjusted to compensate for the sample sectioning thickness (~ 60 nm) as previously described by the Almers group.42 Without considering the effect of sectioning for TEM sample preparation, the radius of vesicles in isotonic condition was 138 ± 8 nm and 121± 3 nm for cells exposed to hypertonic condition. For the following calculations and presented results, compensated vesicle size dimensions have been used. The histogram in Figure 3 illustrates the size distribution of vesicles and shows that vesicles in cells subjected to a high extracellular hypertonic environment have a more narrow size distribution compared to vesicles from cells in isotonic condition. Also previously, it has been shown in pharmacological studies that vesicle volume is correlated with its catecholamine content. 43-46 Translating the change in vesicle size in cells subjected to a high external hypertonic environment, to a change in vesicular volume corresponds to a decrease to 60% of the vesicle volume in cells experiencing isotonic condition (Figure 4). This relative volume reduction is of the same magnitude as the relative decline in the number of catecholamine molecules in vesicles as quantified by intracellular cytometry when cells are exposed to osmotic stress (number of molecules measured by intracellular amperometry in hypertonic decreased to 59% of isotonic condition).

Vesicles maintain the intravesicular catecholamine concentration nearly constant at hyperosmotic induced volume change By combining the information on vesicle size from the TEM imaging with intracellular electrochemical quantification of catecholamine content in the vesicles, the vesicle catecholamine concentration (C) can be estimated using the following equation:

=

where r is average vesicle radius measured by TEM and the other terms are defined above by the Faraday equation. The vesicle concentration of


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catecholamine in cells exposed to isotonic and hypertonic solutions was calculated and determined to be equal to 0.35 M (for average values of Q=1.33 pC and r=168 nm) and 0.31 M (for average values of Q= 0.79 pC and r=147 nm), respectively. These calculated vesicle concentrations of catecholamine in cells at these osmotic environments are slightly lower but still in agreement with previously reported concentrations of catecholamine for large dense core vesicles in chromaffin cells.1, 2 Thus deviation from previous literature most likely stem from the intrinsic limitations of the methods from determining vesicle size. For instance, if we determine the concentration of catecholamine in vesicles without considering the effect of sectioning thickness by TEM sample preparation, the vesicle concentration of catecholamine was calculated to 0.63 M in cells in isotonic condition (for average values of Q=1.33 pC and r=138 nm) and 0.56 M in cells exposed to osmotic stress (for average values of Q= 0.79 pC and r=121 nm), respectively. Anyhow it appears that these large dense core vesicles from chromaffin cells adjust the content of catecholamine in the vesicle compartment to maintain a constant intravesicular catecholamine concentration during the vesicle shrinking process in response to the osmotic stress. This is consistent with the hypothesis of Colliver et al.43 and Gong et al.,44 where the vesicle volume was observed to expand or shrink by pharmacological treatment of chromaffin cells and was correlated to the altered quantal size of neurotransmitter released by exocytosis. Alterations in quantal size of neurotransmitter release from secretory vesicles in chromaffin cells has been observed previously by pharmacological treatment of cells with the catecholamine precursor L-DOPA 3,4-dihydroxyphenylalanine (LDOPA) in order to load vesicles with catecholamine and with a vesicular monoamine transporter (VMAT) blocker, reserpine, that decreases the vesicle catecholamine content.

43, 45-47

The action of these drugs showed that by

stimulating or blocking loading of neurotransmitter into the vesicle compartment, changes of quantal size associated with swelling or shrinking of vesicles occurs. In the case of large dense core vesicles in chromaffin cells, the total vesicle membrane area is altered in order to maintain a constant concentration of catecholamine inside the vesicles.43,


44

These drugs were

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targeted to the VMAT activity, and was suggested that VMAT mediate changes in vesicle

quantal

size

and

vesicular

volume

to

maintain

a

constant

neurotransmitter concentration in the chromaffin vesicle compartments.43 Here we apply a physical force to cells in terms of an osmotic pressure and a theoretical expectation is that this induces diffusion of water molecules out from the cells and possibly also from vesicles in response to this osmotic force. To equilibrate, we observed in addition to vesicles reducing the water content, an adjustment in catecholamine concentration occurred, even though osmotic stress is not targeted to directly influence any of the vesicle transport proteins that control the active transport of neurotransmitter across the vesicle membrane. Alteration in vesicle catecholamine content may be related to norepinephrine leakage from vesicles, which contributes to the metabolism of catecholamine turnover. This point of view was hypothesized by Kopin in 196448 and was mentioned that the leakage of catecholamine from vesicles is compensated by vesicle membrane amine transporter ( VMAT) activity. And later, catecholamine leakage from vesicles was observed in relation to the action of reserpine.49-51 Alternatively, adjustment to equilibrate the vesicle catecholamine concentration may be due to partitioning of free catecholamine molecules from the halo solution to the dense core matrix in order to induce a more condense pool of neurotransmitters inside the large dense core vesicle as hypothesized by Morris et al 19774. Potentially the decline in vesicle neurotransmitter content in cells exposed to osmotic stress might be affected by the experimental conditions of intracellular cytometry. If a portion of the catecholamine molecules are frozen inside the dense core protein matrix and are not releasable during the vesicle rupturing process or perhaps are released at such slow rate that the frozen portion of the vesicle catecholamine content is not detectable in regards to the noise level of the intracellular amperometric measurements. In addition, as the mechanism for the vesicle rupture at the electrode surface in these experiments is not yet fully understood, and might differ for vesicles in cells exposed to isotonic versus hyperosmotic conditions, this may affect the quantification efficiency of the vesicle catecholamine content. Therefore, for a better understanding of the affect on the catecholamine pathway under hypertonic stress further investigation is required. Still the data here shows that applying


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osmotic pressure to cells affects the intracellular water content and the intracellular cytometry recordings suggest that large dense core vesicles respond by regulating the catecholamine

content to maintain a constant

neurotransmitter concentration. To independently investigate whether the decline in the vesicle volume and number of stored catecholamine molecules are related to loss of the catecholamines associated with the dense core protein or loss of the free soluble catecholamines in the halo solution, the size of the dense core proteins in vesicles was measured from TEM images of cells exposed to isotonic and hypertonic conditions. By subtracting the volume of the dense core matrix from the total vesicle volume, the volume of the free catecholamine molecules in the halo was calculated. The measured adjustment in vesicle volume and the respective volume shift of the dense core protein and the surrounding halo solution in cells exposed to isotonic and hypertonic conditions are summarized in Figure 4. The average vesicle halo volume was estimated to be 12.7 ±2.1 aL and 5.7±0.6 aL in cells subjected to isotonic and hypertonic environments, respectively. If we assume that the catecholamine concentration in vesicles is homogenous and the catecholamine concentration is 0.33 M (average of 0.35 M and 0.31 M as determined from isotonic and hypertonic conditions, respectively), the average reduction in the vesicle halo volume is 7.0 aL as monitored in cells exposed hyperosmotic stress versus isotonic condition. This corresponds to a reduction of 1.39 million soluble catecholamine molecules per vesicle. This result is in good agreement with the quantitative drop in catecholamine content attained from the intracellular cytometry recording, demonstrating an average reduction in charge of 0.54 pC per vesicle rupture event, which corresponds to 1.69 million molecules. This shows a strong relationship between the changes in quantal neurotransmitter content of vesicles and vesicle volume, in particular by alteration in the vesicle halo volume when the vesicle is adjusting in size. This points to a novel physical property, that vesicles can respond to changes in neurotransmitter content in order to maintain a constant concentration of catecholamines by alteration in the amount of free soluble catecholamine molecules in the vesicle halo within the vesicle


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compartment when vesicles respond to high osmotic stress.

Conclusion By combining quantification of vesicle catecholamine content in chromaffin cells using

intracellular

vesicle

electrochemical

cytometry

together

with

amperometry recording of exocytotic catecholamine release and vesicle size analysis from TEM imaging of dense core vesicles in chromaffin cells subjected to either an isotonic or a hypertonic environment, novel insights of how osmotic stress affects secretory vesicles in live cells and the exocytosis process was achieved. In contrast with previous assumptions, we observe that vesicles are significantly affected by extracellular hyperosmotic shock in the prior stage of exocytosis release. Our results show that vesicles respond to extracellular osmotic stress by decreasing the vesicle size and by regulating the vesicular neurotransmitter content. Adjustment of vesicle size in cells might also be fast and here we observe that the vesicle shrink within the time frame of 10 min incubation times used in these experiments. We here show that when cells are exposed to a hyperosmotic solution, the vesicle catecholamine content rapidly decreases to about 60% of isotonic condition. In response to extracellular osmotic pressure, the vesicles also demonstrate a fine-tuning of the catecholamine content to the vesicle size as observed by intracellular amperometry measurements and suggests that vesicles adjust the catecholamine content to maintain a constant concentration. This suggests that vesicles are very adaptable in terms of retention of neurotransmitters in the vesicle compartment. TEM imaging of dense core vesicles in chromaffin cells showed that in the presence of hypertonic stress vesicles shrink and mainly by a reduction in vesicle halo volume. This suggest that the decrease in vesicle catecholamine content during vesicle shrinking is equivalent to a decline in the free soluble catecholamine content in the halo part of the vesicles. This suggests that not only the cell can behave as an osmometer, but also organelles in the cytoplams such as secretory vesicles respond to external osmotic stress by adjusting their volume and quantal content to maintain a constant concentration of


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neurotransmitters inside the vesicle in situ. In addition, we show that when cells are exposed to osmotic pressure the exocytosis activity is not affected by the pool of vesicles that are ready releasable, but is then subsequently significantly reduced. This might be related to the obstruction of vesicle transport by the enhanced molecular crowding in the cytoplasm. Hence the data from this study suggest that hyperosmotic stress leads to a change in vesicle size and vesicle catecholamine content rather than affecting the release process.

Experimental section Materials Cell culture materials: Collagenase P (from Clostridium histolyticum) was obtained from Roche, Sweden. Ham’s F12 medium, 5-fuoro-2-deoxyuridine, cytosine β-D-arabinofuranoside, Dulbecco’s modified Eagle’s medium (DMEM)– low glucose, Dulbecco’s Phosphate Buffered Saline (DPBS), Locke’s solution components, penicillin-streptomycin, fetal bovine serum, bovine serum albumin (powder) and percoll were purchased from Sigma-Aldrich, Sweden. Culture dishes (Collagen IV 60 mm TC-treated, Corning Biocoat) and TrypLE Express, GIBCO were purchased from Fisher Scientific, Sweden. T-75 flasks (Vented, polystyrene, Falcon) were purchased from Fisher Scientific, Sweden. Water was purified with Purelab Classic purification system (ELGA, Sweden). Bovine adrenal glands were kindly donated by the slaughterhouse, Dalsjöfors Kött AB, Dalsjöfors, Sweden. TEM sample preparation materials: glutaraldehyde, sodium cacodylate, osmium tetroxide and Agar 100 resin, all from Agar Scientific Ltd, UK were purchased from Oxford Instruments, Sweden. Sodium azide was purchased from BDH, UK and formaldehyde from Sigma-Aldrich, Sweden. Uranyl acetate, Merck, Germany, was obtained from VWR International, Sweden. Reynolds lead citrate was prepared as previously described.52 Cell culture Bovine chromaffin cells were isolated from adrenal medulla by enzymatic


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digestion as described previously.53 The cell yield was approximately 4 million cells/mL. Chromaffin cells were plated at a density of 17.5 × 103 cells/cm2 in collagen IV 60 mm plastic dish and incubated at 37 °C in a 5% CO2 environment for the purpose of exocytosis and intracellular experiments. The experiments were performed in following 1-3 days of cell culture. In order to measure the size of the vesicles by transmission electron microscopy, cells were plated in T-75 flasks (7-8 million cells per flask) and incubated at 37 °C in a 5% CO2 environment for 1 day. Prior to the cell chemical fixation, cells were incubated with isotonic and hypertonic buffers for 10 min at 37 °C in a 5% CO2 environment before chemical fixation was performed. Single cell amperometry experiments Cells were prepared for experiments by changing the medium to isotonic physiological saline (5 mL; 150 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 5 mM glucose, 10 mM HEPES, pH 7.4) with an osmolality of 310 mOsm/kg for isotonic control experiments. Then the buffer was replaced with a hypertonic solution with an osmolality that was adjusted to 730 mOsm/kg by increasing the concentration of NaCl. Amperometry experiments were performed within 10 min incubation time in either isotonic or hypertonic buffer solutions. Electrochemical recordings were performed after positioning the dish onto an inverted microscope (IX81, Olympus), in a Faraday cage. The experiments were performed at 37˚C. The working electrode was held at +700 mV versus a Ag/AgCl reference electrode using an Axon Instruments (Axopatch 200B, Molecular Devices, Sunnyvale, CA). The signal was digitalized at 10 KHz and the output was filtered with an internal low pass Bessel filter at 2 kHz. For exocytosis measurements a 5-μm (in diameter) carbon fiber microelectrode was positioned in close contact with the membrane of a chromaffin cell using a micromanipulator. The position of the electrode on the cell surface was confirmed by a slight deformation of the cell. Then, a glass microcapillary (20–30 μm tip diameter) was positioned with a second micromanipulator at a distance of 20 μm from the cell and used to inject a 5 s pulse of 5 mM BaCl2 solution (Femtojet injector, Eppendorf Inc., Hamburg, Germany) towards the cell surface. Each cell was stimulated once and amperometric recording was performed


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during 3 min. For intracellular vesicle electrochemical cytometry,28 a flame etched 5 μm (in diameter) carbon electrode with the tip size of 50-100 nm and length 30-100 μm was used. This cylindrical electrode was inserted into the cell cytoplasm and membrane was allowed to seal around the electrode before the potential at the amperometric electrode was applied. Method for TEM sample preparation Karnovsky fixative54 containing 0.01 % sodium azide, 1 % formaldehyde and 1.25 % glutaraldehyde, was used to incubate chromaffin cells in suspension at 4 °C for several hours. Fixed cells were washed with 0.15 M sodium cacodylate buffer and post-fixed with 1 % osmium tetroxyde for 2 h at 4 °C and 0.5 % uranyl acetate for 1 h at room temperature in the dark. Cells were dehydrated in ethanol of ascending concentrations and 100 % acetone. Embedding was done in Agar 100 resin and after obtaining cell sections of 60-nm thickness with an ultramicrotome. Uranyl acetate and Reynolds lead citrate were applied for poststaining. The imaging was done with a Leo 912AB Omega TEM operated at 120 kV.

Data analysis The amperometry traces were analysed by IgorPro 6.22 routine originating from David Sulzer’s group55. The threshold for the collected spikes was three times the root-mean square (RMS) standard deviation of the noise. The traces were manually inspected and false spikes were rejected. All statistics were done in MATLAB (R2016a, MathWorks Inc., Natick, MA) using student’s t-test assuming unequal variance. The normality of the data was evaluated using normal probability plots in combination with linear regression with a criteria of R2 > 0.90. The measurements of vesicle size were performed by image analysis of chromaffin cell sections images from transmission electron microscopy imaging using the software Image J.


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Author Contributions

H.F. and S.M. performed the single cell electrochemical experiments. H.F. performed the electron microscopy imaging. H.F. and J.D did the data analysis with support from all co-authors. H.F., J.D and A-S.C. conceived the project, designed the experiments with help from S.M and wrote the paper. All authors discussed the results and commented on the manuscript.

Funding Sources This work has been funded by The Swedish Research Council, The Knut and Alice Wallenberg Foundation and NIH. Acknowledgment The authors would like to thank Professor Andrew Ewing at the Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden for his support and comments on the manuscript, PhD student Jelena Lovric by donation of chromaffin cell culture, and assisting in sample fixation for TEM imaging, and Ms. Yvonne Josefsson from Electron Microscopy Unit, Institute of Biomedicine, University of Gothenburg, Sweden, for the technical assistance in sample preparation for TEM. Also Dalsjö fors Kö tt AB (Dalsjö fors, Sweden) is gratefully acknowledged for donation of bovine adrenal glands. Associated Content Additional information as noted in the text has been provided in the supporting information: Table S1: Kinetics of exocytosis at chromaffin cells in isotonic and hypertonic (730 mOsm/kg) conditions Table S2: Kinetics of prespike foot features from amperometric recording of exocytosis release at chromaffin cells exposed to an isotonic and a hypertonic environment. Figure S1: Representative TEM images of chromaffin cells at isotonic and hypertonic conditions.


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Figure S2: Amperometric recording of catecholamine release from a single chromaffin cell stimulated with Ba2+- solution. Figure S3: Amperometric recording of catecholamine release from vesicle rupture in situ of a chromaffin cell using intracellular electrochemical cytometry.


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Figure 1. a) A schematic showing the experimental set up for amperometric measurements of exocytosis at chromaffin cells by placing of a carbon fiber micro-electrode in close contact to the surface of single chromaffin cells. b) Amperometric quantification of the number of catecholamine molecules released during exocytosis at cells (n=20) exposed to hypertonic condition (730 mOsm/kg) and at cells (n=22) in an isotonic environment (310 mOsm/kg). Values are expressed as group average of averages from each single cell recording (mean ± SEM). Statistical significance of changes was tested using ttest for unpaired data (p-value 0.0003). c) A time profile of the average frequency of exocytosis release events in cells stimulated by barium and exposed either to isotonic or hypertonic conditions.

Figure 2. a) A schematic of the experimental set up for intracellular electrochemical cytometry for quantification of the vesicle catecholamine content prior to exocytosis. b) A scanning electron microscopy image of a nanotip conical carbon fiber micro-electrode used in these experiments. Scale bar is 20 µm. The tip size of the electrodes were in the range of 50-100 nm and with a tip length of 30-100 μm. c) The average number of catecholamine molecules in vesicles prior to exocytosis measured by intracellular electrochemical cytometry in cells (n=19) subjected to isotonic solution (310 mOsm/kg) and cells (n=16) exposed to hypertonic solution (730 mOsm/kg). Values are expressed as group average of averages from each single cell recording (mean ± SEM). Statistical significance of changes was tested using ttest for unpaired data (p-value 0.0108).


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Table 1: Kinetics of catecholamine release from vesicle rupture at the electrode surface in intracellular electrochemical cytometry measurements. N is number of cells expose to measured in isotonic and hypertonic buffers. Values are expressed as group average of averages from each single cell recording (mean ± SEM). Presented p-values between isotonic and hypertonic conditions are results of t-test for unpaired data. t1/2 (ms)

trise (ms)

Imax (pA)

n

Isotonic

11.4±1.2

3.71±0.45

75.5±6.9

19

hypertonic

5.21±0.54

1.52±0.21

86.3±9.3

16

p-value

0.0001

0.0002

0.36

***

***

Figure 3: Size distribution of vesicle radius as determined by transmission electron microscopy imaging. Light color bars are vesicle sizes measured from cells (n=12) subjected to isotonic condition and dark colors are vesicle sizes from cells (n=9) in a hypertonic environment.


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Figure 4: Changes in volume of vesicles, dense core particles and halo solution as determined by transmission electron microscopy (TEM) images from cells (n=12) in isotonic and cells (n=9) in hypertonic solutions. Values are expressed as group average of averages from each single cell recording (mean ± SEM). pvalues are reported from unpaired t-test. p-value is for vesicle volume 0.0385, for dense core volume 0.3967 and for halo volume 0.0074.


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Figure 1 49x13mm (600 x 600 DPI)


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Extracellular Osmotic Stress Reduces the Vesicle Size while

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