From the Department of Biomedical Sciences, Consiglio
Nazionale delle Ricerche, Center of Biomembranes, University of Padova,
Via G. Colombo 3, 35100 Padova Italy and ¶ Division of Clinical
Biochemistry, Department of Internal Medicine, University Medical
Center, CH-1211, Geneva 4, Switzerland
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ABSTRACT |
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The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell line Ins-1 to determine whether the Ca2+ trapped in the secretory granules plays a role in exocytosis. The results demonstrate that a high proportion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the acidic compartment. The latter pool, however, does not significantly contribute to the [Ca2+]i changes elicited by thapsigargin and the inositol trisphosphate-producing agonist carbachol. By monitoring membrane capacitance at the single cell level or by measuring insulin release in cell populations, we show that Ca2+ mobilization from nonacidic Ca2+ pools causes a profound and long lasting increase in depolarization-induced secretion, whereas breakdown of granule pH had no significant effect. In contrast, releasing Ca2+ from the acidic pool markedly reduces secretion. It is suggested that a high Ca2+ concentration in the secretory compartment is needed to sustain optimal exocytosis.
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INTRODUCTION |
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A rise in intracellular Ca2+ concentration ([Ca2+]i) is necessary to induce regulated secretion in most cell types (1, 2). In neurons, [Ca2+]i increases up to several hundred µM are needed to trigger vesicle fusion, whereas in endocrine cells, granule exocytosis appears to require lower [Ca2+]i rises (3-7). The time course of exocytosis also appears different in the two cell types. Synaptic vesicle fusion is very fast (µs) and abrupt, whereas granule fusion is slower and more sustained (3, 8, 9).
Aside from these differences, important similarities exist between
secretory vesicles and granules. Both secretory vesicles and granules
contain large amounts of Ca2+ ions (10-13). The function
traditionally attributed to the high Ca2+ content in the
secretory compartment is the packaging and processing of intravesicular
content (14, 15). More recently a granular localization of the type 3 InsP31 receptor
has been suggested, based on high resolution immunocytochemistry of
pancreatic -cells (16). In the exocrine pancreas, evidence has been
provided suggesting that the intragranular Ca2+ content is
released by opening of low affinity InsP3 receptors (17).
These conclusions, however, have recently been challenged (18, 19), and
the role of granular Ca2+ remains elusive. Another line of
evidence suggesting that intragranular Ca2+ is implicated
in secretion comes from the recent identification of an acidic
Ca2+-binding protein, granule lattice Protein 1 (Grl1p), in
dense core secretory granules of Tetrahymena thermophila
that appears essential for regulated secretion (20).
Another common feature between secretory vesicles and granules is their low luminal pH. They share this characteristic with the lysosomal/endosomal compartment and the trans-Golgi network (21, 22). Indeed, the low pH of the lumen has proven a reliable means for determining the Ca2+ content of the so-called "acidic Ca2+ pool." Since ionophores such as ionomycin or A23187 are largely ineffective in transporting Ca2+ from an acidic environment (28), the pH gradient between lumen and cytosol must be collapsed before they can effectively release the Ca2+ content of this pool into the cytoplasm (23-26).
The aim of the present study was to establish whether the
Ca2+ stored within the acidic pool is important in the late
steps of exocytosis. For this purpose we employed as a model system the
-cell line Ins-1, an insulin-secreting cell line established from a
rat insulinoma that, among different lines, best retains the phenotype
of
-cells (27-29). Among other properties, Ins-1 cells display
temperature-dependent and glucose-responsive secretion and,
as shown here, temperature-dependent increases in membrane capacitance upon depolarizing pulses. Therefore, they can be used as an
alternative to
-cells for studying secretion at the single cell
level. By using capacitance measurements in combination with agents
that mobilize Ca2+ and/or collapse intracellular pH
gradients, the role of different intracellular Ca2+ pools
in secretion has been assessed. We here demonstrate that, although a
low pH in the granules is not required for the late steps of secretion,
the level of intragranular Ca2+ significantly affects the
secretory profile.
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EXPERIMENTAL PROCEDURES |
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Cells-- Ins-1 cells were cultured as described previously (27, 29). Two days before the experiment, cells were trypsinized and plated on poly(L-lysine)-coated coverslips (diameter 24 mm; 105 cells/coverslip).
Ca2+ Measurements in Ins-1 Cells-- Cells were loaded for 30 min at 37 °C with 2 µM fura-2/AM as described previously (26). Coverslips were then bathed in 1 ml of Ringer's solution containing 140 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5.6 mM glucose, 10 mM HEPES, pH 7.4, at 33 °C. Cells were placed on the stage of an inverted microscope equipped with a 40× oil immersion objective (Zeiss, Germany) and connected to a digital video imaging system (Georgia Instruments, Roswell, GA). All experiments were performed at 31-33 °C. Excitation wavelengths were set at 340 and 380 nm. Fluorescence emission at 510 ± 15 nm was collected by a CCD camera, and 8 images were averaged/time point. Time series were acquired with a frame interval of 4 s, and images at both excitation wavelengths were stored on an optomagnetic disc recorder (Panasonic, Japan). All data were normalized to the first min base-line ratio.
The total content of cellular Ca2+ under different conditions was assayed by atomic absorption spectrophotometry. Cells (20 × 107 cells/ml) were suspended in a Ca2+-free medium containing 1 mM EGTA and challenged with ionomycin (10 µM) alone or in combination with monensin (10 µM). Cell aliquots (107) were then centrifuged for 5 min at 14,000 rpm in Eppendorf tubes containing 100 µl of sucrose 12.5% and 400 µl of silicon oil. The pellet was resuspended in 1 ml of a solution containing Triton (0.02%) and NaOH (0.2 N) before measurement.Membrane Capacitance Measurements--
Unless otherwise
specified, during electrophysiological recordings, cells were perfused
at 31-33 °C with an external solution containing 118 mM NaCl, 20 mM tetraethylammonium chloride, 5.6 mM CsCl, 1.2 mM MgCl2, 2 mM CaCl2, 5.7 mM glucose, 10 mM HEPES at pH 7.4. Electrophysiological recordings and
fura-2 fluorescence were performed in the whole-cell configuration of
the patch-clamp technique with a computer-based patch-clamp amplifier
(EPC-9, HEKA, Lambrecht, Germany) controlled by the Pulse software
(HEKA). The internal solution consisted of 145 mM glutamic
acid, 155 mM CsOH, 1 mM MgCl2, 8 mM NaCl, 2 mM MgATP, 0.1 mM cAMP,
0.1 mM fura-2 (or 0.1 mM BCECF), and 10 mM HEPES at pH 7.2. Membrane capacitance (Cm) was measured
with the "sine+dc" mode of the "lock-in" extension of the Pulse
software, based on the Lindau-Neher algorithm (30). An 800 Hz, 40 mV
peak-to-peak sinusoid stimulus was applied to the DC holding potential
of 80 mV. During a depolarizing pulse and 5 ms before and after the
pulse, no sinewave was applied. No leak subtraction was performed on
the evoked currents in the calculations used. After the whole-cell
configuration was established, Cm was recorded and canceled by the
automatic capacitance compensation of the EPC-9. The procedure was
repeated every 180 s to prevent a possible saturation of the
lock-in signal (31).
Insulin Secretion Studies-- Superfusion experiments on Ins-1 cell suspensions were performed as described previously (34). In short, cells were brought into suspension and placed in 1-ml superfusion chambers at a density of 106 cells/chamber. The cells were superfused at a rate of 1 ml/min at 37 °C, and test substances were introduced with the buffer. One-min fractions were collected and subjected to an insulin radioimmunoassay. For presentation, the KCl-induced stimulation of the second pulse was integrated and normalized to that of the first pulse.
Materials-- fura-2/AM, fura-2 free acid, BCECF free acid, and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA); culture media and sera were from Technogenetics (Milan, Italy); and other chemicals were from Sigma.
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RESULTS |
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[Ca2+]i Dynamics in Ins-1 Cells-- When [Ca2+]i was monitored by fura-2 in intact cells bathed in a medium containing CaCl2 (2 mM) and glucose (5.6 mM), approximately 50% of the cells displayed asynchronous oscillations of [Ca2+]i, whose frequency and amplitude largely depended on the cell batch (29). In Fig. 1, the [Ca2+]i kinetics from several individual cells were averaged, leading to a partial masking of the initial oscillations. The addition of EGTA immediately abolished these oscillations and caused a decrease in the base-line ratio, indicating that they depend on Ca2+ influx. The presence of acidic Ca2+ pools and their contribution to [Ca2+]i rises were evaluated with the protocol previously employed in other cell lines (23-26). In Fig. 1A, after EGTA addition, the fast-exchangeable Ca2+ pool was released by the Ca2+ pump inhibitor thapsigargin (Tg) (1 µM). The subsequent addition of the Ca2+ ionophore ionomycin (1 µM) led to a small, further increase in [Ca2+]i, indicating that in these cells the large majority of the ionomycin-sensitive pool is represented by the Tg-sensitive one. Release of the acidic Ca2+ pool was then achieved by the addition of the Na+/H+ exchanger monensin (2 µM). Qualitatively similar data have been obtained by addition of the weak base chloroquine (40 µM), used in place of monensin to dissipate the intraluminal pH gradients. The increase in [Ca2+]i after monensin (or chloroquine) application requires the pretreatment with ionomycin (26). In fact, addition of either drug alone was without appreciable effect on [Ca2+]i (data not shown). Integrating peak areas showed that the amount of Ca2+ residing in acidic compartments was, on average, 51.3 ± 3.6% (n = 4) of total releasable Ca2+. The amount of Ca2+ released from the different Ca2+ pools was also assayed by atomic absorption spectrophotometry. In controls (unstimulated conditions), the total content of cellular Ca2+ was estimated to be 5.7 nmol of Ca2+/mg of protein (n = 3). Ionomycin alone or ionomycin and monensin together released 2.4 ± 1.1 and 4.5 ± 0.4 nmol of Ca2+/mg of protein, respectively (n = 3). Thus, of the total releasable Ca2+, about 54% was released by ionomycin alone; the remaining 46% was then attributed to the Ca2+content selectively released from the acidic pool.
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Capacitance Changes After Activation of Voltage Operated
Ca2+ Channels--
Fig. 2
shows fast changes in Cm, membrane conductance (Gm) as well as series
conductance (Gs) before and after a 200-ms depolarizing pulse from 80
mV holding potential to 0 mV. With 200-ms pulses, individual cells
displayed
Cm ranging from 20 to 400 fF with an average of 49.2 ± 4.4 fF (mean ±S.E., n = 76). Increasing the pulse
duration to 400 ms led to an increase in
Cm of 60% when compared
with a 200-ms depolarizing pulse in 4 of 9 cells (data not shown).
However, these longer depolarizing pulses caused a rapid rundown of the
evoked Ca2+ currents. We therefore decided to perform the
experiments with 200-ms pulse duration.
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Role of Granular Ca2+ Content in Secretion--
We
first tested if drug application by itself induced Cm. From the low
frequency recording of Cm it can be seen that during application of
ionomycin, monensin, or the combination of the two, an increase in Cm
occurred (Fig. 5A-C,
lower panels). Since similar increases were observed when
the solvents ethanol or Me2SO were tested and when
experiments were performed at room temperature to inhibit regulated
secretion (36), we conclude that to a large extent the observed changes
are due to the solvent. Chloroquine, on the other hand, being dissolved
in Ringer's solution, had no effect. We also tested whether drug
application changed the intracellular pH; therefore in some experiments
BCECF was included in the intracellular solution instead of fura-2.
These experiments showed that application of none of the drugs, applied
alone or in combination, significantly altered the cytosolic pH when
cells were kept in the whole-cell configuration (data not shown).
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Insulin Secretion Studies--
To determine whether the reduction
in Cm caused by acidic Ca2+ pool depletion was
attributable, at least in part, to fusion of insulin-containing
granules, we followed the release of insulin in populations of cells
treated with protocols that mimic those used in Fig. 5. Cell
suspensions obtained from monolayers were challenged with two pulses of
30 mM KCl of 1-min duration, 5 min apart. As summarized in
Fig. 8, secretion during the second
depolarizing pulse was 25 ± 4% (n = 3) of that
obtained during the first challenge. This reduction in secretion
probably reflects a reduction in readily releasable insulin granules,
although a rundown of the Ca2+ peak after depolarization
may also contribute to this effect (34). One-min stimulation with 1 µM ionomycin between the first and second KCl pulses
resulted in a less drastic reduction of insulin secretion (62 ± 24% that of initially released, n = 3). When a
combination of ionomycin and chloroquine (or monensin) was employed,
secretion during the second KCl pulse was 25 ± 11 and 24 ± 4% (n = 3), respectively; i.e. the
potentiating effect of ionomycin was completely abolished.
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DISCUSSION |
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In the -cell line Ins-1, as in other secretory cells, a
relatively high proportion of intracellular Ca2+ appears to
be stored in acidic structures. In fact, these cells respond with a
large [Ca2+]i increase to the protocol previously
employed to reveal this compartment, i.e. the application of
drugs that collapse internal acidic pH gradients (monensin or
chloroquine) after addition of the Ca2+ ionophore
ionomycin. The subcellular localization of acidic Ca2+
pools has not been determined with certainty, although it is likely
that in Ins-1 cells, as in other cell types, it is heterogeneous. A
rough estimation of the contribution of insulin granules to the
Ca2+ content of the acidic pool can be obtained by
considering the total releasable Ca2+ of Ins-1 cells (4.5 nmol/mg of protein, this work), the cell volume occupied by the
granules (1.2%),2 and the
releasable granule Ca2+ (about 125 nmol/mg of granule
protein, Ref. 10). By using these parameters, intragranular
Ca2+ mobilization could be as high as 1.5 nmol/mg of
protein. We have shown here that in Ins-1 cells 46% (i.e.
2.4 nmol/mg of protein) of the total releasable Ca2+ is due
to the acidic pool. Although based on a number of assumptions, these
values indicate that insulin granules represent a major part (more than
60%) of the acidic compartment in this cell type.
The main goal of this investigation was to establish the role played by
Ca2+ trapped in the secretory compartment in the process of
secretion. To address this question, we first investigated whether or
not the acidic compartment could contribute to (i) the
[Ca2+]i changes induced in Ins-1 cells by the
muscarinic agonist CCh and (ii) the [Ca2+]i
changes induced by depolarization. The finding that Ca2+
mobilization induced by thapsigargin or InsP3 production
through activation of muscarinic receptors does not affect the acidic pool is meaningful. In fact, given that insulin granules represent a
large proportion of that pool, it confirms by a functional approach the
conclusion of Ravazzola et al. (18) that InsP3
receptors are not expressed on the membrane of the secretory granules
of -cells. Similarly, a role for the acidic pool (and thus for
insulin granules) in Ca2+-induced Ca2+ release
is unlikely since the increase in [Ca2+]i caused
by depolarization was indistinguishable in controls and cells whose
acidic pool had been depleted (Fig. 5).
We next tested the possibility that intragranular Ca2+
plays a role in the secretory process by monitoring membrane
capacitance in single Ins-1 cells under different experimental
conditions. In untreated cells, the magnitude of Cm has a tendency
to decrease during a series of successive pulses; however up to the
fourth pulse (i.e. 300 s),
Cm is fairly constant. On
the contrary, manipulation of intracellular Ca2+ in the
time interval between the first and the second depolarizing pulses
significantly changed the extent of secretion. In fact, depletion of
Ca2+ from nonacidic stores led to a prolonged stimulation
of secretion up to 50%. Such a priming action of ionomycin has been
described previously (4), but the fact that it can last for several min at resting [Ca2+]i is a novel observation.
Releasing Ca2+ from the ionomycin-sensitive compartments
may favor granule recruitment from a distant cytoskeletal-anchored pool
(38) or by promoting priming of granules at a late, post-docking step
(39). Such a priming has been previously described by mechanisms
that cause long lasting phases of moderately elevated
[Ca2+]i (31).
In marked contrast with the potentiating effect of a brief increase in [Ca2+]i, releasing Ca2+ from the acidic compartments led to inhibition of secretion that reached 50% when compared with cells treated only with ionomycin. Since breakdown of the intracellular pH gradients by itself was without effect and the inhibition was observed with both monensin and chloroquine (two agents that act on pH gradients by different mechanisms), it can be concluded that the inhibitory effect is due to the release of Ca2+ from the acidic organelles, including insulin granules. Since our alkalinization protocol is by no means specific for the granules, the question can be raised as to whether the reduction in secretion is due to the decrease in Ca2+ within the granules themselves or in other acidic compartments (trans-Golgi network or lysosomes). The observation that inhibition is maximal within a few tens of seconds after acidic Ca2+ pool discharge argues for a distal site of action, i.e. the granules themselves.
It is probable that both insulin-containing granules and
-aminobutyric acid-containing vesicles contribute to the increases in Cm monitored by us and by other groups (7, 40, 41). However, the
fact that Ca2+ depletion from nonacidic and acidic
compartments affects both Cm increases and insulin secretion in
radioimmunoassay suggests that at least part of the effects seen in our
study reflects fusion of insulin-containing granules.
A high intragranular Ca2+ concentration may be important for docking or priming of the granules for the fusion process itself or for all of the steps. It is noteworthy that a protein called Grl1p, abundant in secretory granules of T. thermophila (20) is sensitive to both Ca2+ and pH.
In conclusion, our experiments show that in Ins-1 cells, a relatively large amount of the stored Ca2+ resides in acidic compartments. A high [Ca2+] in this compartment but not a low pH is needed for optimal exocytosis.
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ACKNOWLEDGEMENTS |
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We are grateful to Clarissa Bartley for performing the superfusion experiments and the radioimmunoassays, M. Mancon for total Ca2+ measurements, and G. Ronconi and M. Santato for skillful assistance. We thank Drs. Aldebaran M. Hofer and Bruce G. Jenks for discussion and for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from Telethon number 845, European Union Programs Human Capital Mobility Network CHRXCT940500, Human Frontier Science Program RG520/95, the Armenise Foundation grant (Harvard) (to T. P.), and in part by Swiss National Science Foundation Grant 32-49755.96 (to C. B. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by EU Grant CHRXCT940500. To whom correspondence should be addressed: Dept. of Cellular Animal Physiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. Tel.: 00.31.24.3653335; Fax: 00.31.24.3652714; E-mail: scheenen{at}sci.kun.nl.
1 The abbreviations used are: InsP3, inositol trisphosphate; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; Cm, membrane capacitance; Gm, membrane conductance; Gs, series conductance; Tg, thapsigargin; CCh, carbachol; F, farads.
2
This value has been estimated considering that
Ins-1 cells contain 10% of the insulin content of -cells (27) and
that in the latter cell type, the percentage volume occupied by
granules is 11.5% based on morphometric analysis (37).
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REFERENCES |
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