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Introduction
Glucose homeostasis is critical to the health or survival of mammals and is maintained, in large part, by pancreatic
b cells, which secrete insulin in response to an increasing
concentration of glucose. Pancreatic b cells sense glucose through
their glucose metabolism and the resulting increase in the ATP/ADP ratio closes the ATP-sensitive potassium channels
(KATP channels)[1]. The closure of
KATP channels depolarizes b cells and opens
voltage-dependent calcium channels to allow
Ca2+ entry. Ca2+ acts on the exocytotic machinery to stimulate fusion of insulin-containing vesicles with the plasma membrane for
releasing insulin into the
bloodstream[2]. As Ca2+ plays a pivotal role in insulin secretion, it is worthwhile to characterize the
Ca2+ channel activity and the relationship between the
Ca2+ signal and the exocytosis in the pancreatic
b cell. In this study, we aimed at establishing an efficient method to isolate healthy pancreatic
b cell from the mouse model, and for characterizing
the relationship between Ca2+ signaling and exocytosis in those cells. The reason for choosing the mouse is to open up the
possibility for future functional studies of insulin secretion employing the genetic modified mouse model.
Materials and methods
Preparation and culture of b cells Pancreatic
b cells from adult male Kunming (KM) mice were prepared as described in
a previous study[3,4] with modifications. In brief, 3_4 week-old KM male mice were killed by cervical dislocation and the
pancreas was quickly removed and placed in a 35-mm dish containing cold HanksĄŻ balanced salt solution (HBSS). After being
washed for 3 times, the pancreas was minced into tiny tissue blocks and transferred to 50 mL Falcon tube with 40 mL cold
HBSS. After removing the supernatant, the tissue blocks were digested by adding 5 mL pre-warmed HBSS containing
collagenase P (5 mg/mL, Roche) into the tube and maintained at 37 °C for 40 min in a shaking bath. The digestion was
terminated by adding 20 mL cold HBSS solution with 10 % serum, and then centrifuged at 1000 rpm to remove supernatant.
The digested tissues were transferred to a dish and the pancreas islets were picked out with a 200 µL pipette tip by hand. The
isolated islets obtained were dissociated into single cells by vigorous shaking in
Ca2+-Mg2+-free HBSS. The dispersed
b cells were plated on glass coverslips pre-coated with
poly-L-lysine and grown in RPMI 1640 medium supplemented with 10 %
(v/v) fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 IU/mL penicillin and 10 mmol/L glucose at 37 °C gassed with a
humidified mixture of 5% CO2 and 95% air. Cells cultured for 3_5 d were used in the experiments.
Solutions and drugs Standard HBSS contained 0.14 g/L
CaCl2, 0.1 g/L
MgCl2.6H2O, 0.1 g/L
MgSO4-7H2O, 0.4 g/L KCl, 0.06 g/L
KH2PO4, 0.35 g/L
NaHCO3, 8 g/L NaCl, 0.48 g/L
Na2HPO4, 1 g/L glucose, 20 mmol/L HEPES and 1 mg/mL BSA
(pH=7.2; adjusted with NaOH, osmolarity=310 mOsm).
Ca2+-Mg2+-free HBSS solution contained 0.4 g/L KCl,
0.06 g/L KH2PO4, 0.35 g/L
NaHCO3, 8 g/L NaCl, 0.48 g/L
Na2HPO4, 1 g/L Glucose, addition of 1 mmol/L EGTA, 20
mmol/L HEPES and 10 mg/mL BSA (pH=7.2, 310 mOsm).
RPMI medium 1640 (Gibco) was supplemented with of 10% FBS (Gibco), 100
µg/mL streptomycin, 100 IU/mL penicillin and 10 mmol/L glucose.
Standard bath solution for the experiments contained 138 mmol/L NaCl, 5.6 mmol/L KCl, 2.6 mmol/L
CaCl2, 1.2 mmol/L MgCl2, 5 mmol/L glucose, and 10 mmol/L HEPES (pH 7.2, 310 mOsm). For depolarization experiments, the bath solution
contained 10 mmol/L CaCl2 and 20 mmol/L tetraethylammonium-Cl (TEA-Cl). For preparing pipette solutions, we generally
prepared 2×buffers, which contained 250 mmol/L Cs-glutamate and 80 mmol/L HEPES (pH 7.2). The standard electrode
pipette solution contained 125 mmol/L Cs-glutamate, 40 mmol/L HEPES, 2 mmol/L MgATP, 0.3 mmol/L
Na2GTP, 1 mmol/L MgCl2 and 0.1 mmol/L EGTA (pH 7.2 adjusted with 1 mol/L CsOH, 300 mOsm). Internal solution for UV-flash photolysis
consisted of 110 mmol/L Cs-glutamate, 2 mmol/L MgATP, 0.3 mmol/L
Na2GTP, 35 mmol/L HEPES, 5 mmol/L NP-EGTA
[nitrophenyl-ethylene glycol-bis
(b-aminoethyl
ether)-N,N,NĄŻ,NĄŻ-tetraacetic acid,NP-FGTA, Molecular Probes, Carlsbad, CA,
USA], and 0.2 mmol/L Ca2+ indicator furaptra (Molecular Probes), and was adjusted to pH 7.2 with either HCl or CsOH and to
osmolarity around 300 mOsm. The free
Ca2+ concentration of the pipette solution was determined to be approximately 200
nmol/L.
Patch clamp recording and membrane capacitance measurement
Patch clamp electrodes with a resistance of 2_3
MW were made from borosilicate glass capillaries, coated with Sylgard 184 (Dow Corning, Midland, Michigan, USA) and
heat-polished. The membrane capacitance (Cm) of primary
b cell was measured in real time using an EPC9 amplifier (Heka
Electronics, Lambrecht, Germany) in conventional whole-cell patch clamp configuration with series resistance (Rs) between
5 to 12 MW. A sine+DC protocol was applied using the Lock-In extension of the Pulse program (Heka Electronics).
The b cells were voltage clamped at a holding potential of
-70 mV and a sine wave voltage command with amplitude of 20 mV and
frequency of 977 Hz was applied. Currents were filtered at 2.9 kHz and sampled at 15.6 kHz.
Flash photolysis Homogenous global
[Ca2+]i elevation was generated by photolysis of the
caged-Ca2+ compound, NP-EGTA with UV light generated from a Rapp flash lamp (Rapp Optoelektronik, Hamburg, Germany).
[Ca2+]i was measured using dual wavelength excitation method. A series of illuminations alternating between 350 and 380 nm were applied, which allowed
ratiometric determination of the Ca2+ concentration according to the equation:
[Ca2+]i=Keff
×(R-Rmin)/(R
max-R). Keff,
Rmin, and Rmax are constants obtained from in vivo calibration. The duration of the illuminations was adjusted to maintain relatively
constant Ca2+ concentrations, as the illumination at 350 and 380 nm also lead to photolytic release of
Ca2+. Trains of light alternating at 350 and 380 nm were generated from a Polychrome IV (Till Photonics, Planegg, Germany). The resulting
fluorescence was recorded with a photodiode (Till
Photonics).
Statistical analysis Data analysis was performed in IGOR Pro 4.02 (WaveMetrics, Lake Oswego, OR, USA), and results
were presented as mean±SEM with the indicated number of experiments.
Results
Ca2+ channel current in KM mouse b cells
We selected cells with diameters 8_10 mm for study, so that >80%_90% of the
cells were expected to be b cells[5]. TEA-containing extracellular solution and intracellular
Cs+ were used to block most of the voltage-gated
K+ channels. From recording in whole-cell configuration with a holding potential of -70 mV,
b cells exhibited inward currents typical for
Ca2+ current in response to depolarizing voltage ramped from -70 to +70 mV (Figure 1A). Figure 1B
shows slowly inactivating L-type Ca2+ currents
(ICa-L) evoked by depolarizing step pulses of 100 ms duration from a holding
potential of -70 mV to +50 mV in 10 mV increments. The rundown
of ICa-L was minimized by intracellular supplement of MgATP
(2 mmol/L) and Na2GTP (0.3 mmol/L). The averaged peak
Ca2+ current measured at +20 mV was -60±6 pA
(n=13). The current-voltage relationships
(I-V, shown in Figure 1C) of L-type
Ca2+ were obtained by 100 ms depolarization applied in 10
s-intervals, from holding potential -70 mV to +50mV in 10 mV increments.
Exocytotic response to pulse depolarization in KM mouse
b cells Exocytosis of a vesicle incorporates its vesicular
membrane into the plasma membrane. This leads to an increase in the cell-surface membrane, which can be monitored
electrically as an increase in Cm as it is proportional to the cell-surface membrane. Cm measurement has the advantage of
monitoring secretion from the whole population of vesicles with millisecond time resolution. Pulse voltage, which
depolarizes cell membrane and elicits
Ca2+ influx, is recognized as a somewhat physiological stimulus. By using capacitance
measurement and simultaneously current recording, the exocytotic response and
Ca2+ current in primary b cells from KM mice were
characterized. A typical response was shown in Figure 2A. Pulse voltage stimuli of 90 mV (from the holding potential -70 mV
to +20 mV) elicited distinct Ca2+ current and exocytotic responses. Increasing the duration of the depolarization resulted in
more Ca2+ influx, and larger Cm increments. For depolarization duration <15 ms, no consistent Cm increase was detected. The
average secretory
responses from 12 cells are summarized in Figure 2B, in which the average amplitudes of Cm increments were displayed
against the duration of the depolarizing pulse. The amplitudes of secretion increased exponentially with a duration shorter
than 100 ms, and reached a plateau at durations from 100 to 300 ms. Depolarization pulse of 500 ms in duration elicited further
increases in Cm (Figure 2).
Immediately releasable pool is situated close to the
Ca2+ channel Ca2+-dependent exocytosis can, at least in the short
term, be functionally divided into the release of vesicles from a readily releasable pool (RRP) and the subsequent refilling of
the RRP from a reserve pool[6]. We hypothesized that this also applied to mouse pancreatic
b cells and that secretion reflects the sequential release of the two pools of secretory granules. A subset of RRP is spatially arranged in close association to
Ca2+-channels, thus forming a so-called immediately releasable pool (IRP) that will exocytose instantaneously upon opening
of the Ca2+-channels.
We explored the relationship between IRP, RRP and
Ca2+ channels by voltage-clamp depolarization shown in Figure 3.
The stimulation protocol consisted of a train of five 30-ms depolarizations followed by nine 100-ms pulses from -70 mV to 20
mV. In response to this stimulation protocol, we observed a small Cm increment (16 fF) after the first five pulses representing
exocytosis from IRP, and a large exocytosis (254 fF) after the following nine pulses representing exocytosis from RRP.
Kinetics of exocytosis and endocytosis in KM mouse
b cells To determine the
[Ca2+]i dependence of vesicles, a
well-defined [Ca2+]i stimulation is necessary. Here we used
Ca2+ uncaging from photolabile
caged-Ca2+ to generate a homogenous
[Ca2+]i elevation, to avoid the complications of
Ca2+ microdomains as a result of local influx,
Ca2+ buffering and
mobilization[7]. As shown in Figure 4, the Cm increase in response to step-like
[Ca2+]i elevation starts with an exocytotic burst component
(within 1 s after flash) followed by a linear sustained release in mouse
b cells. It is generally held that the exocytotic burst
represents the release from RRP and the sustained phase of secretion is thought to reflect the rate-limiting step of refilling of
RRP[6,8]. Detailed examination of the exocytotic burst revealed a double exponential time course, indicating the presence of
two releasable vesicle pools with distinct release
kinetics[6]. The exocytotic burst can be best described by a sum of two
exponential components: a fast burst component with a release rate of approximately 50
s-1, and a slow burst component with a much slower release rate of approximately 5
s-1. The sustained component has a slope of 10 fF/s, corresponding to a
supplying rate of 3 granules/s if we assume approximately 3 fF for one insulin-containing
LDCV[9,10]. The characteristics of these exocytotic components were not significantly different from previous studies on mouse
b cells[10,11].
Secretion needs a balance between vesicle exocytosis and endocytosis to keep constant cell
surface. In b cells, exocytotion
sis is frequently accompanied by endocytosis with a latency of around 300 ms. We measured 22 endocytotic events out
of 28 cells. Figure 4B showed a typical trace. The time course of endocytosis was best fitted by a double exponential
function, indicating that there existed two endocytotic components. The rate
(1/t) of the fast endocytosis was much faster than that of the slow one, as indicated in Figure 4B. The time constants and amplitudes of both components were:
t1=0.10 s, A1=591 fF for fast endocytosis; and
t2=2.41 s, A2=202 fF for slow endocytosis.
Discussion
We present in this paper an improved method for isola
of b cells from the KM mouse pancreas. Compared with other methods described
in previous studies, our method possesses the following advantages: (1) only single enzyme treatment (collagenase P) instead of multiple enzymes is required, therefore,
the isolation becomes simple and easy to manipulate; (2)
Ca2+-Mg2+-free HBSS was used to dissociate the pancreas islets into
a single cell. The relative weak digestion by a single enzyme keeps the membrane protein including receptors and channels
less destroyed. Thus, this method could be widely used in
b cell isolation.
In mouse pancreatic b cells, insulin is released by
Ca2+-dependent exocytosis initiated by
Ca2+ entering through voltage-gated L-type
Ca2+ channels[12]. And
Ca2+ channels have been shown to co-localize with insulin-containing secretory
granules[13]. Bokvist et al demonstrated that the
Ca2+ transient need extend no more than 1 mm from the plasma membrane to
trigger exocytosis[14].
Using trains of short pulse depolarization, an immediately releasable pool (IRP) of vesicles is characterized in
the b cell of KM mouse. The IRP is found in numerous endocrine cells. It is generally recognized that the IRP, which is a distinct subset
of RRP, is situated in the vicinity of the
Ca2+ channel[15]. Thus, vesicles sensor a relative higher
Ca2+ level and release faster in response to trains of depolarization.
Ca2+ is a triggering signal for neurotransmitter and hormone release.
Ca2+ diffusion yields an immediate accumulation of
calcium ions within an area covering tens of nanometers around the mouth of open
Ca2+ channels. This calcium signal can be
restrained by the buffering action of
Ca2+-binding proteins. Local
Ca2+ influx and buffering will result in qualitatively different
types of intracellular signals, depending on the spatial arrangement of
Ca2+ channels relative to each other and to the relevant
Ca2+-binding proteins. The variety of
Ca2+ signals that vesicles sense complicate the analysis of
Ca2+ dependence and kinetics of vesicle release. By employing photolysis of caged
Ca2+ to generate a spatial homogenous
[Ca2+]i elevation as stimuli for secretion, we characterized the kinetics of exocytosis and endocytosis in the
primary b cell of KM mouse. Flash experiments reveal that the exocytotic response to step-like
Ca2+ elevation exhibits three kinetic components, in consistence
with previous studies in pancreatic beta
cells[10,11].
Endocytosis is an important cellular function, by which the increased plasma membrane is taken up into cell, and the
released vesicles are renewed and reused. Fast and slow modes
of endocytosis are found at a number of synapses and in
various cells. Our high-time resolution capacitance measurement demonstrated the existence of two endocytotic
components in b cells of KM mouse. Until now, the molecular mechanism underlying the fast endocytosis still remains
elusive[16]. The fast endocytosis is characterized by a very fast endocytotic rate
(1/t>1 s-1) and amplitude normally exceeding that of
exocytosis[17,18]. The underlying mechanism is still unknown. Further analysis of the
Ca2+-dependent exocytosis and endocytosis in pancreatic
b cells from genetic manipulated mouse model will be necessary for elucidating the underlying
mechanism.
References
1 Eliasson L, Renstrom E, Ding WG, Proks P, Rorsman P. Rapid ATP-dependent priming of secretory granules precedes
Ca2+-induced exocytosis in mouse
pancreatic b-cells. J Physiol 1997; 503: 399_412.
2 MacDonald PE, Rorsman P. Oscillations, intercellular coupling, and insulin secretion in pancreatic beta cells. PLoS Biol 2006; 4: 49.
3 Wan QF, Dong Y, Yang H, Lou X, Ding J, Xu T. Protein kinase activation increases insulin secretion by sensitizing the secretory
machinery to Ca2+. J Gen Physiol 2004; 124: 653_62.
4 Chen L, Koh DS, Hille B. Dynamics of calcium clearance in mouse pancreatic beta-cells. Diabetes 2003; 52: 1723_31.
5 Rorsman P, Trube G. Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions. J Physiol
1986; 374: 531_50.
6 Sorensen JB. Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles. Pflugers Arch 2004; 448: 347_62.
7 Neher E. Vesicle pools and
Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 1998; 20:
389_99.
8 Xu T, Binz T, Niemann H, Neher E. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci
1998; 1:192_200.
9 Gopel S, Zhang Q, Eliasson L, Ma XS, Galvanovskis J, Rorsman P,
et al. Capacitance measurements of exocytosis in mouse pancreatic
alpha-, beta- and delta-cells within intact islets of Langerhans. J Physiol 2004; 556: 711_26.
10 Olofsson CS, Gopel SO, Barg S, Galvanovskis J, Rorsman P, Eliasson L,
et al. Fast insulin secretion reflects exocytosis of docked granules
in mouse pancreatic b-cells. Pflugers Arch 2002; 444: 43_51.
11 Takahashi N, Kadowaki T, Yazaki Y, Miyashita Y, Kasai H. Multiple exocytotic pathways in pancreatic
b cells. J Cell Biol 1997; 138: 55_64.
12 Ammala C, Eliasson L, Bokvist K, Larsson O, Ashcroft FM, Rorsman P. Exocytosis elicited by action potentials and voltage-clamp
calcium currents in individual mouse pancreatic b-cells. J Physiol 1993; 472: 665_88.
13 Wiser O, Trus M, Hernandez A, Renstrom E, Barg S, Rorsman P,
et al. The voltage sensitive Lc-type
Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci USA 1999; 96: 245_53.
14 Bokvist K, Eliasson L, Ammala C, Renstrom E, Rorsman P. Co-localization of L-type
Ca2+ channels and insulin-containing secretory
granules and its significance for the initiation of exocytosis in mouse pancreatic
b-cells. EMBO J 1995; 14: 50_7.
15 Ammala C, Eliasson L, Bokvist K, Larsson O, Ashcroft FM, Rorsman P. Exocytosis elicited by action potentials and voltage-clamp
calcium currents in individual mouse pancreatic b-cells. J Physiol 1993; 472: 665_88.
16 Harata NC, Choi S, Pyle JL, Aravanis AM, Tsien RW. Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at
hippocampal synapses studied with novel quenching methods. Neuron 2006; 49: 243_56
17 Heinemann C, Chow RH, Neher E, Zucker RS. Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of
caged Ca2+. Biophys J 1994; 67: 2546_57.
18 Smith C, Neher E. Multiple forms of endocytosis in bovine adrenal chromaffin cells. J Cell Biol 1997; 139: 885_94.
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