1Laboratory of Islet Cell Physiology, Novo Nordisk, DK-2880 Bagsvaerd; and 2Department of Biological Chemistry, The August Krogh Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
Submitted 24 February 2003 ; accepted in final form 11 March 2003
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ABSTRACT |
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arachidonic acid; ClC-3 chloride channels; insulin exocytosis; lysophospholipids; phospholipase A2
Arachidonic acid is believed to be an important signaling component in insulin secretion from pancreatic -cells. This is supported by the observation that glucose stimulates accumulation of arachidonic acid in islets (22, 29), which amplifies the depolarization-induced rise in [Ca2+]i and insulin secretion (23, 29, 30). Much less information is available on the effects of lysophospholipids on
-cell function and the possible interplay between arachidonic acid and lysophospholipids on insulin secretion.
Here we have used high-resolution capacitance measurements to explore the mechanisms by which cPLA2 and its products arachidonic acid and lysophospholipids modulate exocytosis in single mouse pancreatic
-cells. We demonstrate that intracellular application of cPLA2
promotes exocytosis primarily by increasing the availability of secretory granules for release. This mobilization of secretory granules from a reserve pool to the readily releasable pool (RRP) was dependent on cPLA2
activity and resulted from the combined actions of arachidonic acid and lysophospholipids.
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METHODS |
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Electrophysiology. Patch pipettes were pulled from borosilicate capillaries, coated with Sylgard near their tips, and fire-polished before use. When filled with pipette solution, they had a tip resistance of 34 M. The zero-current potential was adjusted before establishment of the seal with the pipette in the bath. The holding potential in all experiments was -70 mV. Exocytosis was monitored in single
-cells as changes in cell capacitance using the standard whole cell configuration of the patch-clamp technique, which has the advantage of permitting the cell interior to be dialyzed by the pipette-filling solution. An EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) was used, and exocytosis was elicited by 500-ms voltage-clamp depolarizations that went from -70 mV to 0 mV. Changes in cell capacitance were detected using the Pulse software (version 8.31; HEKA Elektronik) with the lock-in module. Briefly, a 2.5-kHz 40-mV peak-to-peak sinusoid stimulus was applied symmetrically around the holding potential of -70 mV immediately before and after the voltage-clamp pulse. The resulting current was processed by the Pulse software to give estimates for the membrane capacitance and conductance, as well as the access resistance of the patch. During the experiments, the cells were situated in an experimental chamber with a volume of 0.4 ml, which was continuously superfused at a rate of 1.52 ml/min to maintain the temperature at
33°C.
Measurements of [Ca2+]i. The [Ca2+]i measurements were made using an Axiovert 135 inverted microscope with a Plan-Neofluar x100/NA 1.30 objective (Carl Zeiss, Göttingen, Germany) and an Ionoptix (Milton, MA) fluorescence imaging system. Excitation was effected at 340 and 380 nm and emitted light recorded at 510 nm with a video camera synchronized to the excitation light source and a computer interface. The [Ca2+]i measurements were combined with voltage-clamp depolarizations, and the experiments were conducted using the standard whole cell configuration of the patch-clamp technique with 150 µM fura 2 (Molecular Probes, Eugene, OR) included in the pipette-filled solution. Calibration of the fluorescence signal was performed by infusing the cells with fura 2 and Ca2+-EGTA buffers (Molecular Probes) with known free Ca2+ concentrations ranging between 0 and 39.8 µM. These data were fitted to the equation described in Ref. 10 and were used to calculate [Ca2+]i.
Solutions. The standard extracellular solution contained (in mM) 118 NaCl, 20 tetraethylammonium-Cl (TEA-Cl), 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.40 using NaOH), and 5 glucose. TEA-Cl was included to block outward K+ currents that persist even after replacement of intracellular K+ with Cs+. The pipette solution consisted of (in mM) 125 CsOH, 125 glutamate, 10 CsCl, 10 NaCl, 1 MgCl2, 5 HEPES, 0.05 EGTA, 3 Mg-ATP, 0.1 cAMP, and 0.01 GTP (pH 7.15 using CsOH). cAMP was included in the pipette-filled solution to increase the exocytotic responsiveness of the cells. After establishment of the standard whole cell configuration, the pipette solution was allowed to exchange with the cytosol for 2 min before stimulation commenced. The molecular mass of cPLA2 is 85 kDa. Experiments using dextran-conjugated fura 2 with a molecular mass of 100 kDa (Molecular Probes) suggest that the solution exchange between the pipette and the cell interior is >85% complete 2 min after establishment of whole cell configuration. Recombinant human cPLA2
was obtained from Roger L. Williams, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. AACOCF3 was purchased from Calbiochem (La Jolla, CA). All other chemicals were from Sigma.
Antisense and sense oligonucleotides. To investigate the role of granular ClC-3 Cl- channels for cPLA2-induced priming of secretory granules, we cotransfected cultures of
-cells with 50 µg/ml of the antisense oligonucleotide (5'-TCCATTTGTCATTGT-3') or sense oligonucleotide (5'-ACAATGACAAATGGA-3') (28) and green fluorescent protein (GFP; 1 µg/ml). The cells were transfected using oligofectamine (Invitrogen) and cultured for 24 h before use. The antisense and sense oligonucleotides were synthesized at TAG Copenhagen (Copenhagen, Denmark) and correspond to the identical initiation codon region (-3 to +12) of the human, rat, and mouse ClC-3 mRNA (28). The first three bases at either end in both oligonucleotides were phosphorothioated.
The effects of cPLA2 on Ca2+-dependent exocytosis were investigated using the antisense oligonucleotide (5'-GTGCTGATAAGGATCTAT-3') directed against codons 49 of the rat and mouse cPLA2
(18). A scrambled sequence oligonucleotide (5'-GTGCTCCTAAGTTTCTAT-3') was used for control (TAG Copenhagen, Copenhagen, Denmark). The first two bases at either end in both oligonucleotides were phosphorothioated. The oligonucleotides (20 µg/ml) were cotransfected with GFP (1 µg/ml) by use of oligofactamin (Invitrogen), and cells were cultured for 24 h before used.
Data analysis. Results are presented as means ± SE for the indicated number of experiments. All current amplitudes are given without compensation for leak conductance. Statistical significance was evaluated using Student's t-test (see Figs. 1, 4, 5, 6, and 8) and Dunnett's test for multiple comparisons to a single control (see Figs. 2, 3, 7, and 9). Experiments commenced when two successive depolarizations or trains of pulses applied at a 2-min interval elicited exocytotic responses of the same magnitude to ascertain that the observed changes are not simply attributable to spontaneous longterm changes of the secretory capacity.
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RESULTS |
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The effect of cPLA2 on exocytosis was dependent on dose (Fig. 2). No effect on exocytosis was observed at concentrations ≤50 nM. At higher concentrations, cPLA2
stimulated exocytosis by 280450%. Approximating the average exocytotic responses observed at the different cPLA2
concentrations to the Hill equation yielded values of the association constant and cooperativity factor of 86 nM and 3.7, respectively.
Figure 3 shows that the stimulatory action of cPLA2 on exocytosis was suppressed by AACOCF3 (50 µM of the inhibitor was included in the pipette-filled solution). AACOCF3 is a trifluoromethyl ketone analog of arachidonic acid and a potent inhibitor of cPLA2
(27). AACOCF3 slightly reduced the exocytotic capacity in the absence of cPLA2
without affecting the integrated Ca2+ current (Fig. 3). These data suggest that cPLA2
-induced exocytosis is secondary to increased enzyme activity after infusion into
-cells. cPLA2
has a role in the regulation of Ca2+-dependent exocytosis in
-cells. The data in Fig. 3 show that inhibition of endogenous cPLA2
with AACOCF3 reduced exocytosis. To further investigate the role of endogenous cPLA2
in the stimulation of depolarization-induced exocytosis, we transfected cells with antisense oligonucleotides against cPLA2
. Figure 4A shows that depolarization-induced exocytosis was inhibited by 50% in cells treated with cPLA2
antisense oligonucleotide, whereas no effect was observed on exocytosis in cells treated with the corresponding sense oligonucleotide. Figure 4B summarizes the average effects on depolarization-evoked exocytosis in control cells as well as in cells treated with either sense or antisense oligonucleotides against cPLA2
. It is clear that cPLA2
has an important role in the regulation of Ca2+-dependent exocytosis of the insulin-containing granules.
Effects of arachidonic acid on depolarization-induced exocytosis. In the following experiments, we investigated whether the stimulatory action of cPLA2 on exocytosis is secondary to phospholipid hydrolysis and accumulation of arachidonic acid. Figure 5A illustrates the finding that inclusion of 50 µM arachidonic acid in the pipette solution doubled the depolarization-induced exocytosis. On average (Fig. 5B), arachidonic acid increased exocytosis by 86% (P < 0.05; n = 5, control and n = 7, arachidonic acid). The effect occurred without affecting the whole cell Ca2+ current (Fig. 5C) or the [Ca2+]i transient (Fig. 5D). The applied arachidonic acid concentration was maximal, because in infusion experiments in which the cell interior was dialyzed with a patch pipette-filled solution with a free Ca2+ concentration of 0.2 µM and increasing arachidonic acid concentrations, we found that the half-maximal stimulatory concentration was 6 µM, whereas ≥50 µM of the lipid was required for maximally stimulating exocytosis (data not shown). This fits well with the endogenous levels of arachidonic acid achieved during glucose stimulation of islets (50100 µM) (29, 30).
Next, we repeated the above experiments in -cells pretreated with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10 µM for 15 min), the cyclooxygenase inhibitor indomethacin (10 µM for 15 min), or 17-octadecynoic acid (17-ODCA; 10 µM for 15 min), an inhibitor of the P-450 system. Figure 5, E-G, shows that these inhibitors did not influence the exocytotic capacity in the absence of arachidonic acid. Furthermore, NDGA and 17-ODCA failed to affect the stimulatory action of arachidonic acid (50 µM) on exocytosis (Fig. 5, E and G). On average, arachidonic acid enhanced exocytosis by 76% (NDGA; P < 0.05; Fig. 5E) and 89% compared with control for 17-ODCA-pretreated cells (P < 0.05; Fig. 5G). These values are close to those obtained in the absence of inhibitors (85% stimulation; Fig. 5B). On the contrary, the exocytotic response to arachidonic acid in cells treated with indomethacin was significantly increased and amounted to 77 ± 9 fF (200% stimulation vs. control; P < 0.05; n = 7; Fig. 5F). These data suggest that the stimulatory action of arachidonic acid on exocytosis is not secondary to metabolism of the lipid by the lipoxygenase or P-450 signaling pathways. However, inhibition of the cyclooxygenase pathway by indomethacin increased the exocytotic response, suggesting that cyclooxygenase-produced metabolites of arachidonic acid can antagonize arachidonic acid-induced stimulation of exocytosis.
Lysophosphatidylcholine stimulates Ca2+-dependent exocytosis. Hydrolysis of phospholipids by cPLA2 results not only in the accumulation of arachidonic acid but also in the production of lysophospholipids. Figure 6, A and B, shows that lysophosphatidylcholine (2 µg/ml) doubled the exocytotic response without affecting the whole cell Ca2+ current (Fig. 6, A and C) or the [Ca2+]i transient (Fig. 6, A and D). The stimulatory action of lysophosphatidylcholine on exocytosis was mimicked by lysophosphatidylserine (2 µg/ml), which increased the exocytotic response from 28 ± 4 fF (n = 7) under control conditions to 63 ± 15 fF in the presence of the lysophospholipid (P < 0.05; n = 8; data not shown).
Effects of arachidonic acid and lysophosphatidylcholine on Ca2+-evoked exocytosis. The data in Figs. 5 and 6 suggest that neither arachidonic acid nor lysophosphatidylcholine alone stimulates exocytosis to an extent similar to that observed in the presence of cPLA2. In Fig. 7A we explored the possibility that both products of the enzyme mediate cPLA2
-induced exocytosis. Indeed, in the simultaneous presence of both arachidonic acid (50 µM) and lysophosphatidylcholine (2 µg/ml), the capacitance increase elicited by a single membrane depolarization was 166 ± 45 fF (n = 6), which is not different from that observed in the presence of cPLA2
[172 ± 47 fF (n = 7)] but is significantly higher than the changes in cell capacitance in the presence of either compound alone (Fig. 7B).
One possible mechanism by which cPLA2 stimulates exocytosis is to accelerate granule mobilization from a reserve pool with resultant overfilling of RRP. This should be detected as an increase in amount of exocytosis that can maximally be elicited during intense and repetitive stimulation. Figure 7C shows the average response of several individual trains consisting of twelve 500-ms depolarizations (1-Hz stimulation) applied from -70 mV to 0 mV under control conditions (0.1 mM cAMP was present in the pipettefilling solution) and in cells preloaded with cPLA2
(250 nM), arachidonic acid (50 µM), lysophosphatidylcholine (2 µg/ml), or a combination of the latter two compounds. Under control conditions, the maximum increase in cell capacitance amounted to 239 ± 84 fF (n = 5). As expected, cPLA2
strongly stimulated exocytosis elicited by the train of depolarizations, and the total increase in cell capacitance amounted to 880 ± 110 fF (P < 0.01; n = 7). It is clear from Fig. 7C that the combination of arachidonic acid and lysophosphatidylcholine on exocytosis elicited by a single depolarization (Fig. 7B) is also reflected in the time course and magnitude of the capacitance increase in response to the train of repetitive membrane depolarizations. In the presence of either compound alone, the total capacitance increase was modest and amounted for arachidonic acid to 478 ± 66 fF (P < 0.05; n = 6) and 473 ± 52 fF in the presence of lysophosphatidylcholine (P < 0.05; n = 12). Interestingly, in the simultaneous presence of both arachidonic acid and lysophosphatidylcholine, the response was indistinguishable from that observed in the presence of cPLA2
(Fig. 7C). On average, the total increase in exocytosis in the combined presence of arachidonic acid and lysophosphatidylcholine amounted to 865 ± 89 fF (P < 0.05; n = 7). Finally, the exocytotic response was dramatically reduced in cells treated with antisense oligonucleotides against cPLA2
(Fig. 7C). Under these experimental conditions, the average increase in cell capacitance amounted to 98 ± 21 fF (P < 0.01; n = 5). On the contrary, the average capacitance increase in sensetreated cells was not different from control (222 ± 45 fF; n = 5; data not shown).
Effects of granular ClC-3 Cl- channels on cPLA2-induced exocytosis. ClC-3 Cl- channels sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) have been proposed to be present on
-cell secretory granules and to participate in sulfonylurea- and Ca2+-dependent modulation of exocytosis (1). Intracellular application of this Cl- channel blocker DIDS (0.1 mM) abolished the ability of cPLA2
(250 nM) to stimulate exocytosis evoked by single 500-ms depolarizations from -70 mV to 0 mV (Fig. 8, A and B) without affecting the whole cell Ca2+ current (Fig. 8C). DIDS itself reduced the depolarization-evoked capacitance increase by 25% (P < 0.05; n = 11; Fig. 8B).
We next confirmed that the stimulatory action of cPLA2 on exocytosis involves granular ClC-3 Cl- channels. Fig. 9A shows that exocytosis was reduced by 40% in cells treated with antisense oligonucleotides against ClC-3 Cl- channels. Furthermore, the stimulatory action of cPLA2
(250 nM) on exocytosis was completely abolished in cells treated with antisense oligonucleotides (Fig. 9B). By contrast, in cells treated with the corresponding sense oligonucleotide against ClC-3 Cl- channels, neither depolarization-evoked (Fig. 9C) nor cPLA2
-stimulated exocytosis was affected (Fig. 9D). Figure 9E summarizes the average effects of cPLA2
on exocytosis in control cells as well as in cells treated with either sense or antisense oligonucleotides. It is clear that cPLA2
produced a 440% increase in Ca2+-induced exocytosis in cells treated with sense oligonucleotide (P < 0.05; n = 7), which is not different from that observed in nontreated cells (450% stimulation; P < 0.05; n = 6). On the contrary, pretreatment with antisense oligonucleotide reduced basal secretion by 40% (P < 0.05; n = 7) and abolished the ability of cPLA2
to stimulate exocytosis. No effect was observed on the integrated Ca2+ current (Fig. 9F).
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DISCUSSION |
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Arachidonate represents >30% of the total esterified fatty acyl mass in phospholipids of rodent and human islets (22, 23). The abundance of arachidonate-containing phospholipid species is higher in islets than in many other tissues, and islet plasma membranes and secretory granule membranes are especially enriched in such species (23, 25). The production of arachidonic acid in -cells leads not only to stimulation of exocytosis but also to inhibition of ATP-sensitive K+ (KATP) channel activity (5), with resulting membrane depolarization and stimulation of Ca2+ influx through the voltage-dependent Ca2+ channels. Arachidonic acid has also been reported to mobilize intracellular Ca2+ stores (2, 16, 30). In the present study we were unable to detect an effect of arachidonic acid on [Ca2+]i. This we attribute to the fact that the cells were voltage clamped, since we have clearly observed that application of arachidonic acid to nonclamped
-cells results in a robust increase in [Ca2+]i, an action we believe is secondary to closure of KATP channels (Juhl K, Efanov AM, and Gromada J, unpublished data).
During glucose stimulation, the intracellular concentration of free arachidonic acid can achieve levels of 100 µM (29, 30), well above the level required for maximally stimulating exocytosis. Our data suggest that the enhanced exocytotic response is most likely mediated by arachidonic acid rather than its metabolites. This is supported by the finding that the increase in exocytosis was unaffected by inhibitors of the lipoxygenase and P-450 signaling pathways and even increased after inhibition of the cyclooxygenase pathway. The latter observation is consistent with previous reports that inhibition of the cyclooxygenase pathway with indomethacin increased insulin secretion from rat islets (9, 17). This suggests that a metabolite of the cyclooxygenase pathway might inhibit glucose-induced insulin secretion by interaction with the exocytotic process itself. The finding that the stimulatory action of arachidonic acid on exocytosis is most likely mediated by arachidonic acid per se rather than by one of its metabolites is supported by the previous observations that a substantial fraction of the arachidonic acid produced after cPLA2 activation is not metabolized in islets but is instead reacylated into phospholipids (12, 15).
Previous data have led to the proposal that firstphase insulin secretion reflects the release of granules belonging to the RRP (6). In this scenario, secondphase insulin secretion is envisaged to result from the ATP-, Ca2+-, and time-dependent mobilization (priming) of granules from a reserve pool. Our data indicate that cPLA2 stimulates exocytosis by accelerating granule mobilization and "overfilling" of the RRP so that more granules are available for release once [Ca2+]i rises to exocytotic levels. Moreover, our data suggest that cPLA2
has an important role for regulation of Ca2+-dependent exocytosis, because the rate of granule mobilization was strongly reduced in cells treated with antisense oligonucleotides against cPLA2
.
It has previously been reported that granular Cl- uptake and acidification are important for priming of insulin-containing granules in mouse -cells (1). Influx of Cl- through granular ClC-3 Cl- channels stimulates the priming of the secretory granules by facilitating H+ pumping into the granules by a V-type H+-ATPase. This Cl- flux prevents the development of a large electrical gradient over the granule membrane that would otherwise prevent H+ pumping and acidification (1). Evidence for a role for cPLA2
-induced stimulation of granular Cl- flux in the mobilization of secretory granules in
-cells is provided by the observations that inclusion of the Cl- channel inhibitor DIDS in the pipette solution and pretreatment of
-cells with ClC-3 Cl- channel antisense oligonucleotides antagonized the stimulatory action of cPLA2
on exocytosis and even reduced Ca2+-dependent exocytosis by 40%. We believe that our findings are of general significance, since it has previously been reported that arachidonic acid and lysophosphatidylcholine stimulate Cl- transport in exocrine pancreatic secretory granules (8), which clearly implies an important function of granular Cl- channels for cPLA2
- and Ca2+-dependent exocytosis in pancreatic
-cells.
The physiological significance of our results is based on the use of the granule phospholipid bilayer as a substrate by cPLA2. Activation of cPLA2
during the
-cell stimulus-secretion coupling would cause translocation of the enzyme to the secretory granules and accumulation of arachidonic acid and lysophospholipids in the membrane. The mechanism by which these products enhance granule Cl- transport is unknown but might involve either a direct effect on the ClC-3 Cl- channels or mediation through bulk changes in membrane structure or fluidity. Previous data suggest that the lipid bilayer fluidity of exocrine secretory granules is variable and that the rate of Cl- transport across these granule membranes is directly correlated to its fluidity (7). Stimulation of ClC-3 Cl- channel activity is required for priming and results in an increase in the number of readily releasable granules. The proposed model allows those granules that do not undergo exocytosis to be recycled back to the reserve pool of granules after termination of the stimulus signal. This could be accomplished by reacylation of the cPLA2
products, which would in turn restore the original membrane composition.
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ACKNOWLEDGMENTS |
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M. Høy holds a scholarship from the Academy of Technical Sciences and Novo Nordisk A/S. H. L. Olsen is supported by a scholarship from Medicon Valley Academy and Novo Nordisk A/S.
Present address of K. Bokvist, A. M. Efanov, and J. Gromada: Lilly Research Laboratories, Essener Strasse 93, D-22419 Hamburg, Germany.
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FOOTNOTES |
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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.
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REFERENCES |
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