cPLA2{alpha}-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic {beta}-cells

Kirstine Juhl,1 Marianne Høy,1 Hervør L. Olsen,1 Krister Bokvist,1 Alexander M. Efanov,1 Else K. Hoffmann,2 and Jesper Gromada1

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2{alpha}) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic {beta}-cells. cPLA2{alpha} dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70–80 to 280–300. cPLA2{alpha}-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2{alpha}. The action of cPLA2{alpha} was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2{alpha} was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2{alpha} has an important role in controlling the rate of exocytosis in {beta}-cells. This effect of cPLA2{alpha} reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.

arachidonic acid; ClC-3 chloride channels; insulin exocytosis; lysophospholipids; phospholipase A2


PHOSPHOLIPASE A2 (PLA2) is a family of lipolytic enzymes that hydrolyze the sn-2 position of glycerophospholipids, leading to generation of nonesterified arachidonic acid and lysophospholipids. Pancreatic islets express low-molecular-weight secretory PLA2 associated with insulin-secretory granules (3, 15, 25) and the cytosolic, Ca2+-dependent enzyme cPLA2{alpha} with a molecular mass of 85 kDa (3, 13, 14, 20). cPLA2{alpha} shows strict substrate specificity for arachidonate-containing phospholipids and is activated in {beta}-cells by physiological increases in free cytosolic Ca2+ concentrations ([Ca2+]i) (11) promoting its translocation from the cytosol to membrane compartments (4). This enzyme is responsible for the component of glucose-induced arachidonic acid accumulation that is dependent on Ca2+ entry from the extracellular space (12) for sustained insulin secretion and maintenance of insulin stores (21). Finally, an 84-kDa cytosolic, Ca2+-independent PLA2, iPLA2, is also expressed in pancreatic islets (14). The iPLA2 has been proposed to be involved in early signaling events in glucose-stimulated islets (14, 24) and in the insulinotropic action of cholecystokinin-8 (26).

Arachidonic acid is believed to be an important signaling component in insulin secretion from pancreatic {beta}-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 {beta}-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{alpha} and its products arachidonic acid and lysophospholipids modulate exocytosis in single mouse pancreatic {beta}-cells. We demonstrate that intracellular application of cPLA2{alpha} 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{alpha} activity and resulted from the combined actions of arachidonic acid and lysophospholipids.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of mouse {beta}-cells. Pancreatic {beta}-cells isolated from female NMRI mice (18–25 g; Bomholtgård, Ry, Denmark) were used throughout this study. The mice were killed by cervical dislocation, and the pancreata were quickly removed. The local ethical committee in Copenhagen approved the methods of euthanasia. The pancreatic islets were isolated by collagenase (3 mg/ml, type XI from Sigma) digestion, and the islets were dispersed into single cells by shaking in a Ca2+-free solution. The resulting cell suspension was plated on glass coverslips in Nunc Petri dishes and maintained for ≤4 days in RPMI 1640 tissue-culture medium (GIBCO, Life Technologies, Paisley, UK) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin.

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 3–4 M{Omega}. 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 {beta}-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 1.5–2 ml/min to maintain the temperature at {approx}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{alpha} 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{alpha} 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{alpha}-induced priming of secretory granules, we cotransfected cultures of {beta}-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{alpha} on Ca2+-dependent exocytosis were investigated using the antisense oligonucleotide (5'-GTGCTGATAAGGATCTAT-3') directed against codons 4–9 of the rat and mouse cPLA2{alpha} (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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Fig. 1. Cytosolic phospholipase A2{alpha} (cPLA2{alpha}) stimulates depolarization-dependent exocytosis in mouse pancreatic {beta}-cells. A: whole cell Ca2+ currents (ICa; middle), cell capacitance ({Delta}Cm; lower middle), and cytoplasmic Ca2+ levels ([Ca2+]i; bottom) recorded in response to 500-ms depolarization from -70 mV to 0 mV as indicated (Vm; top)in a control cell (left) and in a different cell dialyzed with 250 nM cPLA2{alpha} (right). Experiments were performed using the standard whole cell patch configuration, and 0.1 mM cAMP was included in the pipette-filled solution in this and all subsequent experiments. cPLA2{alpha} was allowed to diffuse into the cells for 2 min before initiation of the experiment. Dotted lines, zero current level and prestimulatory capacitance level, respectively. Note that [Ca2+]i transients are depicted on a different time scale. Histograms summarize increases in cell capacitance ({Delta}Cm; B), integrated Ca2+ current (QCa; C), and cytoplasmic Ca2+ levels ({Delta}[Ca2+]i; D) elicited by 500-ms voltage-clamp depolarization in the absence (-) and presence (+) of 250 nM cPLA2{alpha} in the pipette-filled solution. Values are means ± SE of 9 (control) and 7 (cPLA2{alpha}) different cells. **P < 0.01.

 


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Fig. 4. cPLA2{alpha} controls Ca2+-dependent exocytosis in mouse {beta}-cells. A: changes in cell capacitance ({Delta}Cm) and whole cell Ca2+ currents (ICa) elicited by 500-ms voltage-clamp depolarizations from -70 mV to 0 mV (Vm) with standard whole cell configuration. {beta}-Cells had been treated for 24 h with 20 µM antisense oligonucleotides against cPLA2{alpha} (antisense cPLA2{alpha}; right) or the corresponding sense oligonucleotide (sense cPLA2{alpha}; left). Dotted lines, zero-current level and prestimulatory capacitance level, respectively. Histograms show average changes in cell capacitance ({Delta}Cm; B) and integrated Ca2+ current (QCa; C) in the absence (-) and presence (+) of either antisense or sense oligonucleotides against cPLA2{alpha}. Values are means ± SE of 7 (antisense) and 5 (sense) cells. *P < 0.05.

 


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Fig. 5. Arachidonic acid (AA) stimulates exocytosis in single mouse {beta}-cells. A: whole cell Ca2+ currents (ICa), cell capacitance ({Delta}Cm), and cytoplasmic Ca2+ levels recorded in response to 500-ms depolarizations from -70 mV to 0 mV as indicated (Vm) in a control cell (left) and in a different cell dialyzed with 50 µM AA (right) using the standard whole cell configuration. AA was allowed to diffuse into cells for 2 min before initiation of experiments. Dotted lines, zerocurrent level and prestimulatory capacitance level, respectively. Note that [Ca2+]i transients are depicted on a different time scale. Histograms summarize increases in cell capacitance ({Delta}Cm; B), integrated Ca2+ current (QCa; C), and cytoplasmic Ca2+ levels ({Delta}[Ca2+]i; D) elicited by 500-ms voltage-clamp depolarization in the absence (-) and presence (+) of 50 µM AA in pipette-filled solution. Values are means ± SE of 5 (control) and 7 (AA) experiments. E-G: histogram depicts average increases in cell capacitance in the absence (-) and presence of 50 µM AA (+) elicited by a 500-ms voltage-clamp depolarization from -70 mV to 0 mV (Vm) in single {beta}-cells. Cells were pretreated with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 10 µM for 15 min, E), the cyclooxygenase inhibitor indomethacin (Indo; 10 µM for 15 min, F), or an inhibitor of the P-450 system, 17-octadecynoic acid (17-ODCA; 10 µM for 15 min, G). Values are means ± SE of 5–23 different cells. *P < 0.05, **P < 0.01.

 


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Fig. 6. Lysophosphatidylcholine stimulates exocytosis in single mouse {beta}-cells. A: whole cell Ca2+ currents (ICa), cytoplasmic Ca2+ levels ([Ca2+]i), and cell capacitance ({Delta}Cm) evoked by a 500-ms depolarization from -70 mV to 0 mV as indicated (Vm) in a control cell (left) and in a different cell dialyzed with 2 µg/ml lysophosphatidylcholine (LysoPC; right). Experiments were performed with standard whole cell patch configuration. Dotted lines, zero-current level and prestimulatory capacitance level, respectively. Histograms show average changes in cell capacitance ({Delta}Cm; B), integrated Ca2+ current (QCa; C), and cytoplasmic Ca2+ levels ({Delta}[Ca2+]i; D) in the absence (-) and presence of 2 µg/ml lysoPC (+). Values are means ± SE of 5 cells. *P < 0.05.

 


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Fig. 8. The Cl- channel blocker DIDS inhibits Ca2+- and cPLA2{alpha}-stimulated exocytosis in single mouse {beta}-cells. A: whole cell Ca2+ currents (ICa) and cell capacitance ({Delta}Cm) evoked by a 500-ms depolarization from -70 mV to 0 mV, as indicated (Vm) in a control cell (left) and in different cells dialyzed with 100 µM DIDS (middle) or a combination of 100 µM DIDS and 250 nM cPLA2{alpha} (right). Experiments were performed using the standard whole cell patch configuration. Dotted lines, zero-current level and prestimulatory capacitance level, respectively. Histograms show average changes in cell capacitance ({Delta}Cm; B) and integrated Ca2+ current (QCa; C) in the absence and presence of 100 µM DIDS and 250 nM cPLA2{alpha}. Values are means ± SE of 5–11 cells. *P < 0.05.

 


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Fig. 2. Concentration dependence of stimulatory action of cPLA2{alpha} on exocytosis. Increases in exocytosis were measured in response to 500-ms membrane depolarizations from a holding potential of -70 mV to 0 mV by use of the standard whole cell configuration. Different concentrations of cPLA2{alpha} were allowed to diffuse into the cells for 2 min before initiation of experiment. Curve represents a least squares fit of mean data points to the Hill equation. Data are means ± SE of 5–9 experiments. *P < 0.05; **P < 0.01.

 


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Fig. 3. AACOCF3 inhibits cPLA2{alpha}-induced exocytosis in mouse {beta}-cells. Effects of the specific cPLA2{alpha} inhibitor AACOCF3 on cell capacitance ({Delta}Cm) and whole cell Ca2+ currents (ICa) elicited by 500-ms voltage-clamp depolarizations from -70 mV to 0 mV (Vm) by use of the standard whole cell configuration in single mouse {beta}-cells. Changes in cell capacitance were measured under control conditions (A), after inclusion of either 50 µM AACOCF3 (B) or 250 nM cPLA2{alpha} (C) in the pipette-filled solution, or in the simultaneous presence of AACOCF3 and cPLA2{alpha} (D). Histograms show average changes in cell capacitance ({Delta}Cm; E) and integrated Ca2+ current (QCa; F) in the absence (-) and presence (+) of either AACOCF3 or cPLA2{alpha}. Values are means ± SE of 5–9 cells. *P < 0.05.

 


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Fig. 7. Combined actions of AA and lysoPC on exocytosis in mouse {beta}-cells. A: cell capacitance ({Delta}Cm) recorded in response to 500-ms depolarizations from -70 mV to 0 mV, as indicated (Vm) in cells dialyzed with either 50 µM AA, 2 µg/ml LysoPC, or a combination of the 2 reagents. Experiments were performed using the standard whole cell patch configuration. Dotted lines, prestimulatory capacitance level. B: histogram shows average increases in cell capacitance ({Delta}Cm) in the absence (control) and presence of 50 µM AA, 2 mg/ml LysoPC, or the combination of AA and LysoPC. To facilitate comparison, increases in cell capacitance recorded in the presence of 250 nM cPLA2{alpha} have been included in histogram. C: trains of twelve 500-ms voltage-clamp depolarizations from -70 mV to 0 mV were applied at a frequency of 1 Hz (Vm). Experiments were performed under control conditions and in the presence of 50 µM AA, 2 µg/ml LysoPC, the combination of AA and LysoPC, 250 nM cPLA2{alpha}, and in cells treated with 20 µg/ml antisense oligonucleotides against cPLA2{alpha} (antisense cPLA2{alpha}). Trains of depolarizations were applied 3 min after establishment of the whole cell configuration and represent the mean increase in cell capacitance of 5–8 different experiments for each experimental condition. Data are representative of 5 separate experiments in both A and B.

 


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Fig. 9. ClC-3 Cl- channels control Ca2+- and cPLA2{alpha}-induced exocytosis in single mouse {beta}-cells. Whole cell Ca2+ currents (ICa) and cell capacitance ({Delta}Cm) evoked by a 500-ms depolarization from -70 mV to 0 mV as indicated (Vm) in {beta}-cells treated for 24 h with 50 µg/ml antisense oligonucleotide against ClC-3 Cl- channels in the absence (A) and presence of 250 nM cPLA2{alpha} (B) or the corresponding sense oligonucleotide in the absence (C) and presence of 250 nM cPLA2{alpha} (D). Experiments were performed using the standard whole cell patch configuration. Dotted lines, zero-current level and prestimulatory capacitance level, respectively. Note: small dotted line above capacitance trace (A) represents increase in nontreated cells. Histograms show average changes in cell capacitance ({Delta}Cm; E) and integrated Ca2+ current (QCa; F) in the absence and presence of 250 nM cPLA2{alpha} and sense or antisense oligonucleotides against ClC-3 Cl- channels. Values are means ± SE of 5–8 cells. *P < 0.05.

 


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of cPLA2{alpha} on Ca2+ currents, cytoplasmic Ca2+ levels, and exocytosis in mouse {beta}-cells. Figure 1 shows simultaneous recordings of whole cell Ca2+ currents (ICa), changes in cytoplasmic Ca2+ levels ({Delta}[Ca2+]i), and associated increases in cell capacitance ({Delta}Cm) evoked by 500-ms depolarizations ranging from -70 mV to 0 mV in single mouse {beta}-cells. Under control conditions (Fig. 1A, left), the membrane depolarization elicited an integrated Ca2+ current of 14 pC, increased [Ca2+]i to 0.65 µM, and evoked a capacitance increase of 31 fF, corresponding to the release of 10 secretory granules, because each granule contributes ~3 fF of capacitance upon exocytosis (19). Figure 1A, right, shows that, in a single {beta}-cell preloaded for 2 min with 250 nM recombinant human cPLA2{alpha} through the recording pipette, the same depolarization elicited a Ca2+ current of 13 pC but nevertheless evoked a much larger exocytotic response of 149 fF ({approx}5-fold stimulation). The peak amplitude of the [Ca2+]i transient was not different from control and amounted to 0.70 µM. On average, cPLA2{alpha} (250 nM) increased the exocytotic response by 450% (Fig. 1B; P < 0.01; n = 9, control and n = 7, cPLA2{alpha}). The latter effect was not accompanied by a change of the integrated Ca2+ current (Fig. 1C; QCa) or the increase in [Ca2+]i ({Delta}[Ca2+]i; Fig. 1D).

The effect of cPLA2{alpha} on exocytosis was dependent on dose (Fig. 2). No effect on exocytosis was observed at concentrations ≤50 nM. At higher concentrations, cPLA2{alpha} stimulated exocytosis by 280–450%. Approximating the average exocytotic responses observed at the different cPLA2{alpha} 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{alpha} 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{alpha} (27). AACOCF3 slightly reduced the exocytotic capacity in the absence of cPLA2{alpha} without affecting the integrated Ca2+ current (Fig. 3). These data suggest that cPLA2{alpha}-induced exocytosis is secondary to increased enzyme activity after infusion into {beta}-cells. cPLA2{alpha} has a role in the regulation of Ca2+-dependent exocytosis in {beta}-cells. The data in Fig. 3 show that inhibition of endogenous cPLA2{alpha} with AACOCF3 reduced exocytosis. To further investigate the role of endogenous cPLA2{alpha} in the stimulation of depolarization-induced exocytosis, we transfected cells with antisense oligonucleotides against cPLA2{alpha}. Figure 4A shows that depolarization-induced exocytosis was inhibited by 50% in cells treated with cPLA2{alpha} 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{alpha}. It is clear that cPLA2{alpha} 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{alpha} 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 (50–100 µM) (29, 30).

Next, we repeated the above experiments in {beta}-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{alpha} 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{alpha}. In Fig. 7A we explored the possibility that both products of the enzyme mediate cPLA2{alpha}-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{alpha} [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{alpha} 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{alpha} (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{alpha} 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{alpha} (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{alpha} (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{alpha}-induced exocytosis. ClC-3 Cl- channels sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) have been proposed to be present on {beta}-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{alpha} (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{alpha} 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{alpha} (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{alpha}-stimulated exocytosis was affected (Fig. 9D). Figure 9E summarizes the average effects of cPLA2{alpha} on exocytosis in control cells as well as in cells treated with either sense or antisense oligonucleotides. It is clear that cPLA2{alpha} 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{alpha} to stimulate exocytosis. No effect was observed on the integrated Ca2+ current (Fig. 9F).


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although it has been demonstrated that cPLA2{alpha} is expressed in pancreatic {beta}-cells and affects insulin secretion, surprisingly little is known about the cellular mechanisms involved. It has been proposed that the cPLA2{alpha}-evoked stimulation of insulin secretion is mediated by arachidonic acid synthesis and involves interaction with both proximal and distal steps in the {beta}-cell stimulus-secretion coupling. In this study we have investigated the role of cPLA2{alpha} in the control of the exocytotic process in mouse {beta}-cells by use of capacitance measurements. We demonstrate that the secretory response is reduced in the presence of the cPLA2 inhibitor AACOCF3 and in cells pretreated with cPLA2{alpha} antisense oligonucleotides, clearly indicating the importance of this enzyme for the regulation of Ca2+-dependent exocytosis. The ability of cPLA2{alpha} to stimulate exocytosis was not associated with changes in Ca2+ channel activity or cytoplasmic Ca2+ levels but resulted from a direct interference with the priming of secretory granules for release, an effect mimicked by a combination of arachidonic acid and lysophosphatidylcholine.

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 {beta}-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 {beta}-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{alpha} 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{alpha} 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{alpha} 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{alpha}.

It has previously been reported that granular Cl- uptake and acidification are important for priming of insulin-containing granules in mouse {beta}-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{alpha}-induced stimulation of granular Cl- flux in the mobilization of secretory granules in {beta}-cells is provided by the observations that inclusion of the Cl- channel inhibitor DIDS in the pipette solution and pretreatment of {beta}-cells with ClC-3 Cl- channel antisense oligonucleotides antagonized the stimulatory action of cPLA2{alpha} 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{alpha}- and Ca2+-dependent exocytosis in pancreatic {beta}-cells.

The physiological significance of our results is based on the use of the granule phospholipid bilayer as a substrate by cPLA2{alpha}. Activation of cPLA2{alpha} during the {beta}-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{alpha} products, which would in turn restore the original membrane composition.


    ACKNOWLEDGMENTS
 
We thank Prof. Roger L. Williams (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for kindly providing human cPLA2{alpha}.

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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Gromada, Lilly Research Laboratories, Essener Strasse 93, D-22419 Hamburg, Germany (E-mail: gromada_jesper{at}lilly.com)

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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