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J. Biol. Chem., Vol. 278, Issue 37, 35168-35171, September 12, 2003

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Involvement of Protein Kinase C- in Inositol Hexakisphosphate-induced Exocytosis in Mouse Pancreatic -Cells^*

Marianne Høy  , Per-Olof Berggren ¶ and Jesper Gromada  ||

From the Laboratory of Islet Cell Physiology, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark and ^¶The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, S-171 76 Stockholm, Sweden

Received for publication, April 15, 2003 , and in revised form, June 26, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Inositolhexakisphosphate (InsP₆) plays a pivotal role in the pancreatic -cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP₆ on Ca²⁺-dependent exocytosis in single mouse pancreatic -cells. In the presence of inhibitors of the protein phosphatase calcineurin to block endocytosis, intracellular application of InsP₆ produced a dose-dependent stimulation of exocytosis, and half-maximal effect was observed at 22 µM. The stimulatory effect of InsP₆ was dependent on protein kinase C (PKC) activity. Antisense oligonucleotides directed against specific PKC isoforms (, II, , , ) revealed the involvement of PKC- in InsP₆-induced exocytosis. Furthermore, expression of dominant negative PKC- abolished InsP₆-evoked exocytosis, whereas expression of wild-type PKC- led to a significant stimulation of InsP₆-induced exocytosis. These data demonstrate that PKC- is involved in InsP₆-induced exocytosis in pancreatic -cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Inositol hexakisphosphate (InsP₆)¹ levels transiently increase in pancreatic -cells following an elevation of the ambient glucose concentration (1). This elevation in InsP₆ levels plays an important role in controlling exocytosis of the insulin-containing granules and subsequent retrieval of membrane by endocytosis (2, 3). These processes of exocytosis and endocytosis are dependent on protein kinase C (PKC) activity (2, 3). Several PKC isoforms are expressed in pancreatic -cells and include PKC-, -, -, -, and - (4, 5). However, the importance of a particular PKC isoform in controlling InsP₆-evoked exocytosis is unknown. The present study was designed to examine which PKC isoform mediates the stimulatory action of InsP₆ on Ca²⁺-induced exocytosis. Using whole-cell patch clamp techniques and capacitance measurements of exocytosis, we demonstrate that PKC- regulates InsP₆-induced exocytosis in single mouse pancreatic -cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Preparation of Islet Cells—Pancreatic islets were isolated from fed female NMRI mice (18–23 g) by collagenase digestion as described previously (3). The local ethical committee in Copenhagen approved the methods of euthanasia. The islets were dispersed into single cells by shaking in a Ca²⁺-free solution, and the resulting cell suspension was plated on Nunc Petri dishes and maintained for up to 3 days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 international units/ml penicillin, and 100 µg/ml streptomycin.

Electrophysiology—Exocytosis was measured as increases in cell membrane capacitance using an EPC-9 patch clamp amplifier and the Pulse software (v. 8.31; HEKA Elektronik, Lamprecht/Pfalz, Germany) as described previously (3). The interval between two successive points was 0.2 s, and the measurements of cell capacitance were initiated <5 s following establishment of the standard whole-cell configuration. The volume of the recording chamber was 0.4 ml, and the solution entering the bath (1.5 ml/min) was maintained at 33 °C. Exocytosis was elicited by infusion of an electrode solution consisting of 125 mM potassium glutamate, 10 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 5 mM HEPES, 3 mM Mg-ATP, 10 mM EGTA, and 0.01, 5, 8, and 10 mM CaCl₂ (pH 7.15 with KOH). The free Ca²⁺ concentrations ([Ca²⁺]_f) of the resulting buffers were 0.03, 0.22, 0.87, and 2.0 µM, using the binding constants described in Ref. 6. The extracellular solution was composed of 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 5 mM HEPES (pH 7.4 with NaOH), and 5 mM D-glucose. Ins(1,4,5)P₃, deltamethrin, and permethrin were from Alomone Laboratories (Jerusalem, Israel), and (R_p)-cAMP was obtained from Biolog Life Science Institute (Bremen, Germany). Ins(1,3,4,5)P₄, InsP₆, and bisindolylmaleimide were from Calbiochem. Ins(1,4,5,6)P₄ was purchased from Alesis (San Diego, CA). All other chemicals were obtained from Sigma.

Oligonucleotides—To investigate the role of PKC isoforms in InsP₆-evoked exocytosis, the following antisense and sense oligonucleotides were used: PKC- antisense, 5'-CAGCCATGGTTCCCCCAAC-3' (7); PKC-II antisense, 5'-GTTGGAGGTGTCTCT-3' (8); PKC- antisense, 5'-TCCAGGTCAACGCGGCATTC-3' (7); PKC- antisense, 5'-GTCCATGCGATCTTGCGCCC-3' (7); PKC- scrambled, 5'-GCCAGCTCCGATCTTGCGCCC-3' (7); PKC- antisense, 5'-GGCCACACATGTCTCGCACT-3' (9). Cultures of -cells were co-transfected with green fluorescent protein (1 µg/ml) and 5 µM PKC oligonucleotides using Oligofectamine (Invitrogen) and incubated in RPMI 1640 medium supplemented as described above for 48 h before use. The antisense and sense oligonucleotides were synthesized at TAG Copenhagen (Copenhagen, Denmark). The oligonucleotides were phosphothioated at the underlined positions.

Plasmid Construction—The cDNAs for PKC- and PKC-I were kindly provided by Y. Nishizuka (Kobe University, Kobe, Japan). A dominant negative mutant of PKC- was obtained from K. Ridge (Northwestern University, Chicago, IL). Transfection of mouse -cells was performed using 3 µg of PKC cDNA and 1 µg of green fluorescent protein using LipofectAMINE according to the manufacturer's instructions. Following transfection, cells were cultured for 48 h in RPMI 1640 supplemented as described above.

Data Analysis—Results are presented as mean values ± S.E. for the indicated number of experiments. The exocytotic rate (C_m/t) is presented as the increase in cell capacitance measured 30–90 s following establishment of the whole-cell configuration. Statistical significance was evaluated using Student's t test for paired observations or Dunnett's test for multiple comparisons.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Fig. 1A shows the exocytotic responses in single mouse pancreatic -cells obtained using the standard whole-cell configuration in which the pipette-filling electrode solution replaces the cytosol. Exocytosis was stimulated by intracellular dialysis of an electrode solution with a Ca²⁺-EGTA buffer with a free Ca²⁺ concentration of 0.87 µM. Under control conditions, the rate of capacitance increase averaged 21 ± 4 femtofarads/s (n = 5). This corresponds to the release of seven granules/s as every granule contributes 3 femtofarads (10). Inclusion of 100 µM InsP₆ in the pipette-filling solution produced a delayed (23 ± 4 s; n = 5) decrease in cell capacitance, an effect that we attribute to stimulation of endocytosis (3). On average, the increase in cell capacitance was reduced by 62% (p < 0.05; n = 5) when measured after 30–90 s. We have previously reported that InsP₆-induced endocytosis is mediated by the protein phosphatase calcineurin (3). Interestingly, in cells pretreated for 15 min with 1.5 µM of the calcineurin inhibitor cyclosporin A, rather different results were obtained (Fig. 1A). Under these conditions, InsP₆ (100 µM) increased the exocytotic response by 86% (p < 0.01; n = 6). InsP₆ also stimulated exocytosis in the presence of the calcineurin inhibitor deltamethrin (20 nM for 1.5 h) (96% stimulation; p < 0.01; n = 5), an effect that was not mimicked by permethrin, an inactive analogue of deltamethrin (Fig. 1B). In the presence of permethrin, InsP₆-induced exocytosis was inhibited by 65% (p < 0.05; n = 5), which is not different from that observed under control conditions (Fig. 1B). Finally, okadaic acid, an inhibitor of protein phosphatases 1, 2A, and 3, did not affect the InsP₆-induced reduction of exocytosis (Fig. 1B). These data suggest that InsP₆ stimulates both exocytosis and endocytosis in pancreatic -cells and that the endocytotic process dominates under the actual experimental conditions as suggested by the overall decrease in the rate of capacitance increase. In this respect, it is important to emphasize that capacitance measurements reflect net changes in plasma membrane area resulting from the summed activity of all endocytotic and exocytotic processes. The stimulatory action of InsP₆ on exocytosis was revealed following inhibition of endocytosis.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Effect of InsP6 on Ca2+-dependent exocytosis in single mouse -cells. A, representative traces showing increases in cell capacitance elicited by intracellular infusion with a Ca2+-EGTA buffer with a free Ca2+-concentration of 0.87 µM in the absence (Control) or presence of 100 µM InsP6 in the pipette solution during the first 2 min after establishment of the standard whole-cell configuration. The effect of InsP6 was also recorded in -cells pretreated for 15 min with 1.5 µM of the calcineurin inhibitor cyclosporin A (Cyclo). Throughout the recordings, the cells were clamped at –70 mV to avoid activation of the voltage-dependent Ca2+ channels that would otherwise interfere with the measurements. fF, femtofarads. B, histogram depicting the average rates of increase in cell capacitance (Cm/t) measured 30–90 s after establishment of the whole-cell configuration in the absence (–) and presence (+) of 100 µM InsP6 in non-treated cells and following pretreatment of cells with the calcineurin inhibitors cyclosporin A (1.5 µM for >15 min) and deltamethrin (20 nM for >30 min), permethrin, an inactive analogue of deltamethrin (20 nM for >30 min), or okadaic acid (100 nM for >10 min), an inhibitor of the type 1, 2A, and 3 phosphatases. All inhibitors were included in the pipette-filling solution in the same concentrations as indicated above. C, dose-dependent stimulation of exocytosis by InsP6. In this and all following experiments, cells were pretreated with cyclosporin A (1.5 µM for >15 min) to block InsP6-induced endocytosis. The rate of capacitance increase (Cm/t) was measured from 30 to 90 s after establishment of the whole-cell configuration. The line is the best fit of the average data to the Hill equation. D, Ca2+ dependence of stimulatory action of 100 µM InsP6 on exocytosis. Pipette-filling solutions with 0.03, 0.22, 0.87, and 2.0 µM [Ca2+]f were used to elicit exocytosis. E, effects of indicated inositol polyphosphates (all 100 µM) on exocytosis rate measured from 30 to 90 s. The data are mean values ± S.E. of 5–10 different cells. *, p < 0.05; **, p < 0.01. Ins(1,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; Ins(3,4,5,6)P4, inositol 3,4,5,6-tetrakisphosphate.

The stimulatory action of InsP₆ on exocytosis was dependent on dose. No significant stimulation of exocytosis was observed at <10 µM InsP₆, but higher concentrations elicited a dose-dependent acceleration of secretion (Fig. 1C). Approximating the average data points to the Hill equation yielded a half-maximal stimulatory effect at 22 µM and a cooperativity factor of 1.4. Maximal stimulation of exocytosis was seen at concentrations of InsP₆ 100 µM (Fig. 1C), at which exocytosis was nearly doubled over the control rate of capacitance increase.

The ability of InsP₆ to stimulate exocytosis depends on the [Ca²⁺]_f in the pipette-filling solution (Fig. 1D). No stimulation was observed at 30 nM [Ca²⁺]_f, but increasing concentrations of Ca²⁺ elicited a progressive increase in the rate of exocytosis, which was further accelerated in the presence of InsP₆. The lack of effect of InsP₆ at low [Ca²⁺]_f contrasts previous findings in HIT insulinoma cells (2). This difference in Ca²⁺ sensitivity of the stimulatory action of InsP₆ on exocytosis most likely reflects the use of a clonal cell line versus primary -cells but could also result from different experimental approaches (permeabilized cells and single cell capacitance measurements).

Fig. 1E shows that the stimulatory action of InsP₆ on exocytosis was also evoked, although to a lesser extent, by inositol tetrakisphosphate compounds and the inositol pentakisphosphate, Ins(1,3,4,5,6)P₅. These inositol polyphosphates stimulated exocytosis by only 48–66%, as compared with 86% increase in the presence of InsP₆ (Fig. 1D). It is noteworthy that the required stimulatory concentrations of InsP₄, but not InsP₆, are 50–100 times higher than those measured in insulin-secreting cells (11). No stimulation of exocytosis was observed in the presence of 100 µM Ins(1,3,4)P₃ and Ins(1,4,5)P₃ (Fig. 1D). These data suggest that a minimum of four phosphate groups are required for stimulation of exocytosis and that these phosphate groups can be randomly placed on the inositol ring. Furthermore, the data show that all six phosphate groups are required for maximal stimulation of exocytosis.

InsP₆ stimulates exocytosis by activation of PKC in permeabilized insulinoma cells (2). Here we extend this observation to mouse -cells since the stimulatory action of InsP₆ on exocytosis was abolished by the PKC inhibitors calphostin C (1.5 µM for >15 min), bisindolylmaleimide (4 µM for 20 min), and staurosporine (100 nM for >10 min) (Fig. 2). On the contrary, no effect was observed on the stimulatory action of InsP₆ in the presence of the protein kinase A inhibitor (R_p)-cAMP (100 µM for >30 min). Under these conditions, InsP₆ stimulated exocytosis by 90% (p < 0.05; n = 5; Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIG. 2. InsP6-induced exocytosis is dependent on PKC-activity. The graph shows increases in cell capacitance in the absence (–) and presence (+) of 100 µM InsP6 in cells treated with the calcineurin inhibitor cyclosporin A (1.5 µM for >15 min) and the PKC inhibitors calphostin C (1.5 µM for >15 min), bisindolylmaleimide (4 µM for >20 min), and staurosporine (100 nM for >10 min) or following inhibition of protein kinase A with (Rp)-cAMP (100 µM for >30 min). The rate of capacitance increase (Cm/t) was measured from 30 to 90 s. All inhibitors were included in the pipette-filling solution at the concentrations indicated above. The data are mean values ± S.E. for 5–8 different experiments. *, p < 0.05. fF, femtofarads.

To investigate which isoform of PKC mediates the stimulatory action of InsP₆ on exocytosis, cells were treated for 48 h with antisense oligonucleotides against PKC-, -II, -, -, and -. Fig. 3A shows that InsP₆ failed to increase exocytosis in cells exposed to antisense oligonucleotides against PKC-. However, InsP₆ retained its stimulatory action when cells were treated with antisense oligonucleotides against PKC-, -II, -, -, and sense PKC- oligonucleotides. These data argue that InsP₆ stimulates exocytosis by a mechanism that involves activation of PKC-.

View larger version (22K): [in this window] [in a new window]   FIG. 3. PKC- controls InsP6-induced exocytosis in mouse -cells. A, histogram depicting average rates of capacitance increase (Cm/t) measured from 30 to 90 s after establishment of the whole-cell configuration in the absence (–) and presence (+) of 100 µM InsP6. The cells were treated for 48 h with 5 µM antisense oligonucleotides against PKC-, -II, -, -, and - or sense oligonucleotide against PKC-. fF, femtofarads. B, same as in panel A, except that cells were transiently expressing wild-type PKC- (wt-PKC-), dominant negative PKC- (dn-PKC-), and wild-type PKC-I(wt-PKC-I). As control, cells were transfected with pcDNA alone (mock). In all experiments, cells were treated with cyclosporin A (1.5 µM for >15 min). The data are mean values ± S.E. for 6–9 different experiments. *, p < 0.05.

The involvement of PKC- in InsP₆-induced exocytosis was further supported by studies in mouse -cells transiently transfected with either wild-type or a dominant negative mutant of PKC-. Fig. 3B shows that InsP₆ (100 µM) evoked a robust increase in cell capacitance in mock-transfected cells and in cells expressing wild-type PKC-. On the contrary, the dominant negative PKC- isoform abolished the ability of InsP₆ to stimulate exocytosis. Interestingly, the wild-type PKC- isoform significantly increased Ca²⁺-induced exocytosis, whereas the dominant negative isoform reduced the exocytotic response in the absence of InsP₆ by 50% (Fig. 3B). Transfection of the wild-type PKC-I isoform did not influence Ca²⁺- and InsP₆-induced exocytosis (Fig. 3B).

The results from this study show for the first time that PKC- is involved in the stimulatory action of InsP₆ on Ca²⁺-evoked exocytosis. The molecular mechanism for the activation of PKC- by InsP₆ remains to be explored. However, it is pertinent to emphasize that an InsP₆-activated protein kinase has been identified in rat brain (12) and that InsP₆ enhances L-type Ca²⁺ channel activity in vascular smooth muscle cells by a PKC-dependent mechanism (13). The substrate(s) for PKC- are poorly understood, but recent data from our laboratories demonstrate that PKC- associates with insulin-containing granules in response to glucose and clinically used sulfonylureas.² These data suggest that PKC- represents an important component of the secretory network in pancreatic -cells.

A model for the effects of glucose and InsP₆ on exocytosis and endocytosis is presented in Fig. 4. Glucose acts to stimulate ATP synthesis, leading to closure of ATP-sensitive K⁺ channels, cell depolarization, and Ca²⁺ influx. The resulting increase in cytoplasmic Ca²⁺ concentration stimulates exocytosis. Not only activation of the phospholipase C system but also glucose stimulation result in InsP₆ production, which leads to enhanced PKC- activity. PKC- potentiates Ca²⁺-induced exocytosis. An increase in cytoplasmic free Ca²⁺ concentration is also required for stimulation of endocytosis. This process involves activation of calcineurin and PKC-, most likely resulting in a sequential phosphorylation and dephosphorylation of proteins involved in endocytosis (for details, see Ref. 3). This suggests that InsP₆ has an important integral role in pancreatic -cell membrane trafficking, being part of the molecular mechanisms linking exocytosis and endocytosis.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Model for InsP6-regulated exocytosis and endocytosis in pancreatic -cells. See "Results and Discussion" for details. KATP, ATP-sensitive K+ channel; Calci, calcineurin; PLC, phospholipase C; PIP2, phosphatidylinositol bisphosphate.

	FOOTNOTES

 
^* This study was supported in part by the National Institutes of Health (Grant DK 58508), the Swedish Research Council, the Swedish Diabetes Association, the Novo Nordisk Foundation, the Juvenile Diabetes Foundation International, and the Funds of Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A recipient of a scholarship from the Academy of Technical Sciences and Novo Nordisk A/S. Present address: Dept. of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

^|| To whom correspondence may be addressed: Lilly Research Laboratories, Essener Strasse 93, D-22419 Hamburg, Germany. Tel.: 49-40-527-24-323; Fax: 49-40-527-24-615; E-mail: gromada{at}lilly.com.

¹ The abbreviations used are: InsP₆, inositol hexakisphosphate; InsP₄, inositol tetrakisphosphate; Ins(1,3,4)P₃, inositol 1,3,4-trisphosphate; Ins(1,4,5)P₃, inositol 1,4,5-trisphosphate; Ins(1,3,4,5)P₄, inositol 1,3,4,5-tetrakisphosphate; Ins(1,4,5,6)P₄, inositol 1,4,5,6-tetrakisphosphate; Ins(1,3,4,5,6)P₅, inositol 1,3,4,5,6-pentakisphosphate; [Ca²⁺]_f, free Ca²⁺ concentrations; PKC, protein kinase C.

² C. F. Mendez, I. B. Leibiger, B. Leibiger, M. Hoøy, J. Gromada, P. O. Berggren, and A. M. Bertorello, manuscript submitted for publication.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 278/37/35168    most recent M303927200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Høy, M. Articles by Gromada, J. Search for Related Content PubMed PubMed Citation Articles by Høy, M. Articles by Gromada, J. Social Bookmarking                      What's this?