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Involvement of Protein Kinase C-ϵ in Inositol Hexakisphosphate-induced Exocytosis in Mouse Pancreatic β-Cells

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m303927200

1083-351X

Marianne Høy, Per‐Olof Berggren, Jesper Gromada,

Lysosomal Storage Disorders Research

Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic β-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic β-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 (α, βII, δ, ϵ, ξ) revealed the involvement of PKC-ϵ in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-ϵ abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-ϵ led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-ϵ is involved in InsP6-induced exocytosis in pancreatic β-cells. Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic β-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic β-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 (α, βII, δ, ϵ, ξ) revealed the involvement of PKC-ϵ in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-ϵ abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-ϵ led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-ϵ is involved in InsP6-induced exocytosis in pancreatic β-cells. Inositol hexakisphosphate (InsP6) 1The 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. levels transiently increase in pancreatic β-cells following an elevation of the ambient glucose concentration (1Larsson O. Barker C.J. Sjöholm Å. Carlquist H. Michell R.H. Bertorello A. Nilsson T. Honkanen R.E. Mayr G.W. Zwiller J. Berggren P.-O. Science. 1997; 278: 471-474Crossref PubMed Scopus (122) Google Scholar). This elevation in InsP6 levels plays an important role in controlling exocytosis of the insulin-containing granules and subsequent retrieval of membrane by endocytosis (2Efanov A.M. Zaitsev S.V. Berggren P.-O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4435-4439Crossref PubMed Scopus (76) Google Scholar, 3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). These processes of exocytosis and endocytosis are dependent on protein kinase C (PKC) activity (2Efanov A.M. Zaitsev S.V. Berggren P.-O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4435-4439Crossref PubMed Scopus (76) Google Scholar, 3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). Several PKC isoforms are expressed in pancreatic β-cells and include PKC-α, -β, -δ, -ϵ, and -ξ (4Kaneto H. Suzuma K. Bonner-Weir S. King G.L. Weir G.C. J. Biol. Chem. 2002; 277: 3680-3685Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 5Tian Y.-M. Urquidi V. Ashcroft S.J.H. Mol. Cell. Endocrin. 1996; 119: 185-193Crossref PubMed Scopus (80) Google Scholar). 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-ϵ regulates InsP6-induced exocytosis in single mouse pancreatic β-cells. Preparation of Islet Cells—Pancreatic islets were isolated from fed female NMRI mice (18–23 g) by collagenase digestion as described previously (3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). 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 (3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). 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. 6Martell A.E. Smith R.M. Critical Stability Constants, Vols. 1 and 2. Plenum Press, New York1971Google Scholar. 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-α antisense, 5′-CAGCCATGGTTCCCCCAAC-3′ (7Ranganathan G. Song W. Dean N. Monia B. Barger S.W. Kern P.A. J. Biol. Chem. 2002; 277: 38669-38675Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar); PKC-βII antisense, 5′-GTTGGAGGTGTCTCT-3′ (8Simpson R.U. O'Connell D. Pan Q. Newhouse J. Somerman M.J. J. Biol. Chem. 1998; 273: 19587-19591Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar); PKC-δ antisense, 5′-TCCAGGTCAACGCGGCATTC-3′ (7Ranganathan G. Song W. Dean N. Monia B. Barger S.W. Kern P.A. J. Biol. Chem. 2002; 277: 38669-38675Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar); PKC-ϵ antisense, 5′-GTCCATGCGATCTTGCGCCC-3′ (7Ranganathan G. Song W. Dean N. Monia B. Barger S.W. Kern P.A. J. Biol. Chem. 2002; 277: 38669-38675Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar); PKC-ϵ scrambled, 5′-GCCAGCTCCGATCTTGCGCCC-3′ (7Ranganathan G. Song W. Dean N. Monia B. Barger S.W. Kern P.A. J. Biol. Chem. 2002; 277: 38669-38675Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar); PKC-ζ antisense, 5′-GGCCACACATGTCTCGCACT-3′ (9Rzymkiewicz D.M. Tetsuka T. Daphna-Iken D. Srivastava S. Morrison A.R. J. Biol. Chem. 1996; 271: 17241-17246Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Cultures of β-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-ϵ and PKC-βI were kindly provided by Y. Nishizuka (Kobe University, Kobe, Japan). A dominant negative mutant of PKC-ϵ was obtained from K. Ridge (Northwestern University, Chicago, IL). Transfection of mouse β-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 (ΔCm /Δt) 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. Fig. 1A shows the exocytotic responses in single mouse pancreatic β-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 (10Olofsson C.S. Gopel S.O. Barg S. Galvanovskis J. Ma X. Salehi A. Rorsman P. Eliasson L. Pfluegers Arch. Eur. J. Physiol. 2002; 444: 43-51Crossref PubMed Scopus (221) Google Scholar). 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 (3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). 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 (3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). 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 β-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. The stimulatory action of InsP6 on exocytosis was dependent on dose. No significant stimulation of exocytosis was observed at 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). 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-α, -βII, -δ, -ϵ, and -ξ. Fig. 3A shows that InsP6 failed to increase exocytosis in cells exposed to antisense oligonucleotides against PKC-ϵ. However, InsP6 retained its stimulatory action when cells were treated with antisense oligonucleotides against PKC-α, -βII, -δ, -ξ, and sense PKC-ϵ oligonucleotides. These data argue that InsP6 stimulates exocytosis by a mechanism that involves activation of PKC-ϵ. The involvement of PKC-ϵ in InsP6-induced exocytosis was further supported by studies in mouse β-cells transiently transfected with either wild-type or a dominant negative mutant of PKC-ϵ. 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-ϵ. On the contrary, the dominant negative PKC-ϵ isoform abolished the ability of InsP6 to stimulate exocytosis. Interestingly, the wild-type PKC-ϵ 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-βI isoform did not influence Ca2+- and InsP6-induced exocytosis (Fig. 3B). The results from this study show for the first time that PKC-ϵ is involved in the stimulatory action of InsP6 on Ca2+-evoked exocytosis. The molecular mechanism for the activation of PKC-ϵ by InsP6 remains to be explored. However, it is pertinent to emphasize that an InsP6-activated protein kinase has been identified in rat brain (12Hilton J.M. Plomann M. Ritter B. Modregger J. Freeman H.N. Falck J.R. Krishna U.M. Tobin A.B. J. Biol. Chem. 2001; 276: 16341-16347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and that InsP6 enhances L-type Ca2+ channel activity in vascular smooth muscle cells by a PKC-dependent mechanism (13Quignard J.F. Rakotoarisoa L. Mironneau J. Mironneau C. J. Physiol. (Lond.). 2003; 549: 729-737Crossref Scopus (14) Google Scholar). The substrate(s) for PKC-ϵ are poorly understood, but recent data from our laboratories demonstrate that PKC-ϵ associates with insulin-containing granules in response to glucose and clinically used sulfonylureas. 2C. F. Mendez, I. B. Leibiger, B. Leibiger, M. Hoøy, J. Gromada, P. O. Berggren, and A. M. Bertorello, manuscript submitted for publication. These data suggest that PKC-ϵ represents an important component of the secretory network in pancreatic β-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-ϵ activity. PKC-ϵ 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-ϵ, most likely resulting in a sequential phosphorylation and dephosphorylation of proteins involved in endocytosis (for details, see Ref. 3Høy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.-O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (54) Google Scholar). This suggests that InsP6 has an important integral role in pancreatic β-cell membrane trafficking, being part of the molecular mechanisms linking exocytosis and endocytosis.