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• W1989092000 abstract "Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from β-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting β-cell line β-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized β-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized β-TC3 cells. Mastoparan-induced insulin secretion from permeabilized β-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on β-TC3 homogenate and ISG demonstrated that Gαi was dramatically enriched in ISG. Levels of Gαo and Gαq were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of Gαi to ISG was confirmed with electron microscopic immunocytochemistry in β-TC3 cells and β-cells from rat pancreas. In contrast to Gαo and Gαq, Gαi was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein Gαi located in the ISG of β-cells to stimulate insulin secretion. Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from β-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting β-cell line β-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized β-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized β-TC3 cells. Mastoparan-induced insulin secretion from permeabilized β-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on β-TC3 homogenate and ISG demonstrated that Gαi was dramatically enriched in ISG. Levels of Gαo and Gαq were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of Gαi to ISG was confirmed with electron microscopic immunocytochemistry in β-TC3 cells and β-cells from rat pancreas. In contrast to Gαo and Gαq, Gαi was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein Gαi located in the ISG of β-cells to stimulate insulin secretion. Insulin secretion from β-cells can be stimulated by different types of secretagogues (1Ashcroft S.J. Diabetologia. 1980; 18: 5-15Crossref PubMed Scopus (215) Google Scholar). D-Glucose, a fuel secretagogue, is the major physiological stimulus (2Hedeskov C.J. Physiol. Rev. 1980; 60: 442-509Crossref PubMed Scopus (329) Google Scholar, 3Malaisse W.J. Sener A. Herchuelz A. Hutton J.C. Metabolism. 1979; 28: 373-386Abstract Full Text PDF PubMed Scopus (266) Google Scholar). The mechanism by which glucose-induced insulin release occurs is not completely elucidated, although glucose oxidation is essential (3Malaisse W.J. Sener A. Herchuelz A. Hutton J.C. Metabolism. 1979; 28: 373-386Abstract Full Text PDF PubMed Scopus (266) Google Scholar, 4Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 5Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (419) Google Scholar, 6Wollheim C.B. Sharp G.W. Physiol. Rev. 1981; 61: 914-973Crossref PubMed Scopus (693) Google Scholar). Glucokinase is believed to act as a glucose sensor, with phosphorylation of glucose to glucose 6-phosphate serving as the rate-limiting step in glucose oxidation (7Matschinsky F. Liang Y. Kesavan P. Wang L. Froguel P. Velho G. Cohen D. Permutt M.A. Tanizawa Y. Jetton T.L. Niswender K. Magnuson M.A. J. Clin. Invest. 1993; 92: 2092-2098Crossref PubMed Scopus (262) Google Scholar). While inhibition of glucose oxidation inhibits insulin release, the details of the mechanism coupling glucose oxidation to insulin secretion are less clear. It is currently believed that oxidation of fuel secretagogues increases intracellular levels of ATP (8Rajan A.S. Aguilar-Bryan L. Nelson D.A. Yaney G.C. Hsu W.H. Kunze D.L. Boyd A.E. Diabetes Care. 1990; 13: 340-363Crossref PubMed Scopus (135) Google Scholar), although this view has been challenged by some groups (9MacDonald M.J. Diabetes. 1990; 39: 1461-1466Crossref PubMed Scopus (154) Google Scholar). An increased ATP/ADP ratio is believed to close KATP+ channels at the plasma membrane, resulting in decreased K+ efflux and subsequent depolarization of the β-cell (10Misler S. Falke L.C. Gillis K. McDaniel M.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7119-7123Crossref PubMed Scopus (234) Google Scholar, 11Dunne M.J. Petersen O.H. Biochim. Biophys. Acta. 1991; 1071: 67-82Crossref PubMed Scopus (152) Google Scholar, 12Cook D.L. Satin L.S. Ashford M.L. Hales C.N. Diabetes. 1988; 37: 495-498Crossref PubMed Scopus (207) Google Scholar). Depolarization then activates voltage-dependent Ca2+ channels, causing an influx of extracellular Ca2+ into the β-cell and an increase in intracellular Ca2+ levels (6Wollheim C.B. Sharp G.W. Physiol. Rev. 1981; 61: 914-973Crossref PubMed Scopus (693) Google Scholar, 13Prentki M. Matschinsky F.M. Physiol. Rev. 1987; 67: 1185-1248Crossref PubMed Google Scholar). In addition, it has recently been shown that nutrient secretagogues increase β-cell malonyl-CoA levels, which leads to increased cytosolic long-chain acyl-CoA esters that positively modulate insulin secretion (14Corkey B.E. Glennon M.C. Chen K.S. Deeney J.T. Matschinsky F.M. Prentki M. J. Biol. Chem. 1989; 264: 21608-21612Abstract Full Text PDF PubMed Google Scholar, 15Deeney J.T. Tornheim K. Korchak H.M. Prentki M. Corkey B.E. J. Biol. Chem. 1992; 267: 19840-19845Abstract Full Text PDF PubMed Google Scholar, 16Prentki M. Vischer S. Glennon M.C. Regazzi R. Deeney J.T. Corkey B.E. J. Biol. Chem. 1992; 267: 5802-5810Abstract Full Text PDF PubMed Google Scholar). While increased intracellular Ca2+ activates protein kinases such as the Ca2+- and calmodulin-dependent protein kinase (17Turk J. Wolf B.A. McDaniel M.L. Prog. Lipid Res. 1987; 26: 125-181Crossref PubMed Scopus (116) Google Scholar, 18Colca J.R. Wolf B.A. Comens P.G. McDaniel M.L. Biochem. J. 1985; 228: 529-536Crossref PubMed Scopus (31) Google Scholar, 19Easom R.A. Landt M. Colca J.R. Hughes J.H. Turk J. McDaniel M. J. Biol. Chem. 1990; 265: 14938-14946Abstract Full Text PDF PubMed Google Scholar, 20Wollheim C.B. Regazzi R. FEBS Lett. 1990; 268: 376-380Crossref PubMed Scopus (62) Google Scholar, 21Wenham R.M. Landt M. Easom R.A. J. Biol. Chem. 1994; 269: 4947-4952Abstract Full Text PDF PubMed Google Scholar), the mechanism by which insulin exocytosis occurs is not completely elucidated. Over the past few years, factors other than calcium have been proposed to be involved, including GTP and small monomeric GTP-binding proteins such as Rab3A (22Olszewski S. Deeney J.T. Schuppin G.T. Williams K.P. Corkey B.E. Rhodes C.J. J. Biol. Chem. 1994; 269: 27987-27991Abstract Full Text PDF PubMed Google Scholar). In addition to small GTP-binding proteins, heterotrimeric GTP-binding proteins have also been implicated in regulating insulin secretion by pancreatic β-cells (23Robertson R.P. Seaquist E.R. Walseth T.F. Diabetes. 1991; 40: 1-6Crossref PubMed Scopus (80) Google Scholar, 24Hsu W.H. Xiang H.D. Rajan A.S. Kunze D.L. Boyd III, A.E. J. Biol. Chem. 1991; 266: 837-843Abstract Full Text PDF PubMed Google Scholar, 25Baffy G. Yang L. Wolf B.A. Williamson J.R. Diabetes. 1993; 42: 1878-1882Crossref PubMed Google Scholar). Epinephrine is an adrenergic agonist that inhibits insulin secretion by acting through a heterotrimeric G-protein (23Robertson R.P. Seaquist E.R. Walseth T.F. Diabetes. 1991; 40: 1-6Crossref PubMed Scopus (80) Google Scholar, 26Seaquist E.R. Neal A.R. Shoger K.D. Walseth T.F. Robertson R.P. Diabetes. 1992; 41: 1390-1399Crossref PubMed Scopus (39) Google Scholar, 27Ullrich S. Wollheim C.B. J. Biol. Chem. 1988; 263: 8615-8620Abstract Full Text PDF PubMed Google Scholar). Although epinephrine acts through a G-protein-coupled adrenergic receptor located on the plasma membrane of the β-cell to decrease cAMP accumulation, this is not thought to account entirely for its inhibition of insulin secretion (26Seaquist E.R. Neal A.R. Shoger K.D. Walseth T.F. Robertson R.P. Diabetes. 1992; 41: 1390-1399Crossref PubMed Scopus (39) Google Scholar). The ability of epinephrine to inhibit insulin secretion is believed to be due to its action on the distal portion of the insulin exocytotic pathway, since epinephrine is capable of inhibiting calcium-induced insulin secretion from permeabilized β-cells (27Ullrich S. Wollheim C.B. J. Biol. Chem. 1988; 263: 8615-8620Abstract Full Text PDF PubMed Google Scholar). Epinephrine is thought to inhibit insulin release by acting through a pertussis toxin-sensitive G-protein. Epinephrine inhibition of insulin secretion is itself inhibited by pretreatment of cells with pertussis toxin, which ADP ribosylates pertussis toxin-sensitive G-protein α subunits at a cysteine residue located four amino acids from the carboxyl terminus (26Seaquist E.R. Neal A.R. Shoger K.D. Walseth T.F. Robertson R.P. Diabetes. 1992; 41: 1390-1399Crossref PubMed Scopus (39) Google Scholar). This ADP ribosylation renders the G-protein α subunit incapable of interacting with its receptor. In addition to epinephrine, a variety of other compounds are thought to modulate insulin secretion by acting through heterotrimeric G-proteins (23Robertson R.P. Seaquist E.R. Walseth T.F. Diabetes. 1991; 40: 1-6Crossref PubMed Scopus (80) Google Scholar). One class of such compounds consists of amphiphilic peptides. A well known member of this class is mastoparan, a tetradecapeptide found in wasp venom. Mastoparan stimulates insulin secretion from β-cells (28Jones P.M. Mann F.M. Persaud S.J. Wheeler-Jones C.P.D. Mol. Cell. Endocrinol. 1993; 94: 97-103Crossref PubMed Scopus (55) Google Scholar, 29Komatsu M. McDermott A.M. Gillison S.L. Sharp G.W.G. J. Biol. Chem. 1993; 268: 23297-23306Abstract Full Text PDF PubMed Google Scholar) and also stimulates secretion from many other cell types such as platelets (30Wheeler-Jones C.P. Saermark T. Kakkar V.V. Authi K.S. Biochem. J. 1992; 281: 465-472Crossref PubMed Scopus (33) Google Scholar, 31Wheeler-Jones C.P. Saermark T. Kakkar V.V. Authi K.S. Biochem. Soc. Trans. 1991; 19: 114SCrossref PubMed Scopus (3) Google Scholar, 32Ozaki Y. Matsumoto Y. Yatomi Y. Higashihara M. Kariya T. Kume S. Biochem. Biophys. Res. Commun. 1990; 170: 779-785Crossref PubMed Scopus (51) Google Scholar), neutrophils (33Norgauer J. Eberle M. Lemke H.D. Aktories K. Biochem. J. 1992; 282: 393-397Crossref PubMed Scopus (36) Google Scholar), and pneumocytes (34Joyce-Brady M. Rubins J.B. Panchenko M.P. Bernardo J. Steele M.P. Kolm L. Simons E.R. Dickey B.F. J. Biol. Chem. 1991; 266: 6859-6865Abstract Full Text PDF PubMed Google Scholar). Mastoparan has been shown to increase the GTPase activity of many G-proteins including Gi and Go(35Higashijima T. Burnier J. Ross E.M. J. Biol. Chem. 1990; 265: 14176-14186Abstract Full Text PDF PubMed Google Scholar, 36Sukumar M. Higashijima T. J. Biol. Chem. 1992; 267: 21421-21424Abstract Full Text PDF PubMed Google Scholar). Mastoparan is thought to stimulate these G-proteins by inserting itself into the membrane adjacent to G-proteins and mimicking the normal interaction that occurs between G-protein-coupled receptors and their respective G-proteins (37Mousli M. Bueb J.L. Bronner C. Rouot B. Landry Y. Trends. Pharmacol. Sci. 1990; 11: 358-362Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 38Mousli M. Bronner C. Landry Y. Bockaert J. Rouot B. FEBS Lett. 1990; 259: 260-262Crossref PubMed Scopus (284) Google Scholar). Mastoparan is believed to bind to the carboxyl terminus of the G-protein α subunit, resulting in βγ and GDP dissociation from the α subunit, causing the α subunit to assume the active configuration (39Weingarten R. Ransnas L. Mueller H. Sklar L.A. Bokoch G.M. J. Biol. Chem. 1990; 265: 11044-11049Abstract Full Text PDF PubMed Google Scholar, 40Tomita U. Inanobe A. Kobayashi I. Takahashi K. Ui M. Katada T. J. Biochem.(Tokyo). 1991; 109: 184-189Crossref PubMed Scopus (46) Google Scholar). Mastoparan, however, unlike epinephrine, stimulates insulin secretion from β-cells, suggesting that G-proteins, in addition to having a role in negatively modulating insulin secretion, may also positively modulate insulin secretion (28Jones P.M. Mann F.M. Persaud S.J. Wheeler-Jones C.P.D. Mol. Cell. Endocrinol. 1993; 94: 97-103Crossref PubMed Scopus (55) Google Scholar, 29Komatsu M. McDermott A.M. Gillison S.L. Sharp G.W.G. J. Biol. Chem. 1993; 268: 23297-23306Abstract Full Text PDF PubMed Google Scholar). The identity of the G-protein through which mastoparan stimulates insulin secretion from the β-cell is unknown. To better understand if mastoparan stimulates insulin secretion from the β-cell by acting distally in exocytosis, we have examined the effects of mastoparan and its analogues on insulin secretion from digitonin-permeabilized β-cells. We have also examined the effects of various mastoparan analogues on GTPase activity of insulin secretory granules (ISG)1( 1The abbreviations used are: ISGinsulin secretory granuleTES2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acidPBSphosphate-buffered salineMES2-(N-morpholino)ethanesulfonic acidGTPγSguanosine 5′-O-(thiotriphosphate).) prepared from β-cells and have shown that the ability of mastoparan analogues to stimulate ISG GTPase activity correlates with their ability to stimulate insulin secretion. Finally, we have used G-protein antisera for Western blotting and electron microscopic immunocytochemistry in order to determine which G-protein is localized to the ISG of β-cells and is a target for mastoparan. insulin secretory granule 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid phosphate-buffered saline 2-(N-morpholino)ethanesulfonic acid guanosine 5′-O-(thiotriphosphate). Tissue culture medium (CMRL-1066) and 1 M HEPES were from Life Technologies, Inc. Newborn bovine serum was from Hazleton Biologics (Lenexa, KS). β-TC3 cells (passage 34) were obtained through the University of Pennsylvania Diabetes Center from Dr. D. Hanahan (University of California, San Francisco). The following compounds were purchased from Sigma: D-glucose, carbachol, Hanks' balanced salt solution, penicillin, streptomycin, glutamine, Ficoll, bovine serum albumin, ovalbumin, ATP, GTP, NAD, thymidine, and creatine phosphate. Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). Mastoparan; its active analogues mastoparan 7, mastoparan 8, and mastoparan X; and its inactive analogue mastoparan 17 were purchased from Peninsula Laboratories (Belmont, CA). Creatine kinase and adenylyl imidodiphosphate were purchased from Boehringer Mannheim. Digitonin was obtained from Waco Pure Chemical Industries. [32P]GTP (10 Ci/mmol) and [32P]NAD (1000 Ci/mmol) were purchased from Amersham Corp. 125I-Protein A was purchased from ICN Radiochemicals (Irvine, CA). Antisera to selected G-proteins were raised by injecting New Zealand White rabbits with synthetic decapeptides corresponding to the carboxyl-terminal amino acid sequences of the respective G-proteins as described previously (41Williams A.G. Woolkalis M.J. Poncz M. Manning D.R. Gewirtz A.M. Brass L.F. Blood. 1990; 76: 721-730Crossref PubMed Google Scholar, 42Law S.F. Reisine T. Mol. Pharmacol. 1992; 42: 398-402PubMed Google Scholar, 43Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar). In this study, the following antisera were used for Western blotting and electron microscopic immunocytochemical analysis of rat pancreas: anti-Gi 8730 (immunogen (KLH)KNNLKDCGLF), anti-Go 9072 (immunogen (KLH)ANNLRGCGLY), and anti-Gq 946 (immunogen (KLH)QLNLKEYNLV). These same antisera, along with anti-Gi/Go 5296 (immunogen bovine brain Go) were used for electron microscopic immunocytochemical analysis of β-TC3 cells. β-TC3 cells were cultured in 6-well plates or 15-cm dishes in the presence of RPMI 1640 (11 mM glucose) supplemented with 10% fetal bovine serum, penicillin (75 μg/ml), streptomycin (50 μg/ml), and 2 mML-glutamine. Cells were trypsinized and subcloned weekly. Medium was changed twice weekly and the day prior to an experiment. Insulin secretory capacity of the β-TC3 cells in response to glucose and carbachol was monitored regularly. Cells were used between passages 40 and 55. Approximately 24 h prior to the experiment, β-TC3 cells in either 6-well plates or 15-cm dishes were cultured in RPMI 1640 supplemented with 50 ng/ml pertussis toxin. β-TC3 cells in 6-well plates were washed 3 times with 2 ml of Krebs-HEPES buffer (25 mM HEPES, pH 7.40, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, and 0.1% bovine serum albumin). Cells in monolayers were permeabilized in the same media supplemented with 20 μg/ml digitonin for 10 min at 37°C under an atmosphere of 95% air, 5% CO2(44Wolf B.A. Florholmen J. Turk J. McDaniel M.L. J. Biol. Chem. 1988; 263: 3565-3575Abstract Full Text PDF PubMed Google Scholar). Using this method, greater than 99% permeabilization of β-TC3 cells was achieved as assessed by trypan blue exclusion. Lactate dehydrogenase release into the permeabilization medium was found to become maximal at a digitonin concentration of 20 μg/ml. Permeabilized cells were washed 3 times with 2 ml of TES buffer (50 mM TES, pH 7.40, 100 mM KCl, 1 mM EGTA, 2 mM MgCl2, and 0.1% bovine serum albumin). Cells were incubated for 60 min at 37°C under an atmosphere of 95% air, 5% CO2 with fresh buffer supplemented with 1 mM ATP and the appropriate secretagogue. In selected experiments, an ATP regenerating system consisting of 5 mM creatine phosphate and 0.2 mg/ml creatine phosphokinase was included. At the end of the incubation period, a sample of the supernatant was removed for insulin measurement by radioimmunoassay (45Konrad R.J. Jolly Y.C. Major C. Wolf B.A. Biochem. J. 1992; 287: 283-290Crossref PubMed Scopus (56) Google Scholar). β-TC3 cells were fractionated by sequential centrifugation using a sucrose/nycodenz gradient as described previously (46Hutton J.C. Penn E.J. Peshavaria M. Diabetologia. 1982; 23: 365-373Crossref PubMed Scopus (55) Google Scholar). 10-20 confluent 15-cm dishes of β-TC3 cells were washed 3 times with ice-cold 50 mM MES, pH 7.20, containing 0.25 M sucrose and 1 mM EGTA. Cells were scraped and resuspended in 1 ml of the same buffer. Cells were then mechanically homogenized with 10 strokes of a motor-driven Teflon pestle in a Potter-Elvehjem homogenizing tube. The homogenate was centrifuged (600 × g, 5 min) to remove intact cells and nuclear debris. The supernatant was saved, and the pellet was homogenized twice more as above. Supernatants were combined and transferred to a 13-ml Beckman ultracentrifuge tube already underlaid with stepwise gradients (4 ml) of nycodenz stock/homogenization buffer in the following ratios: 16:84, 32:68, 64:36. Tubes were centrifuged at 100,000 × g (SW-40 rotor, Beckman TL-100 centrifuge) for 60 min to yield a band at the lower interface corresponding to ISG. Granules were harvested, resuspended in a 15-ml Corex tube in 14 ml of 10 mM MES, pH 6.50 containing 0.25 M sucrose, and centrifuged at 27,000 × g (Beckman J2-21 centrifuge, JA-20 rotor) for 20 min. ISG were resuspended in 100-200 μl of 10 mM MES, pH 7.40, supplemented with 1 mM EDTA. Insulin enrichment of the ISG relative to the respective homogenate was systematically checked. Enzyme marker studies were performed as described previously (47McDaniel M.L. Colca J.R. Kotagal N. Lacy P.E. Methods Enzymol. 1983; 98: 182-200Crossref PubMed Scopus (119) Google Scholar). For these experiments, 2-25 μg of protein in 20 μl of loading buffer (70 mM Tris, pH 6.7, 16 M urea, 6.0% SDS, 100 mM dithiothreitol, 0.005% bromphenol blue) were loaded onto 10% SDS-polyacrylamide gel electrophoresis gels. Colored rainbow molecular weight markers (Amersham Corp.) were also run on each gel. Proteins were separated for 1 h at 175 V at room temperature using a Bio-Rad Mini-PROTEAN II dual slab cell. Proteins were transferred to nitrocellulose paper (Hybond C, Amersham Corp.) for 1.5 h at 100 V at 4°C using a Bio-Rad mini Trans-Blot electrophoretic transfer cell. Nitrocellulose blots were blocked overnight in blocking buffer (1% ovalbumin, 3% bovine serum albumin, 10 mM Tris, pH 7.40, 150 mM NaCl, and 0.1% sodium azide) at 4°C. The next day, blots were probed with appropriate antisera at a dilution of 1:100 in blocking buffer for 3 h at room temperature. Blots were washed twice with 10 mM Tris, pH 7.40, 150 mM NaCl, and 0.1% sodium azide (TNA) for 10 min, once with TNA supplemented with 0.05% Nonidet P-40 for 5 min, and twice more with TNA for 10 min. Blots were incubated with 5 μCi of 125I-protein A in 10 ml of blocking buffer for 1 h at room temperature. Blots were washed again as above, air dried, and exposed on a PhosphorImager cassette (Molecular Dynamics) overnight at room temperature. The following day, radioactivity bound to the blot was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software. The reaction was initiated by the addition of 5 μg of sample protein to 100 μl of 25 mM HEPES, pH 7.20, supplemented with 1 mM dithiothreitol, 1 mM EGTA, 20 μM MgCl2, 1 mM ATP, 1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 0.2% bovine serum albumin, 100 nM [γ-32P]GTP, and varying concentrations of mastoparan or mastoparan analogues in a 1.5-ml snap-top Eppendorf tube. Tubes were incubated at 30°C in a shaking water bath for 5 min. The reaction was stopped by the addition of 900 μl of ice-cold trichloroacetic acid, pH 2.0, supplemented with 5% charcoal by weight, which was kept continuously stirring on ice. Tubes were centrifuged at 10,000 × g for 30 min at 4°C, and 32Pi in the supernatant was quantitated by liquid scintillation spectrophotometry. Blanks determined in the presence of 50 μM GTP were routinely subtracted, and GTPase activity was calculated as pmol of GTP hydrolyzed per mg of protein/min. Under these conditions, GTPase activity was linear at 30 min. The reaction was initiated by the addition of 5 μg of protein to 100 μl of 25 mM HEPES, pH 7.20, supplemented with 1 mM EDTA, 1 mM ATP, 0.1 mM GTP, 0.1% Lubrol, 0.02% bovine serum albumin, 10 mM thymidine, 10 μM [32P]NAD, and 5 μg/ml activated pertussis toxin. Tubes were incubated at 30°C in a shaking water bath for 30 min. The reaction was stopped by the addition of 900 μl of ice-cold 10% trichloroacetic acid, pH 2.0. Tubes were centrifuged at 10,000 × g for 20 min at 4°C, and membrane pellets were washed once with 1 ml of 10% trichloroacetic acid, pH 2.0. Membrane pellets were then suspended in 1 ml of ice-cold diethyl ether. Ether was evaporated in a Savant concentrator connected to a −90°C cold trap, and membrane pellets were resuspended in 30 μl of loading buffer supplemented with 15 mg/ml dithiothreitol. Samples were then run on 10% SDS-polyacrylamide gels as described above. Gels were dried and exposed on a PhosphorImager cassette overnight. The following day, radioactivity bound to the blot was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software. β-TC3 cells were plated on Costar Transwell cell culture inserts 5 days before use and cultured as described above. Medium was changed the day prior to fixation. On the day of fixation, the medium was aspirated, and 2 ml of ice-cold phosphate-buffered saline (PBS), pH 7.40, was added to wash the cells. Inserts were removed and transferred to a culture plate containing PBS for 1-2 min. PBS was aspirated, and 2 ml of GPA fixative containing 1% glutaraldehyde and 0.2% picric acid in pH 7.40 PBS at 4°C was added. Inserts were transferred to culture plates containing fixative. The cells were allowed to fix for 2-18 h at 4°C. Cells were dehydrated with 70, 80, and 90% ethanol at 4°C for 30 min. Cells were stained with 1% eosin B (to permit visualization of the cells) in 100% ethanol for 8 min at 24°C and washed in 100% ethanol 3-4 times. Inserts were transferred to LR White, 100% ethanol (1:1) for 2 h and through three changes of 100% LR White (Electron Microscopy Sciences, Fort Washington, PA) for 1 h, overnight, and for 1 h the next morning. The membrane on which the cells were attached was removed from the plastic insert with a scalpel. The membrane was gently rolled, with cells on the inside, into a roll approximately 2 mm in diameter. The membrane was secured in two places by tying a short length of hair around the roll. The roll was cut between the two ties and excess membrane on the outer side of the ties was removed and discarded. Membranes were placed in gelatin capsules partially filled with LR White resin with the cut end oriented toward the bottom of the capsule. Capsules were filled with resin, capped, and cured for 18 h at 60°C. Thin sections (600-700 Å thick) were cut perpendicular to the plane of the membrane and mounted on nickel grids. Pancreata were excised from male Sprague-Dawley rats and minced in the GPA fixative. Tissue was fixed 2-18 h, dehydrated, infiltrated, and embedded in LR White resin as described above. Thick sections were cut from randomly selected tissue blocks and stained with 1% toluidine blue in 1% sodium borate to select blocks with islets. Thin sections (600-700 Å thick) were cut from the selected blocks and mounted on nickel grids. Thin sections of β-TC3 cells and rat pancreas were floated section-side down on small drops of 1% ovalbumin, 0.2% cold water fish skin gelatin in PBS for 1 h at 24°C to reduce nonspecific immunoglobulin absorption. Sections of β-TC3 cells were incubated with G-protein antisera diluted one-tenth to one-thousandth with PBS overnight at 4°C in a humidified chamber. Sections were washed by transferring the grids through small drops of 20 mM Tris-HCl-buffered saline with 0.02% Tween-20, pH 7.40, (TBST) four times for 5 min, each at 24°C. β-TC3 cells were incubated with 15-nm diameter gold-labeled protein A prepared as described (48Horisberger M. Clerc M.-F. Histochemistry. 1994; 82: 219-223Crossref Scopus (90) Google Scholar, 49Soler A.P. Thompson K.A. Smith R.M. Jarett L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6640-6644Crossref PubMed Scopus (64) Google Scholar, 50Lewis J.M. Woolkalis M.J. Gerton G.L. Smith R.M. Jarett L. Manning D.R. Cell Regul. 1991; 2: 1097-1113Crossref PubMed Scopus (56) Google Scholar) for 1 h at 24°C to detect bound immunoglobulin. Two staining procedures were used to identify α-cells and β-cells and, by deduction, δ-cells, in islets. In the first, serial sections were cut, and different sections were stained with anti-insulin, anti-glucagon, or anti-G-protein antisera, and the antibodies were detected with gold-labeled protein A as described above. Alternatively, sections were co-incubated overnight at 4°C with anti-G-protein Ig complexed to 18-nm diameter colloidal gold and anti-insulin or anti-glucagon Ig complexed to 8-nm diameter colloidal gold. All gold-labeled immunoglobulins were prepared as described previously (49Soler A.P. Thompson K.A. Smith R.M. Jarett L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6640-6644Crossref PubMed Scopus (64) Google Scholar, 50Lewis J.M. Woolkalis M.J. Gerton G.L. Smith R.M. Jarett L. Manning D.R. Cell Regul. 1991; 2: 1097-1113Crossref PubMed Scopus (56) Google Scholar). All sections were washed twice with TBST, twice with deionized water, and then stained with 2% aqueous uranyl acetate for 3 min. Insulin radioimmunoassay was performed by the University of Pennsylvania Diabetes Endocrine Research Center. Protein concentrations of β-TC3 cell granule fractions and homogenates were calculated using a bicinchoninic acid microassay with bovine serum albumin as the standard (51Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provemano M.A. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Results are expressed as the mean ± S.E. Statistical analysis was performed using version 5.0 of SPSS for Windows. Data were analyzed by one-way analysis of variance or analysis of covariance as appropriate followed by multiple comparisons between means using the least significant difference test. A probability of p < 0.05 was considered statistically significant. An insulinoma cell line was used to study mastoparan-induced insulin secretion and the effects of mastoparan on secretory granule GTPase activity because of the large amount of cells necessary to perform these experiments. Between 30 and 60 rats would need to be sacrific" @default.
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• W1989092000 title "The Heterotrimeric G-protein Gi Is Localized to the Insulin Secretory Granules of β-Cells and Is Involved in Insulin Exocytosis" @default.
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