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. 1995 Apr 1;307(Pt 1):169–174. doi: 10.1042/bj3070169

Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells.

H C Cable 1, A el-Mansoury 1, N G Morgan 1
PMCID: PMC1136759  PMID: 7717971

Abstract

1. Alpha-2-adrenoceptor agonists, such as noradrenaline, are potent inhibitors of insulin secretion, and it has been suggested that they control a late step in the pathway of exocytosis. We have investigated whether this could be related to a change in the extent of actin polymerization in the pancreatic B-cell, since actin microfilaments are implicated in regulating the access of secretory granules to the plasma membrane prior to exocytosis. 2. Cultured HIT-T15 pancreatic B-cells responded to noradrenaline with an increase in F-actin content, as judged by a rise in the fluorescence output after probing of the cells with phalloidin (a toxin which binds specifically to F-actin) conjugated to rhodamine. The response to noradrenaline was rapid, dose-dependent and sustained and could be reproduced by the highly selective alpha-2-agonist UK14,304. Examination of HIT-T15 cells by fluorescence microscopy after treatment with rhodamine-phalloidin, revealed a significant localization of F-actin immediately adjacent to the plasma membrane. The pattern of F-actin distribution in the cells was not altered dramatically by noradrenaline, although the intensity of staining close to the plasma membrane appeared to be slightly reduced. 3. The increase in F-actin content induced by noradrenaline and UK14,304 was inhibited significantly by the alpha-2-antagonist idazoxan but not by the alpha-1-selective antagonist prazosin. Pretreatment of HIT-T15 cells with pertussis toxin did not lead to any direct alteration in F-actin content, although the toxin significantly modified the responses induced by noradrenaline and UK14,304. In each case, cells incubated for 24 h with pertussis toxin responded to the alpha-2-agonist with an enhanced fluorescence output, indicating that F-actin levels had increased still further. This did not correlate with any gross change in the distribution of F-actin as judged by fluorescence microscopy. 4. The results demonstrate that alpha-2-adrenoceptors are coupled to control of actin polymerization in HIT-T15 cells. They suggest that regulation of F-actin formation could be a component of the mechanism by which alpha-2-agonists mediate inhibition of insulin secretion.

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Selected References

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  1. Bengtsson T., Stendahl O., Andersson T. The role of the cytosolic free Ca2+ transient for fMet-Leu-Phe induced actin polymerization in human neutrophils. Eur J Cell Biol. 1986 Dec;42(2):338–343. [PubMed] [Google Scholar]
  2. Berrow N. S., Morgan N. G. Evidence for the presence of low molecular mass GTP-binding proteins in rat islets of Langerhans. Biochem Soc Trans. 1990 Jun;18(3):485–486. doi: 10.1042/bst0180485. [DOI] [PubMed] [Google Scholar]
  3. Burgoyne R. D., Morgan A., O'Sullivan A. J. The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C. Cell Signal. 1989;1(4):323–334. doi: 10.1016/0898-6568(89)90051-x. [DOI] [PubMed] [Google Scholar]
  4. Chan S. L. Role of alpha 2-adrenoceptors and imidazoline-binding sites in the control of insulin secretion. Clin Sci (Lond) 1993 Dec;85(6):671–677. doi: 10.1042/cs0850671. [DOI] [PubMed] [Google Scholar]
  5. Faulstich H., Zobeley S., Heintz D., Drewes G. Probing the phalloidin binding site of actin. FEBS Lett. 1993 Mar 8;318(3):218–222. doi: 10.1016/0014-5793(93)80515-v. [DOI] [PubMed] [Google Scholar]
  6. Fyles J. M., Cawthorne M. A., Howell S. L. The determination of alpha-adrenergic receptor concentration on rat pancreatic islet cells. Biosci Rep. 1987 Jan;7(1):17–22. doi: 10.1007/BF01122723. [DOI] [PubMed] [Google Scholar]
  7. Hall A. Ras-related GTPases and the cytoskeleton. Mol Biol Cell. 1992 May;3(5):475–479. doi: 10.1091/mbc.3.5.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Howell S. L. The mechanism of insulin secretion. Diabetologia. 1984 May;26(5):319–327. doi: 10.1007/BF00266030. [DOI] [PubMed] [Google Scholar]
  9. Howell S. L., Tyhurst M. Interaction between insulin-storage granules and F-actin in vitro. Biochem J. 1979 Feb 15;178(2):367–371. doi: 10.1042/bj1780367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Howell S. L., Tyhurst M. Regulation of actin polymerizaton in rat islets of Langerhans. Biochem J. 1980 Oct 15;192(1):381–383. doi: 10.1042/bj1920381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hsu W. H., Xiang H. D., Rajan A. S., Boyd A. E., 3rd Activation of alpha 2-adrenergic receptors decreases Ca2+ influx to inhibit insulin secretion in a hamster beta-cell line: an action mediated by a guanosine triphosphate-binding protein. Endocrinology. 1991 Feb;128(2):958–964. doi: 10.1210/endo-128-2-958. [DOI] [PubMed] [Google Scholar]
  12. Hurst R. D., Morgan N. G. Evidence for differential effects of noradrenaline and somatostatin on intracellular messenger systems in rat islets of Langerhans. J Mol Endocrinol. 1990 Jun;4(3):231–237. doi: 10.1677/jme.0.0040231. [DOI] [PubMed] [Google Scholar]
  13. Jesaitis A. J., Erickson R. W., Klotz K. N., Bommakanti R. K., Siemsen D. W. Functional molecular complexes of human N-formyl chemoattractant receptors and actin. J Immunol. 1993 Nov 15;151(10):5653–5665. [PubMed] [Google Scholar]
  14. Jones P. M., Fyles J. M., Persaud S. J., Howell S. L. Catecholamine inhibition of Ca2+-induced insulin secretion from electrically permeabilised islets of Langerhans. FEBS Lett. 1987 Jul 13;219(1):139–144. doi: 10.1016/0014-5793(87)81206-1. [DOI] [PubMed] [Google Scholar]
  15. Lacombe C., Viallard V., Paris H. Identification of alpha 2-adrenoceptors and of non-adrenergic idazoxan binding sites in pancreatic islets from young and adult hamsters. Int J Biochem. 1993 Jul;25(7):1077–1083. doi: 10.1016/0020-711x(93)90124-w. [DOI] [PubMed] [Google Scholar]
  16. Laychock S. G., Bilgin S. Alpha 2-adrenergic inhibition of pancreatic islet glucose utilization is mediated by an inhibitory guanine nucleotide regulatory protein. FEBS Lett. 1987 Jun 22;218(1):7–10. doi: 10.1016/0014-5793(87)81007-4. [DOI] [PubMed] [Google Scholar]
  17. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  18. MacDonald M. J., Kowluru A. Calcium-activated factors in pancreatic islets that inhibit actin polymerization. Arch Biochem Biophys. 1982 Dec;219(2):459–462. doi: 10.1016/0003-9861(82)90178-3. [DOI] [PubMed] [Google Scholar]
  19. Malaisse W. J., Svoboda M., Dufrane S. P., Malaisse-Lagae F., Christophe J. Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets. Biochem Biophys Res Commun. 1984 Oct 15;124(1):190–196. doi: 10.1016/0006-291x(84)90935-5. [DOI] [PubMed] [Google Scholar]
  20. Matuoka K., Shibasaki F., Shibata M., Takenawa T. Ash/Grb-2, a SH2/SH3-containing protein, couples to signaling for mitogenesis and cytoskeletal reorganization by EGF and PDGF. EMBO J. 1993 Sep;12(9):3467–3473. doi: 10.1002/j.1460-2075.1993.tb06021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Melamed I., Downey G. P., Aktories K., Roifman C. M. Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. J Immunol. 1991 Aug 15;147(4):1139–1146. [PubMed] [Google Scholar]
  22. Metz S. A., Rabaglia M. E., Stock J. B., Kowluru A. Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins. Biochem J. 1993 Oct 1;295(Pt 1):31–40. doi: 10.1042/bj2950031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morgan N. G., Montague W. Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans. Biochem J. 1985 Mar 1;226(2):571–576. doi: 10.1042/bj2260571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nelson T. Y., Boyd A. E., 3rd Gelsolin, a Ca2+-dependent actin-binding protein in a hamster insulin-secreting cell line. J Clin Invest. 1985 Mar;75(3):1015–1022. doi: 10.1172/JCI111762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Orci L., Gabbay K. H., Malaisse W. J. Pancreatic beta-cell web: its possible role in insulin secretion. Science. 1972 Mar 10;175(4026):1128–1130. doi: 10.1126/science.175.4026.1128. [DOI] [PubMed] [Google Scholar]
  26. Persaud S. J., Jones P. M., Howell S. L. Effects of Bordetella pertussis toxin on catecholamine inhibition of insulin release from intact and electrically permeabilized rat islets. Biochem J. 1989 Mar 15;258(3):669–675. doi: 10.1042/bj2580669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rasmussen H., Zawalich K. C., Ganesan S., Calle R., Zawalich W. S. Physiology and pathophysiology of insulin secretion. Diabetes Care. 1990 Jun;13(6):655–666. doi: 10.2337/diacare.13.6.655. [DOI] [PubMed] [Google Scholar]
  28. Regazzi R., Ullrich S., Kahn R. A., Wollheim C. B. Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues. Biochem J. 1991 May 1;275(Pt 3):639–644. doi: 10.1042/bj2750639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  30. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  31. Rorsman P., Bokvist K., Ammälä C., Arkhammar P., Berggren P. O., Larsson O., Wåhlander K. Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells. Nature. 1991 Jan 3;349(6304):77–79. doi: 10.1038/349077a0. [DOI] [PubMed] [Google Scholar]
  32. Seaquist E. R., Neal A. R., Shoger K. D., Walseth T. F., Robertson R. P. G-proteins and hormonal inhibition of insulin secretion from HIT-T15 cells and isolated rat islets. Diabetes. 1992 Nov;41(11):1390–1399. doi: 10.2337/diab.41.11.1390. [DOI] [PubMed] [Google Scholar]
  33. Sham R. L., Phatak P. D., Ihne T. P., Abboud C. N., Packman C. H. Signal pathway regulation of interleukin-8-induced actin polymerization in neutrophils. Blood. 1993 Oct 15;82(8):2546–2551. [PubMed] [Google Scholar]
  34. Sheth B., Banks P., Burton D. R., Monk P. N. The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2. Biochem J. 1991 May 1;275(Pt 3):809–811. doi: 10.1042/bj2750809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smolen J. E., Stoehr S. J., Kuczynski B., Koh E. K., Omann G. M. Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils. Biochem J. 1991 Nov 1;279(Pt 3):657–664. doi: 10.1042/bj2790657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Snabes M. C., Boyd A. E., 3rd Increased filamentous actin in islets of Langerhans from fasted hamsters. Biochem Biophys Res Commun. 1982 Jan 15;104(1):207–211. doi: 10.1016/0006-291x(82)91960-x. [DOI] [PubMed] [Google Scholar]
  37. Strubbe J. H., Steffens A. B. Neural control of insulin secretion. Horm Metab Res. 1993 Oct;25(10):507–512. doi: 10.1055/s-2007-1002162. [DOI] [PubMed] [Google Scholar]
  38. Stutchfield J., Howell S. L. The effect of phalloidin on insulin secretion from islets of Langerhans isolated from rat pancreas. FEBS Lett. 1984 Oct 1;175(2):393–396. doi: 10.1016/0014-5793(84)80775-9. [DOI] [PubMed] [Google Scholar]
  39. Swanston-Flatt S. K., Carlsson L., Gylfe E. Actin filament formation in pancreatic beta-cells during glucose stimulation of insulin secretion. FEBS Lett. 1980 Aug 11;117(1):299–302. doi: 10.1016/0014-5793(80)80966-5. [DOI] [PubMed] [Google Scholar]
  40. Särndahl E., Bokoch G. M., Stendahl O., Andersson T. Stimulus-induced dissociation of alpha subunits of heterotrimeric GTP-binding proteins from the cytoskeleton of human neutrophils. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6552–6556. doi: 10.1073/pnas.90.14.6552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ullrich S., Wollheim C. B. GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels. J Biol Chem. 1988 Jun 25;263(18):8615–8620. [PubMed] [Google Scholar]
  42. Vara E., Tamarit-Rodriguez J. Does cyclic guanosine monophosphate mediate noradrenaline-induced inhibition of islet insulin secretion stimulated by glucose and palmitate? Biochem J. 1991 Aug 15;278(Pt 1):243–248. doi: 10.1042/bj2780243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wu Y. N., Yang Y. C., Wagner P. D. Modification of chromaffin cells with pertussis toxin or N-ethylmaleimide lowers cytoskeletal F-actin and enhances Ca(2+)-dependent secretion. J Biol Chem. 1992 Apr 25;267(12):8396–8403. [PubMed] [Google Scholar]

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