Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Apr 1;267(1):227–232. doi: 10.1042/bj2670227

The role of cytosolic free Ca2+ and protein kinase C in acetylcholine-induced insulin release in the clonal beta-cell line, HIT-T15.

S J Hughes 1, J G Chalk 1, S J Ashcroft 1
PMCID: PMC1131268  PMID: 2183793

Abstract

We examined the contribution of signal-transduction pathways to acetylcholine-induced insulin release in the clonal beta-cell line HIT-T15. To assess the importance of changes in cytosolic free Ca2+ [( Ca2+]i), we studied time courses of the effects of glucose and acetylcholine on [Ca2+]i and insulin release in quin 2-loaded HIT cells. Incubation in the presence of glucose (2 mM) resulted in a sustained increase in [Ca2+]i in HIT cells from 98 +/- 7 nM to 195 +/- 12 nM measured after 9 min, whereas subsequent addition of acetylcholine (50 microM) produced a transient increase in [Ca2+]i which reached a peak after 30 s (at 274 +/- 10 nM), returning to pre-stimulus levels after 3 min. In contrast, incubation of HIT cells with acetylcholine in the presence of glucose produced a sustained increase in insulin release over and above that stimulated by glucose alone; after 10 min acetylcholine had potentiated glucose-stimulated insulin release by an additional increment of 135%. The transient increase in [Ca2+]i induced by acetylcholine was dose-dependent, and was prevented by omission of glucose or extracellular Ca2+ from the incubation medium. It was also inhibited by inclusion of 50 microM-verapamil in the incubation medium (by 87 +/- 3%) or by decreasing the Na+ concentration in the medium (by 73 +/- 6%). To evaluate the role of the protein kinase C pathway, we have pretreated HIT cells with the phorbol ester 12-O-tetradecanoylphorbol acetate (TPA), to deplete the protein kinase C activity, and have compared their secretory activity with that of control cells. Protein kinase C activity was decreased by 73% in HIT cells cultured in the presence of 200 nM-TPA for 22-24 h. TPA pre-treatment also significantly decreased the insulin content of HIT cells, but had no effect on cell number or the increases in [Ca2+]i induced by glucose or acetylcholine. TPA-pre-treated cells responded comparatively less well to secretagogues than did control cells: glucose-stimulated insulin release was decreased by 40%, whereas potentiation by TPA was significantly decreased by 50% in comparison with control cells (P less than 0.05, n = 24). Acetylcholine (50 microM) potentiated glucose-stimulated insulin release by 61% in control cells. This effect was abolished in HIT cells pre-treated with TPA, whereas these cells still retained their normal secretory response to stimulation by forskolin. These data suggest that an early increase in [Ca2+]i may be important for the initial increase in insulin release induced by acetylcholine in HIT cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Full text

PDF
227

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams J. C., Gullick W. J. Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines. Biochem J. 1989 Feb 1;257(3):905–911. doi: 10.1042/bj2570905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashcroft S. J., Crossley J. R. The effects of glucose, N-acetylglucosamine, glyceraldehyde and other sugars on insulin release in vivo. Diabetologia. 1975 Aug;11(4):279–284. doi: 10.1007/BF00422392. [DOI] [PubMed] [Google Scholar]
  3. Ashcroft S. J., Hammonds P., Harrison D. E. Insulin secretory responses of a clonal cell line of simian virus 40-transformed B cells. Diabetologia. 1986 Oct;29(10):727–733. doi: 10.1007/BF00870283. [DOI] [PubMed] [Google Scholar]
  4. Ashcroft S. J., Stubbs M. The glucose sensor in HIT cells is the glucose transporter. FEBS Lett. 1987 Jul 27;219(2):311–315. doi: 10.1016/0014-5793(87)80242-9. [DOI] [PubMed] [Google Scholar]
  5. Best L., Malaisse W. J. Nutrient and hormone-neurotransmitter stimuli induce hydrolysis of polyphosphoinositides in rat pancreatic islets. Endocrinology. 1984 Nov;115(5):1814–1820. doi: 10.1210/endo-115-5-1814. [DOI] [PubMed] [Google Scholar]
  6. Burr I. M., Slonim A. E., Burke V., Fletcher T. Extracellular calcium and adrenergic and cholinergic effects on islet beta-cell function. Am J Physiol. 1976 Oct;231(4):1246–1249. doi: 10.1152/ajplegacy.1976.231.4.1246. [DOI] [PubMed] [Google Scholar]
  7. Christie M. R., Ashcroft S. J. Substrates for cyclic AMP-dependent protein kinase in islets of Langerhans. Studies with forskolin and catalytic subunit. Biochem J. 1985 May 1;227(3):727–736. doi: 10.1042/bj2270727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gagerman E., Sehlin J., Täljedal I. B. Effects of acetylcholine on ion fluxes and chlorotetracycline fluorescence in pancreatic islets. J Physiol. 1980 Mar;300:505–513. doi: 10.1113/jphysiol.1980.sp013175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gaines K. L., Hamilton S., Boyd A. E., 3rd Characterization of the sulfonylurea receptor on beta cell membranes. J Biol Chem. 1988 Feb 25;263(6):2589–2592. [PubMed] [Google Scholar]
  10. Garcia M. C., Hermans M. P., Henquin J. C. Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets. Biochem J. 1988 Aug 15;254(1):211–218. doi: 10.1042/bj2540211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griffey M. A., Conaway H. H., Whitney J. E. Extracellular calcium and acetylcholine-stimualted insulin secretion. Diabetes. 1974 Jun;23(6):494–498. doi: 10.2337/diab.23.6.494. [DOI] [PubMed] [Google Scholar]
  12. Hellman B., Gylfe E. Mobilization of different intracellular calcium pools after activation of muscarinic receptors in pancreatic beta-cells. Pharmacology. 1986;32(5):257–267. doi: 10.1159/000138178. [DOI] [PubMed] [Google Scholar]
  13. Henquin J. C., Bozem M., Schmeer W., Nenquin M. Distinct mechanisms for two amplification systems of insulin release. Biochem J. 1987 Sep 1;246(2):393–399. doi: 10.1042/bj2460393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henquin J. C., Garcia M. C., Bozem M., Hermans M. P., Nenquin M. Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx. Endocrinology. 1988 May;122(5):2134–2142. doi: 10.1210/endo-122-5-2134. [DOI] [PubMed] [Google Scholar]
  15. Hermans M. P., Henquin J. C. Relative importance of extracellular and intracellular Ca2+ for acetylcholine stimulation of insulin release in mouse islets. Diabetes. 1989 Feb;38(2):198–204. doi: 10.2337/diab.38.2.198. [DOI] [PubMed] [Google Scholar]
  16. Hermans M. P., Schmeer W., Henquin J. C. Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells. Endocrinology. 1987 May;120(5):1765–1773. doi: 10.1210/endo-120-5-1765. [DOI] [PubMed] [Google Scholar]
  17. Hii C. S., Jones P. M., Persaud S. J., Howell S. L. A re-assessment of the role of protein kinase C in glucose-stimulated insulin secretion. Biochem J. 1987 Sep 1;246(2):489–493. doi: 10.1042/bj2460489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hill R. S., Boyd A. E., 3rd Perifusion of a clonal cell line of Simian virus 40-transformed beta cells. Insulin secretory dynamics in response to glucose, 3-isobutyl-1-methylxanthine, and potassium. Diabetes. 1985 Feb;34(2):115–120. doi: 10.2337/diab.34.2.115. [DOI] [PubMed] [Google Scholar]
  19. Hughes S. J., Ashcroft S. J. Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal beta-cell line HIT-T15. J Mol Endocrinol. 1988 Jul;1(1):13–17. doi: 10.1677/jme.0.0010013. [DOI] [PubMed] [Google Scholar]
  20. Hughes S. J., Ashcroft S. J. Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets. Biochem J. 1988 Feb 1;249(3):825–830. doi: 10.1042/bj2490825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hughes S. J., Chalk J. G., Ashcroft S. J. Effect of secretagogues on cytosolic free Ca2+ and insulin release at different extracellular Ca2+ concentrations in the hamster clonal beta-cell line HIT-T15. Mol Cell Endocrinol. 1989 Aug;65(1-2):35–41. doi: 10.1016/0303-7207(89)90162-7. [DOI] [PubMed] [Google Scholar]
  22. Hughes S. J., Christie M. R., Ashcroft S. J. Potentiators of insulin secretion modulate Ca2+ sensitivity in rat pancreatic islets. Mol Cell Endocrinol. 1987 Apr;50(3):231–236. doi: 10.1016/0303-7207(87)90021-9. [DOI] [PubMed] [Google Scholar]
  23. Jones P. M., Stutchfield J., Howell S. L. Effects of Ca2+ and a phorbol ester on insulin secretion from islets of Langerhans permeabilised by high-voltage discharge. FEBS Lett. 1985 Oct 21;191(1):102–106. doi: 10.1016/0014-5793(85)81002-4. [DOI] [PubMed] [Google Scholar]
  24. Keahey H. H., Rajan A. S., Boyd A. E., 3rd, Kunze D. L. Characterization of voltage-dependent Ca2+ channels in beta-cell line. Diabetes. 1989 Feb;38(2):188–193. doi: 10.2337/diab.38.2.188. [DOI] [PubMed] [Google Scholar]
  25. Klip A., Ramlal T. Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture. Biochem J. 1987 Feb 15;242(1):131–136. doi: 10.1042/bj2420131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mathias P. C., Carpinelli A. R., Billaudel B., Garcia-Morales P., Valverde I., Malaisse W. J. Cholinergic stimulation of ion fluxes in pancreatic islets. Biochem Pharmacol. 1985 Oct 1;34(19):3451–3457. doi: 10.1016/0006-2952(85)90717-8. [DOI] [PubMed] [Google Scholar]
  27. Metz S. A. Perspectives in diabetes. Is protein kinase C required for physiologic insulin release? Diabetes. 1988 Jan;37(1):3–7. doi: 10.2337/diab.37.1.3. [DOI] [PubMed] [Google Scholar]
  28. Nelson T. Y., Gaines K. L., Rajan A. S., Berg M., Boyd A. E., 3rd Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells. J Biol Chem. 1987 Feb 25;262(6):2608–2612. [PubMed] [Google Scholar]
  29. Nenquin M., Awouters P., Mathot F., Henquin J. C. Distinct effects of acetylcholine and glucose on 45calcium and 86rubidium efflux from mouse pancreatic islets. FEBS Lett. 1984 Oct 29;176(2):457–461. doi: 10.1016/0014-5793(84)81218-1. [DOI] [PubMed] [Google Scholar]
  30. Niki I., Kelly R. P., Ashcroft S. J., Ashcroft F. M. ATP-sensitive K-channels in HIT T15 beta-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding. Pflugers Arch. 1989 Oct;415(1):47–55. doi: 10.1007/BF00373140. [DOI] [PubMed] [Google Scholar]
  31. Nilsson T., Arkhammar P., Hallberg A., Hellman B., Berggren P. O. Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells. Biochem J. 1987 Dec 1;248(2):329–336. doi: 10.1042/bj2480329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Prentki M., Glennon M. C., Thomas A. P., Morris R. L., Matschinsky F. M., Corkey B. E. Cell-specific patterns of oscillating free Ca2+ in carbamylcholine-stimulated insulinoma cells. J Biol Chem. 1988 Aug 15;263(23):11044–11047. [PubMed] [Google Scholar]
  33. Rorsman P., Abrahamsson H. Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+. Acta Physiol Scand. 1985 Dec;125(4):639–647. doi: 10.1111/j.1748-1716.1985.tb07766.x. [DOI] [PubMed] [Google Scholar]
  34. Santerre R. F., Cook R. A., Crisel R. M., Sharp J. D., Schmidt R. J., Williams D. C., Wilson C. P. Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4339–4343. doi: 10.1073/pnas.78.7.4339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sánchez-Andrés J. V., Ripoll C., Soria B. Evidence that muscarinic potentiation of insulin release is initiated by an early transient calcium entry. FEBS Lett. 1988 Apr 11;231(1):143–147. doi: 10.1016/0014-5793(88)80719-1. [DOI] [PubMed] [Google Scholar]
  36. Tamagawa T., Niki H., Niki A. Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester. FEBS Lett. 1985 Apr 22;183(2):430–432. doi: 10.1016/0014-5793(85)80825-5. [DOI] [PubMed] [Google Scholar]
  37. Wollheim C. B., Biden T. J. Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells. J Biol Chem. 1986 Jun 25;261(18):8314–8319. [PubMed] [Google Scholar]
  38. Wollheim C. B., Dunne M. J., Peter-Riesch B., Bruzzone R., Pozzan T., Petersen O. H. Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels. EMBO J. 1988 Aug;7(8):2443–2449. doi: 10.1002/j.1460-2075.1988.tb03090.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wollheim C. B., Siegel E. G., Sharp G. W. Dependency of acetylcholine-induced insulin release on Ca++ uptake by rat pancreatic islets. Endocrinology. 1980 Oct;107(4):924–929. doi: 10.1210/endo-107-4-924. [DOI] [PubMed] [Google Scholar]
  40. Yamatani T., Chiba T., Kadowaki S., Hishikawa R., Yamaguchi A., Inui T., Fujita T., Kawazu S. Dual action of protein kinase C activation in the regulation of insulin release by muscarinic agonist from rat insulinoma cell line (RINr). Endocrinology. 1988 Jun;122(6):2826–2832. doi: 10.1210/endo-122-6-2826. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES