Histochemie

, Volume 7, Issue 2, pp 116–131 | Cite as

Cytochemical studies of hydrolytic and oxidative enzymes in sections and isolated cells from the endocrine pancreas of guinea-pigs

  • Birger Petersson
Article
Received:

Summary

Histochemical staining methods for phosphatases and dehydrogenases were applied to isolated cells, prepared from microdissected islets of guinea-pigs. The enzyme reactions in these cells were compared with those obtained in cryostat sections. The findings were as follows:
  1. 1.

    Enzyme activity was well preserved in the free islet cells with a more distinct intracellular localization than in the sections. After incubation of the free islet cells for detection of dehydrogenase activity it was still possible after subsequent granule staining, to identify A2 cells.

     
  2. 2.

    Incubations for acid or alkaline phosphatases gave negative reactions in the islet cells, while incubation for glucose-6-phosphatase showed an intense activity in the islet B cells with the reaction products localized to the nuclear membrane and as irregular aggregations dispersed in the cytoplasm. After incubation for diphosphatases no activity was noted with ADP as substrate, while with IDP and TPP there was a distinct reaction in the Golgi zone of most of the islet cells. There was a positive reaction in the islet cells for ATPase and the cytoplasmic reaction product was mainly localized to mitochondria-like structures.

     
  3. 3.

    Intense reactions for NADP-diaphorase and α-glycerophosphatate-Menadion reductase were observed in the islets. Most islet cells displayed moderate activity of LDH and SDH while the LDH activity in the A2 cells was somewhat stronger. The intracellular reaction products of all the oxidative enzymes appeared to be accumulated in dot-like structures dispersed in the cytoplasm.

     

Keywords

Islet Cell Oxidative Enzyme Histochemical Staining Cryostat Section Endocrine Pancreas 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

ADP

adenosine 5′-diphosphate

ATP

adenosine 5′-triphosphate

ATPase

adenosine triphosphatase

IDP

inosine 5′-diphosphate

LDH

lactic dehydrogenase

NAD

nicotinamide-adenine dinucleotide, oxidized form

NADP

nicotinamideadenine dinucleotide phosphate, oxidized form

NADPH

nicotinamide-adenine dinucleotide phosphate, reduced form

Nitro-BT

2,2′-di-p-nitrophenyl-5, 5′-diphenyl-3, 3′-(3,3′-dimetoxy-4, 4′-biphenylene)-ditetrazolium chloride

PMS

phenazine metosulphate

SDH

succino dehydrogenase

TPP

thiamine pyrophosphate chloride

This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Allen, J. M.: The properties of Golgi-associated nuoleoside diphosphatase and thiamine pyrophosphatase: I. Cytochemical analysis. J. Histochem. Cytochem. 11, 529–541 (1963).Google Scholar
  2. Andersson, A., and C. Hellerström: Unpublished observations 1966.Google Scholar
  3. Barter, R., J. P. Danielli, and H. G. Davies: A quantitative cytochemical method for estimating alkaline phosphatase activity. Proc. roy. Soc. B 144, 412–426 (1956).Google Scholar
  4. Björkman, N., C. Hellerström, B. Hellman, and U. Rothman: Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse. Z. Zellforsch. 59, 535–554 (1963).Google Scholar
  5. Brolin, S. E., G. Lundquist, and B. Petersson: Isolation of A2 cells from the pancreatic islets for ultramicrochemical analyses. Acta endocr. (Kbh.) in press (1966).Google Scholar
  6. Burstone, M. S.: Enzyme histochemistry and its application in the study of neoplasms. New York: Academic Press 1962.Google Scholar
  7. Cavallero, C., and E. Solcia: Cytologic and cytochemical studies on the pancreatic islets. In: S. Brolin, B. Hellman, and H. Knutson (eds.), The structure and metabolism of the pancreatic islets, p. 83–97. London: Pergamon Press 1964.Google Scholar
  8. Cohen, S., and S. Way: Histochemical demonstration of pentose shunt activity in smears from the uterine cervix. Brit. med. J. 1966 I, 88–89.Google Scholar
  9. Conklin, J. L., M. M. Dewey, and R. H. Kahn: Cytochemical localization of certain oxidative enzymes. Amer. J. Anat. 110, 19–27 (1962).Google Scholar
  10. Defendi, V., and B. Pearson: Quantitative estimation of succinic dehydrogenase activity in a single microscopic tissue section. J. Histochem. Cytochem. 3, 61–69 (1955).Google Scholar
  11. Dixon, M., and E. C. Webb: Enzymes, 2nd ed. London: Longmans 1964.Google Scholar
  12. Doyle, W. L.: Quantitative evaluation of Gomori histochemical preparations. Science 111, 64–65 (1950).Google Scholar
  13. —, J. H. Omoto, and M. E. Doyle: Estimation of phosphatase in histological preparations. Exp. Cell Res. 2, 20–38 (1951).Google Scholar
  14. Essner, E., J. Fogh, and P. Fabrizio: Localization of mitochondrial adenosine triphosphatase activity in cultured human cells. J. Histochem. Cytochem. 13, 647–656 (1965).Google Scholar
  15. Fahimi, H. D., and C. R. Amarasingham: Cytochemical localization of lactic dehydrogenase in white skeletal muscle. J. Cell Biol. 22, 29–48 (1964).Google Scholar
  16. Gepts, W., and D. Toussaint: Effect of cortisone, growth hormone and hypophysectomy on the enzymatic activity on the pancreatic islets. In: S. Brolin, B. Hellman, and H. Knutson (eds.), The structure and metabolism of the pancreatic islets, p. 357–377. London: Pergamon Press 1964.Google Scholar
  17. Gössner, W.: Zur Enzymhistochemie der Langerhansschen Inseln. Verh. dtsch. Ges. Path. 42, 125–130 (1959).Google Scholar
  18. —: Die Enzymhistochemie der Langerhansschen Inseln. In: K. Oberdisse u. K. Jahnke (eds.), Fortschritte der Diabetesforschung, S. 140–145. Stuttgart: Georg Thieme 1963.Google Scholar
  19. Gomori, G.: Microscopic histochemistry. Chicago: Chicago University Press 1952.Google Scholar
  20. Hellerström, C.: Effects of steroid diabetes on the pancreatic islets of guinea-pigs with special reference to the A1 cells. Acta Soc. Med. upsalien. 68, 1–16 (1963).Google Scholar
  21. —: A method for the microdissection of intact pancreatic islets of mammals. Acta endocr. (Kbh.) 45, 122–132 (1964).Google Scholar
  22. —: Oxygen consumption of isolated pancreatic islets of mice studies with the Cartesian-diver micro-gasometer. Biochem. J. 98, 7c-9c (1966).Google Scholar
  23. —, and B. Hellman: Some aspects of silver impregnation of the islets of Langerhans in the rat. Acta endocr. (Kbh.) 35, 518–532 (1960).Google Scholar
  24. —: Demonstration of oxidative enzymes in the human pancreas with special reference to the islets of Langerhans. Acta path, microbiol. scand. 55, 385–394 (1962).Google Scholar
  25. Hellerström, C., and B. Petersson: Unpublished observations 1966.Google Scholar
  26. Hellman, B.: Microchemical analyses of pancreatic beta cells isolated from mammals. In: B. S. Leibel, and G. A. Wrenshall (eds.), On the nature and treatment of diabetes, p. 58–72. Amsterdam: Excerpta Medica Foundation 1965a.Google Scholar
  27. —: Fluorometric assays of glutamic-pyruvic transaminase activity in microdissected pancreatic islets from obese-hyperglycemic mice. Acta physiol. scand. 65, 357–363 (1965b).Google Scholar
  28. —, and C. Hellerström: Histochemical studies of glucose-6-phosphatase, adenosine triphosphatase and amylo phosphorylase in the pancreatic islets of normal and obese-hyperglycemic mice. Acta endocr. (Kbh.) 39, 474–482 (1962a).Google Scholar
  29. —: Oxidative enzymes in the pancreatic islets of normal and obese-hyperglycemic mice. Z. Zellforsch. 56, 97–106 (1962b).Google Scholar
  30. —, S. Larsson, and S. Brolin: Histochemical observation on the pancreatic islets in normal and obese-hyperglycemic mice. Z. Zellforsch. 56, 235–246 (1961).Google Scholar
  31. Hess, R., D. G. Scarpelli, and A. G. E. Pearse: The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J. biophys. biochem. Cytol. 4, 753–760 (1958).Google Scholar
  32. Holter, H., S. Løvtrup, and I. Rubin: Chemistry of the histochemical phosphatase reaction. Acta chem. scand. 5, 194–195 (1951).Google Scholar
  33. Keen, H., R. Sells, and R. J. Jarrett: A method for the study of the metabolism of isolated mammalian islets of Langerhans and some preliminary results. Diabetologia 1, 28–32 (1965).Google Scholar
  34. Kissane, J. M., and S. E. Brolin: Enzymatic activity of pancreatic islets and acini in normal and tolbutamide-treated rats. J. Histochem. Cytochem. 11, 197–201 (1963).Google Scholar
  35. Lacy, P. E.: Quantitative histochemistry of the islets of Langerhans. I. Lactic, malic, glucose-6-phosphate and 6-phosphogluconic dehydrogenase activities of beta cells and acini. Diabetes 11, 96–100 (1962).Google Scholar
  36. Lazarus, S. S.: Demonstration of glucose-6-phosphatase in mammalian pancreas. Proc. Soc. exp. Biol. (N.Y.) 101, 819–822 (1959).Google Scholar
  37. —, and H. Barden: Histochemistry of rabbit pancreatic B cell enzymes and effect of cortisone and alloxan. Fed. Proc. 20, 192 (1961).Google Scholar
  38. —: Histochemistry and electron microscopy of mitochondrial adenosinetriphosphatase. J. Histochem. Cytochem. 10, 285–293 (1962a).Google Scholar
  39. —: Demonstration of nucleoside diphosphatase in the Golgi apparatus at alkaline pH. J. Histochem. Cytochem. 10, 368–369 (1962b).Google Scholar
  40. —: Ultramicroscopic localization of mitochondrial adenosine-triphosphatase. J. Ultrastruct. Res. 10, 189–193 (1964).Google Scholar
  41. —: Specificity and ultrastructural localization of pancreatic B cell glucose-6-phosphatase. Diabetes 14, 146–156 (1965).Google Scholar
  42. —, and B. W. Volk: Submicroscopic distribution and function of phosphatases in rabbit pancreatic B cells. Excerpta med. (Amst.) Int. Congr. Ser. 74, 68 (1964).Google Scholar
  43. —, and, M. Bradshaw: Oxidative pathways in pancreatic B cells. Proc. Soc. exp. Biol. (N.Y.) 102, 463–468 (1959).Google Scholar
  44. —, and, B. W. Volk: Histochemical and electron microsopic studies of a functioning insulinoma. Lab. Invest. 11, 1279–1294 (1962).Google Scholar
  45. Miller, L. A., and A. Goldfeder: Comparison of adenosine triphosphatase activity of nuclei and mitochondria from mouse mammary adenocarcinoma. Cancer Res. 21, 64–70 (1961).Google Scholar
  46. Nachlas, M. M., A. C. Young, and A. M. Seligman: Problems of enzymatic localization by chemical reactions applied to tissue sections. J. Histochem. Cytochem. 5, 565–583 (1957).Google Scholar
  47. Novikoff, A. B., and E. Essner: Pathological changes in cytoplasmic organelles. Fed. Proc. 21, 1130–1142 (1962).Google Scholar
  48. —, D. H. Hausman, and E. Podber: The localization of adenosine triphosphatase in liver: in situ staining and cell fractionation studies. J. Histochem. Cytochem. 6, 61–71 (1958).Google Scholar
  49. Padykula, H., and E. Herman: Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J. Histochem. Cytochem. 3, 161–169 (1955).Google Scholar
  50. Pearse, A. G. E.: Histochemistry. Theoretical and applied, 2nd ed. London: J. & A. Churchill Ltd. 1960.Google Scholar
  51. Petersson, B.: Isolation and characterization of different types of pancreatic islet cells in guinea-pigs. Acta endocr. (Kbh.) in press (1966).Google Scholar
  52. Petkov, P., J. Verne, and R. Wegmann: Sur l'apparition de glucose-6-phosphatase dans le pancreas du rat au cours diabete alloxanique. Experientia (Basel) 21, 530–531 (1965).Google Scholar
  53. Rosa, C. G.: The use of cytochemical tools for study of oxidative enzymatic activity in cells of vaginal smear preparations of humans. Ann. Histochim. 6, 293–304 (1961).Google Scholar
  54. —, and K. C. Tsou: The use of tetranitro-blue tetrazolium for the cytochemical localization of succinic dehydrogenase. J. Cell Biol. 16, 445–454 (1963).Google Scholar
  55. Rosenbaum, R. M.: The intracellular localization of enzymes. In: W. Graumann, and K. Neumann (eds.), Handbuch der Histochemie, Bd VII/4, S. 1–108. Stuttgart: Gustav Fischer 1964.Google Scholar
  56. Scarpelli, D. G., R. Hess, and A. G. E. Pearse: The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J. biophys. biochem. Cytol. 4, 747–752 (1958).Google Scholar
  57. Shelton, E., and M. E. Rice: Comparison of the reduction of two tetrazolium salts with succinoxidase activity of tissue homogenates. J. nat. Cancer Inst. 18, 117–125 (1957).Google Scholar
  58. Shugar, D., A. Szenberg, and H. Sierakowska: Quantitative histochemistry by means of radioactive indicators-alkaline phosphatase. Exp. Cell Res. 13, 424–426 (1957).Google Scholar
  59. Täljedal, I.-B., B. Hellman, and C. Hellerström: Quantitative studies on isolated pancreatic islets of mammals: Adenosine triphosphatase activity in normal and obesehyperglycemic mice. J. Histochem. Cytochem. 12, 491–497 (1964).Google Scholar
  60. Takeda, Y., A. Ichihara, H. Tanioka, and H. Inoue: The biochemistry of animal cells. I. The effect of corticosteroids on leakage of enzymes from dispersed rat liver cells. J. biol. Chem. 239, 3590–3596 (1964).Google Scholar
  61. Trump, B. F., P. J. Goldblatt, V. S. Waravdekar, C. Griffin, and R. E. Stowell: Electron microscopic and biochemical studies of cellular alterations following rapid freezing and thawing. J. Histochem. Cytochem. 12, 24 (1964).Google Scholar
  62. Wegmann, R., et P. Petkov: Histoenzymologie du pancréas endocrine de l'homme. Ann. Histochim. 10, 93–100 (1965).Google Scholar
  63. Williams, G. Z., and A. C. Peacock: Ultraviolet studies of tetrazolium reduction in living cells. Exp. Cell Res., Suppl. 7, 131–144 (1959).Google Scholar
  64. Wulff, H. R.: Histochemical studies of leukocytes from an inflammatory exudate. II. Succinic dehydrogenase, mitochondrial α-glycerophosphate dehydrogenase and di- and triphosphopyridine nucleotide diaphorases. Acta haemat. (Basel) 29, 208–217 (1963a).Google Scholar
  65. —: Histochemical studies of leukocytes from an inflammatory exudate. III. Di- and triphosphopyridine nucleotide-linked dehydrogenases. Acta haemat. (Basel) 30, 16–24 (1963b).Google Scholar

Copyright information

© Springer-Verlag 1966

Authors and Affiliations

  • Birger Petersson
    • 1
  1. 1.Histological DepartmentUniversity of UppsalaUppsalaSweden

Personalised recommendations