Skip to main content
SpringerLink
Log in

Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade

Untersuchungen zum Metabolismus von Metallothionein und alkalischer Phosphatase adulter primärer Rattenhepatocyten: Rolle von foetalem Kälberserum und Agonisten des Phosphoinositol-Effektorsystems

  • Originalbeiträge
  • Published:
Zeitschrift für Ernährungswissenschaft Aims and scope Submit manuscript

Summary

Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A 23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A 23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue.

In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses.

Zusammenfassung

Am Modell primärer Rattenhepatocytenkulturen wurde die Beteiligung von Agonisten des Phosphoinositol-Effektorsystems am Metabolismus von Metallothionein (MT) und alkalischer Phosphatase (ALP) untersucht. Alle Experimente wurden in DMEM/F12 (Ham)-Medium sowohl nach 24stündiger Vorinkubation mit foetalem Kälberserum (FCS) als auch nach Vorinkubation mit Rinderserumalbumin (BSA) durchgeführt. Die Versuche an den Hepatocytenkulturen wurden mit Dexamethason (DEX), Zink (Zn) und den Agonisten des Phosphoinositol-Effektorsystems, der Calciumionophore A 23187, 1,2-Dioctanoyl-sn-glycerol (DiC8), 12-O-Tetradecanoylphorbol-13-acetat (TPA), Angiotensin II (AT), Platelet Activating Factor (PAF) und Arg8-Vasopressin (VP), durchgeführt. Als Parameter wurden die Konzentrationen an MT und die Aktivität der ALP im Zellmaterial bestimmt. Die Vitalität der Kulturen wurde über die Freisetzung der Aktivität der Laktatdehydrogenase (LDH) ins Kulturmedium, der Induzierbarkeit der Tyrosinaminotransferase (TAT) durch DEX und der Anfärbbarkeit der Zellen mit Trypanblau nachgewiesen. Die Zellvitalität wurde durch die FCS-Vorinkubation und DEX-Supplementierung insgesamt verbessert. Unabhängig davon, ob die Zellen mit FCS oder BSA vorinkubiert wurden, stieg der MT-Gehalt der Zellen durch Zn und DEX, als aus der Literatur bekannte direkte Induktoren von MT, um ein Mehrfaches an. Nach FCS-Vorbehandlung war ein moderater Anstieg der ALP-Aktivität nachzuweisen, der jedoch als Vitalitätseffekt interpretiert werden kann. DEX und Zn führten zu keinen Veränderungen der ALP-Aktivität. Alle getesteten Agonisten des Phosphoinositol-Effektorsystems konnten weder MT noch ALP induzieren. Lediglich A 23187 führte zu einer signifikanten konzentrationsabhängigen Reduktion der beiden Parameter. Diese Beobachtung wurde, durch den Anstieg der LDH-Aktivität im Medium und der Zunahme mit Trypanblau anfärbbaren Zellen, auf einen cytotoxischen Effekt von A 23187 zurückgeführt.

Die vorliegende Untersuchung zeigt, daß Agonisten des Phosphoinositol-Effektorsystems nicht in der Lage sind, den Metabolismus von MT und ALP primärer Rattenhepatocyten zu verändern. Die Ergebnisse früherer in vivo Experimente, in denen Agonisten des Phosphoinositol-Effektorsystems den Zn-Stoffwechsel der Leber modulierten, können somit als indirekter systemischer Effekt gedeutet werden.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ALP:

alkaline phosphatase

AT:

angiotensin II

A 23187:

calciumionophore

BSA:

bovine serum albumin

DEX:

dexamethasone

DiC8 :

1,2-dioctanoyl-sn-glycerol

DMSO:

dimethyl sulfoxide

FCS:

fetal calf serum

HEPES:

N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid

IU:

international unit

LDH:

lactate dehydrogenase

MT:

metallothionein

ns:

not significant

P:

probability (level of significance)

PAF:

platelet activating factor

PBS:

phosphate buffered saline

r:

coefficient of correlation

TAT:

tyrosine aminotransferase

TPA:

12-O-tetradecanoylphorbol-13-acetate

TRIS:

tris(hydroxymethyl)aminomethane

U:

unit

VP:

Arg8-vasopressin

References

  1. Barnes D, Sato G (1980) Methods for growth of cultured cells in serum-free medium. Anal Biochem 102:255–270

    Article  CAS  Google Scholar 

  2. Bissel DM, Guzelian PS (1980) Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann NY Acad Sci 349:85–98

    Article  Google Scholar 

  3. Bracken WM, Klaassen CD (1987) Induction of metallothionein in rat primary hepatocyte cultures: evidence for direct and indirect induction. J Tox Environ Health 22:163–174

    Article  CAS  Google Scholar 

  4. Brady FO, Helvig BS (1984) Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver. Am J Physiol 247:E318-E322

    CAS  Google Scholar 

  5. Brady FO, Helvig BS, Funk AE, Garrett SH (1987) The involvement of catecholamines and polypeptide hormones in the multihormonal modulation of rat hepatic thionein levels. In: Kägi JHR, Kojima Y (eds) Metallothionein II. Experientia Supplementum, Vol 52. Birkhäuser, Basel, pp 555–563

    Google Scholar 

  6. Bremner I, Beattie JH (1990) Metallothionein and trace minerals. Ann Rev Nutr 10:63–83

    Article  CAS  Google Scholar 

  7. Cousins RJ (1985) Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol Rev 65:238–309

    CAS  Google Scholar 

  8. Cousins RJ (1989) Systemic transport of zinc. In: Mills CF (ed) Zinc in Human Biology. Springer, London, pp 79–93

    Chapter  Google Scholar 

  9. Cousins RJ, Coppen DE (1987) Regulation of liver zinc metabolism and metallothionein by cAMP, glucagon glucocorticoids and suppression of free radicals by zinc. In: Kägi JHR, Kojima Y (eds) Metallothionein II. Experientia Supplementum, Vol 52. Birkhäuser, Basel, pp 545–553

    Google Scholar 

  10. Dawson RMC, Elliott DC, Elliott WH, Jones KM (1986) Data for Biochemical Research. 3d ed, Clarrendon Press, Oxford, p 543

    Google Scholar 

  11. Deutsche Gesellschaft für Klinische Chemie (1970) Standardisierung der Methoden zur Bestimmung von Enzymaktivitäten in biologischen Flüssigkeiten. Z Klin Chem Klin Biochem 8:188–198, 658–660

    Google Scholar 

  12. Deutsche Gesellschaft für Klinische Chemie (1972) Standard-Methode zur Bestimmung der alkalischen Phosphatase (AP). Z Klin Chem Klin Biochem 10:191

    Google Scholar 

  13. Eaton DL, Toal BF (1982) Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Tox Appl Pharm 66:134–142

    Article  CAS  Google Scholar 

  14. Exton JH (1988) The roles of calcium and phosphoinositides in the mechanism of α1-adrenergic and other agonists. Rev Physiol Biochem Pharmacol 111:117–224

    Article  CAS  Google Scholar 

  15. Granner DK, Tomkins GM (1970) Tyrosine aminotransferase (rat liver). In: Colowick SP, Kaplan N (eds) Methods in Enzymology, Vol XVIIIA, Academic Press, New York, pp 633–637

    Google Scholar 

  16. Helvig BS, Brady FO (1984) Effects of glucagon, Arg-vasopressin, and angiotensin II on rat hepatic zinc thionein levels. Life Sci 35:2513–2518

    Article  CAS  Google Scholar 

  17. Herz F (1984) Induction of alkaline phosphatase expression. In: Stigbrand T, Fishman WH (eds) Human alkaline phosphatases. Alan Liss, New York, pp 139–166

    Google Scholar 

  18. Hidalgo J, Borrás M, Garvey JS, Armario A (1990) Liver, brain, and heart metallothionein induction by stress. J Neurochem 55:651–654

    Article  CAS  Google Scholar 

  19. Holinka CF, Hata H, Kuramoto H, Guripe E (1986) Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishikawa line). Cancer Res 46:2771–2774

    CAS  Google Scholar 

  20. Horiuti Y, Nakamura Y, Ichihara A (1982) Role of serum in maintenance of functional hepatocytes in primary culture. J Biochem 92:1995–2000

    Article  Google Scholar 

  21. Imbra RJ, Karin M (1987) Metallothionein gene expression is regulated by serum factors and by activators of protein kinase C. Mol Cell Biol 7:1358–1363

    Article  CAS  Google Scholar 

  22. Karasawa M, Hosoi J, Hashiba H, Nose K, Tohyama C, Abe E, Suda T, Kuroki T (1987) Regulation of metallothionein gene expression by 1α,25-dihydroxyvitamin D3 in cultured cells and in mice. Proc Natl Acad Sci USA 84:8810–8813

    Article  CAS  Google Scholar 

  23. Kato S, Aoyama K, Nakamura T, Ichihara A (1979) Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membrane during culture. J Biochem 86:1419–1425

    Article  CAS  Google Scholar 

  24. Kirchgessner M, Roth H-P (1983) Nutritional influence of zinc on the activity of enzymes and hormones. In: Sigl H (ed) Metal Ions in Biological Systems. Vol 15, Zinc and its Role in Biology and Nutrition. Marcel Dekker, New York, pp 363–414

    Google Scholar 

  25. Krämer K, Markwitan A, Montigel C, Pallauf J (1992) Einfluß eines Aktivators der Proteinkinase C (TPA) und eines Ca2+-mobilisierenden Agonisten (A 23187) auf den Zinkmetabolismus der Ratte. Z Ernährungswiss 31:246–257

    Article  Google Scholar 

  26. Krämer K, Markwitan A, Menne A, Pallauf J (1993) Zinc metabolism in fasted rats. J Trace Elem Electrolytes Health Dis (in press)

  27. Laishes BA, Williams GM (1976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12:821–832

    Article  CAS  Google Scholar 

  28. Lehnert V, Weiss C, Weintraut H, Pallauf J (1993) Bestimmung des freien cytosolischen Ca2+-Spiegels in isolierten Rattenhepatocyten mit Hilfe der digitalen Bildanalyse unter Verwendung des Fluoreszenzmarkers Fura 2. Proc Soc Nutr Physiol 1:107

    Google Scholar 

  29. Lindl T, Bauer J (1989) Zell- und Gewebekultur. 2. Aufl., Gustav Fischer, Stuttgart

    Google Scholar 

  30. Maurer HR (1986) Towards chemically-defined, serum-free media for mammalian cell culture. In: Freshney RF (ed) Animal Cell Culture. A Practical Approach. IRL Press, Oxford, pp 13–30

    Google Scholar 

  31. Michell RH, Drummond AH, Downes CP (eds) (1989) Inositol Lipids in Cell Signalling. Academic Press, London

    Google Scholar 

  32. Palmiter RD (1987) Molecular biology of metallothionein gene expression. In: Kägi JHR, Kojima Y (eds) Metallothionein II. Experientia Supplementum, Vol 52. Birkhäuser, Basel, pp 63–80

    Google Scholar 

  33. Sato M, Mehra RK, Bremner I (1984) Measurement of plasma metallothionein-I in the assessment of zinc status of zinc-deficient and stressed rats. J Nutr 114:1683–1689

    CAS  Google Scholar 

  34. Schroeder JJ, Cousins RJ (1991) Maintenance of zinc-dependent hepatic functions in rat hepatocytes cultured without added zinc. J Nutr 121:844–853

    CAS  Google Scholar 

  35. Seglen PO (1976) Preparation of isolated rat liver cells. Meth Cell Biol 13:29–83

    Article  CAS  Google Scholar 

  36. Sorimachi K, Hayashi T, Takaoka T, Niwa A, Yasumura Y (1988) Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in human liver cells continuously cultured in serum-free medium. Cell Struc Funct 13:1–11

    Article  CAS  Google Scholar 

  37. Strom SC, Michalopoulos G (1982) Collagen as a substrate for cell growth and differentiation. Methods Enzymol 82:544–555

    Article  CAS  Google Scholar 

  38. Tanaka K, Sato M, Tomita Y, Ichihara A (1978) Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis. J Biochem 84:937–946

    Article  CAS  Google Scholar 

  39. Tsao M-S, Nelson KG, Grisham JW (1984) Biochemical effects of 12-O-tetradecanoylphorbol-13-acetate, retinoic acid, phenobarbital, and 5-azacytidine on a normal rat liver epithelial cell line. J Cell Physiol 121:1–6

    Article  CAS  Google Scholar 

  40. Watanabe J, Kanamura S, Asada-Kubota M, Kanai K, Oka M (1983) Observation of cytochemical alkaline phosphatase on the plasma membrane of cultured rat hepatocytes by backscattered electron imaging. Anat Rec 207:635–642

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Tierernährung, Justus-Liebig-Universität Gießen, Germany

    K. Krämer, A. Markwitan & J. Pallauf

Authors
  1. K. Krämer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Markwitan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Pallauf
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Krämer, K., Markwitan, A. & Pallauf, J. Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade. Z Ernährungswiss 32, 176–186 (1993). https://doi.org/10.1007/BF01610728

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01610728

Key words

Schlüsselwörter

Search

Navigation