Abstract
O2 plays a dominant role in the metabolism and viability of cells; changes in O2 supply lead to many physiological responses in the cell. Recent reports have shown that hypoxia induces the transcription of a number of genes, among them those for the glycolytic enzymes. We have investigated signalling events that may lead to enhanced activity of lactate dehydrogenase (LDH) in cultured vascular smooth muscle (VSM) cells derived from rat aorta, grown under hypoxic conditions (1% versus 20% O2). LDH was chosen because this enzyme exhibits one of the largest increases in activity among the glycolytic enzymes after hypoxic stimulation of cells. Hypoxic exposure of VSM cells for 24 h resulted in a 2-fold increase in LDH activity and in a 2.5-fold increase in intracellular cAMP levels. Agents that activate adenylate cyclase, such as forskolin, cholera toxin and 1-methyl-3-isobutylxanthine (IBMX), and thus increase cAMP production, significantly induced LDH activity. Moreover, induction of LDH activity by hypoxia was prevented in the presence of the protein kinase A inhibitor N-[2-(methyl-amino)ethyl]-5-isoquinolinsulphonamide dihydrochloride (H-8), and the cyclooxygenase inhibitor indomethacin. In contrast to the cAMP-stimulating agents, stable cGMP analogues (dibutyryl-cGMP, 8 bromo-cGMP), activators of protein kinase C [12-O tetradecanoylphorbol-13-acetate (TPA), and 1-oleoyl-2 acetyl-glycerol (OAG), and the calcium ionophore ionomycin did not alter LDH activity in VSM cells kept at 20% O2. A dose-dependent increase in LDH activity was also observed in normoxic cells exposed to cobalt chloride (50–200 μM), indicating that a metal binding protein might be involved in this signalling cascade. This transition metal does not seem to act by interfering with cellular oxidative phosphorylation, because 10−5–10−4M cyanide, a potent inhibitor of cell respiration, had no effect on LDH activity, as has been also shown for the production of erythropoietin (EPO). Thus, we suggest that the phosphorylation potential is not crucial to the O2-sensing mechanism regulating LDH activity and EPO production. Our results suggest that the “metabolic indicator” leading to an enhanced LDH activity under hypoxic conditions in VSM cells is represented by cAMP.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bar-Tana J, Rose G, Shapiro B (1975) Long chain fatty acyl-CoA synthetase from rat liver microsomes. In: Lowenstein JM (ed) Methods in enzymology, vol. XXXV. Academic. New York, pp 117–122
Bergmeyer HU, Bernt E (1974) Lactat-Dehydrogenase. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse, vol. 1. Verlag Chemie, Weinheim/Bergstr, pp 607–612
Brown BL, Ekins RP, Albano JDM (1972) Saturation assay for cyclic AMP using endogenous binding protein. In: Greengard P, Robinson GA (eds) Advances in cyclic nucleotide research, vol. 2. Raven, New York, p 25
Chamley-Campell J, Campell GR, Ross R (1979) The smooth muscle cell in culture. Physiol Rev 59:1–61
Fukasawa KM, Li SS-L (1987) Complete nucleotide sequence of the mouse lactate dehydrogenase-A function gene: comparison of the exon-intron organization of dehydrogenase genes. Genetics 116:99–105
Goldberg MA, Dunning SP, Bunn HF (1988) Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242:1412–1415
Helfman T, Falanga V (1993) Gene expression in low oxygen tension. Am J Med Sci 306:37–41
Hidaka H, Inagaki M, Kawamoto S, Sasaki J (1984) Isoquinoline sulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase A and protein kinase C. Biochemistry 23:5036–5041
Jelkmann W (1992) Erythropoietin: structure, control of production, and function. Physiol Rev 72:449–489
Jungmann RA, Kelley DC, Miles MF, Milkowski DM (1983) Cyclic AMP regulation of lactate dehydrogenase. J Biol Chem 258:5312–5318
Kinnula VL (1975) Rat liver mitochondrial enzyme activities in hypoxia. Acta Physiol Scand 95:54–59
Kurtz A, Jelkmann W, Pfeilschifter J, Bauer C (1985) Role of prostaglandins in hypoxia-stimulated erythropoietin production. Am J Physiol 249:C3-C8
Maxwell PH, Pugh CW, Ratcliffe PJ (1993) Inducible operation of the erythropoietin 3′ enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc Natl Acad Sci USA 90:2423–2427
Mellon PL, Clegg CH, Correll LA, McKnight GS (1989) Regulation of transcription by cyclic AMP-dependent protein kinase. Proc Natl Acad Sci USA 86:4887–4891
Michiels C, Arnould T, Knott I, Dieu M, Remacle J (1993) Stimulation of prostaglandin synthesis by human endothelial cells exposed to hypoxia. Am J Physiol 264:C866-C874
Montminy MR, Bilezikjian LM (1987) Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328:175–178
Osborn M, Debus E, Weber K (1983) Monoclonal antibodies to desmin, the muscle specific intermediate filament protein. EMBO J 2:2305–2312
Ptashne KA, Theodore J, Robin ED (1983) Increased phosphofructokinase content during chronic hypoxia in cultured skeletal muscle (L8) cells. Biochim Biophys Acta 763:169–174
Ptashne KA, Morin ME, Hance A, Robin ED (1985) Increased biosynthesis of pyruvate kinase under hypoxic conditions in mammalian cells. Biochim Biophys Acta 844:19–23
Robin ED (1980) Of men and mitochondria: coping with hypoxic dysoxia. Am Rev Respir Dis 122:517–531
Robin ED, Murphy BJ, Theodore J (1984) Coordinate regulation of glycolysis by hypoxia in mammalian cells. J Cell Physiol 118:287–290
Roesler WJ, Vandenbark GR, Hanson RW (1988) Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 263:9063–9066
Rozengurt E, Stroobant P, Waterfield MD, Deuel TF, Keehan M (1983) Platelet-derived growth factor elicits cyclic AMP accumulation in swiss 3T3 cells: role of prostaglandin production. Cell 34:265–272
Spiro S, Guest R (1990) FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev 6:399–428
Tan CC, Ratcliffe PJ (1991) Effect of inhibitors of oxidative phosphorylation on erythropoietin mRNA in isolated perfused rat kidneys. Am J Physiol 261:F982-F987
Terrados N, Jansson E, Sylvén C, Kaijser L (1990) Is hypoxia a stimulus for synthesis of oxidative enzymes and myoglobin? J Appl Physiol 68:2369–2372
Wang GL, Semenza GL (1993) General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci USA 90:4304–4308
Wang GL, Semenza GL (1993) Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82:3610–3615
Webster KA (1987) Regulation of glycolytic enzyme RNA transcriptional rates by oxygen availability in skeletal muscle cells. Mol Cell Biochem 77:19–28
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Marti, H.H., Jung, H.H., Pfeilschifter, J. et al. Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells. Pflugers Arch. 429, 216–222 (1994). https://doi.org/10.1007/BF00374315
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00374315