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. 2001 Apr 15;355(Pt 2):389–395. doi: 10.1042/0264-6021:3550389

Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects.

W Wieser 1, G Krumschnabel 1
PMCID: PMC1221750  PMID: 11284726

Abstract

The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells [Buttgereit and Brand (1995) Biochem. J. 312, 163-167]. Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived. We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na(+)/K(+)-ATPase, measuring their activity directly by [3H]leucine incorporation and Rb(+)-flux respectively. We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively). On the other hand, Na(+)/K(+)-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species. We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na(+)/K(+)-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na(+)/K(+)-ATPase of the two fish species. A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used. It was found that at 15 mM cycloheximide [three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited] total oxygen consumption was suppressed by 71-75%, whereas the measured rate of [3H]leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx. 10% of the total oxygen consumption. On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium. Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a 'hierarchy' of ATP-consumers may reflect the lifestyle and physiology of the species studied.

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Selected References

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  1. Arthur P. G., Hogan M. C., Bebout D. E., Wagner P. D., Hochachka P. W. Modeling the effects of hypoxia on ATP turnover in exercising muscle. J Appl Physiol (1985) 1992 Aug;73(2):737–742. doi: 10.1152/jappl.1992.73.2.737. [DOI] [PubMed] [Google Scholar]
  2. Buttgereit F., Brand M. D. A hierarchy of ATP-consuming processes in mammalian cells. Biochem J. 1995 Nov 15;312(Pt 1):163–167. doi: 10.1042/bj3120163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buttgereit F., Burmester G. R., Brand M. D. Therapeutically targeting lymphocyte energy metabolism by high-dose glucocorticoids. Biochem Pharmacol. 2000 Mar 15;59(6):597–603. doi: 10.1016/s0006-2952(99)00273-7. [DOI] [PubMed] [Google Scholar]
  4. Ch'ih J. J., Duhl D. M., Faulkner L. S., Devlin T. M. Regulation of mammalian protein synthesis in vivo. Simulated transport of nuclear ribonucleoprotein complexes to the cytoplasm after cycloheximide treatment. Biochem J. 1979 Mar 15;178(3):643–649. doi: 10.1042/bj1780643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Culic O., Gruwel M. L., Schrader J. Energy turnover of vascular endothelial cells. Am J Physiol. 1997 Jul;273(1 Pt 1):C205–C213. doi: 10.1152/ajpcell.1997.273.1.C205. [DOI] [PubMed] [Google Scholar]
  6. Fuery C. J., Withers P. C., Guppy M. Protein synthesis in the liver of Bufo marinus: cost and contribution to oxygen consumption. Comp Biochem Physiol A Mol Integr Physiol. 1998 Feb;119(2):459–467. doi: 10.1016/s1095-6433(97)00452-2. [DOI] [PubMed] [Google Scholar]
  7. Fuller M. F., Cadenhead A., Mollison G., Seve B. Effects of the amount and quality of dietary protein on nitrogen metabolism and heat production in growing pigs. Br J Nutr. 1987 Sep;58(2):277–285. doi: 10.1079/bjn19870095. [DOI] [PubMed] [Google Scholar]
  8. Gronostajski R. M., Pardee A. B., Goldberg A. L. The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts. J Biol Chem. 1985 Mar 25;260(6):3344–3349. [PubMed] [Google Scholar]
  9. Guppy M., Withers P. Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol Rev Camb Philos Soc. 1999 Feb;74(1):1–40. doi: 10.1017/s0006323198005258. [DOI] [PubMed] [Google Scholar]
  10. Haller T., Ortner M., Gnaiger E. A respirometer for investigating oxidative cell metabolism: toward optimization of respiratory studies. Anal Biochem. 1994 May 1;218(2):338–342. doi: 10.1006/abio.1994.1188. [DOI] [PubMed] [Google Scholar]
  11. Hand S. C., Hardewig I. Downregulation of cellular metabolism during environmental stress: mechanisms and implications. Annu Rev Physiol. 1996;58:539–563. doi: 10.1146/annurev.ph.58.030196.002543. [DOI] [PubMed] [Google Scholar]
  12. Hochachka P. W. Solving the common problem: matching ATP synthesis to ATP demand during exercise. Adv Vet Sci Comp Med. 1994;38A:41–56. [PubMed] [Google Scholar]
  13. Krumschnabel G., Biasi C., Schwarzbaum P. J., Wieser W. Acute and chronic effects of temperature, and of nutritional state, on ion homeostasis and energy metabolism in teleost hepatocytes. J Comp Physiol B. 1997 May;167(4):280–286. doi: 10.1007/s003600050075. [DOI] [PubMed] [Google Scholar]
  14. Krumschnabel G., Biasi C., Schwarzbaum P. J., Wieser W. Membrane-metabolic coupling and ion homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes. Am J Physiol. 1996 Mar;270(3 Pt 2):R614–R620. doi: 10.1152/ajpregu.1996.270.3.R614. [DOI] [PubMed] [Google Scholar]
  15. Krumschnabel G., Biasi C., Wieser W. Action of adenosine on energetics, protein synthesis and K(+) homeostasis in teleost hepatocytes. J Exp Biol. 2000 Sep;203(Pt 17):2657–2665. doi: 10.1242/jeb.203.17.2657. [DOI] [PubMed] [Google Scholar]
  16. Land S. C., Buck L. T., Hochachka P. W. Response of protein synthesis to anoxia and recovery in anoxia-tolerant hepatocytes. Am J Physiol. 1993 Jul;265(1 Pt 2):R41–R48. doi: 10.1152/ajpregu.1993.265.1.R41. [DOI] [PubMed] [Google Scholar]
  17. Mandel L. J., Balaban R. S. Stoichiometry and coupling of active transport to oxidative metabolism in epithelial tissues. Am J Physiol. 1981 May;240(5):F357–F371. doi: 10.1152/ajprenal.1981.240.5.F357. [DOI] [PubMed] [Google Scholar]
  18. Müller M., Siems W., Buttgereit F., Dumdey R., Rapoport S. M. Quantification of ATP-producing and consuming processes of Ehrlich ascites tumour cells. Eur J Biochem. 1986 Dec 15;161(3):701–705. doi: 10.1111/j.1432-1033.1986.tb10496.x. [DOI] [PubMed] [Google Scholar]
  19. Pannevis M. C., Houlihan D. F. The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss). J Comp Physiol B. 1992;162(5):393–400. doi: 10.1007/BF00258960. [DOI] [PubMed] [Google Scholar]
  20. Reeds P. J., Cadenhead A., Fuller M. F., Lobley G. E., McDonald J. D. Protein turnover in growing pigs. Effects of age and food intake. Br J Nutr. 1980 May;43(3):445–455. doi: 10.1079/bjn19800112. [DOI] [PubMed] [Google Scholar]
  21. Schmidt H., Siems W., Müller M., Dumdey R., Rapoport S. M. ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases. Exp Cell Res. 1991 May;194(1):122–127. doi: 10.1016/0014-4827(91)90140-p. [DOI] [PubMed] [Google Scholar]
  22. Siems W. G., Schmidt H., Gruner S., Jakstadt M. Balancing of energy-consuming processes of K 562 cells. Cell Biochem Funct. 1992 Mar;10(1):61–66. doi: 10.1002/cbf.290100110. [DOI] [PubMed] [Google Scholar]
  23. Siems W., Dubiel W., Dumdey R., Müller M., Rapoport S. M. Accounting for the ATP-consuming processes in rabbit reticulocytes. Eur J Biochem. 1984 Feb 15;139(1):101–107. doi: 10.1111/j.1432-1033.1984.tb07982.x. [DOI] [PubMed] [Google Scholar]
  24. Siems W., Müller M., Dubiel W., Dumdey R., Rapoport S. ATP production and consumption of rabbit reticulocytes increase in an amino-acid-enriched medium. Biomed Biochim Acta. 1986;45(5):585–591. [PubMed] [Google Scholar]
  25. Soltoff S. P., Mandel L. J. Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump. J Gen Physiol. 1984 Oct;84(4):643–662. doi: 10.1085/jgp.84.4.643. [DOI] [PMC free article] [PubMed] [Google Scholar]

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