Effects of growth hormone and glucagon on ornithine decarboxylase activity and adenosine 3‘,5‘-Monophosphate levels in isolated rat hepatocytes

Mark R. Klingensmith, Alison G. Freifeld, Anthony E. Pegg, Leonard S. Jefferson

Research output: Contribution to journalArticle

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Abstract

l-Ornithine decarboxylase (l-ornithine carboxylase; E.C. 4.1.1.17) activity was studied in isolated rat hepatocytes maintained as suspensions of free cells. Activity in freshly prepared hepatocytes was reduced to approximately 10% of the amount found in the intact liver. Activity increased to the same level as that found in the intact liver when the cells were incubated for 4 h in medium containing a mixture of 20 amino acids at concentrations 10 times those found in normal rat plasma. The increase in activity between 2 and 4 h of incubation was substantially augmented when the medium also contained 25 μg/ml of a crude preparation of bovine GH (bGH; NIH-GHB18). The increase in activity and the hormone effect were not observed in the presence of normal plasma levels of amino acids. The addition of 1 mm putrescine caused enzyme activity to fall to very low levels even in the presence of the higher amino acid concentrations, and the effect of the bGH preparation was lost. After incubation in the presence of putrescine, a nondiffusible inhibitor of ornithine decarboxylase was found in extracts of the liver cells. Chromatography of the NIH-GH-B18 preparation on Sephadex G-100 yielded two major fractions, dimeric bGH and a higher molecular weight fraction that was excluded by the gel matrix (fraction I). bGH retained the growth-promoting activity of NIH-GH-B18, as determined in hypophysectomized rats. However, bGH had no effect on ornithine decarboxylase activity in isolated hepatocytes, while fraction I produced effects similar to those observed with the NIH-GH-B18 preparation. bGH administered to intact rats caused some increase in hepatic ornithine decarboxylase activity, but fraction I produced a much greater increase. Compared to either NIH-GH-B18 or fraction I, glucagon caused a much greater increase in ornithine decarboxylase in isolated hepatocytes with enzyme activity at 2 h of incubation, being 40-fold greater than that observed in freshly prepared cells. Most of the increase caused by glucagon occurred between 1-2 h of incubation and was preceded by a 6-fold increase in cellular cAMP levels. cAMP attained a maximal level 5-10 min after the addition of glucagon and returned to control levels by 2 h. Either NIH-GH-B18 or fraction I, added in combination with glucagon, blunted both the increase in enzyme activity and the rise in cAMP produced by glucagon alone, while bGH had no influence on the glucagon effects. cAMP was not altered from control levels when NIH-GH-B18, fraction I, or bGH was added alone. In conclusion, these studies demonstrate the ability of NIH-GH-B18 to directly stimulate hepatic ornithine decarboxylase but suggest that this effect is due to the presence of factors other than bGH in the preparation. The active factor does not appear to mediate its effect through cAMP, since it does not by itself alter the level of this nucleotide and since it impairs the ability of glucagon to elevate cAMP levels and increase enzyme activity.

Original languageEnglish (US)
Pages (from-to)125-132
Number of pages8
JournalEndocrinology
Volume106
Issue number1
DOIs
StatePublished - Jan 1980

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Ornithine Decarboxylase
Glucagon
Adenosine
Growth Hormone
Hepatocytes
Putrescine
Liver
Enzymes
Amino Acids
Liver Extracts
Ornithine
Cell Extracts
Chromatography
Suspensions
Nucleotides
Molecular Weight
Gels
Hormones

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

@article{8d3c021ab30547718a47e7ac4b379749,
title = "Effects of growth hormone and glucagon on ornithine decarboxylase activity and adenosine 3‘,5‘-Monophosphate levels in isolated rat hepatocytes",
abstract = "l-Ornithine decarboxylase (l-ornithine carboxylase; E.C. 4.1.1.17) activity was studied in isolated rat hepatocytes maintained as suspensions of free cells. Activity in freshly prepared hepatocytes was reduced to approximately 10{\%} of the amount found in the intact liver. Activity increased to the same level as that found in the intact liver when the cells were incubated for 4 h in medium containing a mixture of 20 amino acids at concentrations 10 times those found in normal rat plasma. The increase in activity between 2 and 4 h of incubation was substantially augmented when the medium also contained 25 μg/ml of a crude preparation of bovine GH (bGH; NIH-GHB18). The increase in activity and the hormone effect were not observed in the presence of normal plasma levels of amino acids. The addition of 1 mm putrescine caused enzyme activity to fall to very low levels even in the presence of the higher amino acid concentrations, and the effect of the bGH preparation was lost. After incubation in the presence of putrescine, a nondiffusible inhibitor of ornithine decarboxylase was found in extracts of the liver cells. Chromatography of the NIH-GH-B18 preparation on Sephadex G-100 yielded two major fractions, dimeric bGH and a higher molecular weight fraction that was excluded by the gel matrix (fraction I). bGH retained the growth-promoting activity of NIH-GH-B18, as determined in hypophysectomized rats. However, bGH had no effect on ornithine decarboxylase activity in isolated hepatocytes, while fraction I produced effects similar to those observed with the NIH-GH-B18 preparation. bGH administered to intact rats caused some increase in hepatic ornithine decarboxylase activity, but fraction I produced a much greater increase. Compared to either NIH-GH-B18 or fraction I, glucagon caused a much greater increase in ornithine decarboxylase in isolated hepatocytes with enzyme activity at 2 h of incubation, being 40-fold greater than that observed in freshly prepared cells. Most of the increase caused by glucagon occurred between 1-2 h of incubation and was preceded by a 6-fold increase in cellular cAMP levels. cAMP attained a maximal level 5-10 min after the addition of glucagon and returned to control levels by 2 h. Either NIH-GH-B18 or fraction I, added in combination with glucagon, blunted both the increase in enzyme activity and the rise in cAMP produced by glucagon alone, while bGH had no influence on the glucagon effects. cAMP was not altered from control levels when NIH-GH-B18, fraction I, or bGH was added alone. In conclusion, these studies demonstrate the ability of NIH-GH-B18 to directly stimulate hepatic ornithine decarboxylase but suggest that this effect is due to the presence of factors other than bGH in the preparation. The active factor does not appear to mediate its effect through cAMP, since it does not by itself alter the level of this nucleotide and since it impairs the ability of glucagon to elevate cAMP levels and increase enzyme activity.",
author = "Klingensmith, {Mark R.} and Freifeld, {Alison G.} and Pegg, {Anthony E.} and Jefferson, {Leonard S.}",
year = "1980",
month = "1",
doi = "10.1210/endo-106-1-125",
language = "English (US)",
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Effects of growth hormone and glucagon on ornithine decarboxylase activity and adenosine 3‘,5‘-Monophosphate levels in isolated rat hepatocytes. / Klingensmith, Mark R.; Freifeld, Alison G.; Pegg, Anthony E.; Jefferson, Leonard S.

In: Endocrinology, Vol. 106, No. 1, 01.1980, p. 125-132.

Research output: Contribution to journalArticle

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T1 - Effects of growth hormone and glucagon on ornithine decarboxylase activity and adenosine 3‘,5‘-Monophosphate levels in isolated rat hepatocytes

AU - Klingensmith, Mark R.

AU - Freifeld, Alison G.

AU - Pegg, Anthony E.

AU - Jefferson, Leonard S.

PY - 1980/1

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N2 - l-Ornithine decarboxylase (l-ornithine carboxylase; E.C. 4.1.1.17) activity was studied in isolated rat hepatocytes maintained as suspensions of free cells. Activity in freshly prepared hepatocytes was reduced to approximately 10% of the amount found in the intact liver. Activity increased to the same level as that found in the intact liver when the cells were incubated for 4 h in medium containing a mixture of 20 amino acids at concentrations 10 times those found in normal rat plasma. The increase in activity between 2 and 4 h of incubation was substantially augmented when the medium also contained 25 μg/ml of a crude preparation of bovine GH (bGH; NIH-GHB18). The increase in activity and the hormone effect were not observed in the presence of normal plasma levels of amino acids. The addition of 1 mm putrescine caused enzyme activity to fall to very low levels even in the presence of the higher amino acid concentrations, and the effect of the bGH preparation was lost. After incubation in the presence of putrescine, a nondiffusible inhibitor of ornithine decarboxylase was found in extracts of the liver cells. Chromatography of the NIH-GH-B18 preparation on Sephadex G-100 yielded two major fractions, dimeric bGH and a higher molecular weight fraction that was excluded by the gel matrix (fraction I). bGH retained the growth-promoting activity of NIH-GH-B18, as determined in hypophysectomized rats. However, bGH had no effect on ornithine decarboxylase activity in isolated hepatocytes, while fraction I produced effects similar to those observed with the NIH-GH-B18 preparation. bGH administered to intact rats caused some increase in hepatic ornithine decarboxylase activity, but fraction I produced a much greater increase. Compared to either NIH-GH-B18 or fraction I, glucagon caused a much greater increase in ornithine decarboxylase in isolated hepatocytes with enzyme activity at 2 h of incubation, being 40-fold greater than that observed in freshly prepared cells. Most of the increase caused by glucagon occurred between 1-2 h of incubation and was preceded by a 6-fold increase in cellular cAMP levels. cAMP attained a maximal level 5-10 min after the addition of glucagon and returned to control levels by 2 h. Either NIH-GH-B18 or fraction I, added in combination with glucagon, blunted both the increase in enzyme activity and the rise in cAMP produced by glucagon alone, while bGH had no influence on the glucagon effects. cAMP was not altered from control levels when NIH-GH-B18, fraction I, or bGH was added alone. In conclusion, these studies demonstrate the ability of NIH-GH-B18 to directly stimulate hepatic ornithine decarboxylase but suggest that this effect is due to the presence of factors other than bGH in the preparation. The active factor does not appear to mediate its effect through cAMP, since it does not by itself alter the level of this nucleotide and since it impairs the ability of glucagon to elevate cAMP levels and increase enzyme activity.

AB - l-Ornithine decarboxylase (l-ornithine carboxylase; E.C. 4.1.1.17) activity was studied in isolated rat hepatocytes maintained as suspensions of free cells. Activity in freshly prepared hepatocytes was reduced to approximately 10% of the amount found in the intact liver. Activity increased to the same level as that found in the intact liver when the cells were incubated for 4 h in medium containing a mixture of 20 amino acids at concentrations 10 times those found in normal rat plasma. The increase in activity between 2 and 4 h of incubation was substantially augmented when the medium also contained 25 μg/ml of a crude preparation of bovine GH (bGH; NIH-GHB18). The increase in activity and the hormone effect were not observed in the presence of normal plasma levels of amino acids. The addition of 1 mm putrescine caused enzyme activity to fall to very low levels even in the presence of the higher amino acid concentrations, and the effect of the bGH preparation was lost. After incubation in the presence of putrescine, a nondiffusible inhibitor of ornithine decarboxylase was found in extracts of the liver cells. Chromatography of the NIH-GH-B18 preparation on Sephadex G-100 yielded two major fractions, dimeric bGH and a higher molecular weight fraction that was excluded by the gel matrix (fraction I). bGH retained the growth-promoting activity of NIH-GH-B18, as determined in hypophysectomized rats. However, bGH had no effect on ornithine decarboxylase activity in isolated hepatocytes, while fraction I produced effects similar to those observed with the NIH-GH-B18 preparation. bGH administered to intact rats caused some increase in hepatic ornithine decarboxylase activity, but fraction I produced a much greater increase. Compared to either NIH-GH-B18 or fraction I, glucagon caused a much greater increase in ornithine decarboxylase in isolated hepatocytes with enzyme activity at 2 h of incubation, being 40-fold greater than that observed in freshly prepared cells. Most of the increase caused by glucagon occurred between 1-2 h of incubation and was preceded by a 6-fold increase in cellular cAMP levels. cAMP attained a maximal level 5-10 min after the addition of glucagon and returned to control levels by 2 h. Either NIH-GH-B18 or fraction I, added in combination with glucagon, blunted both the increase in enzyme activity and the rise in cAMP produced by glucagon alone, while bGH had no influence on the glucagon effects. cAMP was not altered from control levels when NIH-GH-B18, fraction I, or bGH was added alone. In conclusion, these studies demonstrate the ability of NIH-GH-B18 to directly stimulate hepatic ornithine decarboxylase but suggest that this effect is due to the presence of factors other than bGH in the preparation. The active factor does not appear to mediate its effect through cAMP, since it does not by itself alter the level of this nucleotide and since it impairs the ability of glucagon to elevate cAMP levels and increase enzyme activity.

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