scholarly journals Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

1980 ◽  
Vol 192 (2) ◽  
pp. 395-402 ◽  
Author(s):  
Perumana R. Sudhakaran ◽  
Wolfgang Sinn ◽  
Kurt von Figura
Keyword(s):  
Cyclic Amp ◽  
Heparan Sulphate ◽  
Molecular Size ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Dibutyryl Cyclic Amp ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Compartments ◽  
Sulphated Glycosaminoglycans ◽  
Chain Synthesis

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E1, and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of 35SO42− into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of 35SO42− or [14C]glucosamine into heparan sulphate was decreased to 40–50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse–chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by β-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, 35SO42− incorporation was decreased to 5%. Addition of p-nitrophenyl β-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl β-d-xylosides. Incorporation of [3H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.

scholarly journals Different modulatory effects of elevated cyclic AMP on cytosolic Ca2+ spikes induced by phenylephrine or vasopressin in single rat hepatocytes

1993 ◽  
Vol 291 (1) ◽  
pp. 163-168 ◽  
Author(s):  
A Sanchez-Bueno ◽  
I Marrero ◽  
P H Cobbold
Keyword(s):  
Cyclic Amp ◽  
Rat Hepatocytes ◽  
Specific Information ◽  
Dibutyryl Cyclic Amp ◽  
Isolated Rat Hepatocytes ◽  
Frequency Peak ◽  
Isolated Rat

We show here, by aequorin measurements in single isolated rat hepatocytes, that elevation of cyclic AMP, by dibutyryl cyclic AMP, forskolin or glucagon, has different effects on oscillations in cytosolic concentration of free Ca2+ (‘free Ca’) induced by phenylephrine or vasopressin. Elevated cyclic AMP does not itself induce free Ca oscillations, but enhances both the peak free Ca and the frequency of spikes induced by phenylephrine. In contrast, elevated cyclic AMP has no effect on peak free Ca of vasopressin-induced spikes, but markedly prolongs the falling phase, with the result that spiking frequency (peak to peak) falls, although the period between spikes of resting free Ca is usually decreased. The data provide another example of receptor-specific information being retained in the oscillator mechanism, with implications for models of the hepatocyte calcium oscillator.

Effect of multiplication-stimulating activity (MSA) on the cyclic AMP level and proteoglycan synthesis in cultured chondrocytes

1983 ◽  
Vol 104 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Mitsuko Tsuji ◽  
Yukio Kato ◽  
Yoshio Nomura ◽  
Masahiko Kinoshita ◽  
Yuichi Kumahara ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Human Fibroblasts ◽  
Inhibitory Effect ◽  
Proteoglycan Synthesis ◽  
Prostaglandin E ◽  
Dibutyryl Cyclic Amp ◽  
Cyclic Amp Accumulation ◽  
Rat Liver Cells ◽  
Sulphated Glycosaminoglycans ◽  
Cultured Chondrocytes

Abstract. Multiplication-stimulating activity (MSA), a somatomedin purified from conditioned medium of Buffalo rat liver cells, had little effect on the intracellular level of cyclic AMP when it markedly enhanced the synthesis of sulphated glycosaminoglycans in rabbit chondrocytes in culture. In addition, MSA did not inhibit prostaglandin E1- or parathyroid hormone-induced accumulation of cyclic AMP in the chondrocytes. On the contrary, MSA slightly decreased stimulation of cyclic AMP accumulation by prostaglandin in human fibroblasts. Dibutyryl cyclic AMP and MSA increased the incorporation of [35S]sulphate and [3H]serine into proteoglycans synthesized by rabbit chondrocytes, and their effects were additive. These findings suggest that somatomedin and dibutyryl cyclic AMP enhance sulphate proteoglycan synthesis through different mechanisms. The lack of inhibitory effect of MSA on cyclic AMP accumulation may be favourable for producing additive effects with cyclic AMP on proteoglycan synthesis and DNA synthesis in chondrocytes.

scholarly journals 4 β-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes

1986 ◽  
Vol 238 (3) ◽  
pp. 737-743 ◽  
Author(s):  
J M Staddon ◽  
R G Hansford
Keyword(s):  
Protein Kinase C ◽  
Cyclic Amp ◽  
Phosphodiesterase Inhibitor ◽  
Rat Hepatocytes ◽  
Dibutyryl Cyclic Amp ◽  
Isolated Rat Hepatocytes ◽  
Kinase C ◽  
Subsequent Addition ◽  
Isolated Rat ◽  
Sustained Increase

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [(Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.

scholarly journals Inhibition of phosphatidylethanolamine synthesis by glucagon in isolated rat hepatocytes

1989 ◽  
Vol 257 (3) ◽  
pp. 645-650 ◽  
Author(s):  
L B M Tijburg ◽  
M Houweling ◽  
M J H Geelen ◽  
L M G Van Golde
Keyword(s):  
Cyclic Amp ◽  
De Novo ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Isolated Rat Hepatocytes ◽  
Ethanolamine Phosphate ◽  
Isolated Rat

Exposure of isolated rat hepatocytes to glucagon or chlorophenylthio cyclic AMP led to an inhibition of the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-chase experiments and measurement of the activities of the enzymes involved in the CDP-ethanolamine pathway provided evidence that the inhibitory effect of glucagon on the synthesis de novo of phosphatidylethanolamine was not caused by a diminished conversion of ethanolamine phosphate into CDP-ethanolamine. The observations suggested that the glucagon-induced inhibition of the biosynthesis of phosphatidylethanolamine is probably due to a decreased supply of diacylglycerols, resulting in a decreased formation of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerols.

scholarly journals Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes

1986 ◽  
Vol 236 (1) ◽  
pp. 131-136 ◽  
Author(s):  
E P Brass ◽  
P V Fennessey ◽  
L V Miller
Keyword(s):  
Palmitic Acid ◽  
Propionic Acid ◽  
Oxidative Metabolism ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Acid Oxidation ◽  
Isolated Rat Hepatocytes ◽  
Pyruvate Oxidation ◽  
Metabolic Basis ◽  
Isolated Rat

The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements (‘carnitine insufficiency’).

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