scholarly journals The effects of glucagon and insulin on adenosine 3′:5′-cyclic monophosphate concentrations in an organ culture of mature rat liver

1973 ◽  
Vol 132 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Kenneth Siddle ◽  
Barbara Kane-Maguire ◽  
Anthony K. Campbell
Keyword(s):  
Cyclic Amp ◽  
Organ Culture ◽  
Rat Liver ◽  
Incubation Medium ◽  
Radioactive Tracer ◽  
Maximal Increase ◽  
Low Concentrations ◽  
Glucagon And Insulin ◽  
Millipore Filters ◽  
Glucagon Concentration

1. A modified radioimmunoassay for cyclic AMP was developed from the method of Steiner et al. (1969). Cyclic [3H]AMP was used as the radioactive tracer. Free and antibody-bound nucleotides were separated by adsorption of protein to Millipore filters. The assay was used to measure amounts of cyclic AMP down to 0.1pmol in 50μl. 2. The effect of glucagon on cyclic AMP content in pieces of mature rat liver maintained for 6 days in organ culture was studied. 3. Cyclic AMP content in the tissue reached a maximum in 5–15min and then decreased. This may have been partly due to an inhibitor of glucagon action formed in the tissue. Small amounts of cyclic AMP were released into the incubation medium. 4. The maximal increase in cyclic AMP content produced by glucagon decreased over 6 days in culture. However, liver pieces cultured for 2 and 6 days were more sensitive to low concentrations of glucagon than were fresh liver pieces. Glucagon concentrations for half-maximal effects were approx. 1μm and 0.05μm for fresh liver and 2-day cultured liver respectively. 5. Insulin (3.5μm) lowered the cyclic AMP content by 30% in the presence of a submaximal glucagon concentration in liver cultured for 2 days. No effect of insulin was demonstrated on fresh liver pieces. 6. Insulin and glucagon were rapidly destroyed by fresh liver pieces.

scholarly journals Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver

1986 ◽  
Vol 234 (2) ◽  
pp. 325-334 ◽  
Author(s):  
N J Pyne ◽  
M E Cooper ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Integral Membrane Protein ◽  
Soluble Enzyme ◽  
Cyclic Amp Phosphodiesterase ◽  
Apparent Homogeneity ◽  
Low Concentrations ◽  
Considerable Homology ◽  
Hydrolysis Of

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a ‘particulate enzyme’ found as an integral membrane protein associated with the plasma membrane, and a ‘soluble’ enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

EFFECT OF DIBUTYRYL CYCLIC AMP ON THE RELEASE OF MELANOCYTE-STIMULATING HORMONE FROM RAT NEUROINTERMEDIATE LOBES IN VITRO

1974 ◽  
Vol 63 (3) ◽  
pp. 533-538 ◽  
Author(s):  
BRIDGET I. BAKER
Keyword(s):  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Incubation Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Dibutyryl Cyclic Amp ◽  
Melanocyte Stimulating Hormone ◽  
Low Concentrations ◽  
Stimulating Hormone

SUMMARY A method for measuring melanocyte-stimulating hormone (MSH) in rat neurointermediate lobe in vitro and in incubation medium, using polyacrylamide gel electrophoresis, is described. Using this technique, it was shown that dibutyryl cyclic AMP increased the release of MSH in vitro, the degree of stimulation depending on the concentration of the nucleotide. The effect of low concentrations of the nucleotide was potentiated by theophylline.

scholarly journals Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3′:5′-cyclic monophosphate in foetal rat liver maintained in organ culture

1976 ◽  
Vol 160 (2) ◽  
pp. 159-162 ◽  
Author(s):  
E Edkins ◽  
N C R Rïhä
Keyword(s):  
Cyclic Amp ◽  
Organ Culture ◽  
Rat Liver ◽  
Urea Cycle ◽  
Defined Medium ◽  
Urea Synthesis ◽  
Dibutyryl Cyclic Amp ◽  
Carbamoyl Phosphate ◽  
Foetal Rat ◽  
Ornithine Transcarbamoylase

Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2′-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.

scholarly journals Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver

1975 ◽  
Vol 150 (1) ◽  
pp. 51-58 ◽  
Author(s):  
F J Moreno ◽  
L Sánchez-Urrutia ◽  
J M Medina ◽  
F Sánchez-Medina ◽  
F Mayor
Keyword(s):  
Cyclic Amp ◽  
Nicotinic Acid ◽  
Rat Liver ◽  
Hormonal Regulation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Actinomycin D ◽  
De Novo ◽  
Glycogen Depletion ◽  
Perfusion Medium ◽  
Low Concentrations

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably < the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.

Purification and Properties of Rat Liver Nuclear Protein Kinases

1972 ◽  
Vol 50 (12) ◽  
pp. 1249-1259 ◽  
Author(s):  
P. R. Desjardins ◽  
P. F. Lue ◽  
C. C. Liew ◽  
A. G. Gornall
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Nuclear Protein ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Ph Optimum ◽  
Heat Stable ◽  
Low Concentrations

Two protein kinases, designated NI and NII, have been isolated from rat liver nuclei. These enzymes have a similar pH optimum and phosphorylate phosvitin and casein more readily than histone. Both enzymes require magnesium for activity. In the absence of Mg2+, other divalent cations such as Ca2+, Co2+, and Mn2+ can substitute partially for Mg2+ when the reaction is catalyzed by NI. With NII, only Co2+ showed any activity in the absence of Mg2+. Magnesium decreased the apparent Km for ATP of protein kinase NI without changing the Vmax of the reaction, and decreased the apparent Km's for both ATP and casein, while increasing the Vmax of the reaction threefold with protein kinase NII. Both enzymes are stimulated about twofold by low concentrations (0.1–0.3 M) of NaCl, KCl, and sodium acetate, whereas higher concentrations (> 0.5 M) inhibit their activities. Both enzymes are inhibited by low concentrations of NaF (0.02 M) and (NH4)2SO4 (0.1 M). NI and NII were found to have sedimentation coefficients of 3.6 S and 10.8 S, respectively. The nuclear protein kinases are not activated by cyclic AMP or cyclic GMP, and are not inhibited by the heat-stable cyclic AMP-dependent protein kinase inhibitor.

STIMULATORY EFFECT OF FSH IN VITRO ON THE EXTRACELLULARLY ACTIVE CYCLIC AMP PHOSPHODIESTERASE IN THE PREPUBERTAL RAT OVARY

1976 ◽  
Vol 81 (2) ◽  
pp. 563-573 ◽  
Author(s):  
Gunnar Selstam ◽  
Sten Rosberg
Keyword(s):  
Cyclic Amp ◽  
Incubation Medium ◽  
Inorganic Phosphate ◽  
Kinetic Studies ◽  
Bicarbonate Buffer ◽  
Low Concentrations ◽  
Rat Ovary ◽  
Camp System ◽  
Burk Plot

ABSTRACT Intact prepubertal rat ovaries were incubated with radioactively labelled adenosine 3,′5′-cyclic monophosphate (cAMP) in Krebs bicarbonate buffer containing glucose. The rate of degradation of cAMP was determined by measuring the radioactivity in the medium after precipitation with Ba(OH)2 and ZnSO4. The fate of the nucleotide was followed by measuring the products in the incubation medium. Paper chromatography was used for the separation and identification of these products. It was found that cAMP was degraded to AMP, which in turn was degraded to inorganic phosphate (Pi) and adenosine. An uptake of labelled products was also observed. NIH-FSH-S9 (10 and 100 μg/ml), but not NIH-LH-B8 (0.1–100 μg/ml), increased the degradation of cAMP. Concomitantly, an increased accumulation of labelled adenosine and Pi as well as an increased uptake of labelled products were seen. Kinetic studies with low concentrations of cAMP (0.125–0.025 μmol/l) revealed an apparent Km value of 0.12 μmol/l for the phosphodiesterase (PDE) activity. FSH significantly changed the slope of the curve in the Lineweaver-Burk plot by increasing the PDE activity. The increased PDE activity in the presence of FSH is discussed in relation to earlier findings of differences in action between LH and FSH on the cAMP system in the prepubertal rat ovary.

scholarly journals Regulation of tyrosine aminotransferase in foetal rat liver

1980 ◽  
Vol 186 (2) ◽  
pp. 609-612 ◽  
Author(s):  
S M Andersson ◽  
N C Räihä ◽  
J J Ohisalo
Keyword(s):  
Enzyme Activity ◽  
Methyl Ester ◽  
Cyclic Amp ◽  
Organ Culture ◽  
Rat Liver ◽  
Tyrosine Aminotransferase ◽  
Dibutyryl Cyclic Amp ◽  
Adult Rat ◽  
Foetal Rat

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

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