AMP-dependent Protein Kinase Activity Ratio Research Articles

Mechanism of anti-lipolytic action of acipimox in isolated rat adipocytes.

Acipimox is commonly used to treat hypertriglyceridaemia in non-insulin-dependent diabetic patients, but its precise mechanism of action has yet to be elucidated. We examined the in vitro effects of acipimox on the lipolytic regulatory cascade in epididymal adipocytes isolated from Wistar rats. Acipimox inhibited the lipolytic rate stimulated by adenosine deaminase (1 U/ml) in a concentration-dependent manner, reaching a near-basal value at 10 mumol/l acipimox. Lipolysis activated by sub-maximal levels of isoproterenol in combination with adenosine deaminase (20 mU/ml) was significantly (p < 0.05) decreased by 100 mumol/l acipimox, whereas, in the absence of adenosine deaminase, 100 mumol/l acipimox showed no significant (p > 0.05) inhibition. These findings suggested that the anti-lipolytic mechanism regulated by adenosine may also be regulated by acipimox. Acipimox diminished the intracellular cyclic AMP level produced by 25 nmol/l isoproterenol in the presence of adenosine deaminase (20 mU/ml) in a concentration-dependent manner. At the same level of stimulation, acipimox inhibited the cyclic AMP-dependent protein kinase activity ratio and lipolytic rate over the same concentration range, with significant (p < 0.05) reductions occurring at and above, 0.5 mumol/l and 10 mumol/l acipimox, respectively. Western blotting showed that upon lipolytic stimulation (1 U/ml adenosine deaminase; 100 nmol/l isoproterenol) a threefold increase in the lipolytic rate was accompanied by a significant (p < 0.05) rise in hormone-sensitive lipase associated with the lipid fraction. Acipimox (1 mmol/l) and insulin (1 nmol/l) re-distributed hormone-sensitive lipase back to the cytosol, with a corresponding significant (p < 0.05) loss from the fat cake fraction of adipocyte homogenates. In conclusion, the anti-lipolytic action of acipimox is mediated through suppression of intracellular cyclic AMP levels, with the subsequent decrease in cyclic AMP-dependent protein kinase activity, leading to the reduced association of hormone-sensitive lipase with triacylglycerol substrate in the lipid droplet of adipocytes.

Octimibate, a potent non‐prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP-inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.

Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues

1. The cyclic AMP-dependent protein kinase activity-ratio assay was investigated by comparing histone and a synthetic peptide, malantide [Malencik & Anderson (1983) Anal. Biochem. 132, 32-40], as substrates. 2. In several tissues the activity ratio was higher when assayed with histone as the substrate; this result was obtained in control tissues and also in those incubated with agents known to increase cyclic AMP. The effect of these agents to increase the activity ratio was more clearly demonstrated with malantide. 3. The higher activity ratios observed with histone are due to: (a) measurement of phosphorylation not catalysed by cyclic AMP-dependent protein kinase; (b) activation of cyclic AMP-dependent protein kinase by histone during the assay. 4. When tissues were homogenized in buffers without NACl, lower activity ratios were found, owing to the catalytic subunit being artifactually removed from the supernatant. 5. We conclude that the measured activity ratio more faithfully reflects that in the tissue when NaCl is included in the homogenization buffer and malantide is used in the assay. This was confirmed in experiments where cyclic AMP-dependent protein kinase was added to the tissue before homogenization, and no dissociation of the exogenous enzyme was observed.

Effects of forskolin on contractile responses and protein phosphorylation in the isolated perfused rat heart.

Continuous perfusion of rat hearts with concentrations of forskolin between 0.1 and 12 microM resulted in transient increases in tension after 45 s, followed by a return to the control value after 5 min. In contrast, the content of cyclic AMP increased linearly with time over this period, reaching values up to 35 times control after 5 min. Increases in contractile force, intracellular cyclic AMP concentration and the proportion of phosphorylase in the a form were dependent on the concentration of forskolin when measured 45 s and 120 s after initiation of perfusion. In hearts perfused for 45 s with various concentrations of forskolin, the measured cyclic AMP-dependent protein kinase activity ratio and phosphorylase a content for a given measured intracellular cyclic AMP concentration were both much less than the corresponding values in hearts perfused for 30 s with various concentrations of isoprenaline. The phosphorylation of the contractile proteins troponin-I and C-protein also showed a concentration-dependent increase in hearts perfused with forskolin. There was a strong correlation between the cyclic AMP-dependent protein kinase activity ratios and the phosphorylation of the contractile proteins under all perfusion conditions. These results suggest that cyclic AMP is compartmented in perfused rat heart, and that much of the cyclic AMP produced in response to forskolin is unavailable to activate cyclic AMP-dependent protein kinase.

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H + secretion, as measured by 14C- aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10 6 cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H + secretion, but that the acid response elicited by histamine requires another intracellular pathway.

Regulation of adenosine 3′:5′-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 μM) and forskolin (20 μM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 μM) while isoproterenol at 100 μM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4–6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60–70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.

Age-related changes in pituitary/thyroid function in chickens

Pituitary cyclic AMP-dependent protein kinase (CAMP-PK) activity ratios are not increased in response to thyrotropin-releasing hormone (TRH) in old chickens (retired White Leghorn breeders). This reduced responsiveness may be due to reduced hypothalamic function, reduced thyrotrope function, or to a reduction in TRH membrane receptors. The thyroid cAMP-PK activity ratio of old males does not respond to TRH treatment but the thyroid of old females does have an increased activity ratio after TRH injection. Both males and females have a much higher basal CAMP-PK activity ratio than young birds. This higher basal level is thought to be due to an increased thyroid-stimulating hormone (TSH) stimulation, and the failure of old males to increase activity after TRH injections may be due to a loss of the thyroid's ability to respond to direct TRH stimulation.

Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm.

Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4Cl to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li+ or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCl result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCl in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCl, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism of mammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.

Modulation of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase in skin.

Incubation in vitro of full-thickness guinea-pig skin with epinephrine or histamine resulted in the formation of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) and increase in the cyclic AMP-dependent protein kinase activity ratio. The effect was dose-dependent between 10(-5) and 10(-3) M concentration of the agents. Histamine antagonists, diphenhydramine (H1) and cimetidine (H2) prevented stimulation by histamine in a dose-dependent manner suggesting the presence of both types of receptors in full-thickness skin. Cyclic AMP accumulation with a concomitant increase in protein kinase activity denotes that phosphoproteins may have an important role in skin biology.

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities

cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7–10-fold higher than surface cells. Without a phosphodiesterase inhibitor, proliferative cells had reduced basal cyclic AMP-, cyclic GMP- and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E 2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E 2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particullate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high K m soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4–7-fold higher than those in superficial cells. Moreover, the apparent low K m soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E 2 or vasoactive intestinal peptide inhibited [ 3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.

Effects of trifluoperazine on function and structure of toad urinary bladder. Role of calmodulin vasopressin-stimulation of water permeability.

Calcium ion plays a major regulatory role in many hormone-stimulated systems. To determine the site of calcium's action in the toad urinary bladder, we examined the effect of trifluoperazine, a compound that binds specifically to the calcium binding protein, calmodulin, and thereby prevents activation of enzymes by the calcium- calmodulin complex. 10 microM trifluoperazine inhibited vasopressin stimulation of water flow, but did not alter vasopressin's effects on urea permeability or short-circuit current. Trifluoperazine also blocked stimulation of water flow by cyclic AMP and methylisobutylxanthine, implying a "postcyclic AMP" site of action. Consistent with these results, trifluoperazine did not decrease epithelial cyclic AMP content or the cyclic AMP-dependent protein kinase activity ratio. Assay of bladder epithelial supernate demonstrated calmodulin-like activity of 1.5 U/microgram protein. Morphologic studies of vasopressin-treated bladders revealed that trifluoperazine did not alter the volume density of cytoplasmic microtubules or significantly decrease the number of fusions between cytoplasmic, aggregate-containing, elongated vesicles and the luminal membrane. Nonetheless, the frequency of luminal membrane aggregates, structures that correlate well with luminal membrane water permeability, was decreased by greater than 50%. Thus, trifluoperazine appears to inhibit the movement of intramembranous particle aggregates from the fused intracellular membranes to the luminal membrane, perhaps by blocking an effect of calcium on microfilament function.

Separate mechanisms of induction for ornithine decarboxylase by triiodothyronine and aminophylline

A single dose of aminophylline (200 μmol/kg, i.p.) or triiodothyronine (T 3, 300 μg/kg, i.p.) resulted in the induction of ornithine decarboxylase (ODC) in rat liver with maximal activity 10-fold and 6-fold above controls, respectively, 4 hr after the administration of the drug or hormone. After either agent, the induction of ODC was blocked by either cycloheximide or actinomycin D. The same concentrations of aminophylline and T 3 administered simultaneously produced an additive 16-fold increase in ODC activity. After T 3 administration, the cyclic AMP-dependent protein kinase activity ratio was unaltered at all times measured. After aminophylline, the protein kinase activity ratio was elevated by 15 min and remained elevated for 2 hr. Somatostatin administration (50 μg/100 g), which lowers plasma growth hormone to 30% of control, had no effect on the ability of T 3 to induce ODC. These data suggest separate routes of induction of ODC in response to aminophylline and T 3. Aminophylline induction occurs via cycyclic AMP-mediated event whereas T 3 does not involve ccyclic AMP but results from a direct nuclear interaction.

Alterations in type I and type II protein kinases in the thyroid and adenohypophysis in response to a dietary goitrogen

The goitrogen, propylthiouracil (PTU), when administered in the diet of white leghorn chickens resulted in a marked increase in weight of the thyroid gland within 7 days which was maximal (160% of control) within 14 days. This system was used to determine the alterations in cyclic AMP-dependent protein kinase activity ratios and to assess the amounts of type I and type II cyclic AMP-dependent protein kinases present in the thyroid as well as any changes related to hypertrophy and hyperplasia of the thyroid. There was a marked elevation in the cyclic AMP-dependent protein kinase activity ratio ( −cAMP +cAMP ) 7 days after initiation of treatment as well as an increase in the total amount of supernatant cyclic AMP-dependent protein kinase activity (+cAMP) which doubled within 14 days of dietary PTU. Both type I and type II cyclic AMP-dependent protein kinase activities were detectable in the thyroid and adenohypophysis. Type I comprised 10% and type II 90% of the total cyclic AMP-dependent protein kinase activity in both tissues. The total amount of type I protein kinase activity increased significantly by 7 days. By 14 days, both type I and II protein kinases were increased twofold. The amount of type I returned to a control value by 21 days whereas type II remained elevated. Adenohypophysial type II protein kinase activity decreased to 70% of control by 14 days. Cyclic AMP is known to exert its effect on trophic responses through the activation of cyclic AMP-dependent protein kinase. These data implicate both activation of the enzymes as well as increased amounts of types I and II protein kinase activity in the thyroid in response to goitrogen-induced hypertrophy and hyperplasia.

Inhibitory effect of a lethal toxic fragment of staphylococcal α-toxin on cyclic amp-dependent protein kinase activity

The effect of a lethal toxic fragment of staphylococcal α-toxin on the activity of adenosine 3′,5′-monophosphate(cyclic AMP)-dependent protein kinase was examined. 1. 1. The lethal toxic fragment produced a dose-dependent decrease in both the binding of cyclic AMP to the regulatory subunit and phosphorylation activity of cyclic AMP-dependent protein kinase obtained from rabbit-skeletal muscles upto a plateau at a 50% inhibitory effect. The decrease in the activity of protein kinase observed with low doses of the lethal toxic fragment (0.1 μM) resulted from a competitive inhibition, probably by its interaction with the cyclic AMP-binding site in the regulatory subunit molecule. 2. 2. The effects of a lethal toxic fragment and epinephrine on the cyclic AMP level and protein kinase activity were investigated in the perfused rabbit heart slices. The lethal toxic fragment attenuated the stimulation of cyclic AMP-dependent protein kinase activity ratio by epinephrine. 3. 3. It is suggested that the specific action of a lethal toxic fragment on the cellular membrane enzymes may be attributable to the inhibition of the cyclic AMP-dependent protein kinase activity.

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium

Embryonic chick (7–9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionicf strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7–9-day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio in newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanined by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E 1 or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue. In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.

G 1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3′:5′-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G 1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G 1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G 1 phase (4–5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4–6 h after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G 1 or early S phase. Puromycin (either 10 μg/ml or 50 μg/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 μg/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G 1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cyclic AMP as Chinese hamster ovary cells traversed G 1 phase and entered S phase. These results suggest that cyclic AMP acts during G 1 phase to regulate the activation of cyclic AMP-dependent protein kinase.