U. Schwabe

Adenosine release from isolated fat cells and its significance for the effects of hormones on cyclic 3′,5′-AMP levels and lipolysis

SummaryThe effect of lipolytic stimulants on cyclic AMP levels and glycerol production is strongly dependent on the concentration of fat cells in the incubation medium. After addition of noradrenaline cyclic AMP levels in diluted fat cell suspensions (20 000 cells/ml) reached 10 fold higher levels and declined much more slowly than in concentrated cell suspensions (100 000 cells/ml). An inhibitory substance appeared in the incubation medium which, after addition to a suspension of fresh fat cells caused a dose-dependent inhibition of hormone effects. The inhibitor was produced during preincubation periods of the fat cells and was removed by washing the cells with fresh medium.Purification of the medium by acid extraction, gel filtration, thin layer chromatography and the identification by gas chromatography showed that the inhibitor released represents adenosine. After incubation with adenosine deaminase the inhibitory activity of the medium disappeared. No inhibitory activity was obtained from the incubation medium by solvent extraction, a procedure which fully recovered added PGE2. Adenosine appeared in the medium after 5 min of incubation of fat cells and reached maximal levels (0.2 nmol/ml/105 cells) after 20 min. Noradrenaline (10 μM) did not stimulate the release of adenosine from fat cells. Addition of 0.01 to 0.1 μM of adenosine to fat cells effectively inhibited cyclic AMP accumulation and lipolysis induced by noradrenaline. Hence, a delayed rise and the secondary decline of cyclic AMP levels after hormonal stimulation can be accounted for by adenosine released from fat cells.

An improved assay of cyclic 3′,5′-nucleotide phosphodiesterases with QAE-Sephadex columns

SummaryA method for the assay of cyclic nucleotide phosphodiesterases is described which employs chromatographical separation of cyclic nucleotides and nucleosides on QAE A-25 Sephadex columns and 5′-nucleotidase as an auxiliary enzyme. The assay allows quantitative recovery of adenosine, guanosine and their metabolites from the anion exchanger and thus is suitable for use in crude phosphodiesterase preparations containing purine catabolizing enzymes. in comparative studies, this method was found to be considerably more sensitive than previous reported methods because of lower assay blanks and higher recoveries of the nucleoside catabolites. The method is suitable for kinetic analysis of crude enzyme preparations from micromolar to millimolar substrate concentrations.

Different effects of lipolytic hormones and phosphodiesterase inhibitors on cyclic 3′,5′-AMP levels in isolated fat cells

SummaryCyclic AMP levels of isolated fat cells of rats were increased about 50-fold by noradrenaline (1 μM) and isoprenaline (1 μM) within 4 min of incubation and had declined markedly after 10 min. The effect of a variety of lipolytic hormones on cyclic AMP levels was dose-dependent over a wide range of concentrations, ACTH and glucagon being most potent on a molar basis. Among the adrenergic compounds tested isoprenaline elicited the grea test response. Glucagon, in maximally effective concentrations, caused only half the increase in cyclic AMP values produced by other hormones. This pattern was largely paralleled by the lipolytic effects of the hormones as measured by glycerol production. Various inhibitors of cyclic AMP phosphodiesterase had only small effects on cyclic AMP accumulation. Methylxanthines caused only a 3- to 5-fold elevation of cyclic AMP levels at concentrations which induced maximal lipolytic effects. Papaverine (0.5 mM) and phentolamine (0.1 mM) had virtually no effect and did not potentiate hormone effects on cyclic AMP production. However, theophylline (1 mM) caused a nearly tenfold increase in the effects of isoprenaline when added to 100 000 cells per ml in the medium. At a concentration of 20 000 fat cells per ml isoprenaline alone increased cyclic AMP levels 300-fold and the addition of theophylline had no further stimulatory effect. Our results suggest that lipolysis induced by hormones is mainly mediated by an accumulation of cyclic AMP, whereas methylxanthines must have additional effects. The potentiation of lipolytic hormones by methylxanthines cannot be attributed to an inhibition of phosphodiesterase alone, but seems to be due mainly to their antagonism with an inhibitory factor, which is produced by fat cells and released into the incubation medium.

Studies on the antilipolytic effect of adenosine and related compounds in isolated fat cells

SummaryBasal lipolysis in isolated fat cells of rats was increased by adenosine in a dose-dependent manner. Low concentrations of this nucleoside (1–10 μM) inhibited noradrenaline-induced glycerol production by about 50% and completely blocked the effect of theophylline on fat cells. Glycerol release, induced by dibutyryl cyclic AMP, was increased by 5 μM adenosine. Inosine, hypoxanthine, and xanthine had weaker antilipolytic properties, whereas adenine was virtually without effect.Although dipyridamole (20 μM) strongly decreased the uptake of adenosine into fat cells, it did not counteract the antilipolytic action of this nucleoside. Low concentrations of adenosine (0.1 μM), which by themselves were without any effect, greatly enhanced the effect of insulin on lipolysis. It is tentatively suggested that adenosine may be involved in the physiological control of lipolysis and that this nucleoside has its site of action on the cell membrane.

Specific binding of 3H-adenosine to rat brain membranes

SummaryThe binding of 3H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of 3H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (Kd) were 1.7 μM and 13.6 μM and the maximal number of binding sites (Bmax) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with 3H-adenosine binding. The phosphorylated adenosine compounds 5′-AMP, cyclic AMP and ATP were most potent in displacing 3H-adenosine from its binding sites and the IC50-values ranged from 11–25 μM. N6-Phenylisopropyladenosine produced only partial inhibition (30%) of 3H-adenosine binding and no stereospecific difference between the (−)-and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the 3H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.

Stimulation of cyclic adenosine 3′,5′-monophosphate accumulation and lipolysis in fat cells by adenosine deaminase

SummaryThe basal lipolytic activity of isolated fat cells of the rat was greatly enhanced in the presence of 0.01 to 30 μg adenosine deaminase (ADA) per ml. This effect was more pronounced in dilute (20000 cells/ml) than in concentrated cell suspensions (100000 cells/ml); this is possibly due to the presence, in the incubation medium, of a high concentration of inosine which is formed by the deamination of the large amounts of adenosine released from high concentrations of fat cells. Inosine, although less potent than adenosine as an antilipolytic agent, markedly inhibited ADA-induced lipolysis at concentrations between 10 to 100 μM. The lipolytic effect of ADA was identical with the stimulation of lipolysis by 1 μM noradrenaline or 1 mM theophylline, while 1 mM dibutyryl cyclic AMP yielded two-fold higher values. The effects of ADA and lipolytic agents at maximally stimulating concentrations were not additive.After 5 min of incubation maximally effective concentrations of ADA which were also maximal with respect to lipolysis caused a 3- to 6-fold elevation of cyclic AMP levels in fat cells. A similar increase was observed with maximally effective concentrations of theophylline, whereas noradrenaline produced a 100- to 200-fold elevation. This indicates that a small accumulation of cyclic AMP may be sufficient to trigger the full lipolytic response. Furthermore, ADA, like theophylline, acted synergistically with noradrenaline and prevented the fall of cyclic AMP levels during 30 min of incubation.Insulin (100 μU/ml) and nicotinic acid (0.1 μM) decreased cyclic AMP accumulation and glycerol production induced by ADA.The results support the hypothesis that adenosine is released from isolated fat cells and that this nucleoside may serve as an inhibitor of adenyl cyclase activity, thus regulating cyclic AMP-dependent processes in adipose tissue.

Activation and inhibition of lipolysis in isolated fat cells by various inhibitors of cyclic AMP phosphodiesterase

SummaryThe effects of methylxanthines, papaverine, dipyridamole and imipramine on lipolysis and phosphodiesterase activity of rat adipose tissue were investigated. Lipolysis in isolated fat cells was stimulated by theophylline and caffeine whereas papaverine, dipyridamole and imipramine had no substantial effect on the basal lipolytic rate. Lipolysis induced by noradrenaline was potentiated by theophylline, but blocked by papaverine, dipyridamole and imipramine at concentrations between 0.02 to 0.2 mM. These agents also depressed lipolysis induced by theophylline and dibutyryl cyclic AMP and reduced the lipolytic activity of homogenates of adipose tissue. The activity of phosphodiesterase assayed over a wide range of substrate concentrations revealed two different Michaelis constants. Both types of phosphodiesterase were inhibited by theophylline, papaverine, dipyridamole and imipramine in a competitive manner, the low Km enzyme being more sensitive for inhibition than the high Km enzyme. On both types of phosphodiesterase papaverine and dipyridamole proved to be 10 to 100 times more potent inhibitors than theophylline and imipramine. To explain the antilipolytic effect of phosphodiesterase inhibitors it is assumed that they do not only affect substrate binding of cyclic AMP to phosphodiesterase but also displace cyclic AMP from the binding site on protein kinase, thus acting as inhibitors of the activation process within the lipolytic system.

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

SummaryThe effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 μmoles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 μmoles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 μmoles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 μmoles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 μmoles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 μmoles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 μmoles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 μmoles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 μmoles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 μM ZK 62711 increased the level of cyclic AMP but 100 μM histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 μM ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 μM cimetidine and enhanced the stimulatory effect of 100 μM histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC50=18 μM) and guinea pig gastric mucosa (IC50=1.5 μM). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.

Biphasic effect of 5'-guanylylimidodiphosphate on fat cell adenylate cyclase.

Summary5′-Guanylylimidodiphosphate (GMP-PNP) had a biphasic effect on basal adenylate cyclase activity of rat fat cell ghosts, being inhibitory and stimulatory while GTP was mainly inhibitory. At low concentrations of GMP-PNP a transient inhibitory phase preceded the onset of activation. This initial inhibition was overcome by higher concentrations of GMP-PNP, ATP or magnesium.The stimulatory effects of GMP-PNP were increased by high concentrations of ATP or magnesium, the apparent Km for activation being a function of time. After 5 min of incubation half-maximal activation was obtained at 3 μM GMP-PNP, after 20 min of incubation the Km for GMP-PNP was found to be between 0.1 and 0.3 μM. After 20 min of incubation a 15fold increase of cyclase activity above basal level was observed in the presence of 1 μM GMP-PNP. GTP competitively inhibited the stimulant effect of GMP-PNP. On the other hand, it activated basal activity only under carefully selected conditions.GMP-PNP and noradrenaline had a synergistic action on cyclase activity. At high substrate concentrations (1 mM ATP) GMP-PNP shifted the apparent Km for activation by noradrenaline from 3 μM to 0.1 μM. At low substrate and high magnesium concentrations 1 μM noradrenaline was unable to stimulate adenylate cyclase. Under these conditions GMP-PNP facilitated the stimulation by the hormone, although GMP-PNP itself inhibited basal activity.It is suggested that GMP-PNP activates the adenylate cyclase by competing at a common nucleotide binding site with inhibitory agents such as free ATP or GTP. Moreover, the guanyl nucleotide analogue may initiate conformational changes of the enzyme system which facilitate the response to hormones.

Sensitive determination for adenylate cyclase activity by cyclic adenosine 3′,5′-monophosphate protein binding assay

SummaryA sensitive method for measurement of adenylate cyclase activity in fat cell ghosts is described; it applies the protein binding assay for cyclic AMP of Gilman (1970). Unlabelled ATP is used as substrate in the presence of an ATP-regenerating system containing 5 mM creatine phosphate and 0.1 mg creatine kinase per ml. Measurement of ATP levels showed that at least 80% of the substrate level is maintained during the standard assay procedure. Chromatographic separation of cyclic AMP can be omitted, since the high specificity of the binding protein allows a dilution of the samples below the concentrations at which ATP and other nucleotides interfere with the binding of cyclic AMP. Thus, the measurement of nM concentrations of cyclic AMP in the presence of mM concentrations of ATP is achieved.The main advantage of the method lies in the use of low protein concentrations; it reduces interfering effects of membrane-bound ATPases and phosphodiesterases. No precautions such as addition of phosphodiesterase inhibitors are needed, since cyclic AMP degradation is negligible during standard incubation conditions.