Stimulation of cartilage macromolecule synthesis by adenosine 3',5'-monophosphate.

The role of cyclic AMP in the regulation of cartilage macromolecule synthesis in vitro was studied in pelvic cartilage from 10-12 day chick embryos. Incubation of cartilages in medium containing 0.5 mM cyclic AMP resulted in a 30% inhibition of 35SO4-2, [3H]leucine and [3H]uridine incorporation into proteoglycan, total protein and RNA, respectively. Higher concentrations of cyclic AMP had no greater effects. In contrast, butyrylated cyclic AMP derivatives (0.5-5.0 mM) added to the incubation medium stimulated (50-100%) the incorporation of these radiolabeled precursors into cartilage macromolecules. Theophylline, in concentrations (0.1-0.5 mM) which raise intracellular cyclic AMP, also increases the incorporation of radiolabeled precursors into macromolecules. The data indicate that exogenous cyclic AMP and butyrylated cyclic AMP derivatives have paradoxical effects on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives, not exogenous cyclic AMP, mimic the effects of intracellular cyclic AMP. Incubation of embryonic chicken cartilage with exogenous cyclic AMP results in the extracellular degradation of the cyclic AMP to adenosine. Adenosine (0.125 mM) inhibits precursor incorporation into cartilage macromolecules. The metabolism of exogenous cyclic AMP generates sufficient adenosine to account for the observed inhibitory effects of exogenous cyclic AMP on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives are not degraded during incubation with cartilage. The data indicate that cartilage is a tissue in which the effect of cyclic AMP is to stimulate anabolic processes.     

Regulation of adenosine 3':5'-monophosphate content of rous sarcoma virus-transformed human astrocytoma cells. Effects of cholera toxin on the responsiveness to catecholamines and prostaglandins.

Human astrocytoma cells (EH118MG) respond to catecholamines and prostaglandins with a marked increase in the rate of formation of cyclic AMP. Treatment of EH118MG cells with cholera toxin (10 to 100 ng/ml) for 45 to 60 min caused an increase in cellular cyclic AMP content (5- to 10-fold over basal). Cholera toxin also decreased the K0.5 for isoproterenol 10- to 50-fold and decreased the K0.5 for prostaglandin E1 (PGE1)30- to 100-fold, while increasing the maximal response to PGE1 by 1.5- to 3-fold. Treatment with cholera toxin did not change the K1 values for beta-adrenergic receptor antagonists such as propranolol, alprenolol, and sotalol. Direct binding studies using [125I]iodohydroxybenzylpindolol indicated no significant changes in the number of beta-receptors or in the kinetics of the interaction of the radioligand with receptors after treatment of cells with the toxin. Competition binding studies with propranolol and sotalol revealed no toxin-induced change in Kd values for these antagonists. Treatment with cholera toxin caused only small decreases (2- to 3-fold) in the Kd values for binding of isoproterenol and norepinephrine. It is concluded that cholera toxin has little direct effect on the binding of agonists or antagonists to beta-receptors, but instead increases the efficiency of coupling of receptor and catalytic moieties of adenylate cyclase.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Inhibition of Escherichia coli growth by cyclic adenosine 3', 5'-monophosphate

Cyclic AMP inhibits growth rate of E. coli Hfr 3000. Doubling times in glucose minimal medium increased from 60 to about 90 minutes with the addition of 5 mM cAMP. This effect is specific since it was not observed when the cyclic nucleotide was replaced by 5′ AMP, ADP, ATP or adenosine. Half maximal inhibition was obtained with 1 to 3 mM cyclic AMP. This inhibition occurs only with those carbon sources which are known to decrease intracellular cyclic AMP levels, i.e. glucose and pyruvate. No inhibition was observed with succinate, malate or glycerol.     

Regulation of adenosine 3':5'-monophosphate efflux from animal cells.

Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.     

Stimulation of cyclic adenosine 3':5'-monophosphate and corticosterone formation in isolated rat adrenal cells by cholera enterotoxin. Comparison with the effects of ACTH.

1. The production of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and corticosterone isolated ratadrenal cells was increased by cholera enterotoxin. Both responses were accompanied by a lag period which is characteristic of other known actions of enterotoxin. The duration of the lag period in the production of corticosterone depended on the concentration of enterotoxin; with the maximally stimulating amounts it was 30-45 min. 2. Maximum rates of cyclic AMP and corticosterone synthesis, after the lag period, were constant for at least 1 h. Although the maximum rate of corticosterone formation was the same as that obtained adrenocorticotropic hormone, the maximum rate of cyclic AMP formation was only 8-10% of that with adrenocorticotropic hormone. 3. Pretreatment of the cells with enterotoxin ahd no effect on their subsequent steroidogenic response to maximally stimulating amounts of adrenocorticotropic hormone. 4. Cycloheximide inhibited the effect of both enterotoxin and adrenocorticotropic hormone on corticosterone production. 5. Enterotoxin stimulation of both cyclic AMP and corticosterone formation was dependent on the presence of Ca2+ in the medium although the Ca2+ requirement was not same as that for adrenocorticotropic hormone. Thus, EGTA at concentrations which completely abolished the effect of adrenocorticotropic hormone caused only a partial reduction in the effects of enterotoxin. 6. Exogenously added choleragenoid and gangliosides abolished the effects of enterotoxin without having any significant effect on the response of the cells to adrenocorticotropic hormone. 7. After treatment with neuraminidase, the adrenal cells showed an increased response to enterotoxin in terms of both cyclic AMP and corticosterone formation which was due to a combination of two effects: (a) increased rate of synthesis of both compounds and (b) shortening of the characteristic lag period. This is in sharp contrast to the results obtained with adrenocorticotropic hormone where neuraminidase-treatment made the cells less sensitive to adrenocorticotropic hormone.     

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.     

Inhibition of adenosine 3',5'-monophosphate phosphodiesterase by nicotinamide and its homologues in vitro.

Inhibition of cAMP phosphodiesterase from rat liver by nicotinamide and its homologues was studied. Among the several compounds tested, such agents as nicotinamide, N2-ethylnicotinamide, N2-methylnicotinamide, N,N-diethylnicotinamide, 3-acetylpyridine, methylnicotinate, and ethylnicotinate showed potent inhibition of cAMP phosphodiesterase, with an over 90% inhibition by 5 mM ethylnicotinate when cAMP was used as substrate at a 0.48 x 10(-7) M concentration. A comparison of the inhibitory curves of theophylline, papaverine, and ethylnicotinate on enzyme activity showed them to be approximately coincident. Furthermore, the plots with abscissa of equal increments per concentration of ethylnicotinate in the presence of theophylline or papaverine coincided with that in the absence of these agents.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Guanosine 3',5'-monophosphate receptor protein: separation from adenosine 3',5'-monophosphate receptor protein.

A specific cGMP receptor protein has been identified and separated from the cAMP receptor protein by chromatography on 8-(6-aminohexyl)-amino-cAMP-Sepharose. Scatchard analysis of cGMP binding indicates a single affinity class of receptor sites with KD = 1.4 × 10^?8 M. The specificity of the cGMP receptor site has been defined by using a number of nucleotides as competitors for cGMP binding. The cGMP receptor protein sediments at 7S in glycerol density gradients.     

Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Mediation by adenosine 3':5'-monophosphate.

The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of alpha-amino [1-14C] isobutyrate or [1-14C] cycloleucine than control cartilages incubated in medium alone.Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate amino-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r=0.977). Theophylline and prostaglandin E1, WHICH RAISE CARTILAGE CYCLIC AMP also increase amino-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyryl cyclic AMP and n6, 02'-dibutyryl cyclic AMP increase cartilage amino-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.