A simple radioimmunoassay for 3',5', cyclic adenosine monophosphate.

A radioimmunoassay for 3′,5′ cAMP has been developed in which [³H]3′,5′ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is 0.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotides. Samples are prepared by ethanol precipitation. Tissue levels of 3′,5′ cAMP are comparable to those reported by others.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

Effect of adenosine 3',5' monophosphate on the mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine

The effect of increased cellular concentrations of adenosine 3',5' monophosphate (cAMP) upon mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in V79 Chinese hamster lung cells. Incubation with either forskolin, which increased the accumulation of cAMP, or 8BrcAMP, an analogue of cAMP, resulted in an increase in the mutation frequency which was concentration-dependent, regardless of whether these agents were added before or after mutagen treatment. Increased cAMP concentrations were shown in these cells to inhibit growth; however, this does not seem to be the mechanism responsible for the increase in mutation frequency as low serum concentrations which also retard growth reduced the mutation frequency observed with MNNG.     

Chlorpropamide and tolbutamide inhibition of adenosine 3'5' cyclic monophosphate phosphodiesterase

These sulfonylurea agents inhibit the cyclic AMP phosphodiesterase, and thereby could increase the steady state level of cyclic AMP in various tissues, depending upon the tissue concentrations achieved after oral or parental administration.     

Effects of ethanol on gastric mucosal adenosine 3', 5' monophosphate (cAMP)

The effects of ethanol on the gastric mucosal adenosine 3′, 5′-monophosphate (cAMP) system were evaluated. The activity of adenylate cyclase (AC), phosphodiesterase (PDE), and tissue content of cAMP were determined in the presence of ethanol. NaF stimulated AC in rat gastric mucosa was inhibited in vitro and in vivo by 20% ethanol. Basal AC activity was so low (0.05 ± 0.10 pmoles cAMP formed/min/mg protein) that consistent results without NaF could not be obtained. The PDE activity (172 ± 11 pmoles cAMP consumed/min/mg protein) was approximately 350 fold greater than the basal AC activity. All levels of ethanol tested (2.0–20.0%) significantly inhibited (p<0.05) PDE in vitro. Gastric mucosal levels of cAMP are not measurably altered by ethanol in vivo (5–20%).     

Methylxanthine effects on cyclic adenosine 3':5' monophosphate phosphodiesterase activity in preparations of neonatal rat cerebellum: modification by trifluoperazine

Paradoxically, caffeine was found to stimulate the activity of 3′:5′-cyclic AMP phosphodiesterase at substrate concentrations of 14μM, in cerebellar tissue from 10-day-old rats. Pretreatment with trifluoperazine, an inhibitor of calmodulin-dependent enzyme activation, converted the stimulatory effect of caffeine to the expected inhibitory action. Trifluoperazine pretreatment also increased the inhibitory action of theophylline on the cerebellar phosphodiesterase, but had no effect on the inhibitory action of 3-isobutyl-1-methylxanthine. It is suggested that caffeine and to a lesser extent theophylline in addition to their intrinsic phosphodiesterase inhibitory activity can also cause calmodulin dependent effects on cerebellar phosphodiesterase due to calcium mobilisation.     

Induction of lysozyme activity by adenosine 3':5' cyclic monophosphate in cultured mouse myeloid leukemic cells.

Mouse myeloid leukemic cells(Ml) could be induced by glucocorticoids to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. Adenosine 3′:5′ cyclic monophosphate also induced lysosomal enzyme activities, but not the other differentiation-associated properties. The induction of lysozyme activity was marked, the activity reaching about 400 times the initial activity at 5 days after treatment. This suggests that adenosine 3′:5′ cyclic monophosphate may be important in induction of lysozyme activity during differentiation of the cells.     

Opposite effects of dibutyryl adenosine 3':5' cyclic monophosphate and serum on growth of Chinese hamster cells

The effect of dibutyryl adenosine cyclic 3′:5′ monophosphate and testosterone on growth, response to serum and transport properties of Chinese hamster ovary cells was studied. There was a marked depression of growth in the presence of both compounds. A change of medium was sufficient to permit a partially synchronized burst of growth of the treated cells either in the presence or absence of dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone. However, in the presence of these compounds a second round of cell division was prevented. Initiation of the cell cycle by cells exposed to dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone displayed a greater serum requirement than untreated cells. It is concluded that serum and cyclic AMP could have antagonistic interactions in growth regulation. The treated cells had a reduced ability to accumulate amino-isobutyrate and glutamine, but no difference was observed with uridine uptake. The data suggest that a functional alteration of the membrane is induced by the exposure to dibutyrl adenosine cyclic 3′:5′ monophosphate plus testosterone.     

Prostaglandin-E2 and cyclic adenosine 3'-5' monophosphate levels in the hypertrophied rat heart.

To assess whether prostaglandin-E2 (PGE2) and cyclic adenosine 3'-5'-monophosphate (cAMP) are involved in the cardiac response to chronic pressure overload, we measured by specific radioimmunoassay method the cardiac tissue and plasma concentrations of PGE2 and cAMP in an animal model of left ventricular hypertrophy. The cardiac hypertrophy was accompanied by a significant increase in PGE2 content, and a significant decrease in cAMP content, in the heart. In addition, we found elevated PGE2 and cAMP levels in arterial plasma samples from the rats with hypertrophied hearts compared to normal rats. These findings suggest a link between cardiac and vascular PGE2 and cAMP generation and the hemodynamic stresses of advanced cardiac overload.     

Elevation of cyclic 3'5' adenosine monophosphate levels by cholera toxin inhibits the generation of interleukin 2 activity

Several molecules can interact with membrane receptors on mononuclear cells to increase intracellular levels of cyclic adenosine monophosphate (cAMP). We used the cholera toxin (CT), a cAMP elevating agent, to study the influence of this nucleotide on the production of interleukin 2 (IL-2) by human peripheral blood mononuclear cells stimulated by phytohemagglutinin and phorbol myristate acetate. Stimulated generation of IL-2 activity was inhibited by CT but not by its B subunit. The inhibition was potentiated by addition of theophylline. Therefore the synthesis and/or release of IL-2 is controlled by intracellular cAMP levels and may be modulated by agents active on this nucleotide system, such as bacterial toxins, glycoprotein hormones, or neurotransmitters.