low-Km cAMP Phosphodiesterase Research Articles

Modulation of cAMP and phosphodiesterase activity by flavonoids which induce spore germination of Nectria haematococca MP VI (Fusarium solani)

Flavonoids exuded from legume roots stimulate spore germination of a number of soilborne fungi which interact with these plants, and so may act as host cues to initiate interaction. Macroconidia of Nectria haematococca MPVI (anamorph Fusarium solani), a pathogen of pea (Pisum sativum), germinate in response to the same flavonoids which induce nod gene expression in pea-specific rhizobia. Pisatin, the isoflavonoid phytoalexin of pea, also induces germination. The present study found that cAMP levels in macroconidia were transiently induced by flavonoid treatment. Combined with previous pharmacological studies, this indicates that flavonoid signalling utilizes the cAMP pathway. A hypothesis that flavonoids modulate cAMP levels through direct inhibition of N. haematococca cAMP phosphodiesterase was tested. A low KMcAMP phosphodiesterase activity from macroconidia was tested for inhibition by 10 flavonoids. Naringenin, a strong inducer of germination, was a strong inhibitor of phosphodiesterase (apparent Ki33 μm). There was a general correlation between strength of induction and inhibition of phosphodiesterase. Pisatin, structurally distinct from the others, appeared to be an exception to this trend. The results suggest that the ability of specific flavones and flavanones to inhibit cAMP phosphodiesterase is a potential mechanism through which they can induce cAMP levels and so promote germination.

Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein.

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

Separation and some characteristics of two factors from rat liver particulate fraction which stimulate and inhibit the low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase of rat fat cells.

Two factors were separated from rat liver particulate fraction treated with insulin, one of them having a stimulating effect on low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase activity of crude microsomal fraction (P-2 fraction) and the other having an inhibiting effect on the activity of low-Km cAMP phosphodiesterase solubilized with 0.3% Brij 58 from P-2 fraction. Trypsin and heat treatments had essentially no effect on these two factors. The stimulating factor did not significantly change the apparent Km value of enzyme in P-2 fraction but increased the maximal velocity of the reaction. The inhibiting factor raised the Km value of solubilized enzyme without affecting the maximal velocity of the reaction. The stimulating factor level in diabetic rat was larger than that in normal rat while the inhibiting factor level in diabetic rat was smaller than that in normal rat. Possible participation of both factors in insulin action is discussed.

Mechanism of inhibition by glucagon of low-Km cAMP phosphodiesterase (cGi-PDE) activity in frog ventricles

Mechanism of inhibition by glucagon of low-Km cAMP phosphodiesterase (cGi-PDE) activity in frog ventricles

Effects of isozyme‐selective phosphodiesterase inhibitors on rat aorta and human platelets: smooth muscle tone, platelet aggregation and cAMP levels

The inhibitors of the cGMP-inhibited, low-Km cAMP phosphodiesterase--milrinone and OPC 3911--and an inhibitor of a non-cGMP-inhibited low-Km cAMP phosphodiesterase--rolipram--were used to evaluate the functional importance of the two cAMP phosphodiesterase activities in vascular smooth muscle and in platelets. Vinpocetine, an inhibitor of a calcium-calmodulin-dependent phosphodiesterase was also studied. OPC 3911 and milrinone relaxed the contracted rat aorta, inhibited ADP-induced platelet aggregation and also enhanced isoprenaline-induced relaxation as well as the antiaggregatory effects of adenosine. In platelets, OPC 3911 and milrinone increased cAMP levels, but in the rat aorta the increase was significant only for milrinone (OPC 3911 P = 0.062). In both tissues OPC 3911 and milrinone enhanced the increase in cAMP caused by activators of adenylate cyclase (isoprenaline/adenosine). Rolipram had no effects on aggregation or cAMP levels in platelets and no overadditive effects in combination with adenosine. Rolipram had little effect on relaxation and cAMP levels, did not alter isoprenaline-induced relaxation of guanfacin-contracted rat aorta, but showed synergistic effects with isoprenaline in raising cAMP levels. In PGF2 alpha-contracted aorta rolipram enhanced relaxation caused by isoprenaline. Vinpocetine had a relaxant effect without affecting cAMP levels, but had no effect on platelets. These results support the concept that the cGMP-inhibited phosphodiesterase is an important modulator of vascular smooth muscle tone and platelet function. The role of the non-cGMP-inhibited phosphodiesterase in these tissues is less obvious.

Cloning and Expression of cDNA for a Human Low-Km, Rolipram-Sensitive Cyclic AMP Phosphodiesterase

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.

Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation.

Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.

Relaxant Effects of the Selective Phosphodiesterase Inhibitors Milrinone and OPC 3911 on Isolated Human Mesenteric Vessels

Several new positive inotropic drugs with vasodilating properties for which a major mechanism of action is believed to be inhibition of phosphodiesterase (PDE) have been introduced in the treatment of congestive heart failure. Hydrolysis of cyclic nucleotides is catalyzed by multiple forms of PDE, which may vary between organs and cell-types. These enzymes can be selectively inhibited by various agents, theoretically making it possible to produce tissue-selective responses. An enzyme, which belongs to a subclass of cGMP inhibited low Km cAMP PDE, was recently purified from rat adipose tissue. The enzyme was specifically and potently inhibited by the cilostamide derivative OPC 3911 (OPC) and milrinone (mil). We studied the relaxant effects of OPC and mil on isolated human mesenteric arteries and veins in vitro. In preparations contracted by noradrenaline (NA), both agents produced about 60% maximum relaxation; OPC was 3-4 times more potent than mil. Both OPC and mil caused rightward displacement of the NA concentration-response curve and depressed the maximum responses. Arteries, as compared to veins, were more sensitive to this inhibition of NA contraction. Both drugs relaxed arteries contracted by 30 mM K+, but not 127 mM K+; maximum relaxation was between 60 and 70%. OPC was 10 times more potent than mil. The interactions between mil/OPC and isoprenaline, forskolin and ouabain were also studied. In preparations pretreated with isoprenaline or forskolin, the relaxant effects of mil and OPC were additive to those of isoprenaline and forskolin. Ouabain pretreatment did not affect the actions of the phosphodiesterase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

CAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

Effect of somatomedin C on insulin-sensitive phosphodiesterase in rat fat cells

Membrane-bound low-Km cAMP phosphodiesterase (PDE) was activated when intact rat fat cells were incubated with somatomedin C. Somatomedin C rapidly stimulated the enzyme, reaching a maximum reaction in 5 to 10 minutes. By kinetic analysis, somatomedin C activated PDE by increasing the maximal velocity (Vmax) values without altering the Michaelis-Menten constant (Km) values (0.24 ± 0.03 μmol/L). The ED 50 value of the activation by somatomedin C was very high (38.0 ± 3.2 nmol/L) compared with that of insulin (0.22 ± 0.07 nmol/L). This indicates that somatomedin C was about 173 times less potent than insulin in the stimulation of PDE. This potency ratio is similar to those that have been reported on lipid formation or on the other biologic insulinlike activities. When the insulin receptors were destroyed by trypsin treatment, effects of somatomedin C on the enzyme activation were abolished. This finding suggests that activation of PDE by somatomedin C was mediated through the insulin receptor.

Changes in low-Km cAMP phosphodiesterase activity in liver Golgi fractions from hyper- and hypoinsulinemic rats.

Acute insulin treatment in rats has recently been shown to cause a rapid increase in liver low-Km cAMP phosphodiesterase (PDE) activity, which selectively affects Golgi fractions. To assess the physiological significance of this observation, the cAMP PDE activity associated with liver Golgi fractions has been measured in genetically obese Zucker rats, which spontaneously develop hyperinsulinemia, in rats receiving a continuous infusion of insulin, and in rats treated with anti-insulin serum. In genetically obese Zucker rats, a significant increase in Golgi-associated cAMP PDE relative to age-matched lean animals occurred after 3 wk, coinciding with the development of hyperinsulinemia. This change was maximal at 5-8 wk and affected the light (Gl) and intermediate (Gi) Golgi fractions (100-110% increase) to a greater extent than the heavy (Gh) fraction (30% increase). After 7 wk, despite the further increase in insulinemia, the increase in Golgi-associated cAMP PDE became progressively less marked, and at 18 wk it was no longer detectable except in Gh, suggesting the development of a hepatic insulin resistance. Infusion of insulin through chronically implanted intracardiac catheters led to a 30-50% increase in Golgi-associated cAMP PDE, which occurred earlier in Gi (3 h) than in Gh (7 h) and persisted for greater than 96 h. Injection of anti-insulin serum led to a 30-50% decrease in Golgi-associated cAMP PDE, which occurred sequentially in Gl (5 min), Gi (15 min), and Gh (30 min) and affected predominantly Gl and Gh. These results suggest that the cAMP PDE associated with Golgi fractions is a physiological effector of plasma insulin in vivo.

Changes in low-Km cAMP phosphodiesterase activity in liver Golgi fractions from hyper- and hypoinsulinemic rats

Changes in low-Km cAMP phosphodiesterase activity in liver Golgi fractions from hyper- and hypoinsulinemic rats

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.

Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinguishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60-kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15-2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6, and 110 microM, respectively.

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions.

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.

Activation of insulin-sensitive phosphodiesterase by lectins and insulin-dextran complex in rat fat cells

Membrane-bound low-Km cAMP phosphodiesterase was activated by concanavalin A, wheat germ agglutinin, and insulin-dextran complex under conditions of incubation with intact rat fat cells. Concanavalin A rapidly stimulated the enzyme activities and maximum was reached at 10 to 15 minutes. As little as 10 μg/mL concanavalin A activated the enzyme and a maximal response was obtained at 100 to 300 μg/mL, but concanavalin A and wheat germ agglutinin were less potent than insulin. Specific saccharide inhibitors completely abolished activation of the enzyme by lectins, but had no effect on the activation of insulin. Digestion of fat cells with 1 mg/mL trypsin for 15 minutes completely inhibited activation of the enzyme by insulin. However, concanavalin A was less sensitive to trypsinization. The insulin-dextran complex, which did not penetrate the plasma membrane, activated the enzyme and was one tenth as effective as the native insulin. These results suggest that the insulin-like actions of these lectins are provoked through coupling with the carbohydrate moiety on the cell membrane close to insulin receptors.

Synthesis and characterization of iodopropyl derivatives of adenosine 3′,5′-cyclic-monophosphate: Potential affinity labels of cAMP binding sites in enzymes

The syntheses of two potential cAMP affinity lables, 1,N 6-(3-iodopropyleno)adenosine 3′,5′-cyclic-monophosphate and 2′-O-(2-iodo-3-hydroxypropyl) adenosine 3′,5′-cyclic-monophosphate, by a two-step chemical procedure are described. TheN 6- and 2′-O-allyl intermediates were prepared selectively by alkylation of cAMP in organic and alkaline aqueous solutions, respectively. Treatment of theN 6-allyl derivative withN-iodosuccinimide resulted in iodine addition to the double bond and cyclization to theN 1 position of the purine ring. The iodohydrin analog was synthesized by reaction of 2′-O-allyl-cAMP with potassium iodide and thallium trichloride in acetate buffered solution. The products were isolated by column chromatography and characterized by thin-layer chromatography, elemental analysis, and ultraviolet,13C, and1H NMR spectroscopy. The cAMP analogs were found to react with lysine and cysteine. Both cAMP derivatives were tested for their reaction with the low-K m cAMP phosphodiesterase of human platelets. The ribose-substituted analog functioned as a competitive inhibitor (K I =0.72 μM) and caused a time-dependent irreversible inactivation of the phosphodiesterase. In contrast, the purine-substituted derivative acted neither as a reversible competitive inhibitor nor as an irreversible inactivator of the enzyme. These results indicate the specificity of these potential cAMP analogs in their interaction with the phosphodiesterase.