Adenosine 3',5'-cyclic monophosphate phosphodiesterase in guinea-pig lung--properties and effect of adrenergic drugs

The hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP) by adenosine 3',5'-monophosphate phosphodiesterase (PDE) was studied in whole homogenates and cell fractions of guinea-pig lung. Approximately 60 per cent of the activity of the whole homogenate was contained in the 10,000 g supernatant. This cell fraction contained PDE with two separate affinities for cAMP: PDE I with low affinity. K_m ? 3.1 × 10^?4 M; V_max ? 90 nmoles cAMP hydrolyzed/mg protein/20 min and PDE II with high affinity K_m ? 5.7 × 10^?5 M; V_max ? 35 nmoles cAMP hydrolyzed/mg protein/20 min. Both forms required magnesium which could be replaced by manganese and cobalt but not by copper. Aminophylline and theophylline inhibited both PDE forms. Calcium only inhibited PDE I but not PDE II. Adrenergic drugs inhibited PDE I and at 0.1 mM decreased in potency in the following order: isoproterenol  epinephrine > norepinephrine > methoxamine. Butoxamine was ineffective on PDE I. Only norepinephrine inhibited PDE II but not to the same extent as its effect on PDE I. Chelation of added magnesium and endogenous calcium was not responsible for the inhibition of PDE I by the adrenergic drugs used. Propranolol (0.1 mM) and phentolamine (0.05 to 0.001 mM) alone did not effect PDE I. Propranolol (0.1 mM) failed to reverse the inhibitory effect of isoproterenol (0.1 mM) and epinephrine (0.1 mM). Thus a β-adrenergic mechanism could not explain the inhibitory effect of these agents. Phentolamine (0.05 to 0.001 mM) reversed the inhibition by epinephrine bitartrate but not by epinephrine hydrochloride. These data are inconclusive to support an α-adrenergic mechanism for the inhibition of cAMP phosphodiesterase by epinephrine.     

Effects of benzydamine on the lipolytic system of fat cells.

Benzydamine dose-dependently increased cyclic 3',5'-AMP levels in isolated fat cells and competitively inhibited phosphodiesterase activity (K_i = 1.1 mM). ATP levels of isolated cells and cyclic 3',5'-AMP-dependent protein kinase activity were not affected. However, benzydamine caused a dose-dependent inhibition of lipolysis in isolated fat cells stimulated by norepinephrine or dibutyryl cyclic 3',5'-AMP. The enhancement of cyclic 3',5'-AMP formation may be due to the local anaesthetic properties of benzydamine. The antilipolytic effect appears to result from the direct inhibition of hormone sensitive triglyceride lipase.     

Reassessment of the interactions of guanylate kinase and 6-thioguanosine 5'-phosphate.

Contrary to published studies, 6-thioguanosine 5'-phosphate (6-thioGMP) was found to be a relatively weak inhibitor of the phosphorylation of GMP catalyzed by guanylate kinase. The inhibition constant for 6-thioGMP was 2.3 mM for the enzyme from human erythrocytes. This K_i value is 30-fold larger than previously reported values. The weak inhibition by 6-thioGMP is related to the alternate substrate activity of this compound. It has an apparent K_m value of 2.1 mM and a maximal velocity of 3 per cent of that attainable with GMP as the saturating substrate. The discrepancy between these findings and those previously reported was found to be due to a spectrophotometric artifact that was associated with the high absorbance of 6-thioGMP at the wavelength previously used for the velocity measurements. Guanylate kinase from rat liver, hog brain, Sarcoma 180 tumor cells and Ehrlich ascites tumor cells was also inhibited by 6-thioGMP with a K_i in the mM range.     

Cyclic nucleotide metabolism in Walker carcinoma cells resistant to alkylating agents.

Walker carcinoma cells resistant to growth inhibition by 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) also show some degree of cross-resistance to the cytotoxic effect of N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dbcAMP). Using DEAE-cellulose chromatography and a linear salt gradient the cAMP-dependent protein kinase, binding protein and phosphodiesterase from sensitive and resistant cells has been resolved into multiple forms. A type 1 enzyme which elutes at 0.03 M KCl is present only in sensitive cells and those with a low resistance to CB 1954. A type 2 enzyme which elutes between 0.15 and 0.28 M KCl is present in all cell lines. There is a decrease in specific activity of the cAMP-dependent protein kinase, binding protein and phosphodiesterase with increasing resistance to CB 1954. The binding proteins from resistant cells were more sensitive to temperature than those from sensitive cells, suggesting a difference in conformation of the receptor. Both 2-mercaptoethanol and 5,5′-dithiobis(2-nitrobenzoic acid) increase the temperature sensitivity of the proteins with 2-mercaptoethanol producing a greater effect on the proteins from the resistant lines. The cAMP-dependent protein kinase of resistant Walker cells exhibits an apparent K_a for activation by cAMP 2.5-fold greater than that of sensitive cells. Heterologous reconstituted enzymes using separated subunits from sensitive and resistant cells show defects in both R and C subunits in resistant cells.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

The interaction of 4-alkyl derivatives of 2,6,7-trioxa-1-phosphabicyclo [2,2,2] octanes with cyclic adenosine 3',5'-monophosphate phosphodiesterase, and with cyclic adenosine 3',5'-monophosphate binding proteins.

4-Alkyl-2,6,7-trioxa-1-phosphabicyclo [2,2,2] octanes are weak competitive inhibitors of the high-K_m form of cyclic adenosine 3′,5′-monophosphate phosphodiesterase (PDE) and non-competitive inhibitors of the low-K_m form enzyme. The possibility that inhibition of the low-K_m form enzyme by the bicyclic organo-phosphates may contribute to their toxic action is discussed. The compounds do not affect the binding of adenosine 3′,5′-monophosphate (cyclic AMP) with specific binding proteins, and are unlikely to have any effect on cyclic AMP receptors.     

Accumulation of adenosine 3′, 5′-monophosphate induced by prostaglandin E1 binding to mastocytoma P-815 cells

Addition of prostaglandin E₁ (PGE₁) to intact mouse mastocytoma P-815 cells stimulated adenosine 3', 5'-monophosphate (cAMP) accumulation and retarded cellular growth. To study the effects of prostaglandin (PG) binding on cAMP accumulation, specific [³H]PG binding to intact mastocytoma cells was examined. Intact mastocytoma cells have two types of binding sites for PGE₁ with high (K_d = 2.14 × 10^?9 M) and low (k_d = 1.05 × 10^?8 M) affinities and one type of binding site for PGF_2α. Mastocytoma cells, however, did not have a binding site for PGA₁ or PGD₂. The order of potencies of nonradioactive prostaglandins in competing with [³H]PGE₁ for binding sites was as follows: PGE₁ ? PGE₂ ? PGF_2α ? PGA₁ ? PGD₂ ? PGI₂. In contrast, the relative potencies of the prostaglandins in enhancing cAMP accumulation were PGI₂ ? PGE₁ ? PGE₂ ? PGA₁ ? PGF_2α = PGD₂, indicating that the receptors for E type and I type of PGs were different. Refractoriness of mastocytoma cells to PGE₁ stimulation of cAMP accumulation developed within 1 min of incubation of the cells at 37°, but disappeared immediately after decreasing the temperature to below 23°. A change in the number of PGE₁ receptors was not observed. cAMP concentrations were quickly restored by increasing temperatures from below 23° to 37°. This refractoriness did not develop in the presence of phosphodiesterase inhibitors. Furthermore, the activity of phosphodiesterase in mastocytoma cells was enhanced within 2 min by PGE₁ treatment.     

Differential effects of cyclic nucleotides and their analogs and various agents on cyclic GMP-specific and cyclic AMP-specific phosphodiesterases purified from guinea pig lung.

The differential effects of various compounds on the activities of cyclic GMP-specific phosphodiesterase (cyclic GMP-PDE) and cyclic AMP-specific phosphodiesterase (cyclic AMP-PDE) purified from the guinea pig lung were examined. Cyclic IMP, 2'-deoxy cyclic GMP and 2'-O-monobutyryl cyclic GMP were found to be the most potent inhibitors of the cyclic GMP-PDE, with I₅₀ (concentrations of the compounds inhibiting 50 per cent of the activity) values of 1.5, 4.5 and 35 μM respectively. These compounds, however, were at least 30–600 times less potent in inhibiting the cyclic AMP-PDE. In contrast, 2'-O-monobutyrul cyclic AMP and 2'-deoxy cyclic AMP were the most potent inhibitors of cyclic AMP-PDE, with I₅₀ values of 10 and 45 μM respectively; these compounds, however, were at least 30–100 times less potent in inhibiting cyclic GMP-PDE. Ethanol (13%, v/v) stimulated cyclic GMP-PDE 80 per cent while conversely inhibiting cyclic AMP-PDE 75 per cent. Most of the phosphodiesterase inhibitors studied were found to be more selective for cyclic AMP-PDE, with the possible exception of 1-methyl-3-isobutylxanthine, which was more specific for inhibiting cyclic GMP-PDE. The differential inhibition of the two classes of phosphodiesterases by a wide variety of compounds shown in the present study suggests a possibility of selective regulation of the tissue levels of respective cyclic nucleotides through specific or preferential inhibition of their enzyme activities.     

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

Summary The 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 K _m enzyme being more sensitive for inhibition than the high K _m 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 bioamines on the cellular differentiation of Hartmannella culbertsoni

Epinephrine, 5-hydroxytryptamine, dopamine and tyramine induce axenic encystation of the free living amoeba Hartmannella culbertsoni. Cycloheximide inhibits encystation of amoeba mediated by the amines. The amines uniformly stimulate 3 to 4-fold the ability of the cells to synthesize cyclic 3′,5′-adenosine monophosphate from adenosine triphosphate formed in situ. Epinephrine[¹⁴C] is bound to membranes of the amoeba (K_mof binding = 6.5 mM epinephrine). When amoebae are exposed to epinephrine, tyramine, dopamine or cAMP in a non-nutrient medium for 6 hr protein kinase activity is stimulated as evident from increased incorporation of ³²P into cellular proteins.     

Phosphodiesterase in the liver fluke, Fasciola hepatica.

Phosphodiesterase was found in homogenates of the liver fluke, Fasciola hepatica, and was distributed between a supernatant and particulate fraction after centrifugation at 2000 g. Mg²⁺ was necessary for enzyme activity; Ca²⁺ in the presence of Mg²⁺ did not affect enzyme activity. Enzyme kinetics followed the Michaelis-Menten model with a K_m of 8 μM for cAMP and 300 μM for cGMP as the substrate. The most potent inhibitor tested was 1-ethyl-4-(isopropylidenehydrazino)-1 H-pyrazolo- (3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HC1 (SQ 20009) which had a K_i of 26 μM. The K_i for isobutyl methyl xanthine (IBMX) was 45 μM; for 6,7 dimethyl-4 ethylquinazoline (Quazodine) 75 μM; papaverine. 100 μM; theophylline, 550 μM; and for caffeine or D-lysergic acid diethylamide (LSD), 800 μM. The effects on fluke motility of these phosphodiesterase inhibitors were tested. All phosphodiesterase inhibitors except caffeine stimulated the rhythmical movement of the flukes. None of the inhibitors tested significantly increased the endogenous cAMP concentrations of fluke heads. IBMX potentiated the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT) but SQ 20009, LSD, and papaverine prevented it. The latter results could not be explained on the basis of phosphodies-terase inhibition, but might be attributed to interference with the stimulation of adenylate cyclase by 5-HT.     

The effect of alkylating agents on adenosine 3',5'-monophosphate metabolism in Walker carcinoma.

The effect of some alkylating agents on the activity of the enzymes adenylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase has been studied using Walker carcinoma cells in tissue culture. The monofunctional agent 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), which has previously been shown to elevate the level of adenosine 3′,5′-monophosphate (cyclic AMP) in sensitive Walker cells, has been shown to have no effect on the activity of adenylate cyclase either in the presence or absence of the protecting agent 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl-AlC). Chlorambucil (p-N,N(di-2-chloroethylamino)phenylbutyric acid) (5 μg/ml) while having no effect on either the basal or fluoride-stimulated adenylate cyclase activity caused an inhibition of the high affinity form of the cyclic AMP phosphodiesterase which reached a maximum after 1 hr. This was accompanied by an increase in the intracellular level of cAMP which was proportional to the dose of chlorambucil up to a maximal 2-fold increase at 6.4 μg/ml, a dose which caused complete inhibition of cell growth. Further increases in the concentration of chlorambucil up to 100 μg/ml caused no further increase in cAMP level. Merophan ($\text{dl}\text{-}\text{o}\text{-N,N(}\text{di}\text{-2-}\text{chloroethylamino}\text{)}\text{phenylalanine}$) (0.5 μg/ml) similarly caused an inhibition of the low K_m form of the phosphodiesterase, but the rate of inhibition was slower than that observed with chlorambucil. The molecular forms of the cAMP phosphodiesterase in Walker cells sensitive or resistant to chlorambucil have been resolved using Sepharose 6B gel chromatography. The resistant lines displayed a reduction in the specific activity of the high affinity form of the enzyme which was accompanied by a shift to lower molecular weight forms. This could explain the lack of effect of chlorambucil on cAMP levels in Walker cells with acquired resistance to this agent.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.     

Modes of action of hypoxanthine, inosine and inosine 5'-monophosphate on cyclic nucleotide phosphodiesterase from bovine brain

A purified bovine brain cyclic nucleotide phosphodiesterase catalyzed the hydrolysis of both cyclic AMP and cyclic GMP. Alternative substrate inhibition experiments indicated that cyclic AMP and cyclic GMP were hydrolyzed by the same enzyme and that they shared a common binding site. Inosine, inosine 5'-monophosphate and hypoxanthine competitively inhibited the hydrolysis of cyclic AMP and cyclic GMP by bovine brain cyclic nucleotide phosphodiesterase. This inhibition of cyclic nucleotide hydrolysis by the purines was affected by the pH of the mixture. The inhibition constants of inosine, inosine 5'-monophososphate and hypoxanthine when inhibiting enzymatic hydrolysis of cyclic AMP were 0.36 ± 0.05, 1.3 ± 0.2 and 3.2 ± 0.5 mM, respectively. With cyclic GMP as substrate, the inhibition constants were 0.50 ± 0.09, 1.8 ± 0.2 and 4.5 ± 0.7 mM for inosine, inosine 5'-monophosphate and hypoxantine respectively. The per cent inhibition by inosine, inosine 5'-monophosphate or hypoxanthine of the cyclic nucleotide phosphodiesterase activity was not altered by the addition of calmodulin (calcium-dependent protein activator of cyclic nucleotide phosphodiesterase) to the enzyme. The effect of calmodulin was not changed by these purine inhibitors. These results suggest that the binding site of calmodulin differed from that of the inhibitors and from that of cyclic AMP and cyclic GMP.     

Inhibition of heart and brain cyclic adenosine 3',5'-monophosphate phosphodiesterase by new non-steroidic compounds structurally related to natural cardenolides.

AP 10 and related compounds are non-steroidic analogs of cardenolides which exhibit cardiotonic effects on mammals and amphibians isolated hearts. These synthetic compounds were effective inhibitors of the high affinity cyclic AMP phosphodiesterase of beef heart, rat heart and rat brain. AP 10 was the best inhibitor among them. It was approximately ten times as potent as theophylline and three times less effective than MIX and papaverine. Its affinity for the heart enzyme was eight to ten times higher than for the brain enzyme. The inhibition produced by AP 10 was competitive, reversible and was not reversed by high concentrations of magnesium ions. AP 10 slightly affected the binding of cyclic AMP with specific binding protein, but it had a far lower affinity for the binding sites than cyclic AMP. The possibility that inhibition of the low K_m phosphodiesterase by AP 10 and related compounds may contribute to their cardiotonic action is discussed.     

Alterations in adenosine 3', 5'-monophosphate-binding protein in Walker carcinoma cells sensitive or resistant to alkylating agents.

The extent of binding of 8-[³H]adenosine 3′,5′-monophosphate (cyclic AMP) to specific sites was measured in Walker carcinoma cells of tissue culture lines, having different degrees of resistance to the cytotoxic effects of alkylating agents. When compared with sensitive cells, the resistant lines showed a loss of binding activity with increasing resistance, when measured at either pH 4.0 or pH 6.5. This applied to cell lines made resistant either to 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) or chlorambucil, although the total loss of binding activity was greater in the former cell lines. Scatchard analysis of binding suggested the presence of two sites in all cell lines with K_DI ～ 1–5 × 10^?9M and K_DII ～ 3 × 10^?8M. The decreased binding activity was not due to an increased cAMP phosphodiesterase activity in the resistant cell lines, since the activity of the high affinity form of this enzyme was either the same as (CB 1954-resistant) or less than (chlorambucil-resistant) that found in sensitive cells. Walker cells with acquired resistance to either CB 1954 or chlorambucil showed a degree of cross resistance to the cytostatic effect of cAMP analogues and of other alkylating agents.     

Cyclic AMP independent inhibition by papaverine of histamine release induced by compound 48/80.

Compound 48/80-induced histamine release from isolated rat peritoneal mast cells was inhibited in a dose-dependent manner by papaverine (ic₅₀ approx 20 μM). This effect of papaverine was not influenced by PGE₁ (14–140 μM), even though PGE₁ markedly increased must cell cAMP levels. Papaverine (0.5 mM) completely inhibited histamine release without causing any change in cAMP levels. Theophylline (0.1 and 0.5 mM) potentiated histamine release induced by submaximal concentrations of compound 48/80, while cAMP levels were increased. IBM X was as potent as papaverine in causing inhibition of mast cell phosphodiesterase. IBM X (0.14–0.7 mM) had no effect on histamine release but caused a 6–20 fold increase in mast cell cyclic AMP. Papaverine inhibition of histamine release was gradual at the onset and was parallelled by a depletion of mast cell ATP content. The inhibition of 48/80-induced histamine release and depletion of mast cell ATP levels was reversed by glucose. It is concluded that papaverine induced inhibition of 48/80-induced histamine release is independent of cAMP, is unrelated to phosphodiesterase inhibition but is dependent upon inhibition of energy production.     

Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator.

A new, potent vasodilator (YC-93), 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride, competitively inhibited cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase in the 105,000 g supernatant solutions from canine basilar, carotid, coronary and femoral arteries. The inhibition constant (K_i) of YC-93 for these enzyme preparations was in the range of 2.0–4.3 μM at substrate concentrations near the low K_m (about 1 μM for each enzyme preparation), and was 4.0–12 μM at substrate concentrations near the high K_m (50–70 μM). The potency of YC-93 for inhibition of coronary phosphodiesterase at 1 μM cyclic AMP and 50 μM cyclic AMP was much greater than that of papaverine and 3-isobutyl-l-methyl xanthine (IBMX). Commercially available cyclic AMP phosphodiesterase purified from beef heart was also strongly inhibited by YC-93 in a competitive manner and its K_i value was 2.0 μM in the wide range of substrate concentrations tested. Studies on the structure-activity relationship using low K_m phosphodiesterase from canine coronary artery and high K_m phosphodiesterase from beef heart, demonstrated that 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-collidine, the simplest 1,4-dihydropyridine derivative (tested in the present studies) resulted in slightly less inhibition than papaverine, and the inhibitory potency of the former compound was greatly increased mainly by two structural modifications. Firstly, addition of a nitrophenyl group at position 4 of the 1,4-dihydropyridine ring, secondly, the replacement of ethylester at position 3 of the 1,4-dihydro-pyridine ring by N-benzyl-N-methylaminoethyl ester. A few dihydropyridine derivatives together with YC-93 were the most potent inhibitors of cyclic AMP phosphodiesterase among the compounds tested. The finding that the level of cyclic AMP in canine arterial strips was increased by 64 per cent (P < 0.01) even after 1 min exposure to 1 μM YC-93 supports the possibility of at least a partial involvement of phosphodiesterase inhibition in vasodilation by the drug.     

cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A

BACKGROUND AND PURPOSE

Changes in airway smooth muscle (ASM) phenotype may contribute to the pathogenesis of airway disease. Platelet-derived growth factor (PDGF) switches ASM from a contractile to a proliferative, hypo-contractile phenotype, a process requiring activation of extracellular signal-regulated kinase (ERK) and p70^S6 Kinase (p70^S6K). The effects of cAMP-elevating agents on these processes is unknown. Here, we investigated the effects of cAMP elevation by prostaglandin E₂ (PGE₂) and the activation of the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (Epac) on PDGF-induced phenotype switching in bovine tracheal smooth muscle (BTSM).

EXPERIMENTAL APPROACH

Effects of long-term treatment with the PGE₂ analogue 16,16-dimethyl-PGE₂, the selective Epac activator, 8-pCPT-2′-O-Me-cAMP and the selective PKA activator, 6-Bnz-cAMP were assessed on the induction of a hypo-contractile, proliferative BTSM phenotype and on activation of ERK and p70^S6K, both induced by PDGF.

KEY RESULTS

Treatment with 16,16-dimethyl-PGE₂ inhibited PDGF-induced proliferation of BTSM cells and maintained BTSM strip contractility and contractile protein expression in the presence of PDGF. Activation of both Epac and PKA similarly prevented PDGF-induced phenotype switching and PDGF-induced activation of ERK. Interestingly, only PKA activation resulted in inhibition of PDGF-induced phosphorylation of p70^S6K.

CONCLUSIONS AND IMPLICATIONS

Our data indicate for the first time that both Epac and PKA regulated switching of ASM phenotype via differential inhibition of ERK and p70^S6K pathways. These findings suggest that cAMP elevation may be beneficial in the treatment of long-term changes in airway disease.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.