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.     

Inhibition of cyclic 3',5'-nucleotide phosphodiesterase-a possible mechanism of action of bifunctional alkylating agents.

The effect of anti-tumour alkylating agents on the adenosine 3′,5′-monophosphate (cyclic AMP) levels of sensitive and resistant Walker carcinoma cells in tissue culture has been investigated. Chlorambucil caused a two-fold elevation of cyclic AMP, in the sensitive line only, 8 hr after treatment with a dose of 5 μg/ml. A comparable increase is produced by the cyclic nucleotide phosphodiesterase inhibitor aminophylline at a dose which produces the same degree of inhibition of cell growth. The therapeutically-inactive monofunctional N-ethyl analogue of chlorambucil had no effect on the cyclic AMP level of the sensitive cells at a dose of 250 μg/ml after 8 hr, while the highly selective monofunctional alkylating agent, CB 1954, at 1 μg/ml caused an elevation of cyclic AMP in the sensitive line 24 hr after treatment. This is not a non-specific effect caused by an inhibition of cell growth for the antimetabolite methotrexate had no effect on the intracellular cyclic AMP of sensitive Walker cells at doses which produced complete inhibition of cell growth. The effect of the alkylating agents on cyclic AMP levels is probably due to a specific inhibition of the cyclic nucleotide phosphodiesterase with a low K_m value, since only this form of the enzyme is inhibited, and only in the sensitive cells, when an effect on cell growth and cyclic AMP content is observed. In the case of CB 1954, however, there was no inhibition of either form of the phosphodiesterase at a time when cyclic AMP levels were elevated. A linear relationship exists between the reciprocals of the intracellular cyclic AMP and the percentage growth inhibition produced by a given dose of chlorambucil.     

Inhibitory Effect of NZ-105, a 1,4-Dihydropyridine Derivative,on Cyclic Nucloeotide Phosphodiesterase Activity

Abstract— The effects of NZ-105, a 1,4-dihydropyridine calcium antagonist, on the intracellular cyclic nucleotide system were investigated in-vitro. In rabbit isolated aorta, both NZ-105 (1 and 10 μm ) and nicardipine significantly and in a concentration-dependent manner increased intracellular cyclic AMP and cyclic GMP content. NZ-105 inhibited bovine cardiac phosphodiesterase activity (K_i 30 μm ) by competitive antagonism. The concentration ranges for inhibition were consistent with the range of increases in cyclic nucleotides.     

Dihydro- and tetrahydroisoquinolines as inhibitors of cyclic nucleotide phosphodiesterases from dog heart. Structure-activity relationships.

A series of nineteen closely related dihydro- and tetrahydroisoquinolines was examined for inhibitory effects on soluble and particulate preparations of cyclic AMP and cyclic GMP phosphodiesterases from dog heart. Dose-response curves for all the compounds were approximately parallel. 6, 7-Dimethoxy-1 [3-(trifluoromethyl)phenyl]-3, 4-dihydroisoquinoline hydrochloride (USV 2776), the most potent inhibitor in this series, was a competitive inhibitor of all preparations tested, with K_i values of 2–3 μM for membrane cyclic AMP and cyclic GMP phosphodiesterases and 1, 10 and 5μM for soluble “low K_m” cyclic AMP, “high K_m” cyclic AMP and cyclic GMP phosphodiesterases respectively. 6-Methoxy-7-benzyloxy-I-phenyl-3, 4 dihydroisoquinoline hydrochloride (USV 2469) was about equipotent to USV 2776; both 2776 and 2469 were two to four times more potent than papaverine and 1-methyl-3-isobutylxanthine. Each dihydroisoquinoline was much more potent than its tetrahydroisoquinoline counterpart. Inhibitory potency was influenced by 6, 7-substitutions. e.g. at the C-6 position, benzyloxy > methoxy > hydroxy > hydrogen or methyl. These compounds represent a group of phosphodiesterase inhibitors spanning a 3000-fold activity range, which are related to each other by single structural modifications; they are potentially useful in denning the role of phosphodiesterase inhibition in the various pharmacologic effects elicited by isoquinolines, including papaverine.     

Vasodilator profile of a new 1,4-dihydropyridine derivative, 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 new 1,4-dihydropyridine derivative, 2,6-dimethyl-4-(3-nitrophenyl)-4,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)]-ethyl ester 5-methyl ester hydrochloride (YC-93), when i.v. injected into anesthetized dogs, exhibited not only a greated vasodilation in both cerebral and coronary than in femoral vessels, but also about 100 to 300 times higher potency as well as longer durability than any of reference drugs such as isoxsupurine, papaverine and cinnarizine. YC-93 was also effective in vasodilation by i.m. and i.d. administration. When administered into vertebral and coronary arteries, YC-93 caused vasodilation at the doses that did not affect systemic blood pressure. YC-93 did not potentiate the vasodilator effect of adenosine, and vasodilation by YC-93 was influenced by neither propranolol, atropine, diphenhydramine nor aminophylline. Acute toxicity (LD50) of YC-93 was almost the same as that of papaverine in mice and rats. Thus, YC-93 is a potent bu low-toxic vasodilator agent acting preferentially and perhaps directly on cerebral and coronary vascular beds and is well absorbed from gastrointestinal tract into blood stream.     

Effects of enprofylline on A1 and A2 adenosine receptors

The effects of enprofylline were studied on A₁ adenosine receptors of rat fat cells and on A₂ adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5′-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a K_B of 130 μM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A₂ receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N⁶-R-(−)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a K_B of 32 μM. The K_i value for inhibition of [³H]PIA binding to fat cell membranes was 45 μM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with K_i values of 15, 130 and 110 μM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A₁ and A₂ adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline.     

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.     

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.     

Effect of cyclic nucleotides on activity of cyclic 3',5'-adenosine monophosphate phosphodiesterase

A series of cyclic 2′,3′-nucleotides, cyclic 3′,5′-nucleotides and derivatives of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with a substituent at the C-8 position were investigated as inhibitors of partially purified cyclic AMP phosphodiesterases (PDE) of cat heart and rat brain. The assays were carried out at a substrate concentration (0.06 μM) where the contribution to the total enzyme activity by phosphodiesterases with K_m values for cyclic AMP above 100 μM was insignificant; consequently the activity measured was that of low K_m enzymes.Cyclic 3′,5′-guanosine monophosphate (cyclic GMP) and cyclic 3′,5′-inosine monophosphate (cyclic IMP) were shown to be the most potent inhibitors of PDE of cat heart (I₅₀ = 1 and 2 μM, respectively). Of the cyclic AMP derivatives tested that have a substituent at the C-8 position, 8-bromo cyclic AMP was the most potent inhibitor; next most potent were the derivatives with a sulfur atom, whereas derivatives with oxygen- or nitrogen-containing substituents were the least potent inhibitors of PDE of cat heart or rat brain. Most of the cyclic nucleotides that were tested were more potent inhibitors of the PDE of cat heart than that of PDE of rat brain.The kinetic properties of PDE of cat heart were also investigated in the presence of cyclic GMP, cyclic IMP and 8-bromocyclic AMP. All three compounds were found to be competitive inhibitors, with apparent K_i values of 0.51, 2.3 and 20 μM respectively. The possible pharmacologic role of cyclic nucleotides is discussed.     

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.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Interaction of Hydroflumethiazide and 2,4-Disulfamyl-5-trifluoromethylaniline with Cyclic AMP Phosphodiesterase and Carbonic Anhydrase

Abstract: The inhibitory effect of hydroflumethiazide (HFT) and its metabolite, 2,4-disulfamyl-5-trifluoro-methylaniline (DTA) on cyclic AMP phosphodiesterase and the binding of HFT and DTA to carbonic anhydrase was studied in vitro. Significant inhibition of rat kidney low-K_m cyclic AMP phosphodiesterase was observed with DTA concentration above 2.5 × 10^?4 mol/1 and with HFT concentration above 1 × 10^?4 mol/1. 50% inhibition was observed at a DTA concentration of 1 × 10^?3 mol/1. Binding of DTA and HFT to commercially obtained bovine erythrocyte carbonic anhydrase was demonstrated by equilibrium dialysis. Data were consistent with one class of binding sites. The product of n (number of binding sites) and K_ass (association constant) was 5 × 10⁵ M for DTA and 3.3 × 10⁴ M for HFT at 2°. In human blood in vitro at 37°, the equilibrium erythrocyte/plasma concentration ratio was 18 for DTA and 1.6 for HFT. It is concluded that HFT and DTA have approximately the same potency as cyclic AMP phosphodiesterase inhibitors, whereas DTA is more extensively bound by erythrocyte carbonic anhydrase.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

Inhibition of guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone

Milrinone is a positive inotrope/vasodilator that inhibits cardiovascular low K_m cAMP phosphodiesterase (PDE) and not Na⁺?K⁺ ATPase activity. To explore other possible mechanisms of action, we quantitated the effects of milrinone on Ca²⁺-stimulated Mg²⁺ ATPase activity in guinea pig aortic smooth muscle plasma membranes. Milrinone inhibited Ca²⁺- stimulated activity, but not basal activity, in aortic microsomes. Maximum inhibition (70%) occurred at 1 μM, which coincided with the inflection of a parabolic dose-response curve. In a sarcolemmal-enriched (F1) aortic preparation, 1 μM cAMP, 1 μM Cl-930 (another low K_m cAMP PDE inhibitor), and 100 μM W-7 (a calmodulin antagonist) all inhibited Ca² -stimulated Mg²⁺ ATPase activity. This F1 preparation contained cAMP PDE activity which was inhibited by 1 μM milrinone (26%) and 1 μM Cl-930 (40%) but not by 100 μM W-7. The inhibition of F1 Ca²⁺ -Mg²⁺ ATPase activity by 1 μM milrinone could be diminished by increasing the concentration of CaCl₂ in reaction mixtures. In sum, these studies show that milrinone can inhibit vascular sarcolemmal Ca²⁺ -stimulated Mg²⁺ ATPase activity. However, inhibition may be direct or direct or may be secondary to cAMP PDE inhibition in vascular sarcolemma, since inhibition also occurs with cAMP and another low-K_m cAMP PDE inhibitor, Cl-930.     

Effect of glucagon, dibutyryl adenosine 3',5'-cyclic monophosphate and phosphodiesterase inhibitors on rat liver phosphorylase activity and adenosine 3',5'-cyclic monophosphate levels

Theophylline, papaverine and 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester HCl (SQ 20,009) were found to inhibit both high K_m (200 μM) and low K_m(3 μM) cyclic 3′,5′-AMP phosphodiesterase activity in 100,000 g supernatant fraction from rat liver. Theophylline was the least potent and SQ 20,009 the most potent inhibitor of both activities. In the liver slice preparation, papaverine (5 × 10^?4), theophylline (10^?3 M) and SQ 20,009 (10^?3 M) failed to elevate cyclic 3′,5′-AMP, while glucagon (10^?6 M) produced a significant (P < 0·05) elevation in the tissue levels of the cyclic nucleotide. All three phosphodiesterase inhibitors significantly (P < 0·05) augmented the ability of glucagon to elevate cyclic 3′,5′-AMP levels. Glucagon and papaverine stimulated phosphorylase activity, while theophylline inhibited both basal and glucagon-stimulated phosphorylase activity. SQ 20,009 was without effect. It is concluded that no cause and effect relationship can be assumed between an ability to inhibit phosphodiesterase and to alter phosphorylase activity.     

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.     

Inhibition of 3',5'-nucleotide phosphodiesterase and the stimulation of cerebral cyclic AMP formation by biogenic amines in vitro and in vivo.

A number of compounds that have been shown to be potent inhibitors of phosphodiesterase in brain homogenates were examined for their ability to potentiate biogenic amine-stimulated cyclic AMP formation in mouse cerebral cortex slices and in chick cerebral hemispheres in vivo. Of the drugs examined at a concentration of 200 μM only 4-(3-butoxy-4-methoxy)-2-imidazolidinone (Ro20-1724) and 2-amino-6-methyl-5-oxo-4-n-propyl-4,5,-dihydro-s-triazolo(1,5-a)pyrimidine (ICI63197) significantly potentiated the cyclic AMP response to biogenic amines. Theophylline. papaverine and medazepam were ineffective both in vivo and in vitro. Histamine and dopamine were completely inactive in stimulating cyclic AMP formation in mouse cerebral cortex slices but in the presence of 200 μM Ro20-1724 or ICI63197, significant (2–3 fold) accumulations of the nucleotide were produced by both of these amines. Prostaglandin E₁ and 5-hydroxytryptamine did not enhance cyclic AMP formation either in the presence or absence of Ro20-1724. However, this phosphodiesterase inhibitor greatly potentiated the effect of adenosine. It is suggested that the discrepancy between the effects of drugs on phosphodiesterase in broken cell and whole cell preparations may be related to differential inhibition of different forms of phosphodiesterase in brain or to the relative lipophilic nature of the compounds examined.     

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.     

Ethanol Dependent Interaction between Prostaglandins and Lipoxygenase Products in Human Peripheral Lymphocytes

The effect of lipoxygenase products, 12-L-HETE and 15-L-HPETE, on cyclic AMP levels in human peripheral lymphocytes was examined in the absence and in the presence of a prostaglandin of the E-type (0.6–3.0 μM) or isoprenaline (33 μM). The studies were performed either in the absence or in the presence of 6 per cent ethanol. For comparison the effect of arachidonic acid and linolenic acid were studied. In the absence of ethanol 12-L-HETE and 15-L-HPETE had no significant effect on cyclic AMP accumulation. However, in the presence of ethanol 12-L-HETE (above 1 μM) inhibited prostaglandin E₁ but not isoprenaline induced cyclic AMP accumulation. 15-L-HPETE had a biphasic effect on prostaglandin E₂ induced cyclic AMP accumulation. Concentrations below 1 μM stimulated, those above inhibited. Virtually complete inhibition was seen at 15 μM. The two other fatty acids inhibited both prostaglandin E₂ and isoprenaline induced cyclic AMP accumulation in the presence, but not in the absence of ethanol. The results show that lipoxygenase products have little or no effect on cyclic AMP accumulation in human peripheral lymphocytes unless ethanol is present. In the presence of ethanol both 12-L-HETE and 15-L-HPETE appeared to selectively affect the cyclic AMP accumulation stimulated by PGE.