Potent Phosphodiesterase Research Articles

Cyclic nucleotide phosphodiesterases and vascular smooth muscle.

At least 30 different phosphodiesterase (PDE) enzymes have now been identified in mammalian tissues and cells, many of which are products of separate genes. These different isoenzyme forms can be subdivided into seven families based on their genetic and functional characteristics. Relatively specific inhibitors are available for at least five of these PDE families. A functional classification based on substrate specificity, regulatory properties, and sensitivity to inhibition by isozyme- and tissue-selective inhibitors can be used in describing the PDEs of vascular smooth muscle. Inhibition of these PDEs, especially with inhibitors of the PDE3 isoform, promotes vascular relaxation, particularly if the preparation of smooth muscle has been preconracted. For the most part, the drugs appear to act directly on smooth muscle; their effects are usually observed in endothelium-denuded preparations. In addition to their cardiotonic properties, many PDE3 inhibitors possess antiplatelet and thrombolytic activities, thereby suggesting the potential benefit of these drugs in treating diseases of the cardiovascular system. Isozyme- and cell-specific drugs have been shown to alter the synthetic state (i.e. proliferative phenotype) of smooth muscle cultures toward the appearance of the contractile phenotype. This suggests the possible use of selective PDE inhibitors to minimize the problem of restenosis seen after angioplasty. The development of novel methods to deliver more potent and selective PDE inhibitors to individual cell types and subcellular locales will lead to new therapeutic uses for this class of drugs in diseases of the cardiovascular system.

Characterization of a novel potent and specific inhibitor of type v phosphodiesterase

Guanosine cyclic 3′:5′-monophosphate (cGMP) plays a crucial role in regulating vascular smooth muscle contractile state. In rat aortic smooth muscle cells (RSMC) three isozymes of phosphodiesterase (PDE) may be involved in the degradation of cGMP, namely PDE I, PDE III, and PDE V. To study the effective contribution of PDE V to the control of intracellular cGMP levels, a specific and potent PDE V inhibitor 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3,4d]-pyrimidin-4-(5H)-one (DMPPO) was synthesized. DMPPO is a competitive inhibitor with respect to cGMP ( K i = 3 nM) and displayed high selectivity for PDE V as compared to other PDE isozymes. DMPPO strongly potentiated the cGMP response of atrial natriuretic peptide- or sodium nitroprusside-treated RSMC ( EC 50 = 0.5 μM). In addition, similar intracellular cGMP levels were obtained in the presence of a saturating concentration of DMPPO or 3-isobutyl-1-methylxanthine, a nonspecific PDE inhibitor, suggesting that cGMP is almost exclusively hydrolyzed by PDE V in RSMC. Stimulation of RSMC with atrial natriuretic factor resulted in accumulation of cGMP in the extracellular media. This egression was shown to be proportional to the intracellular level of cGMP and a first-order rate constant of 0.04 min −1 was determined for the egression process. DMPPO did not interfere with the efflux and allowed us to show that intracellular cGMP levels are mainly controlled by PDE V, rather than by egression in RSMC. DMPPO is, therefore, a useful tool for determining the role of PDE V in the control of cGMP levels in living cells and tissues.

Decreased sensitivity to beta-adrenergic stimulation of the ventricular cells isolated from the spontaneously hypertensive rat heart.

1. The stimulatory effects of isoproterenol on the L-type Ca2+ current (ICa) were compared between the control (WKY) and hypertensive (SHR) rat heart cells, using the patch-clamp method. 2. The current density and the shape of the current-voltage relationship for ICa were not different between the two groups. However, the maximal percentage increase in response to isoproterenol was smaller in SHR (+91% in SHR and +81% in WKY), and the ED50 was significantly higher in SHR (0.081 mu mol/L in SHR and 0.020 mu mol/L in WKY). IBMX, a potent phosphodiesterase inhibitor, significantly increased the isoproterenol-stimulated ICa in SHR, but not in WKY. These results suggest an impaired cAMP production in SHR heart cells.

Effect of cilostazol on experimental diabetic neuropathy in the rat.

Two proposed mechanisms of diabetic neuropathy are microvascular ischaemia and a reduction in Na,K-ATPase activity. We evaluated the effect of cilostazol, a drug that is both a potent phosphodiesterase inhibitor that normalizes nerve Na,K-AT-Pase and a vasodilator, on nerve blood flow (NBF) to determine whether it would improve experimental diabetic neuropathy. We examined whether epineurally applied cilostazol acted as a vasodilator on the peripheral nerve of normal and diabetic rats, and whether feeding the rats a cilostazol-supplemented diet could improve diabetic neuropathy. Cilostazol increased nerve blood flow (NBF) in a dose-dependent fashion with an EC50 of 10(-5.74) mol/l. Cilostazol also normalized NBF in experimental diabetic neuropathy with a 10(-4) mol/l local application on the sciatic nerve. In diabetic neuropathy, a cilostazol-supplemented diet improved both NBF and nerve conduction in a dose- and time-dependent fashion. Potential mechanisms of action of cilostazol on the nerve include its effect on NBF, Na, K-ATPase, and restoration of the thromboxane:prostacyclin ratio. Cilostazol may have potential in the treatment of diabetic neuropathy.

Studies on new phosphodiesterase inhibitors. I. Synthesis of 1-(2,3-epoxypropoxy)-2(4)-fluorobenzenes and 1-(2-hydroxy-3-morpholinopropoxy and piperazino)fluorobenzene derivatives.

A series of 1-(2,3-epoxypropoxy)-2(4)-fluorobenzenes and their corresponding 1-(2-hydroxy-3-morpholinopropoxy and piperazino)fluorobenzene derivatives (8a--f) were synthesised via a short synthetic route in good chemical yields and were evaluated for inotropic and chronotropic activity in isolated guinea-pig atria preparation. The compounds act as potential phosphodiesterase (PDE) inhibitors with desirable biological activity and have considerable promise in heart therapy.

モルモット乳頭筋におけるPDE阻害薬の特異的作用と非特異的作用

The inotropic actions of xanthine derivatives, having long alkyl chains, were investigated in guinea-pig ventricular papillary muscle. A potent and nonselective phosphodiesterase (PDE) inhibitor, 3-isobutyl-l-methylxanthine, elicited a positive inotropy and inhibited the negative inotropic effects of calcium channel inhibitors, as well as a selective PDE III inhibitor, amrinone, these effects which were canceled by a protein kinase inhibitor, N-[2-(ρ-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). However, 1, 3-di-n-butyl-7-(2'-oxopropyl)xanthine (denbufylline) and 1-n-butyl-3-n-propylxanthine (XT-044), which possess potent and selective PDE IV inhibitory activities, showed negative inotropic actions which became more potent in the presence of H-89. Denbufylline allowed to disappear late restoration phase induced by ryanodine. This xanthine derivative attenuated the both effects of calcium channel acting agents, Bay K 8644 and verapamil, without interaction with caffeine and dihydropyridine calcium channel inhibitors. A nonxanthine PDE IV inhibitor, Ro 20-1724, did not affect the inotropic actions of calcium channel inhibitors. The attenuation by denbufylline or XT-044 of the negative inotropic action of verapamil was not influenced by pretreatment with H-89. These results suggest that these xanthine derivatives elicit negative inotropy through acting on a verapamil-sensitive site of calcium channel without involving of their PDE inhibitory activitiy, in the ventricular papillary muscle.

Clinical evaluation on combined administration of oral prostacyclin analogue beraprost and phosphodiesterase inhibitor cilostazol

Among various oral antiplatelets, a combination of a novel prostacyclin analogue beraprost (BPT) and a potent phosphodiesterase inhibitor cilostazol (CLZ) may result in untoward clinical effects due to possible synergistic elevation of intracellular cAMP (cyclic adenosine 3′,5′-monophosphate). Thereby, a clinical study of the combined administration of the two agents was attempted. Twelve healthy volunteers were assigned to take BPT/CLZ in the following schedule; BPT: 40 μg at day I and 120 μg t.i.d. from day 7 to 14, CLZ: 200 mg t.i.d. from day 3 to 14. At various time intervals, physical examination and blood collection for ex vivo platelet aggregation and determination of intraplatelet cAMP were performed. Throughout the observation period, no significant alteration in vital signs was observed. Seven out of 12 subjects experienced headache of a short duration accompanying facial flush in one and nausea in one, especially after ingestion of CLZ. All of these symptoms, probably caused by the vasodilating effect of the two agents, were of mild degree and no special treatment was required. Intraplatelet cAMP content was gradually but significantly increased to 9.84± 4.59 pmol per 10 9 platelets at day 14 in comparison with the initial value (6.87±2.25 pmol). The platelet aggregability was significantly suppressed at various time intervals but no additive or synergistic inhibitory effect by the combined administration was noted. In conclusion, the combined administration of BPT/CLZ is safe at doses used in the study, though the beneficial clinical effect of the combined administration has yet to be elucidated.

Stereoselectivity of the positive inotropic effects of newer diazinone-cardiotonics

In 5-methyl-dihydropyridazinones, a stereoselectivity is observed only with respect to the phosphodiesterase III-inhibition. The (−)-enantiomers are very strong inhibitors whereas their (+)-counterparts exhibit only a weak activity. In the case of the thiadiazinone EMD 53998 a pronounced stereoselectivity regarding Ca-sensitivity and phosphodiesterase inhibition is apparent. It is concluded that Levosimendan exerts its positive inotropism almost exclusively through its potent phosphodiesterase III-inhibitory effect.

Effects of a novel, selective and potent phosphodiesterase type V inhibitor, E4021, on myocardial ischemia in guinea pigs

The anti-ischemic effects of a new, selective and potent cyclic 3′,5′-guanosine monophosphate-specific phosphodiesterase (phosphodiesterase type V) inhibitor, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl]piperidine-4-carboxylate (E4021), in a vasopressin-induced guinea pig anginal model were examined and compared with those of coronary vasodilators with a guanylate cyclase-activating action. An intravenous injection of vasopressin (0.2 IU/kg) into anesthetized guinea pigs produced ST segment elevation on the electrocardiogram (an index of myocardial ischemia) of 0.28 ± 0.02 mV (n = 10) from the baseline within 30 s. E4021 administered intravenously at doses of 0.03 and 0.1 mg/kg, 5 min before the injection of vasopressin, significantly inhibited the ST segment elevation to 0.15 ± 0.03 mV ( n = 6, P < 0.01) and 0.17 ± 0.02 mV ( n = 6, P < 0.01), respectively. Three guanylate cyclase activators, isosorbide dinitrate (0.1 mg/kg), nicorandil (0.1 mg/kg), and FK409 (0.3 mg/kg), also significantly reduced the ST segment elevation to 0.18 ± 0.03, 0.11 ± 0.02 and 0.17 ± 0.02 mV, respectively. In a second experiment, E4021 was administered intraduodenally 30 min before the injection of vasopressin to examine its oral effectiveness. Intraduodenal E4021, at doses of 1.0 and 3.0 mg/kg, also significantly inhibited the ST segment elevation to 0.16 ± 0.02 mV ( n = 6, P < 0.01) and 0.13 ± 0.02 mV ( n = 6, P < 0.01), respectively. It is concluded that the potent phosphodiesterase type V inhibitor, E4021, administered intravenously or intraduodenally, ameliorated myocardial ischemia similarly to guanylate cyclase activators. Thus, E4021 may be an orally effective drug in the treatment of angina pectoris.

Determination of a novel and potent cyclic GMP phosphodiesterase inhibitor, 4-([3,4-(methylenedioxy)-benzyl]amino)-6,7,8-trimethoxyquinazoline, in dog plasma by high-performance liquid chromatography.

A simple and accurate method for determining levels of 4-([3,4-(methylenedioxy)benzyl]amino)-6,7,8-trimethoxyquinazoline, a novel cyclic GMP phosphodiesterase inhibitor, in dog plasma was developed. This method involves extraction with diethyl ether and reversed-phase high-performance liquid chromatography with ultraviolet detection. Regression analysis showed that the method was linear over the range 1.0-1000 ng/ml. The method was employed for the analysis of plasma samples in a preliminary pharmacokinetic study in beagle dogs.

P-556 - Cardiohemodynamic characteristics of a novel selective and potent phosphodiesterase V inhibitor, E4021, in the pig.

P-556 - Cardiohemodynamic characteristics of a novel selective and potent phosphodiesterase V inhibitor, E4021, in the pig.

P-555 - Inhibitory effect of a novel selective and potent phosphodiesterase type V inhibitor, E4021, on ST segment elevation induced by vasopressin in anesthetized guinea pigs.

P-555 - Inhibitory effect of a novel selective and potent phosphodiesterase type V inhibitor, E4021, on ST segment elevation induced by vasopressin in anesthetized guinea pigs.

Effects of Combinations of Methylxanthines and Adenosine Analogs on Locomotor Activity in Control and Chronic Caffeine-Treated Mice.

Chronic caffeine ingestion (CCI) by male NIH Swiss strain mice results in a prolonged reduction in locomotor activity and alterations in response to caffeine, other xanthines, and adenosine analogs. Caffeine, the A1 selective 8-cyclopentyltheophylline (CPT), and the A2-selective 3,7-dimethyl-1-propargylxanthine (DMPX) remain stimulatory and the bell-shaped locomotor dose-response curves are left-shifted after CCI. The depressant effects of methylxanthines that are potent phosphodiesterase inhibitors remain after CCI. After CCI, mice became more sensitive to depressant effects of A1, mixed A1/A2, and A2 agonists. In the presence of caffeine the A1-selective agonist N6-cyclohexyladenosine (CHA), the mixed A1/A2 agonist 5'-N-ethylcarboxamidoadenosine and the A2-selective agonist 2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC) all have dose-response curves, appearing to consist of initial depressant effects, then stimulatory effects, and finally pronounced depressant effects. The phasic character is reduced or absent after CCI. Synergistic depressant effects of combinations of CHA and APEC also appear reduced after CCI.

Activation of metabotropic glutamate receptors induces an outward current which is potentiated by methylxanthines in rat cerebellar Purkinje cells

The responses of slice-cultured Purkinje cells to trans- dl-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) were examined by intracellular recording techniques and fura-2 microfluorometry. Bath-application of t-ACPD (100 μM, 30 s), a selective agonist of metabotropic glutamate receptors (mGluRs), to Purkinje cells voltage-clamped near their resting potential −65 to −60 mV) consistently induced a transient inward current, followed by a slower outward current (I out). This outward current was characterized by a linear current-voltage relationship in the range from −130 to −60 mV and accompanied by a significant decrease in membrane conductance. The extrapolated reversal potential of I out was positive to 0 mV. When t-ACPD was applied for 60 s or more it became apparent that I out emerged in parallel to the wash-out of t-ACPD. Microfluorometric fura-2 measurements in combination with electrophysiological recordings were used to assess the relation between I out and intracellular free calcium concentration ([Ca 2+] i). In contrast to the inward current that was associated with a transient elevation in [Ca 2+] i. I out was not correlated with an elevated [Ca 2+] i. When t-ACPD was applied in the presence of caffeine (5 mM), I out was reversibly enhanced in amplitude. Caffeine affected neither the t-ACPD-induced calcium signal nor the resting [Ca 2+] i. While longer applications of caffeine alone induced outward currents with a current-voltage relationship similar to that of I out, short applications (30 s) of caffeine had no detectable effect per se but still were effective in enhancing I out when applied in conjunction with t-ACPD. 3-Isobutyl-1-methylxanthine (IBMX, 0.5 mM), a more selective and potent phosphodiesterase inhibitor than caffeine, exhibited caffeine-like effects at a 10-fold lower concentration. We propose that I out is generated by a transient inhibition of an inward current that is tonically active at rest and largely voltage-independent in the range tested. Our observations provide evidence for an involvement of cyclic nucleotide second messenger systems in the regulation of this current.

Cyclic adenosine 3',5'-monophosphate enhances sodium, potassium-adenosine triphosphatase activity in the sciatic nerve of streptozotocin-induced diabetic rats.

We have investigated the relationship between cAMP and sodium,potassium-ATPase (Na+,K(+)-ATPase) activity in the sciatic nerves of rats treated with cilostazol, a potent phosphodiesterase inhibitor; iloprost, a stable prostacyclin analog; or (Bu)2cAMP, a cAMP analog, which increase cAMP content by different mechanisms. In in vivo studies, administration of cilostazol (20 mg/kg BW.day), iloprost (4 micrograms/kg BW.day), or (Bu)2cAMP (4 mg/kg BW.day) for 4 weeks restored decreased cAMP content and Na+,K(+)-ATPase activity in the sciatic nerves of diabetic rats and further improved motor nerve conduction velocities without alteration of myo-inositol contents. There was a positive correlation between cAMP contents and Na+,K(+)-ATPase activities in the sciatic nerves. In in vitro experiments, cAMP accumulation and Na+,K(+)-ATPase activity in the desheathed sciatic nerve blocks obtained from both normal and diabetic rats were significantly increased by incubation with cilostazol, iloprost, or (Bu)2cAMP. In addition, cAMP accumulation and Na+,K(+)-ATPase activities in endoneurial preparations incubated in both normal and high glucose buffer were also significantly increased by cilostazol, iloprost, and (Bu)2cAMP. These results strongly suggest that there is a close relationship between cAMP content and Na+,K(+)-ATPase activity in rat sciatic nerves. Therefore, cAMP content may play an important role in the development of diabetic neuropathy by modulating Na+,K(+)-ATPase activity in the peripheral nerves.

Cardiac effects of R 79595 and its isomers (R 80122 and R 80123) in an acute heart failure model. A new class of cardiotonic agents with highly selective phosphodiesterase III inhibitory properties.

R 79595 (N-cyclohexyl-N-methyl-2-[[[phenyl (1,2,3,5-tetrahydro-2 oxoimidazo [2,1-b]-quinazolin-7-yl) methylene] amin] oxy] acetamide) and its isomers represent a novel class of compounds with phosphodiesterase (PDE) inhibitory and cardiotonic (positive inotropic) actions. The cardiac effects of this class of compounds were investigated in the hexobarbital-depressed heart-lung preparation of the guinea-pig. After induction of heart failure (reduction of cardiac output to 25% of the initial value) cumulative addition of R 79595 or its isomers R 80122 (E-isomer) and R 80123 (Z-isomer) concentration-dependently reversed the cardiac depressant effects of hexobarbitone-Na. With regard to reconstitution of contractility and cardiac function R 80122 (E-isomer) was 10 fold more potent than R 79595 (1:1 mixture of the isomers) and nearly 100 fold more potent than R 80123 (Z-isomer). Furthermore, the cardiotonic action of the most potent isomer (R 80122) was compared to the effects of several positive inotropic reference compounds. The order of cardiotonic potency was as follows: (-)-adrenaline > R 80122 = adibendan > digitoxin > milrinone = enoximone > theophylline. Adibendan (EC50 value: 6.7 +/- 1.8 x 10.-8 mol/l), which showed cardiotonic effects in the same concentration range as R 80122 (EC50 value: 6.1 +/- 1.3 x 10(-8) mol/l), was significantly (p < 0.01) less effective than R 80122 with respect to the maximally induceable increase in cardiac output (CO). The cardiotonic effects of R 80122 could be observed in the low concentration range of 3 x 10(-8) to 1 x 10(-6) mol/l, whereas enoximone (EC50 value: 1.2 +/- 0.1 x 10(-5) mol/l) and milrinone (EC50 value: 8.9 +/- 3.5 x 10(-6) mol/l) elicited positive inotropic effects at 100 fold higher concentrations. Digitoxin was 10 fold less and theophylline was 300 fold less potent than R 80122 with regard to reconstitution of heart function. The cardiotonic effects of R 80122 were not accompanied by an increase in heart rate as found with milrinone, theophylline or (-)-adrenaline in this model. Furthermore, the PDE inhibitory effect of R 79595 and its E-isomer R 80122 were investigated in partially purified isoenzymes from guinea-pig ventricles. The IC50 values of R 79595 and R 80122 on PDE I-IV were compared to the IC50 values of adibendan, milrinone, enoximone and theophylline. The selectivity of an inhibitor for PDE III was evaluated by division of its IC50 values on PDE I, II and IV by the IC50 value on PDE III. R 80122 was the most potent and selective PDE III inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes

Thirty-one flavonoids were tested for their effects on low K m phosphodiesterase with cyclic AMP as the substrate. Quercetin, luteolin, scutellarein, phloretin and genistein showed inhibitory potencies comparable to or greater than 3-isobutyl-2-methylxanthine (EC 50 30–50 μM). Only four compounds namely, catechin, epicatechin, taxifolin and fustin stimulated the enzyme activity (stimulatory EC 50 130–240 μM). The most potent phosphodiesterase (PDE) inhibitors were aglycones that had a C 2,3 double bond, a keto group at C 4 and hydroxyls at C 3′ and/or C 4′. However, when the C-ring is opened then the requirement for the C 2,3 double bond is eliminated. The same series of flavonoids were also tested for their lipolytic activity. The structural features required for effective synergistic lipolysis (with epinephrine) were generally similar to that required for potent PDE inhibition except that, for lipolytic activity, an intact C-ring was necessary. Fisetin and quercetin having the above-mentioned structure showed a dose- and time-dependent increase in lipolysis which was synergistic with epinephrine. Only butein and hesperetin showed inhibition of epinephrine-induced lipolysis, and their effect was dose-dependent. A time-course study indicated that hesperetin was able to delay the lipolytic action of epinephrine. It is most likely that the lipolytic effects of these compounds were not a result of PDE inhibition, as the orders of potency for the two activities had poor correlation. Apparently, the effective lipolytic flavonoids were also potent PDE inhibitors but not all the PDE inhibitors were able to induce lipolysis.

Absence of 1-Methyladenine Production in Follicle Cells Obtained from Starfish Ovaries in the Post-Spawning Season: (starfish follicle celis/1-MeAde/GSS/cAMP/post-spawning season).

Follicle cells from the ovaries of starfish Asterina pectinifera collected in the breeding season and then kept in an aquarium for three months did not produce 1-methyladenine (1-MeAde) in response to genad-stimulating substance (GSS). The cyclic AMP content of follicle cells of the ovaries was much lower in the post-spawning season than in the spawning season. In the post-spawning season, the cyclic AMP level was increased slightly by GSS, but nor insufficiently for production of 1-MeAde. Addition of 3-isobutyl-1-methylxanthine, a potent phosphodiesterase inhibitor, stimulated the productions of GSS-induced 1-MeAde and cyclic AMP. Adenylate cyclase in membrane preparations of follicle cells from ovaries in the post-spawning season was stimulated by nonhydrolyzable GTP analogs and forskolin. An experiment on ADP-ribosylation with [α-32 P]NAD in the presence of cholera toxin and pertussis toxin showed the presence of two types (stimulatory and inhibitory) of guanine nucleotide-binding regulatory proteins (G-proteins). However, GSS has no effect on the adenylate cyclase activity regardless of the presence of GTP. These findings suggest that GSS does not bind to its receptor in follicle cells of the overy in the post-spawning season, although these cells possess G-proteins and adenylate cyclase. Thus the absence of 1-MeAde production by follicle cells obained from ovaries in the post-spawning season appears to be due to lack of receptor protein for GSS.

Characterization of guinea-pig eosinophil phosphodiesterase activity: Assessment of its involvement in regulating superoxide generation

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O − 2) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 ± 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics ( K m = 1.3 and 31.4 μM) and was unaffected by cGMP ( ic 50 > 100 μ M) or CaCl 2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 ( ic 50 = 0.92 ± 0.43 μ M) , rolipram ( ic 50 = 0.20 ± 0.04 μ M) and denbufylline ( ic 50 = 0.20 ± 0.01 μ M) , potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin ( ic 50 = 0.11 ± 0.02 μ M) and AH-21-132 ( ic 50 = 2.57 ± 0.02 μ M) . Eosinophil cAMP PDE was resistant to SK&F 94120 ( ic 50 > 1000 μM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active ( ic 50 = 35.33 ± 10.74 μ M) . O − 2 release from resting cells was potently inhibited by rolipram ( ic 50 = 0.05 ± 0.03 μ M) and denbufylline ( ic 50 = 0.06 ± 0.04 μ M) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin ( ic 50 = 8.0 ± 2.7 μ M) . AH-21-132 ( ic 50 > 10 μ M) , SK&F 94120 ( ic 50 > 10 μ M) and zaprinast ( ic 50 > 10 μ M) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 μM isoprenaline, potently increased intracellular accumulation ( ec 50 = 0.45 ± 0.16 and 0.28 ± 0.08 μ M , respectively). Trequinsin and AH21–132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 μM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-201724-inhibited cAMP PDE plays a role in regulating eosinophil. O − 2 generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting. O − 2 suggests the existence of a barrier impeding access to the enzyme.

Excitation of Limulus photoreceptors by hydrolysis-resistant analogs of cGMP and cAMP

cGMP was proposed to be the agent which directly opens the light-sensitive channel in Limulus ventral photoreceptors. However, the light-sensitive rhabdomeral-lobe (R-lobe) is not uniformly responsive to cGMP injection; that is, there are ‘hot spots’ of sensitivity. A potent phosphodiesterase which rapidly hydrolyses cGMP was suggested to explain the existence of these ‘hot spots’. To test this idea we injected ventral photoreceptors with hydrolysis-resistant cyclic nucleotide analogues. Whereas there can be a lack of a response to cGMP injection into the R-lobe, we find a reproducible depolarization upon R-lobe injection of hydrolysis-resistant cGMP analogues. Furthermore, the depolarization resulting from the injection of cGMP analogues does not require a rise in intracellular Ca 2+, as it is not blocked by the Ca 2+ buffer, BAPTA. Surprisingly, R-lobe injection of a hydrolysis-resistant cAMP analogue also depolarized ventral photoreceptors. Further work will be needed to resolve the role of cGMP in Limulus visual transduction.