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.     

Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase. III. Inhibition of cyclic AMP phosphodiesterase

Acylpeptides, APD-I, -II and -III, were inhibitors of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase, and their inhibition types were non-competitive. The inhibitory activity of APD-II was the most potent among them. Opening of the lactone linkage reduced the inhibitory activity to about half. The activity almost disappeared when an inhibitor or a derivative with opened lactone linkage was methylated with diazomethane. The activity was, however, restored by the addition of metal ions such as Ca2+, Mn2+, Fe2+, and Co2+. This suggests that the inhibition may be caused by a chelating action of the free carboxyl groups of glutamic acid and aspartic acid in the peptide.     

Cardioprotection with sildenafil,a selective inhibitor of cyclic 3',5'-monophosphate-specific phosphodiesterase 5

The effects of sildenafil (Viagra), a specific inhibitor of phosphodiesterase 5, on ischemic myocardium was examined using an isolated rat heart model. Rats were pretreated with sildenafil at doses ranging from 0.001 mg to 0.5 mg/kg body weight. After 60 min, isolated hearts were subjected to ischemia for 30 min followed by 2 h of reperfusion. The results demonstrated that at 0.05 mg/kg (and to some extent at 0.01 mg/kg), sildenafil provided significant cardioprotection as evidenced by improved ventricular recovery, a reduced incidence of ventricular fibrillation and decreased myocardial infarction. At higher doses, it caused a significant increase in the incidence of ventricular fibrillation while at very low doses it had no effect on cardiac function. As expected, sildenafil increased cyclic 3',5'-monophosphate (cGMP) content in the heart. The results demonstrate for the first time that within a narrow dose range, sildenafil can protect the heart from ischemia/reperfusion injury, probably through a cGMP-signaling pathway.     

8-(4-Chlorophenyl)thio-cyclic AMP is a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA).

8-(4-Chlorophenyl)thio-cyclic AMP (8-CPT-cAMP), extensively used as selective activator of cyclic AMP-dependent protein kinase, has been found to be a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Indeed, 8-CPT-cAMP (IC50 = 0.9 microM) inhibited PDE VA with a potency identical to that of zaprinast. 8-CPT-cAMP was also metabolized by PDE VA at a rate half that of cyclic GMP. The cyclic GMP-inhibited phosphodiesterase (PDE III) (IC50 = 24 microM) and the cyclic AMP-specific phosphodiesterase (PDE IV) (IC50 = 25 microM) were also inhibited by 8-CPT-cAMP. In contrast, most of the other cAMP-derivative studies showed little inhibition of any phosphodiesterase isoenzyme. These observations provide further reasons why the mechanism of the physiological effects of 8-CPT-cAMP should be interpreted with caution.     

Inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by chlorpromazine and related compounds.

The effects of chlorpromazine and related compounds upon soluble and particulate Ins(1,4,5)P3-5-phosphatase were investigated in rat GH3 pituitary and in human IMR-32 neuroblastoma cells. Chlorpromazine inhibited both soluble and particulate activities with an IC50 of 1 mM after a preincubation time of 10 min at 37 degrees C. The inhibition was time-dependent and could not be reversed by washing the membranes. Inhibition of soluble activity was found at mM concentrations with a number of phenothiazine derivatives with an apparent requirement for a-Cl, an-SCH3 or a-CF3 (compared with an-H or a-COCH3) substituent at the ring position 2. Such a requirement was also seen in other tricyclic compounds, although clozapine was not active and methixene was active. No such requirement was seen for the particulate enzyme.     

Modulation of human platelet aggregation by the phosphodiesterase type 5 inhibitor sildenafil

The aim of this study was to investigate if the phospodiesterase type 5 inhibitor sildenafil inhibits collagen- or ADP-induced human platelet aggregation and bleeding time. To investigate this, two studies were designed. In the first, a single oral dose of sildenafil, 100 mg, was administered to healthy men. Bleeding time was determined and agonist (ADP and collagen)-induced platelet aggregation (ex vivo in platelet rich plasma) was measured 0, 1, and 4 h after application. In the second, a single oral dose of sildenafil, 50 mg, was administered and, in addition to the parameters in the first study, we also determined the platelet aggregation after 24 h and measured the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP]) in combination to mimic a physiologic nitric oxide release from the endothelium. The bleeding time of 1 h after sildenafil medication (100 mg) was significantly prolonged but recovered toward control values after 4 h, whereas application of sildenafil at a lower dose (50 mg) did not alter the bleeding time. Sildenafil (100 and 50 mg) did not inhibit the ADP-induced aggregation, whereas the collagen-induced aggregation (100 mg) was markedly reduced after 1 h and significantly inhibited 4 h after application. This inhibitory effect was overcome by higher concentrations of collagen. SNAP (0.5 microM) induced an inhibition of platelet aggregation that was potentiated after taking sildenafil (50 mg, 1 and 4 h afterward) and abrogated after 24 h. These data indicates that sildenafil may inhibit collagen-induced platelet aggregation ex vivo. After co-administration of nitric oxide, collagen- and ADP-induced platelet aggregation was significantly inhibited, which may reflect physiologic conditions of an in vivo system.     

The pharmacology of sildenafil, a novel and selective inhibitor of phosphodiesterase (PDE) type 5

Sildenafil (1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d]pyrimidin-5-yl) phenylsulphonyl]-4-methylpiperazine) has been shown to be an effective oral treatment for male erectile dysfunction. Sildenafil is a potent competitive inhibitor of PDE5 (IC50 3.5 nM) and is selective over PDE1 to 4 (80 to 19,000-fold) and retinal PDE6 (10-fold). Sildenafil enhanced cGMP accumulation driven with sodium nitroprusside in the corpus cavernosum of rabbits without affecting cAMP formulation. In the absence of nitric oxide drive, sildenafil had no functional effect on the human and rabbit isolated corpus cavernosum, but potently potentiated the relaxant effects of nitric oxide on these tissues. In the anaesthetised dog, sildenafil (ED50: 12 to 16 micrograms/kg i.v.) enhanced the increase in intracavernosal pressure induced by electrical stimulation of the pelvic nerve or intracavernosal injection of sodium nitroprusside in the absence of meaningful effects on blood pressure. Consistent with its mode of action, sildenafil potentiated the vasorelaxant effects of glyceryl trinitrate on rabbit isolated aortic rings. However, unlike milrinone, sildenafil had no inotropic effects on the dog isolated trabeculae carneae. Thus it is unlikely to have the deleterious effects on cardiac function associated with PDE3 inhibitors. As a consequence of inhibition of PDE6 in the retina, sildenafil (1 to 100 microM) altered the kinetics of the light response of the dog isolated retina. In the anaesthetised dog, sildenafil modified the a- and b-wave of the electroretinogram induced by a flash of blue light. These effects were proportional to plasma concentrations, were fully reversible and only occurred following plasma concentrations higher (approximately 30-fold) than those active on intracavernosal pressure. These studies have shown that sildenafil is a potent and selective inhibitor of PDE5. It enhances the effect of nitric oxide on the corpus cavernosum and has been shown to be an effective oral treatment of erectile dysfunction.     

Inhibition of bacterial DNA cytosine-5-methyltransferase by S-adenosyl-L-homocysteine and some related compounds

S-Adenosyl-L-homocysteine and five related compounds have been evaluated as inhibitors of a DNA cytosine-5-methyltransferase. DNA methylation was assayed in cell extracts from E. coli strain J6-2 dcm+, proficient in DNA cytosine-5-methyltransferase activity, containing substrate DNA isolated from E. coli strain J6-2 dcm-, a strain deficient in DNA cytosine-5-methyltransferase. S-Adenosyl-L-homocysteine and its 7-deaza analogue, S-tubercidinylhomocysteine, were competitive inhibitors of DNA cytosine-5-methyltransferase with Ki's of 14.2 and 17.6 microM, respectively, in the above enzyme assay.     

Inhibition of lymphocyte-mediated cytolysis and cyclic AMP phosphodiesterase by erythro-9-(2-hydroxy-3-nonyl)adenine

The adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), at low concentrations (less than 10 microM), enhances the inhibitory activity of adenosine against lymphocyte-mediated cytolysis (LMC) without itself being inhibitory. At higher concentrations, EHNA alone is inhibitory to LMC with an IC50 of 160 microM. This inhibition is reversible upon washout, appears to affect an early stage of the lytic process, and does not appear to involve changes in basal levels of cyclic AMP (cAMP), ribonucleoside 5'-triphosphate pool sizes, S-adenosylhomocysteine levels, or protein carboxymethylation. EHNA does enhance the cAMP response of cytolytic lymphocytes (CL) to activators of adenylate cyclase such as prostaglandin E1. EHNA inhibits lymphocyte high-affinity cAMP phosphodiesterase at immunosuppressive levels, exhibiting hyperbolic mixed-type inhibition (Ki = 83 microM, alpha = 0.47, beta = 0.18). Whereas inhibition of intralymphocytic ADA is complete at low concentrations (less than 25 microM) of EHNA, inhibition of LMC and intralymphocytic cAMP phosphodiesterase increases linearly with EHNA concentration to at least 200 microM. The presence of 200 microM EHNA during the centrifugation of mixtures of CL and EL4 leukemia target cells leads to increased CL cAMP levels. 2'-Deoxycoformycin, a more potent ADA inhibitor than EHNA, is not inhibitory to LMC and shows none of these cAMP-related effects. These results suggest that CL-target cell contact stimulates adenylate cyclase in the CL and that EHNA inhibits LMC due to its enhancement of this target cell-stimulated elevation of cAMP.     

Inhibition of cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA receptor antagonism.

Rolipram, a selective inhibitor of type 4 cyclic AMP phosphodiesterase (PDE4), completely reversed the amnesic effects of MK-801 on working and reference memory (F[4,64] = 11.10; p <.0001 and F[4,64] = 2.53; p <.05, respectively) at doses of 0.01-0.1 mg/kg in the radial-arm maze task. Similar antagonism by rolipram of the effects of MK-801 was observed on inhibitory avoidance behavior (F[3,35] = 190.8; p <.0001). In vitro evidence suggests that an increase in cAMP concentrations may mediate the observed behavioral effects of rolipram. In the absence of PDE4 inhibition, NMDA did not increase cAMP concentrations in primary cultures of rat cerebral cortical neurons. However, when PDE4 was inhibited with rolipram, NMDA markedly elevated cAMP. These observations suggest that PDE4 is an integral component of the NMDA receptor-mediated signal transduction pathway involved in memory processes. Inhibitors of PDE4 may act on this pathway to produce their effects on memory and may represent a new class of cognitive enhancers.