Inhibited cGMP Phosphodiesterase Research Articles

The Effect of Nitroxyalkyl Succinimides on Activity of Cyclic Guanosine Monophosphate Phosphodiesterase.

The effect of N-nitroxymethyl succinimide (1), N-(2-nitroxyethyl) succinimide (2) and N-(3-nitroxypropyl) succinimide (3) on enzymatic activity of cyclic guanosine monophosphate (cGMP) phosphodiesterase was studied and crystal structure of compound (2) was determined. It was shown that all studied N-nitroxy succinimides inhibited cGMP phosphodiesterase in a concentration range of 0.1-0.001 mM. Compound (2) noncompetitively and reversibly inhibited hydrolytic function of enzyme with Ki=1.7×10-5 М. Inhibition constant for the reference compound N-(2-nitroethyl) nicotinamide (nicorandil) was 3×10-5 М.

Effects of Fullerene Derivatives on Activity of Ca2+-ATPase of the Sarcoplasmic Reticulum and cGMP Phosphodiesterase.

We studied the effects of new water-soluble polysubstituted fullerene C60 (PFD) derivatives on activity of Ca2+-Mg2+ ATPase of the sarcoplasmic reticulum and cGMP phosphodiesterase. All examined fullerene derivatives inhibited activity of both enzymes. For instance, PFD-I, PFD-II, PFD-III, PFD-V, PFD-IX, PFD-X, and PFD-XI in a concentration of 5×10-5 M completely inhibited hydrolytic and transport functions of Ca2+-ATPase. These compounds in a concentration of 5×10-6 suppressed active transport of calcium ions by 51±5, 77±8, 52±5, 52±5, 100±10, 80±8, and 100±10%, respectively, and inhibited ATP hydrolysis by 31±3, 78±8, 18±2, 29±3, 78±8, 63±7, and 73±9%, respectively, uncoupling the hydrolytic and transport functions of the enzyme. PFD-I noncompetitive and reversibly reduced activity of Ca2+-ATPase (Ki=2.3×10-6 M). All the studied fullerene derivatives (except for PFD-VII) inhibited cGMP phosphodiesterase by more than 80% in concentration of 10-4 M and higher and by more than 50% in concentration of 10-5 M. PFD-I is a non-competitive reversible inhibitor of cGMP phosphodiesterase (Ki=7×10-6 M). These results allow us to expect antimetastatic, antiaggregatory, antihypertensive and vasodilative activity of the studied compounds.

Effects of Nitrosyl Iron Complexes with Thiocarbamide and Its Aliphatic Derivatives on Activities of Ca2+-ATPase of Sarcoplasmic Reticulum and cGMP Phosphodiesterase.

We studied the effects of water-soluble cationic dinitrosyl iron complexes with thiocarbamide and its aliphatic derivatives, new synthetic analogs of natural NO donors, active centers of nitrosyl [1Fe-2S]proteins, on activities of Ca2+-ATPase of sarcoplasmic reticulum and cGMP phosphodiesterase. Nitrosyl iron complexes [Fe(C3N2H8S)Cl(NO)2]0[Fe(NO)2(C3N2H8S)2]+Cl- (I), [Fe(SC(N(CH3)2)2(NO)2]Cl (II), [Fe(SC(NH2)2)2(NO)2Cl×H2O (III), and [Fe(SC(NH2)2)2(NO)2]2SO4×H2O (IV) in a concentration of 10-4 M completely inhibited the transporting and hydrolytic functions of Ca2+-ATPase. In a concentration of 10-5 M, they inhibited active Ca2+ transport by 57±6, 75±8, 80±8, and 85±9% and ATP hydrolysis by 0, 40±4, 48±5, and 38±4%, respectively. Complex II reversibly and noncompetitively inhibited the hydrolytic function of Ca2+-ATPase (Ki=1.7×10-6 M). All the studied iron-sulphur complexes in a concentration of 10-4 M inhibited cGMP phosphodiesterase function. These data suggest that the studied complexes can exhibit antimetastatic, antiaggregation, vasodilatatory, and antihypertensive activities.

Correction: A Novel Sulindac Derivative that Potently Suppresses Colon Tumor Cell Growth by Inhibiting cGMP Phosphodiesterase and β-Catenin Transcriptional Activity

Correction: A Novel Sulindac Derivative that Potently Suppresses Colon Tumor Cell Growth by Inhibiting cGMP Phosphodiesterase and β-Catenin Transcriptional Activity

A Novel Sulindac Derivative that Potently Suppresses Colon Tumor Cell Growth by Inhibiting cGMP Phosphodiesterase and β-Catenin Transcriptional Activity

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3',5',-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of β-catenin as evident by its ability to reduce β-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.

Abstract 3707: Sulindac sulfide inhibits growth and induces apoptosis of human colon tumor cells by a cGMP-dependent pathway leading to suppression of β-catenin transcription activity

Abstract Sulindac and other nonsteroidal anti-inflammatory drugs have shown promise for chemoprevention of colorectal cancer, but toxicities arising from cyclooxygenase (COX) inhibition limit their clinical use. Previous reports suggest that sulindac may exert its antitumor effects by a COX-independent target involving cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition, although the specific isozymes involved and mechanism have not been well defined. In this study, we show that sulindac sulfide (SS) inhibits the growth of human colon tumor cells, HCT116, HT29 and SW480 with IC50 values of 60-80μmol/L. Within this concentration range, SS did not affect the growth of human-derived normal colonic epithelial cells, NCM460. Furthermore, SS induced apoptosis of colon tumor cells but not NCM460 cells. SS inhibited cGMP PDE activity in colon tumor cell lysates with minimal effect on cGMP PDE activity in NCM460 cell lysates. By determining the sensitivity of all eleven PDE isozymes to SS, the cGMP-specific PDE5 was found to be the most sensitive with an IC50 value for enzyme inhibition of 38µmol/L. In addition, PDE5 was found to be overexpressed in colon tumor cell lines compared with NCM460 cells. Knockdown of PDE5 expression in colon tumor cells with siRNA resulted in a significant reduction of tumor cell growth and increased apoptosis. Moreover, SS increased intracellular cGMP levels, activated protein kinase G (PKG) and suppressed the expression of β-catenin in colon tumor cells. SS also inhibited the transcriptional activity of β-catenin and reduced the expression of the β-catenin regulated proteins cyclin D1 and survivin, which are important mediators of tumor cell proliferation and apoptosis, respectively. Our results suggest that the chemopreventive properties of sulindac are closely associated with PDE5 inhibition and involve the activation of cGMP signaling to suppress β-catenin mediated transcriptional activity. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2011-3707

Sulindac Sulfide Selectively Induces Apoptosis of Human Breast Tumor Cells by Inhibiting PDE5A, Elevating cGMP, and Activating PKG.

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2 selective inhibitors display promising antineoplastic activity, but the gastrointestinal, renal, and cardiovascular toxicities that result from COX inhibition limit their clinical utility for cancer chemoprevention. Previous studies suggest that the pro-apoptotic activity of these drugs may be due to a COX-independent mechanism involving cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition. However, the specific PDE isozyme(s) responsible has not yet been identified. Here we used the NSAID sulindac sulfide (SS) to further study cGMP PDE as a potential molecular target of the NSAIDs for breast cancer chemoprevention. SS inhibited growth and induced apoptosis of human SK-BR-3 and MDA-MB-231 breast tumor cells, while normal human mammary epithelial cells (HMEC) were less sensitive. SS also inhibited cGMP PDE activity from tumor cell lysates at concentrations comparable to those required for apoptosis, while appreciably higher concentrations were necessary to inhibit activity from HMEC lysates or cAMP PDE activity from any of the cell lysates. Additionally, SS increased intracellular cGMP concentrations and induced protein kinase G (PKG) activity in MDA-MB-231 cells as determined by ELISA and increased levels of phosphorylated VASP, a known PKG substrate, respectively. Moreover, pretreatment with the guanylyl cyclase inhibitor LY83583 desensitized the breast tumor cells to the growth-inhibitory activity of SS, likely by suppressing intracellular cGMP levels. The guanylyl cyclase activator NOR-3 and the cGMP PDE inhibitors MY5445 and trequinsin inhibited tumor cell growth, while the adenylyl cyclase activator forskolin and other PDE inhibitors had no effect. HMEC were found to express several cGMP hydrolyzing PDE isozymes (PDE1, 5, and 9), while the breast tumor cells expressed predominantly PDE5, which was confirmed to be sensitive to inhibition by SS using a recombinant enzyme. This work demonstrates that inhibition of PDE5A, elevation of cGMP, and activation of PKG are responsible for the pro-apoptotic effects of SS in human breast tumor cells and that differential expression of PDE isozymes between normal and tumor cells may explain the tumor selectivity of NSAIDs. These data also suggest that it may be feasible to develop NSAID derivatives with increased selectivity for inhibiting PDE5 and decreased COX-inhibitory activity, which would potentially improve safety and efficacy for breast cancer chemoprevention. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1041.

Involvement of cGMP in cellular melatonin responses*

Melatonin can enhance and suppress constitutive protein secretion from murine melanoma M2R cells in vitro in a cholera‐toxin (CTX) sensitive process. In a number of tissues melatonin has been shown to modulate cGMP levels. The involvement of cGMP in melatonin responses in the melanoma cells was investigated. The effects of melatonin on melanoma cells cGMP and cGMP‐phosphodiesterase activity and the effects of cGMP analogs on the melatonin‐mediated modulation of protein secretion were studied. Melatonin reduced cGMP levels in the melanoma cells. CTX treatment had a similar and non‐additive effect. The effects of melatonin on protein secretion were abrogated by activation of cGMP‐dependent protein kinases. In addition, melatonin inhibited cGMP phosphodiesterase activity in these cells. The data presented indicate that inhibition of cGMP via a CTX sensitive G protein may be a major signal transduction pathway used by melatonin in melanoma cells.

Involvement of cGMP in cellular melatonin responses

Melatonin can enhance and suppress constitutive protein secretion from murine melanoma M2R cells in vitro in a cholera-toxin (CTX) sensitive process. In a number of tissues melatonin has been shown to modulate cGMP levels. The involvement of cGMP in melatonin responses in the melanoma cells was investigated. The effects of melatonin on melanoma cells cGMP and cGMP-phosphodiesterase activity and the effects of cGMP analogs on the melatonin-mediated modulation of protein secretion were studied. Melatonin reduced cGMP levels in the melanoma cells. CTX treatment had a similar and non-additive effect. The effects of melatonin on protein secretion were abrogated by activation of cGMP-dependent protein kinases. In addition, melatonin inhibited cGMP phosphodiesterase activity in these cells. The data presented indicate that inhibition of cGMP via a CTX sensitive G protein may be a major signal transduction pathway used by melatonin in melanoma cells.

Effect of pharmacological manipulation of endogenous atriopeptin activity on renal function.

Atrial stretch causes the release of atriopeptin (AP, ANF) from preformed vesicular storage sites. The circulating hormone acts on unique receptor sites (containing guanylate cyclase) to release guanosine 3',5'-cyclic monophosphate (cGMP) that mediates the natriuresis and vasodilation and probably the suppression of renin, aldosterone, and vasopressin. The biological effects of atriopeptin are transient because of the rapid inactivation of the circulating hormone (by neutral endopeptidase or clearance receptors) or the second messenger (by cGMP-phosphodiesterase). Heart failure due to chronic cardiac volume overload [aortovenocaval (A-V) fistula] exhibits markedly elevated circulating AP blood levels and urinary cGMP levels, accompanied by induction of ventricular AP gene and protein expression and release. Pharmacological manipulation of endogenous AP, either by inhibiting cGMP phosphodiesterase (i.e., mediator prolongation) or neutral endopeptidase (i.e., prolongation of hormone half-life) in A-V fistula animals results in profound natriuresis and diuresis without hypotension. These pharmacological maneuvers bypass the suppressed renal response to exogenous AP seen in heart failure and provide a rational therapeutic strategy based on our understanding of the underlying physiological and pathological mechanisms.

Pharmacological evaluation of GS-389, a novel tetrahydroisoquinoline analog related to higenamine, on vascular smooth muscle

The effects of GS-389, a novel tetrahydroisoquinoline analog, on isolated rat and mouse thoracic aorta rings, were investigated. Both GS-389 and papaverine induced endothelium-independent, concentration-dependent relaxations of the rat and mouse aortae precontracted with phenylephrine (PE). The GS-389-induced inhibition of the contractile response to PE was noncompetitive. The initial phasic contraction to PE elicited in Ca 2+-free media was also attenuated by pretreatment with GS-389, indicating that GS-389 may interfere with the release of intracellular Ca 2+ and/or the effects of intracellular Ca 2+ release. GS-389 potentiated the vasodilatory effects of isoproterenol and sodium nitroprusside in rat and mouse aortae. GS389 significantly increased cGMP levels in the rat aorta and inhibited cGMP phosphodiesterase from the rabbit brain. Methylene blue, but not propranolol, inhibited the vasodilatory effect of GS-389. These results suggest that the vasorelaxant effect of GS-389 may be due, at least in part, to inhibition of cGMP metabolism.

A structurally novel inhibitor of cGMP phosphodiesterase with vasodilator activity

A novel, potent, competitive inhibitor of smooth muscle cGMP phosphodiesterase is described (Compound I, [4-[2- n-butyl-5-chloro-1-(2-chlorobenzyl)imidazolyl]methyl] acetate). The compound is highly selective for inhibiting cGMP phosphodiesterase compared with cAMP phosphodiesterase. Compound I inhibits the contraction of smooth muscle in response to a variety of agonists in the same concentration range to that which inhibits the enzyme. Compound I produced a dose-related reduction in the pressor responses to angiotensin II infusion while not inhibiting the responses to bolus doses of angiotensin II. Two structural analogues of Compound I which did not inhibit cGMP phosphodiesterase failed to inhibit smooth muscle contraction in vitro and did not affect angiotensin II pressor responses in vivo. We propose a mechanism to account for the effects of a cGMP phosphodiesterase inhibitor on smooth muscle contraction in vitro and in vivo.

Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY‐5445, vinpocetine and 1‐methyl‐3‐isobutyl‐8‐(methylamino)xanthine

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.

Alteration of cyclic GMP metabolism by CD-349, a novel calcium antagonist, and by sodium nitroprusside in bovine intrapulmonary artery and vein

The effects of 2-nitratopropyl 3-nitratopropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (CD-349) and sodium nitroprusside (NP) on cyclic GMP (cGMP) metabolism in bovine intrapulmonary artery (BPA) and vein (BPV) were examined. CD-349 inhibited cGMP phosphodiesterase (PDE) activity in BPA and BPV. In the latter, about 40% of the cGMP PDE activity was Ca 2+ dependent. The inhibition of cGMP PDE activity by CD-349 also depended on Ca 2+. The inhibitory effect of CD-349 was more potent than that of nicardipine or nifedipine. The conversion of cGMP from GTP in the homogenates of BPA and BPV was stimulated by NP in a concentration-dependent manner. The NP-induced cGMP formation was stimulated further by CD-349. This effect of CD-349 depended on Ca 2+ in the BPV but not in the BPA. The NP-induced elevation of cGMP levels in the tissue preparations of BPA and BPV was also potentiated by CD-349. These results suggest that CD-349 inhibited Ca 2+-dependent cGMP PDE activity and that the levels of cGMP were elevated in vascular smooth muscle, particularly when guanylate cyclase was activated.

MORPHINE: SITES OF ACTION IN GUANOSINE NUCLEOSIDE PATHWAY: 38

Though cyclic GMP (cGMP) has been strongly implicated in morphine's analgetic action, the relationship between pharmacologic activity of morphine and brain levels of cGMP is not yet well understood. Here, we investigated effects of morphine on cGMP metabolism. Brain slices were incubated in tonometer at 37°C with Krebs-Ringer bicarbonate/glucose buffer, pH 7.35, constant flow of O2/CO2 (20:5) and 2.90 × 10−1 mM of substrate cGMP or 5'-GMP. Sequentially withdrawn aliquots of incubation mixture were analyzed by HPLC. Control brain slices yielded primary metabolic products, GMP, guanosine (GUS), guanine, xanthine, and inosine. Accumulation of guanosine agreed with evidence demonstrating that nucleoside phosphorylase is rate limiting. Chromatographic findings of inosine from samples of human brain slices incubated with cGMP or 5'-GMP as substrate extended our earlier report of 5'-GMP reductase activity in rat brain. No uric acid was detected. Morphine (1.46 × 10−2 mM) significantly reduced rate of cGMP and 5'-GMP metabolisms resulting in latent GUS accumulation substantially increased over control values. Our data suggest that morphine alters purine metabolism by inhibiting cGMP phosphodiesterase and 5'-nucleotidase. This may ultimately assist in establishing both biological significance of cGMP metabolites and multiple actions of morphine on the central nervous system. Supported by NIH/MBRS Grant RR 0809-12.

Modulatory effect of guanosine-3':5' cyclic monophosphate on macrophage susceptibility to Trypanosoma cruzi infection.

The effects of agents that elevate intracellular levels of cGMP on macrophage internalization of the unicellular parasite Trypanosoma cruzi and latex particles were examined in an attempt to define characteristics of the infective process. Presence of imidazole, a drug that prevents degradation of the cGMP by inhibiting cGMP phosphodiesterase activity, during macrophage-T. cruzi interaction resulted in a marked increase in the number of parasites associated with the cells and the percentage of infected cells. Similar results were obtained when sodium nitroprusside (SNP), which increases cGMP levels by an as yet undefined mechanism, dibutyryl-cGMP, or both imidazole and dibutyryl-cGMP were added to the system. In contrast, the presence of imidazole, SNP, or dibutyryl-cGMP had no significant consequence on latex particle uptake by the macrophages. Whereas pretreatment of macrophages with imidazole plus dibutyryl-cGMP readily increased T. cruzi infection, pretreatment of the parasite with these drugs had no significant effect on the interaction. Furthermore, results of radioimmunoassays showed that imidazole and SNP indeed elevated cGMP levels in the macrophages but not in the parasites. Taken together, these results indicate that cGMP plays a facilitating role in macrophage infection by T. cruzi that contrasts with the lack of effect on the uptake of inert latex particles and the previously reported inhibitory effect of cAMP in the same system. Thus, cyclic nucleotides appear to play a role in modulating internalization of the parasite but not in the uptake of an inert particle by macrophages.