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The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells.

CB-5083 is an inhibitor of p97/valosin-containing protein (VCP), for which phase I trials for cancer were terminated because of adverse effects on vision, such as photophobia and dyschromatopsia. Lower dose CB-5083 could combat inclusion body myopathy with early-onset Paget disease and frontotemporal dementia or multisystem proteinopathy caused by gain-of-function mutations in VCP. We hypothesized that the visual impairment in the cancer trial was due to CB-5083's inhibition of phosphodiesterase (PDE)-6, which mediates signal transduction in photoreceptors. To test our hypothesis, we used in vivo and ex vivo electroretinography (ERG) in mice and a PDE6 activity assay of bovine rod outer segment (ROS) extracts. Additionally, histology and optical coherence tomography were used to assess CB-5083's long-term ocular toxicity. A single administration of CB-5083 led to robust ERG signal deterioration, specifically in photoresponse kinetics. Similar recordings with known PDE inhibitors sildenafil, tadalafil, vardenafil, and zaprinast showed that only vardenafil had as strong an effect on the ERG signal in vivo as did CB-5083. In the biochemical assay, CB-5083 inhibited PDE6 activity with a potency higher than sildenafil but lower than that of vardenafil. Ex vivo ERG revealed a PDE6 inhibition constant of 80 nM for CB-5083, which is 7-fold smaller than that for sildenafil. Finally, we showed that the inhibitory effect of CB-5083 on visual function is reversible, and its chronic administration does not cause permanent retinal anomalies in aged VCP-disease model mice. Our results warrant re-evaluation of CB-5083 as a clinical therapeutic agent. We recommend preclinical ERG recordings as a routine drug safety screen. SIGNIFICANCE STATEMENT: This report supports the use of a valosin-containing protein (VCP) inhibitor drug, CB-5083, for the treatment of neuromuscular VCP disease despite CB-5083's initial clinical failure for cancer treatment due to side effects on vision. The data show that CB-5083 displays a dose-dependent but reversible inhibitory action on phosphodiesterase-6, an essential enzyme in retinal photoreceptor function, but no long-term consequences on retinal function or structure.

Medical plants, including Vitex agnus-castus L., Lamiaceae, were used in folk medicine for a long time; however, V. agnus-castus mechanisms of actions on trachea are not known. This study was attempted to give evidence for medicinal use of V. agnus-castus as a relaxant and to identify its underlying mechanisms. To this end, V. agnus-castus extract was extracted with ethanol and tested in rabbit tracheal smooth muscle using isolated organ baths. The V. agnus-castus extract (0.15–60 mg/ml) relaxed against acetylcholine-induced constriction in isolated rabbit tracheal smooth muscle, in vitro. Further, V. agnus-castus extract (0.15–75 mg/ml) shown relaxing effects in carbachol and high K-induced contraction of rabbit tracheal smooth muscle, implying antimuscarinic and calcium channel blocking activities. The addition (preincubation) of L-NAME (inhibitor of nitric oxide synthases), as well as indomethacin (cyclooxygenase inhibitor), inhibited (102%, 95% respectively) relaxing effects induced by V. agnus-castus extract precontracted with carbachol. On the other hand, bradykinin (stimulator of nitric oxide synthases) and zaprinast (phosphodiesterase inhibitor) further potentiated (40%, 41%, respectively) relaxing-induced effects by the V. agnus-castus extract. Our data suggest that V. agnus-castus extract airway-relaxing properties have muscarinic antagonism, are dependent on calcium blockade, and are at least partly mediated by the nitric oxide/cyclic guanosine monophosphate- and cyclooxygenase-1-prostaglandin E2-dependent mechanisms. These effects justify V. agnus-castus extract use in various respiratory disorders.

Background The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naive and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain. Methods The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively. Results Our results have shown, for the first time, that administration of CXCL17 in naive mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy. Conclusions Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain.

It is well known that osteoporosis is a significant chronic disease with the increase of the aging population. Here, we report that expression of G protein-coupled receptor 35 (GPR35) in bone marrow mesenchymal stem cells (BMSCs) is suppressed in diagnosed osteoporosis patients and osteoporotic mice. The expression of GPR35 on BMSCs is enhanced during osteogenic differentiation. GPR35 knockout suppresses the proliferation and osteogenesis of BMSCs and deteriorates bone mass in both sham-treated and ovariectomized mice. Moreover, GPR35 deficiency reduces β-catenin activity in BMSCs. In contrast, the overexpression of GPR35 contributes to these processes in BMSCs. Finally, using zaprinast, a synthetic GPR35 agonist, we show that zaprinast rescues OVX-induced bone loss and promotes bone generation in mice. Thus, GPR35 may as a new target and its agonist zaprinast may serve as a novel treatment for osteoporosis.

Phosphodiesterase-5 inhibitors (PDE-5Is) exert positive effects on bone healing and mineralization by activation the nitric oxide/cyclic guanosine monophosphate/protein kinase-G (NO/cGMP/PKG) signaling pathway. In this study, the effects of zaprinast and avanafil, two PDE-5Is, on the NO signaling pathway, estrogen levels, selected bone formation and destruction marker levels, whole-body bone mineral density (WB-BMD), right femur trabecular bone thickness (RF-TBT) and epiphyseal bone width, angiogenesis in the bone-marrow, and selected oxidative stress parameter levels were investigated in rats with ovariectomy-induced osteoporosis. Twenty four adult rats (8 months old) were equally divided into four groups. The first group was the sham operated group. Groups 2, 3 and 4 included ovariectomized rats. At six months after ovariectomy, the 3rd and 4th groups were administered 10 mg/kg zaprinast and avanafil daily as a single dose for 60 days, respectively. Increases in the activity of the NO/cGMP/PKG signalling-pathway, C-terminal collagen peptide levels, angiogenesis in the bone marrow, RF-TBT, epiphy seal bone width and WB-BMD were observed compared to the ovariectomized positive control group (OVX), while the pyridinoline and deoxypyridinoline levels were decreased in the OVX+zaprinast and OVX+avanafil groups (p

Dopamine replacement represents the standard therapy for Parkinson's disease (PD), a common, chronic, and incurable neurological disorder; however, this approach only treats the symptoms of this devastating disease. In the search for novel disease-modifying therapies that target other relevant molecular and cellular mechanisms, Drosophila has emerged as a valuable tool to study neurodegenerative diseases due to the presence of a complex central nervous system, the blood-brain barrier, and a similar neurotransmitter profile to humans. Human PD-related genes also display conservation in flies; DJ-1 beta is the fly ortholog of DJ-1, a gene for which mutations prompt early-onset recessive PD. Interestingly, flies mutant for DJ-1 beta exhibit PD-related phenotypes, including motor defects, high oxidative stress (OS) levels and metabolic alterations. To identify novel therapies for PD, we performed an in vivo high-throughput screening assay using DJ-1 beta mutant flies and compounds from the Prestwick (R) chemical library. Drugs that improved motor performance in DJ-1ss mutant flies were validated in DJ-1-deficient human neural-like cells, revealing that zaprinast displayed the most significant ability to suppress OS-induced cell death. Zaprinast inhibits phosphodiesterases and activates GPR35, an orphan G-protein-coupled receptor not previously associated with PD. We found that zaprinast exerts its beneficial effect in both fly and human PD models through several disease-modifying mechanisms, including reduced OS levels, attenuated apoptosis, increased mitochondrial viability, and enhanced glycolysis. Therefore, our results support zaprinast as a potential therapeutic for PD in future clinical trials.

Objective: This study investigated effects of zaprinast and avanafil on angiogenesis, vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP) 2, 4 and 7. Methods: Female rats were randomly divided into four groups (n = 6). Sham; abdomen was approximately 2 cm opened and closed. Ovariectomised (OVX); abdomen was opened 2 cm and the ovaries were cut. OVX + zaprinast and OVX + avanafil groups; after the same procedure with OVX, 10 mg/kg zaprinast and avanafil were orally administered for 2 month, respectively. Angiogenesis and the levels of VEGF, BMP2, 4 and 7 were determined. Results: VEGF, BMP2, 4 and 7 levels in OVX + zaprinast and especially OVX + avanafil groups were higher than the sham and OVX (p < .05). However, only VEGF and BMP2 levels in OVX + zaprinast group were significant according to sham (p < .05). Also, angiogenesis in OVX + zaprinast and OVX + avanafil groups was dominant according to sham and OVX (p < .05). Conclusions: Zaprinast and avanafil induced BMP2, 4 and 7 levels synergistically with increased VEGF and angiogenesis in renal tissue.

Objective: This study investigated effect of zaprinast and avanafil on vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP) 4 and 7, and vitamin D-3 levels against the negative effect of dexamethazone. Method: Rats were randomly divided into four groups (n = 6). Control: Empty a syringe was immersed and removed subcutaneously. Dexamethasone (DEX): 120 mu g/kg DEX was injected subcutaneously once a day for 28 days. DEX + zaprinast and DEX + avanafil groups: 10 mg/kg zaprinast and avanafil were administrated to rats in addition to the same procedure in the DEX, respectively. VitaminD(3), VEGF, BMP4 and 7 levels by enzyme linked immunosorbent assay (ELISA) and angiogenesis by histopathological/immunohistochemical were evaluated. Results: BMP4 values in the DEX were lower than the other groups (p < .05). DEX + zaprinast and DEX + avanafil exhibited an increase in all the parameters compared to the control and DEX (p < .05). However, these were not significant for the DEX + zaprinast (p > .05). Also, there was a significant increase in angiogenesis in the DEX + zaprinast and DEX + avanafil. Conclusion: Zaprinast and significantly avanafil induced vitamin D-3, BMP4 and 7 levels by increasing angiogenesis in renal.

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. PDEs control numerous cellular processes making them promising targets for the development of therapeutic agents. Unfortunately, many PDE inhibitor molecules are non-selective among PDE classes and efficient methods for quantitative studies on the isoform-specificity of PDE inhibitors in the natural environments of PDEs are unavailable. The PDE in photoreceptors, PDE6, mediates the conversion of photon information into electrical signals making the retina an exceptional model system for examinations of the pharmacological effects of PDE inhibitors on PDE6. Here we introduce electroretinography-based methods for determining the inhibition constants of PDE inhibitors towards the naturally occurring light-activated and spontaneously activated forms of PDE6. We compare our results to earlier biochemical determinations with trypsin-activated PDE6 with disintegrated PDE6 γ-subunit. The potencies of PDE inhibitors were determined by stimulating the photoreceptors of isolated mouse retinas with light and tracking the inhibitor-induced changes in their electrical responses. The methods were tested with three PDE inhibitors, 3-isobutyl-1-methylxanthine (IBMX), sildenafil, and zaprinast. The inhibition constants towards light-activated, spontaneously activated, and trypsin-activated PDE6 differed significantly from each other for sildenafil and zaprinast but were closely similar for IBMX. We hypothesize that this is due to the ability of the PDE6 γ-subunit to compete with sildenafil and zaprinast from the same binding sites near the catalytic domain of PDE6. The introduced methods are beneficial both for selecting potent molecules for PDE6 inhibition and for testing the drugs targeted at other PDE isoforms against adverse effects on visual function.

GPR35 has long been considered an orphan G protein-couple receptor, and although no endogenous ligand of GPR35 has been discovered, synthetic agonists and antagonists have been developed. CXCL17 (a chemokine) has been reported to be an endogenous ligand of GPR35, and it has even been suggested that it be called CXCR8 (Journal of Immunology 2015; 194:29-33). However, no supporting report on the CXCL17-GPR35 relation has been issued since, although GPR35 is supposed to be a potential therapeutic target in several pathologic conditions. In the present study, we applied two assay systems to explore the relationship. An AP-TGF-α shedding assay in GPR35 over-expressing HEK293 cells was used as a gain-of-function assay. Lodoxamide, a well-known synthetic GPR35 agonist, was confirmed to be a more potent agonist than other reported agonists, that is, pamoic acid, kynurenic acid, and zaprinast. However, human and mouse CXCL17s had no effect on GPR35. The G proteins Gαi/o and Gα12/13 were found to couple with GPR35, which is consistent with previous reports. In addition, lodoxamide-induced activation of GPR35 was concentration-dependently inhibited by CID2745687 (a selective GPR35 antagonist). GPR35 knock-down by siRNA transfection was performed in endogenously GPR35-expressing THP-1 cells, and in these cells, lodoxamide concentration-dependently inhibited migration. Furthermore, the inhibitory effect of lodoxamide was blocked by CID2745687 treatment and by GPR35 siRNA transfection. On the other hand, CXCL17 stimulated the migration of THP-1 cells, which is consistent with a previous report. However, the CXCL17-induced migration stimulatory effect was not blocked by CID2745687 or GPR35 siRNA. Accordingly, the present study suggests GPR35 functions as a migration inhibitory receptor and that CXCL17 stimulates migration in a GPR35-independent manner in THP-1 cells.

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.

Recent work conducted in knockout mouse models or using small molecule modulators of the mitochondrial pyruvate carrier (MPC) have suggested that targeting this protein complex may have efficacy for treating diabetes as insulin sensitizers. To begin to identify other small molecules that interact with the MPC and could potentially be new therapeutic leads, we used a BRET-based MPC reporter assay (reporter sensitive to pyruvate; RESPYR) system that responds to molecules that interact with the pyruvate binding site of the complex. Screening of a 1600 molecule chemical library generated a number of hits for compounds that activated RESPYR activity. Some hits in this unbiased screen confirmed previous reports of MPC interacting drugs, including the insulin-sensitizing thiazolidinediones, pioglitazone and rosiglitazone. Another hit in this screen was Zaprinast, an inhibitor of phosphodiesterase 5 (PDE5). Zaprinast affected RESPYR activity in a dose-dependent manner and at high nM concentrations. Zaprinast also potently inhibited pyruvate-mediated respiration in isolated WT, but not MPC-deficient mitochondria, demonstrating a direct mitochondrial effect and MPC-dependence for this inhibition of pyruvate metabolism. Analyses conducted using other PDE5 inhibitors (Sildenafil, Tadalafil, and Vardenafil) showed no effect on RESPYR activity or mitochondrial respiration. Consistent with the concept that hepatic gluconeogenesis from pyruvate requires mitochondrial pyruvate import, Zaprinast reduced production of glucose from pyruvate in WT, but not MPC-deficient hepatocytes in vitro. Studies are ongoing to determine whether Zaprinast treatment will lower blood glucose in vivo. In conclusion, these data demonstrate the feasibility of screening for novel MPC interacting compounds to identify novel therapeutics for treating diabetes and other metabolic diseases and also suggests that the orphan drug Zaprinast may be a novel antidiabetic agent. Disclosure K.S. McCommis: None. W.T. Hodges: None. Y. Chen: None. K. Chambers: None. B.N. Finck: Board Member; Self; Cirius Therapeutics.

The mechanism of neuropathic pain is complex and unclear. Based on our results, we postulate that an intensification of the kynurenine pathway occurs as a consequence of nerve injury. The G protein-coupled receptor 35 (GPR35) is important for kynurenine pathway activation. Cyclic GMP-specific phosphodiesterase inhibitors have also been shown to have beneficial effects on neuropathic pain. Therefore, the aims of our research were to elucidate how a substance that acts as both an agonist of GPR35 and an inhibitor of phosphodiesterase influences neuropathic pain in a rat model. Here, we demonstrated that preemptive and repeated intrathecal (i.t.) administration (16 h and 1 h before injury and then after nerve ligation daily for 7 days) of zaprinast (1 μg/5 μl) significantly attenuated mechanical (von Frey test) and thermal (cold plate test) hypersensitivity measured on day 7 after chronic constriction injury, and the effect of even a single injection lasted up to 24 h. Our data indicate that zaprinast diminished the number of IBA1-positive cells and consequently attenuated the levels of IL-1beta, IL-6, IL-18, and NOS2 in the lumbar spinal cord and/or dorsal root ganglia. Our results also demonstrated that zaprinast potentiated the analgesic properties of morphine and buprenorphine. In summary, in a neuropathic pain model, zaprinast significantly reduced pain symptoms and enhanced the effectiveness of opioids. Our data provide new evidence that modulation of both GPR35 and phosphodiesterase could be an important strategy for innovative pharmacological treatments designed to decrease hypersensitivity evoked by nerve injury.

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 μM) and Bay 60-7550 (10 μM), and cAMP accumulation, induced by Forskolin (20 μM) and Rolipram (50 μM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 μM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

Renal fibrosis is an important factor for end-stage renal failure. However, only few therapeutic options for its treatment are established. Zaprinast, a phosphodiesterase 5 inhibitor, and serelaxin, the recombinant form of the naturally occurring hormone relaxin, are differently acting modulators of cyclic guanosine monophosphate (cGMP) signaling. Both agents enhance cGMP availability in kidney tissue. These substances alone or in combination might interfere with the development of kidney fibrosis. Therefore, we compared the effects of combination therapy with the effects of monotherapy on renal fibrosis. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) for 7 days in wild-type (WT) and cGKI knockout (KO) mice. Renal antifibrotic effects were assessed after 7 days. In WT, zaprinast and the combination of zaprinast and serelaxin significantly reduced renal interstitial fibrosis assessed by α-SMA, fibronectin, collagen1A1, and gelatinases (MMP2 and MMP9). Intriguingly in cGKI-KO, mRNA and protein expression of fibronectin and collagen1A1 were reduced by zaprinast, in contrast to serelaxin. Gelatinases are not regulated by zaprinast. Although both substances showed similar antifibrotic properties in WT, they distinguished in their effect mechanisms. In contrast to serelaxin which acts both on Smad2 and Erk1, zaprinast did not significantly diminish Erk1/2 phosphorylation. Interestingly, the combination of serelaxin/zaprinast achieved no additive antifibrotic effects compared to the monotherapy. Due to antifibrotic effects of zaprinast in cGKI-KO, we hypothesize that additional cGKI-independent mechanisms are supposed for antifibrotic signaling of zaprinast.

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

The mitogenic and vasoconstrictive properties of the vascular system are attributed to endothelin-1 (ET-1). ET-1 serum concentration increases in a number of pathological conditions, particularly in those associated with blood vessel constriction. ET-1 is also associated with the underlying pathomechanisms of primary pulmonary hypertension, arterial hypertension and eclampsia. The aim of this study was to compare the vasodilating properties of selected phosphodiesterase (PDE) inhibitors and celecoxib in human mesenteric arteries constricted with ET-1, and investigate the role of the endothelium in relaxation. Perfused human mesenteric arteries were collected and stored under the same conditions as organs for transplantation. The mesenteric arteries (with and without the endothelium) were constricted by the addition of ET-1 and treated with one of the following: sildenafil (PDE5 inhibitor), zaprinast (PDE5 and 6 inhibitor), rolipram (PDE4 inhibitor) and celecoxib [cyclooxygenase-2 (COX-2) inhibitor]. Based on the observed changes of the perfusion pressure, concentration response curves (CRCs) were prepared for the respective inhibitors and the EC50 (concentration causing an effect equal to half of the maximum effect), pD2 (negative common logarithm of EC50) and relative potency (RP) were calculated. The results suggested that all the inhibitors triggered a concentration-dependent decrease in the perfusion pressure in isolated human superior mesenteric arteries with endothelium constricted by the addition of ET-1. In the arteries without endothelium, CRCs for celecoxib and rolipram were shifted to the right without a significant decrease in the maximum dilating effect. Moreover, CRCs for sildenafil and zaprinast were shifted to the right with a simultaneous significant decrease in the maximum dilating effect and with an increased inclination angle in reference to the concentration axis. In the presence of the endothelium, all of the evaluated PDE inhibitors, as well as celecoxib, reduced the reactivity of the mesenteric arteries caused by ET-1. Sildenafil indicated the lowest efficacy in the presence of the endothelium, but showed a higher potency compared to that of the other compounds. Removing the endothelium significantly reduced the vasodilating efficacy of PDE5 and 6 inhibitors and a statistically significant influence on the vasodilating efficacy of PDE4 inhibitor and celecoxib was observed. The high vasorelaxing efficacy of celecoxib at the background of the PDE inhibitors was observed, not only in the presence, but also in the absence of the endothelium and may be evidence for the relaxation induced by this COX-2 inhibitor in the cAMP- and cGMP-dependent pathways.

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3'5'-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.

Background Phosphodiesterase (PDE) inhibitors in the central nervous system have been shown to stimulate neuronal functions and increase neurogenesis in Alzheimer disease (AD) patients. Material/Methods The aim of this study was to investigate the effects of zaprinast, a PDE5 inhibitor, and rolipram, a PDE4 inhibitor, on learning and memory in elevated plus maze (EPM) and passive avoidance (PA) tests in naive mice. Male Balb-c mice received short-term treatment with zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) before the acquisition trial of the EPM and PA tests. The exploratory activity of the animals was also investigated in the Hughes box test. Results Both zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased second-day latency compared to the control group in the EPM test, while only rolipram (0.1 mg/kg) significantly increased second-day latency in the PA test. Both zaprinast (10 mg/kg) and rolipram (0.1 mg/kg) significantly decreased the number of entries to new areas and time spent in new areas in the Hughes box test. Conclusions Our study revealed that both zaprinast and rolipram enhanced spatial memory in EPM, while rolipram seemed to have more emotional memory-enhancing effects in the PA test compared to zaprinast. Both zaprinast and rolipram diminished exploratory activity in the Hughes box test, which can be attributed to the drugs’ anxiogenic effects.

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.

Muscarinic acetylcholine receptors MAChRs from Bovine Tracheal Smooth Muscle (BTSM) plasma membranes are responsible for the cGMP rise and signal-amplitude peaks associated with smooth muscle contraction present in bronchial asthma. These MAChRs bind [3H]QNB and exhibit the classic G Protein Coupled-Receptor (GPCR) behavior towards muscarinic agonist and antagonists that is sensitive to sensitive to GTP analogs. Interestingly, the [3H]QNB binding activity was stimulated by cGMP and ATP, and was enhanced by IBMX and Zaprinast, inhibitors of cGMP-PDE. Cyclic GMP plus ATP affected the agonist-antagonist muscarinic binding activities. Thus, the high affinity agonist (Carbamylcholine) binding sites disappeared, whereas, 4-DAMP, a M3 selective antagonist displayed an additional high affinity-binding site. In contrast, non-selective (atropine) and M2-selective (methoctramine and gallamine) antagonists revealed one low binding site. Moreover, the 4-DAMP-mustard alkylation of the MAChRs blocked the cGMP ef...

Prostacyclin (PGI2) and phosphodiesterase 5 (PDE5) inhibitors are potent vasodilators that are used alone and in combination for the treatment of pulmonary arterial hypertension (PAH). Although these vasodilators are known to stimulate relaxation of vascular smooth muscle directly, other cells in circulation, including erythrocytes, express prostacyclin receptor (IPR) and contain PDE5. The binding of PGI2 analogs to the erythrocyte IPR results in activation of a signaling pathway that increases cyclic adenosine 3′,5′ monophosphate (cAMP), a requirement for adenosine 3′5′ triphosphate (ATP) release. Within this pathway, cAMP levels are regulated by phosphodiesterase 3 (PDE3), a PDE that is inhibited by cGMP, a cyclic nucleotide regulated by the activity of PDE5. Since inhibition of PDE3 enhances ATP release in response to PGI2 analogs, we investigated if the selective PDE5 inhibitors, zaprinast (ZAP) and tadalafil (TAD), would similarly increase cAMP and ATP release from human erythrocytes in response to the same stimulus. We determined that pretreatment of erythrocytes with one of two chemically dissimilar PDE5 inhibitors (ZAP or TAD, 10 µM) potentiated increases in cAMP and ATP release in response to incubation of human erythrocytes with the PGI2 analog, UT-15C (100 nM). These results suggest that a heretofore unrecognized synergism exists between IPR agonists and PDE5 inhibitors that could provide a new rationale for the co-administration of these agents as vasodilators in humans with PAH.

Phosphodiesterase 11 (PDE11) is the latest isoform of the PDEs family to be identified, acting on both cyclic adenosine monophosphate and cyclic guanosine monophosphate. The initial reports of PDE11 found evidence for PDE11 expression in skeletal muscle, prostate, testis, and salivary glands; however, the tissue distribution of PDE11 still remains a topic of active study and some controversy. Given the sequence similarity between PDE11 and PDE5, several PDE5 inhibitors have been shown to cross-react with PDE11. Accordingly, many non-selective inhibitors, such as IBMX, zaprinast, sildenafil, and dipyridamole, have been documented to inhibit PDE11. Only recently, a series of dihydrothieno[3,2-d]pyrimidin-4(3H)-one derivatives proved to be selective toward the PDE11 isoform. In the absence of experimental data about PDE11 X-ray structures, we found interesting to gain a better understanding of the enzyme-inhibitor interactions using in silico simulations. In this work, we describe a computational approach based on homology modeling, docking, and molecular dynamics simulation to derive a predictive 3D model of PDE11. Using a Graphical Processing Unit architecture, it is possible to perform long simulations, find stable interactions involved in the complex, and finally to suggest guideline for the identification and synthesis of potent and selective inhibitors.

The C-type natriuretic peptide (CNP) exerts its action via stimulation of the cyclic GMP (cGMP) signalling pathway, which includes the activation of cGMP-dependent protein kinases. The pathway can also be activated by inhibitors of phosphodiesterases (PDE) that hydrolyse cGMP. The present report shows that activation of the cGMP pathway by CNP, by bromo-cGMP, a cell-permeant cGMP analogue, or by the PDE inhibitor zaprinast dose dependently reduces intravenous cocaine self-administration by rats. The effect was found when the compounds were injected in situ into the prefrontal cortex, but not when they were injected into the nucleus accumbens. A decrease in the number of cocaine infusions performed by rats was obtained under the fixed ratio-1 schedule of reinforcement as well as under a progressive ratio schedule, which evaluates the motivation of the animals for the drug. Decrease in cocaine self-administration was accompanied with reduced expression of the epigenetic markers methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in dopaminergic projection areas. An increase in the acetylation level of histone H3, but not of histone H4, was also noticed. Since MeCP2 and HDAC2 are known to modulate dynamic functions in the adult brain, such as synaptic plasticity, our results showing that activation of the cGMP signal transduction pathway decreased both cocaine intake and expression of the epigenetic markers strongly suggest that the MeCP2/HDAC2 complex is involved in the analysis of the reinforcing properties of cocaine in the prefrontal cortex.

Objectives To investigate whether 7-[2-[4-(2-chlorophenyl) piperazinyl] ethyl]-1,3-di-methylxanthine (KMUP-1) inhibits the effects of testosterone on the development of benign prostatic hyperplasia and sensitizes prostate contraction. Methods A benign prostatic hyperplasia animal model was established by subcutaneous injections of testosterone (3 mg/kg/day, s.c.) for 4 weeks in adult male Sprague–Dawley rats. Animals were divided into six groups: control, testosterone, testosterone with KMUP-1 (2.5, 5 mg/kg/day), sildenafil (5 mg/kg/day) or doxazosin (5 mg/kg/day). After 4 weeks, the animals were killed, and prostate tissues were prepared for isometric tension measurement and western blotting analysis. KMUP-1, Y27632, zaprinast, doxazosin or tamsulosin were used at various concentrations to determine the contractility sensitized by phenylephrine (10 μmol/L). Results KMUP-1 inhibited testosterone-induced phosphorylation of extracellular signal-regulated phosphorylated protein kinase and mitogen-activated protein kinase kinase and Rho kinase-II activation. Sildenafil and doxazosin significantly decreased benign prostatic hyperplasia-induced mitogen-activated protein kinase kinase and Rho kinase-II activation. The decreased expressions of soluble guanylate cyclase α1 was reversed by KMUP-1, doxazosin and sildenafil. Soluble guanylate cyclase β1 and protein kinase G were increased by KMUP-1, doxazosin, and sildenafil in the testosterone-treated benign prostatic hyperplasia group. Phosphodiesterase-5A was increased by testosterone and inhibited by KMUP-1 (5 mg/kg/day) or sildenafil (5 mg/kg/day). KMUP-1 inhibited phenylephrine-sensitized prostate contraction of rats treated with testosterone. Conclusions Mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase, soluble guanylate cyclase/cyclic guanosine monophosphate, protein kinase/protein kinase G and Rho kinase-II are related to prostate smooth muscle tone and proliferation induced by testosterone. KMUP-1 inhibits testosterone-induced prostate hyper-contractility and mitogen-activated protein kinase 1/extracellular regulated protein kinases kinase-phosphorylation, and it inactivates Rho kinase-II by cyclic guanosine monophosphate, protein kinase and α1A-adenergic blockade. Thus, KMUP-1 might be a beneficial pharmacotherapy for benign prostatic hyperplasia.

Various studies have demonstrated the role of the nitric oxide (NO)/cGMP pathway in pain processing. Our group has also shown that this system participates in opioid-induced antinociception during peripheral inflammation. We have previously observed that inflammation mobilizes an endogenous opioidergic system to control hyperalgesia. Here, we investigated whether the NO/cGMP pathway underlies peripheral endogenous nociception control during inflammation. In this study, a pharmacological approach was used in conjunction with the rat paw pressure test to assess the effects of intraplantar NO synthase inhibitor NG-Nitro-l-arginine (NOArg), guanylyl cyclase inhibitor methylene blue (MB), phosphodiesterase-5 inhibitor zaprinast (ZP), or NO precursor l-arginine injection on carrageenan-induced hyperalgesia, which mimics an inflammatory process, or by prostaglandin E(2) (PGE(2)), which directly sensitizes nociceptors. Intraplantar carrageenan (62.5, 125, 250 or 500μg) or PGE(2) (0.1, 0.5 or 2μg) administration produced hyperalgesia, which manifested as a reduction in the rat nociceptive threshold to mechanical stimuli. NOArg (25, 50 or 100μg/paw) and MB (125, 250 or 500μg/paw) induced significant and dose-dependent reductions in the nociceptive threshold of carrageenan-induced (125μg/paw) hyperalgesia, but not PGE(2)-induced (0.5μg/paw) hyperalgesia. This was a local effect because it did not produce any modifications in the contralateral paw. Both Zaprinast (100, 200 or 400μg/paw) and l-arginine (100, 200 or 400μg/paw) significantly counteracted carrageenan-induced hyperalgesia (250μg/paw), yielding an increase in the nociceptive threshold compared with the control. Zaprinast (200μg/paw) or l-arginine (400μg/paw) did not produce an antinociceptive effect in the contralateral paw, indicating local action. In addition, at the same dose that was able to modify carrageenan-induced hyperalgesia, neither zaprinast nor l-arginine modified PGE(2) (2μg) injection-induced hyperalgesia of the rat paw. Taken together, these results indicate that the l-arginine/NO/cGMP pathway functions as an endogenous modulator of peripheral inflammatory hyperalgesia.

We recently reported that administration of the non-selective cyclic GMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast to cortically cryoinjured rats results three days post-lesion in reduced neuronal cell death that was associated to decreased macrophage/microglial activation and oxidative stress and increased astrogliosis and angiogenesis. Similar effects have been observed in cryoinjured animals overexpressing metallothioneins I/II (MT-I/II), metal-binding cysteine-rich proteins that are up-regulated in response to injury. In this work we have examined the effect of administration of the selective PDE5 inhibitor sildenafil (10mg/kg, sc) 2h before and 24 and 48h after induction of cortical cryolesion in wild-type and MT-I/II-deficient mice. Our results show that in wild-type animals sildenafil induces similar changes in glial reactivity, angiogenesis and antioxidant and antiapoptotic effects in the cryolesioned cortex as those observed in rats with zaprinast, indicating that inhibition of PDE5 is responsible for the neuroprotective actions. However, these effects were not observed in mice deficient in MT-I/II. We further show that sildenafil significantly increases MT-I/II protein levels in homogenates of lesioned cortex and MT-I/II immunostaining in glial cells around the lesion. Taken together these results indicate that cGMP-mediated pathways regulate expression of MT-I/II and support the involvement of these proteins in the neuroprotective effects of sildenafil in focal brain lesion.

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Activation of the NO-cGMP pathway or adenosine receptors depresses reversibly synaptic transmission in the hippocampus. Here we demonstrate, using the selective A1 receptor antagonist DPCPX, a convergence in the mechanisms of action of the NO donor SNAP, the cGMP phosphodiesterase inhibitor zaprinast and adenosine.

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues.

The interactions between certain α-synuclein (SNCA) conformations and dopamine (DA) metabolism cause selective DA neuron degeneration in Parkinson's disease (PD). Preclinical research on PD took advantage of increasing studies involving different animal models which express different forms of mutated SNCA. Transgenic animals expressing mutant α-synucleins such as mice transgenic for A53T-SNCA (TG) are considered valuable models to assess specific aspects of the pathogenesis of synucleinopathies and PD. In this study we performed electrophysiological recordings in corticostriatal slice preparations from young TG overexpressing mice, in which extracellular striatal DA levels appeared to be normal, and in old TG mice, characterized by abnormalities in striatal DA signaling and impaired long-term depression (LTD). We report no difference in TG mice from the two groups of age of either the basal membrane properties and synaptic striatal excitability in respect to age-matched wild-type mice. Furthermore, in old TG mice, showing plastic abnormalities and motor symptoms, we investigated the mechanisms at the basis of the altered LTD. In old TG mice LTD could not be restored by treatments with acute application of DA or by subchronic treatment with L-3,4-dihydroxyphenylalanine (L-DOPA). Conversely, the application of the phosphodiesterase inhibitor zaprinast fully restored LTD to normal conditions via the stimulation of a cyclic guanosine monophosphate (GMP)-protein kinase G-dependent intracellular signaling pathway. These results suggest that, in addition to the dopaminergic alterations reported in this genetic model of PD, other signal transduction pathways linked to striatal synaptic plasticity are altered in an age-dependent manner.

Previously, the authors reported that zaprinast, an inhibitor of cGMP-selective phosphodiesterases, induced the secretions of TNF-α and IL-1β by microglia and enhanced the induction of iNOS by lipopolysaccharide (LPS). In this study, the signaling mechanism responsible for microglial activation by zaprinast was investigated and the effects of zaprinast and LPS on microglial activation were compared. Zaprinast was found to activate ERK1/2, p38 MAPK, JNK, NFκB, and PI3K/Akt, and subsequently, induce the mRNA expressions of IL-1α, IL-1β, TNF-α, CCL2, CCL4, CXCL1, CXCL2, and CD14. Associations between signaling pathways and gene expressions were examined by treating microglia with signal inhibitors. PDTC inhibited the induction of all the above genes by zaprinast, and SB203580 inhibited all genes except CXCL1. SP600125, PD98059, and LY294002 inhibited the induction of at least CCL2. Microglial activation by zaprinast was then compared with full-blown activation by LPS. The zaprinast-induced phosphorylations of MAPKs and IκB were less prompt than LPS-induced phosphorylations. IκB degradation by LPS was significant at 10min and did not return to normal, whereas zaprinast induced a later, transient degradation. LPS induced the mRNA expressions of IL-1β, TNF-α, IL-6, CCL2, iNOS, and COX-2, and although zaprinast significantly induced the expressions of all except IL-6 and iNOS, these inductions were far less than those induced by LPS. Collectively, zaprinast was found to upregulate microglial activity mainly via NFκB and p38 MAPK signaling and the subsequent expressions of inflammatory genes. Although, zaprinast was found to have obvious effects on microglia, these were weaker than the effects of LPS.

Hyperammonemia is a major pathophysiological factor in encephalopathies associated with acute and chronic liver failure. On mouse brain slice preparations, we analyzed the effects of ammonia on the characteristics of corticostriatal long-term depression (LTD) induced by electrical stimulation of cortical input or pharmacological activation of metabotropic glutamate receptors. Long exposure of neostriatal slices to ammonium chloride impaired the induction and/or expression of all studied forms of LTD. This impairment was reversed by the phosphodiesterase inhibitor zaprinast implying lowered cGMP signaling in LTD suppression. Polyphenols from green tea rescued short-term corticostriatal plasticity, but failed to prevent the ammonia-induced deficit of LTD. Zaprinast counteracts the ammonia-induced impairment of long-term corticostriatal plasticity and may thus improve fine motor skills and procedural learning in hepatic encephalopathy.

N-palmitoyl-ethanolamine (PEA) is an endogenous substance that was first identified in lipid tissue extracts. It has been classified as a CB2 receptor agonist. Exogenous PEA has the potential to become a valid treatment for neuropathic and inflammatory pain. In spite of the welldemonstrated antiinflammatory properties of PEA, its involvement in controlling pain pathways remains poorly characterized. The participation of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in peripheral antinociception has been established by our group to the l-, j -o rd-opioid receptor agonists, nonsteroidal analgesics, a2C-adrenoceptor agonists, and even nonpharmacological electroacupuncture. The aim of this study was to verify whether the peripheral antinociception effects of PEA involve the activation of this pathway. All drugs were locally administered to the right hind paw of male Wistar rats. The paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2. PEA elicited a local peripheral antinociceptive effect that was antagonized by the nonselective NO synthase (NOS) inhibitor L-NOARG and the selective neuronal NOS (nNOS) inhibitor L-NPA. Selective inhibition of endothelial (eNOS) and inducible (iNOS) NOS via L-NIO and L-NIL, respectively, was ineffective at blocking the effects of a local PEA injection. In addition, the dosage of nitrite in the homogenized paw, as determined by colorimetric assay, indicated that exogenous PEA is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ antagonized the PEA effect, whereas the cGMP-phosphodiesterase inhibitor zaprinast potentiated the antinociceptive effect of low-dose PEA. This study provides evidence that PEA activates nNOS, thus initiating the NO/cGMP pathway and inducing peripheral antinociceptive effects. V C 2012 Wiley Periodicals, Inc.

Despite the classical peripheral pronociceptive effect of noradrenaline (NA), recently studies showed the involvement of NA in antinociceptive effect under immune system interaction. In addition, the participation of the NO/cGMP/KATP pathway in the peripheral antinociception has been established by our group as the molecular mechanism of another adrenoceptor agonist xylazine. Thus the aim of this study was to obtain pharmacological evidences for the involvement of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect induced by exogenous noradrenaline. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E(2) (2μg/paw). All drugs were locally administered into the right hind paw of male Wistar rats. NA (5, 20 and 80ng/paw) elicited a local inhibition of hyperalgesia. The non-selective NO synthase inhibitor l-NOarg (12, 18 and 24μg/paw) antagonized the antinociception effect induced by the highest dose of NA. The soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100μg/paw) antagonized the NA-induced effect; and cGMP-phosphodiesterase inhibitor zaprinast (50μg/paw) potentiated the antinociceptive effect of NA low dose (5ng/paw). In addition, the local effect of NA was antagonized by a selective blocker of an ATP-sensitive K(+) channel, glibenclamide (20, 40 and 80μg/paw). On the other hand, the specifically voltage-dependent K(+) channel blocker, tetraethylammonium (30μg/paw), Ca(2+)-activated K(+) channel blockers of small and large conductance types dequalinium (50μg/paw) and paxilline (20μg/paw), respectively, were not able to block local antinociceptive effect of NA. The results provide evidences that NA probably induces peripheral antinociceptive effects by activation of the NO/cGMP/KATP pathway.

The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are important second messengers. They are useful biomarkers to indicate biological activity of drugs such as phosphodiesterase (PDE) inhibitors which block the degradation of these nucleotides. Here, we established a fast and sensitive method for the simultaneous analysis of cAMP and cGMP by LC-MS/MS with broad applicability. The limit of detection is 50 pM. Linearity is given in a range of 0.5-500 nM for both nucleotides, with a high intra- and inter-assay precision and accuracy and an analysis time of 3.5 min. We validated the suitability of the method by pharmacological modulation of cAMP or cGMP concentrations in mice with the PDE4 inhibitor rolipram and the PDE5 inhibitor zaprinast. Rolipram significantly increased cAMP concentrations in plasma, CSF and brain tissue. Zaprinast increased cGMP concentrations in plasma but not in brain tissue, which is in accordance with its blood brain barrier permeability. In conclusion, the LC-MS/MS method described here could be a valuable analytical tool for investigating pharmacodynamic effects of PDE inhibitors and to monitor disease-related changes of cAMP and cGMP in the periphery as well as in the central nervous system.

The aim of this study was to investigate the mechanism of H2 S-induced relaxation in mouse corpus cavernosal tissue. l-cysteine (10(-6) × 10(-3) mol/L) and exogenous H2 S (NaHS; 10(-6) to 10(-3) mol/L) induced concentration-dependent relaxation. l-cysteine-induced relaxations was reduced by d,l-propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l-cysteine induced relaxations, but not of those of H2 S diminished in endothelium-denuded tissues. N(ω) -nitro-l-arginine (l-NA; 10(-4) mol/L), a nitric oxide synthase inhibitor, and ODQ (10(-4) mol/L), a guanylyl cyclase inhibitor, increased the H2 S-induced relaxation. Zaprinast (5 × 10(-6) mol/L) and sildenafil (10(-6) mol/L), phosphodiesterase inhibitors, inhibited H2 S-induced relaxation. Adenylyl cyclase inhibitors N-ethylmaleimide (2.5 × 10(-5) mol/L) and SQ22536 (10(-4) mol/L) reduced relaxation to H2 S. Also, H2 S-induced relaxation was reduced by KCl (50 mmol/L), 4-aminopyridine (10(-3) mol/L), a Kv inhibitor, glibenclamide (10(-5) mol/L), a KATP inhibitor or barium chloride (10(-5) mol/L), a KIR inhibitor. However, H2 S-induced relaxation was not influenced by apamin (10(-6) mol/L), a SKC a (2+) inhibitor, charybdotoxin (10(-7) mol/L), an IKC a (2+) and BKC a (2+) inhibitor or combination of apamin and charybdotoxin. Nifedipine (10(-6) mol/L), an L-type calcium channel blocker and atropine (10(-6) mol/L), a muscarinic receptor blocker, inhibited H2 S-induced relaxation. However, H2 S-induced relaxation was not influenced by ouabain (10(-4) mol/L), a Na(+) /K(+) -ATPase inhibitor. This study suggests that H2 S endogenously synthesizes from l-cysteine by CSE endothelium-dependent in mouse corpus cavernosum tissue, and exogenous H2 S may cause endothelium-independent relaxations via activation of K channels (KATP channel, KV channels, KIR channels), L-type voltage-gated Ca(2+) channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H2 S and NO/cGMP.

The effect of 1-cyclopentyl-3-ethyl-6-(3-ethoxypyrid-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-one (SR 265579), a potent inhibitor of guanosine 3’,5’-cyclic monophosphate (cyclic GMP) phosphodiesterase (PDE5), was examined regarding its specificity toward the other cyclic nucleotide phosphodiesterases, the effect on cyclic nucleotide levels and the bronchodilatory activity, both in vitro and in vivo in guinea-pigs. The effects were compared to those obtained with zaprinast (CAS 37762-06-4), a known PDE5 inhibitor. Anion-exchange chromatography of the soluble fraction of guinea-pig homogenates revealed 5 peaks which corresponded to PDE1, PDE2, PDE3, PDE4 and PDE5. SR 265579 produced a potent and competitive inhibition, with respect to cyclic GMP, of PDE5 with a Ki of 6.4 nmol/l. The compound was 25 fold more potent than zaprinast and demonstrated selectivity toward PDE5. The selectivity index was 14 and 33 with respect to PDE4 and 3, respectively. PDE1 and 2 were only inhibited at considerably higher concentrations. SR 265579 specifically increased the intracellular cyclic GMP levels in guinea-pig tracheal epithelial cells (EC 50 = 117 nmol/l). Moreover, in the guinea pig, plasma cyclic GMP levels were significantly increased after the intravenous or oral administration of doses as low as 1 mg/kg. Isolated guinea-pig trachea were relaxed by the addition of SR 265579 as evaluated by measuring either spontaneous tone or relaxation of histamine and acetylcholine-precontracted preparations. PD 2 values were of 7.64, 6.52 and 5.25, respectively. In vivo, after i.v. administration, bronchodilatory activity was demonstrated in an artifi- cially-ventilated guinea-pig histamine-induced bronchospasm model with an ED 50 of 0.63 mg/ kg. In all experiments, SR-265579 was proved to be more active than zaprinast. These results demonstrate that SR 265579 is an orally active, potent and specific inhibitor of PDE5.

BACKGROUND The involvement of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in antinociception has been implicated as a molecular mechanism of antinociception produced by several antinociceptive agents, including μ-, κ-, or δ-opioid receptor agonists, nonsteroidal analgesics, cholinergic agonist, and α2C adrenoceptor agonist. In this study, we investigated whether ketamine, a dissociative anesthetic N-methyl-D-aspartate receptor antagonist, was also capable of activating the L-arginine/NO/cGMP pathway and eliciting peripheral antinociception. METHODS The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were locally administered into the right hindpaw of male Wistar rats. RESULTS Ketamine (10, 20, 40, 80 μg/paw) elicited a local antinociceptive effect that was antagonized by the nonselective NOS inhibitor L-NOARG (12, 18, and 24 μg/paw) and by the selective neuronal NOS inhibitor L-NPA (12, 18, and 24 μg/paw). In another experiment, we used the inhibitors L-NIO and L-NIL (24 μg/paw) to selectively inhibit endothelial and inducible NOS, respectively. These 2 drugs were ineffective at blocking the effects of the peripheral ketamine injection. In addition, the level of nitrite in the homogenized paw indicated that exogenous ketamine is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ (25, 50, and 100 μg/paw) blocked the action of ketamine, and the cGMP-phosphodiesterase inhibitor zaprinast (50 μg/paw) enhanced the antinociceptive effects of low-dose ketamine (10 μg/paw). CONCLUSIONS Our results suggest that ketamine stimulates the L-arginine/NO/cyclic GMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC(50) of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango β-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.

The NO and cGMP signaling pathways are of broad physiological and pathological significance. We compared the NO/soluble guanylyl cyclase (sGC)/cGMP pathway in human glioma tissues and cell lines with that of healthy control samples and demonstrated that sGC expression is significantly lower in glioma preparations. Our analysis of GEO databases (National Cancer Institute) further revealed a statistically significant reduction of sGC transcript levels in human glioma specimens. On the other hand, the expression levels of particulate (membrane) guanylyl cyclases (pGC) and cGMP-specific phosphodiesterase (PDE) were intact in the glioma cells that we have tested. Pharmacologically manipulating endogenous cGMP generation in glioma cells through either stimulating pGC by ANP/BNP, or blocking PDE by 3-isobutyl-1-methylxanthine/zaprinast caused significant inhibition of proliferation and colony formation of glioma cells. Genetically restoring sGC expression also correlated inversely with glioma cells growth. Orthotopic implantation of glioma cells transfected with an active mutant form of sGC (sGCα1β1(Cys105)) in athymic mice increased the survival time by 4-fold over the control. Histological analysis of xenografts overexpressing α1β1(Cys105) sGC revealed changes in cellular architecture that resemble the morphology of normal cells. In addition, a decrease in angiogenesis contributed to glioma inhibition by sGC/cGMP therapy. Our study proposes the new concept that suppressed expression of sGC, a key enzyme in the NO/cGMP pathway, may be associated with an aggressive course of glioma. The sGC/cGMP signaling-targeted therapy may be a favorable alternative to chemotherapy and radiotherapy for glioma and perhaps other tumors.

There is considerable support for the concept that insulin-mediated increases in microvascular blood flow to muscle impact significantly on muscle glucose uptake. Since the microvascular blood flow increases with insulin have been shown to be nitric oxide-dependent inhibition of cGMP-degrading phosphodiesterases (cGMP PDEs) is predicted to enhance insulin-mediated increases in microvascular perfusion and muscle glucose uptake. Therefore, we studied the effects of the pan-cGMP PDE inhibitor zaprinast on the metabolic and vascular actions of insulin in muscle. Hyperinsulinemic euglycemic clamps (3 mU·min−1·kg−1) were performed in anesthetized rats and changes in microvascular blood flow assessed from rates of 1-methylxanthine metabolism across the muscle bed by capillary xanthine oxidase in response to insulin and zaprinast. We also characterized cGMP PDE isoform expression in muscle by real-time PCR and immunostaining of frozen muscle sections. Zaprinast enhanced insulin-mediated microvascular perfusion by 29% and muscle glucose uptake by 89%, while whole body glucose infusion rate during insulin infusion was increased by 33% at 2 h. PDE2, -9, and -10 were the major isoforms expressed at the mRNA level in muscle, while PDE1B, -9A, -10A, and -11A proteins were expressed in blood vessels. Acute administration of the cGMP PDE inhibitor zaprinast enhances muscle microvascular blood flow and glucose uptake response to insulin. The expression of a number of cGMP PDE isoforms in skeletal muscle suggests that targeting specific cGMP PDE isoforms may provide a promising avenue for development of a novel class of therapeutics for enhancing muscle insulin sensitivity.

Background Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3′-5′-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3′-5′-guanylosine monophosphate. Methods Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. Results In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5′hydroxymethyl-2′furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1–induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. Conclusions Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

The similarity between Plasmodium falciparum phosphodiesterase enzymes (PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

Aims During postnatal development large amplitude spontaneous activity of the neonatal rat bladder changes to a low amplitude adult pattern of activity that leads to improved storage function. Previously, we have shown that spontaneous activity in neonatal rat bladder strips is inhibited by activation of the nitric oxide (NO)–cGMP signaling pathway. In the present experiments we determined if this inhibitory pathway is altered during postnatal development or spinal cord injury. Methods Baseline tone and amplitude and frequency of spontaneous contractions were measured in bladder strips from male or female neonatal (days 10–21), juvenile (days 24–39) and adult female spinal cord intact or chronic spinal cord injured Sprague–Dawley rats. Results The inhibitory effects of an NO donor (SNAP) and a PDE-5 inhibitor (zaprinast) on spontaneous activity of bladder strips decreased during postnatal development, while an inhibitory effect of 8-bromo-cGMP, which was blocked by a protein kinase G inhibitor, was detected at all ages tested. However, the effect of NO–cGMP signaling to reduce baseline tone emerged during postnatal development. The inhibition induced by the NO donor was blocked by an inhibitor of soluble guanylyl cyclase (sGC). Chronic spinal cord injury (cSCI), which causes the re-emergence of a neonatal-like pattern of spontaneous activity, did not restore sensitivity to NO-mediated inhibition in adult rat bladders. Conclusions These data indicate that while cGMP signaling inhibits activity in young and adult bladders as well as after cSCI, there is a developmental decrease in the sensitivity of bladder to NO-mediated inhibition. Neurourol. Urodynam. Neurourol. Urodynam. 30: 1666–1674, 2011. © 2011 Wiley Periodicals, Inc.

BACKGROUND AND PURPOSE 8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), formed nitric oxide (NO)-dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8-nitro-cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non-diabetic and diabetic mice. EXPERIMENTAL APPROACH Vascular tension recording was performed in thoracic aortic rings from wild-type (C57BL/6), non-diabetic db/+ and obese/diabetic db/db mice. Endothelial NO synthase (eNOS) uncoupling and superoxide were tested by Western blot and dihydroethidium fluorescence respectively. KEY RESULTS 8-Nitro-cGMP, at concentrations up to 10 µM, enhanced phenylephrine-induced contractions in aortas from C57BL/6 and db/+ mice, but not from db/db mice. This enhancement was not observed with 8-bromo-cGMP. Pretreatment of aortas from C57BL/6 and db/+ mice with l-NAME (100 µM), superoxide dismutase (100 U·mL−1) or tiron (1 mM), abolished 8-nitro-cGMP-induced enhancement of the phenylephrine contraction. In 8-nitro-cGMP (10 µM)-treated C57BL/6 aortas, eNOS dimer/monomer ratio was significantly decreased and vascular superoxide production increased, suggesting that 8-nitro-cGMP-induced superoxide production via eNOS uncoupling may mediate the enhancement of the phenylephrine contraction. At higher concentrations (>10 µM), 8-nitro-cGMP produced relaxation of the phenylephrine-contracted aortas from C57BL/6, db/+ and db/db mice. The 8-nitro-cGMP-induced relaxation in db/db mouse aortas was found to be resistant to a phosphodiesterase 5 inhibitor, zaprinast (1 µM). CONCLUSIONS AND IMPLICATIONS The vasodilator effect of 8-nitro-cGMP may contribute to amelioration of the vascular endothelial dysfunction in diabetic mice, representing a novel pharmacological approach to prevent the complications associated with diabetes.

We previously demonstrated that exposure to red light (550–670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.

BACKGROUND AND PURPOSE GPR35 is a poorly characterized G protein-coupled receptor at which kynurenic acid has been suggested to be the endogenous ligand. We wished to test this and develop assays appropriate for the study of this receptor. EXPERIMENTAL APPROACH Human and rat orthologues of GPR35 were engineered and expressed and assays developed to assess interaction with β-arrestin-2, activation of Gα13 and agonist-induced internalization. KEY RESULTS GPR35-β-arrestin-2 interaction assays confirmed that both the endogenous tryptophan metabolite kynurenic acid and the synthetic ligand zaprinast had agonist action at each orthologue. Zaprinast was substantially more potent than kynurenic acid at each and both agonists displayed substantially greater potency at rat GPR35. Two novel thiazolidinediones also displayed agonism and displayed similar potency at each GPR35 orthologue. The three ligand classes acted orthosterically with respect to each other, suggesting overlapping binding sites and, consistent with this, mutation to alanine of the conserved arginine at position 3.36 or tyrosine 3.32 in transmembrane domain III abolished β-arrestin-2 recruitment in response to each ligand at each orthologue. CONCLUSIONS AND IMPLICATIONS These studies indicate that β-arrestin-2 interaction assays are highly appropriate to explore the pharmacology of GPR35 and that Gα13 activation is an alternative avenue of signal generation from GPR35. Arginine and tyrosine residues in transmembrane domain III are integral to agonist recognition and function of this receptor. The potency of kynurenic acid at human GPR35 is sufficiently low, however, to question whether it is likely to be the true endogenous ligand for this receptor.

Summary Background Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP. Material/Methods Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP). Results Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP. Conclusions PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.