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Potentially prebiotic chemical reactions leading to RNA nucleotides involve periods of UV irradiation, which are necessary to promote selectivity and destroy biologially irrelevant side products. Nevertheless, UV light has only been applied to promote specific stages of prebiotic reactions and its effect on complete prebiotic reaction sequences has not been extensively studied. Here, we report on an experimental and computational investigation of the photostability of 2-thiooxazole (2-TO), a potential precursor of pyrimidine and 8-oxopurine nucleotides on early Earth. Our UV-irradiation experiments resulted in rapid decomposition of 2-TO into unidentified small molecule photoproducts. We further clarify the underlying photochemistry by means of accurate

Pathogens such as

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the mitigation of cell cytotoxicity. Further systematic structure-activity relationship (SAR) efforts driven by structural biology studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders. Herein, based on the existing PDE2 inhibitors and their binding modes, a series of purin-6-one derivatives were designed, synthesized and evaluated for PDE2 inhibitory activities, which led to the discovery of the best compounds 6p and 6s with significant inhibitory potency (IC50: 72 and 81 nM, respectively). Docking simulation was performed to insert compound 6s into the crystal structure of PDE2 at the active site to determine the binding mode. Furthermore, compound 6s significantly protected HT-22 cells against corticosterone-induced cytotoxicity and rescued corticosterone-induced decreases in cAMP and cGMP levels. It also produced anxiolytic-like effect in the elevated plus-maze test and exhibited favorable pharmacokinetic properties in vivo. These results might bring significant instruction for further development of potent PDE2 inhibitors.

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.

This work scrutinizes the relaxation mechanism of 2-oxopurine. Contrary to its ancestor, purine, which is a UVC chromophore, 2-oxopurine shows a red-shifted absorption spectrum centered in the UVA region. In 2-oxopurine, relaxation along the ππ* spectroscopic state directs the population from the Franck-Condon (FC) region towards a minimum, which acts as a crossroad for the further decay of the system either to triplet states or, alternatively, to the ground state through a C6-puckered S1/S0 funnel. A comparison of the optical properties and excited state potential energy surfaces of purine, 2-oxopurine, 2-aminopurine, 6-oxopurine and adenine, allows establishing how the position and nature of substituent tune the photophysics of purine. For this series, we conclude that both C2 and C6 substitution redshift the absorption spectrum of purine, with 2-oxo substitution exhibiting the largest shift. An important exception is the canonical nucleobase adenine, which presents a blue shifted absorption spectrum. The topography of purine's ππ* potential energy surface experiences major changes when functionalized at the C6 position. In particular, the disappearance of the minimum along the ππ* potential energy surface efficiently funnels the excited state population from the FC region to the ground state and increases the photostability of 6-aminopurine (adenine) and 6-oxopurine (hypoxanthine) nucleobases.

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.

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.

Bradykinin (BK) promotes insulin sensitivity and glucose uptake in adipocytes and other cell types. We demonstrated that in rat adipocytes BK enhances insulin-stimulated glucose transport via endothelial nitric oxide synthase, nitric oxide (NO) generation, and decreased activity of the mitogen-activated protein kinase (MAPK) JNK (c-Jun NH2-terminal kinase). In endothelial cells, NO increases soluble guanylate cyclase (sGC) activity, which, in turn, activates protein kinase G (PKG) by increasing cGMP levels. In this study, we investigated whether BK acts via the sGC-cGMP-PKG pathway to inhibit the negative effects of JNK on insulin signaling and glucose uptake in rat adipocytes. BK augmented cGMP concentrations. The BK-induced enhancement of insulin-stimulated glucose uptake was mimicked by the sGC activator YC-1 and a cell-permeable cGMP analog, CPT-cGMP, and inhibited by the sGC inhibitor ODQ and the PKG inhibitor KT 5823. Transfection of dominant-negative PKG reduced the BK augmentation of insulin-induced Akt phosphorylation. The activation of JNK and ERK1/2 by insulin was attenuated by BK, which was mediated by the sGC-cGMP-PKG pathway. Whereas insulin-stimulated phosphorylation of upstream activators of JNK and ERK, i.e., MKK4 and MEK1/2, was unaffected, BK augmented insulin-mediated induction of MKP-5 mRNA and protein levels. Furthermore, zaprinast, a phosphodiesterase inhibitor, enhanced cGMP and MKP-5 and prolonged the action of BK. These data indicate that BK enhances insulin action by inhibition of negative feedback by JNK and ERK via upregulation of MKP-5, mediated by the sGC-cGMP-PKG signaling pathway.

Highlights • ZBD-2 significantly attenuated the symptoms of chronic SCI-pain and pain-induced depressive-like behaviors. • ZBD-2 inhibited the decreases in the expression of synaptic plasticity-related signaling proteins. • ZBD-2 reversed chronic, SCI-induced gliocyte activation at the lesion site., In addition to debilitating sensory and motor deficits, patients with spinal cord injury (SCI) may experience chronic hyperpathic pain (SCI-pain). Recent studies have revealed that translocator protein (TSPO) is involved in repairing neural cells as well as reducing anxiety and depression. However, the role of TSPO in SCI-pain and pain-induced depression remains unknown. The present study aimed to determine the effects of a new TSPO ligand, ZBD-2, on SCI-pain and consequent pain-induced depressive-like behaviors in mice. Treatment with ZBD-2 at either dose significantly attenuated the symptoms of chronic SCI-pain and pain-induced depressive-like behaviors. ZBD-2 reversed SCI-induced elevation of serum corticosterone levels, an index of hyper-activation of the hypothalamic–pituitary–adrenal (HPA) axis. Additionally, administration of ZBD-2 inhibited decreases in the expression of synaptic plasticity-related signaling proteins, including brain-derived neurotrophic factor (BDNF) and cyclic AMP-responsive element binding protein (CREB). Moreover, ZBD-2 administration reversed chronic, SCI-induced gliocyte activation at the lesion site. Therefore, ZBD-2 may improve chronic SCI-pain and pain-induced depressive-like behaviors via suppression of gliocyte activation and restoration of the synaptic plasticity-related signaling systems.

Alzheimer’s disease (AD), characterized by a progressive impairment of memory and cognition, is a major health problem in both developing and developed countries. Currently, no drugs can reverse the progression of AD. Phosphodiesterase 5 (PDE5) is a critical component of the cyclic guanosine monophosphate/protein kinase G (cGMP/PKG) signaling pathway in neurons, the inhibition of which has produced neuroprotective effects, and PDE5 inhibitors have recently been thought to be potential therapeutic agents for AD. In this paper, we summarized the outstanding progress that has been made in PDE5 inhibitors as anti-AD agents with encouraging results in animal studies, clinical trials and the investigations on the underlying mechanisms. The novel PDE5 inhibitors reported recently in the treatment of AD were also reviewed and discussed.

Aim: DNA damage response plays an eminent role in patients’ response to conventional chemotherapy and radiotherapy. Its inhibition is of great interest as it can overcome cancer cell resistance and reduce the effective doses of DNA damaging agents. Results & methodology: We have focused our research on phosphatidylinositol 3-kinase-related kinases and prepared 35 novel compounds through a scaffold hopping approach. The newly synthesized inhibitors were tested on a panel of nine cancer and one healthy cell lines alone and in combination with appropriate doses of doxorubicin. Conclusion: Five novel compounds 4f, 10b, 15g, 7e and 15f in combination with doxorubicin showed significant antiproliferative effect on seven cancer cell lines while not affecting the cell growth alone.

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

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.

The 18 kDa translocator protein (TSPO) is primarily localized in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. One of the protein’s main functions is transporting substrate cholesterol into the mitochondria in a prerequisite process for steroid synthesis. Clinical trials have indicated that TSPO ligands might be valuable in treating some neuropathies and psychopathies. However, limited information is known about the role of TSPO in postpartum depression (PPD). The TSPO ligand ZBD-2, a derivative of XBD173, was synthesized in our laboratory. Behavioral tests, enzyme linked immunosorbent assay, and Western blot were employed to evaluate ZBD-2’s efficacy against PPD and to elucidate the potential underlying molecular mechanism. The TSPO levels significantly decreased in the basolateral amygdala of PPD models. After treatment for 2 weeks, ZBD-2 alleviated depression-like behaviors and enhanced the TSPO level in a PPD animal model. The underlying mechanisms of ZBD-2 were related to regulate the hypothalamic-pituitary-adrenal axis, enhance 5-HT and BDNF secretion, and maintain the excitatory and inhibitory synaptic protein expression to normal levels. Our results directly confirm that ZBD-2 exerts a therapeutic effect on PPD, which provides a new target for anti-PPD drug development.

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery., Author summary Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei. Current treatments suffer from low efficacy, toxicity issues and complex medication regimens. Moreover, an alarming number of these parasites are demonstrating resistance to current drugs. For these reasons, there is a renewed effort to develop new classes of modern therapeutics based upon the unique T. brucei cellular processes. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRTase), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. We demonstrated that T. brucei encodes two isoforms of hypoxanthine-guanine PRTases (HGPRT) and one hypoxanthine-guanine-xanthine PRTase (HGXPRT). The concurrent activity of these enzymes is required for the normal cell growth in vitro. Moreover, acyclic nucleoside phosphonates represent a promising class of potent and selective compounds as they inhibit the enzymes with Ki values in nanomolar range and exert cytotoxic effects on T. brucei cells grown in vitro with EC50 values in the single digit micromolar range. Our results provide a new foundation for further investigations of these compounds in vivo and suggest that 6-oxopurine salvage pathway represents a possible target for future drug discovery efforts directed at eliminating HAT.

A novel butanehydrazide derivatives of purine-2,6-dione designed using a ligand-based approach were synthesized and their in vitro activity against both PDE4B and PDE7A isoenzymes was assessed. The 7,8-disubstituted purine-2,6-dione derivatives 31, 34, 37, and 40 appeared to be the most potent PDE4/7 inhibitors with IC50 values in the range of that of the reference rolipram and BRL-50481, respectively. Moreover, docking studies explained the importance of N-(2,3,4-trihydroxybenzylidene)butanehydrazide substituent in position 7 of purine-2,6-dione core for dual PDE4/7 inhibitory properties. The inhibition of both the cAMP-specific PDE isoenzymes resulted in a strong anti-TNF-α effect. Compounds 31, 34, and 37 in the in vivo study in rats with LPS-induced endotoxemia decreased the maximum concentration of this proinflammatory cytokine by 53, 84 and 88%, respectively.

Background The aim of this study was to evaluate the effects of the phosphodiesterase-5 (PDE-5) inhibitors, zaprinast and avanafil, on NO signalling pathway, bone mineral density (BMD), epiphyseal bone width, bone marrow angiogenesis, and parameters of oxidative stress in a rat model of glucocorticoid-induced osteoporosis (GIOP). Material/Methods Twenty-four 8-month-old male rats in four groups were given a single daily treatment during a 30-day period: an (untreated) control group (n=6): a dexamethasone-treated group (120 μ/kg) (n=6); a group treated with dexamethasone (120 μ/kg) and zaprinast (10 mg/kg) (n=6): and a group treated with dexamethasone (120 μ/kg) and avanafil (10 mg/kg) (n=6). Rat whole body bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA), and bone histology was performed. Also, selected oxidative stress parameters by HPLC method and the other biochemical parameters by ELISA method were measured. Results The GIOP model rats treated with zaprinast and avanafil showed a significant increase in NO, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) (NO/cGMP/PKG) signaling-pathway components, and in C-terminal telopeptide of type I collagen (CTX-1), bone marrow angiogenesis, BMD, and epiphyseal bone width, compared with the (untreated) control rats (p

Graphical abstract, Eukaryotic mRNA contains a 3′ poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg2+ ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

Summary Ligands of the translocator protein (18 kDa) (TSPO) have demonstrated rapid anxiolytic efficacy in stress responses and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids including pregnenolone, dehydroepiandrosterone, and progesterone. These neurosteroids promote γ-aminobutyric acid-mediated neurotransmission in the central neural system (CNS). A TSPO ligand, N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was recently synthesized. The purpose of the present study was to investigate the neuroprotective effects of ZBD-2 and. In cultured cortical neurons, treatment with ZBD-2 attenuated excitotoxicity induced by N-methyl-d-aspartate (NMDA) exposure. It significantly decreased the number of apoptotic cells by downregulating GluN2B-containing NMDA receptors (NMDARs), the ratio of Bax/Bcl-2, and levels of pro-caspase-3. Systemic treatment of ZBD-2 provided significant neuroprotection in mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that neuroprotection by ZBD-2 is partially mediated by inhibiting GluN2B-containing NMDA receptor-mediated excitotoxicity.

A set of novel pyridine-annulated analogs of purinones, adenines and their oxo/thio congeners, xanthines, guanines, and purine-2,4-diamines as potential anticancer agents was considered based on the scaffold-hopping and hybridization of known anticancer agents/drugs, purine derivatives and our recently developed imidazo-pyridine derivatives. Towards the synthesis of these compounds, a new approach involving a convenient preparation of 3-amino-2-carboxyethyl substituted imidazo[1,2-a]-pyridine and its use as a building block for the construction of fused rings was developed. The approach enabled the preparation of a number of compounds with relevant substitutions for each class. Several of pyridine-annulated adenine and its oxo/thio analogs, xanthine and purine-2,4-diamine were found to possess significant anticancer activities in kidney cancer cells and relatively less cytotoxicity to normal cells. They were relatively more active than the anticancer drugs etoposide and doxorubicin. A representative pyridine-annulated adenine derivative compound(22) was found to exert significant apoptosis.

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.

G protein-coupled receptor 35 (GPR35), a receptor for lysophosphatidic acid, is highly expressed in the gastrointestinal tract. Recently, GPR35 has been implicated in the onset of inflammatory bowel disease (IBD), but its role in physiological and pathological processes in the colon remains undefined. In this study, we investigated the contribution of GPR35-mediated signalling to mucosal repair of colonic epithelium in IBD. GPR35 function was examined in a wound healing model, using young adult mouse colon epithelium (YAMC) cells, and in a dextran sulphate sodium (DSS)-induced mouse model of colitis. Cell proliferation, mRNA expression, extracellular signal-regulated kinase (ERK) activation, and protein localization were determined by MTT assay, quantitative RT-PCR, western blotting, and immunohistochemistry, respectively. GPR35 agonists (YE120, zaprinast, and pamoic acid) promoted wound repair in a concentration-dependent manner independently of cell proliferation, whereas a specific GPR35 antagonist CID2745687, forskolin, and pertussis toxin reversed the YE120-induced effect. YE120 increased the mRNA expression of fibronectin and its receptor integrin α5, and ERK1/2 phosphorylation, but these responses were attenuated by CID2745687 and forskolin. Furthermore, the severity of DSS-induced colitis was significantly reduced by daily injections of pamoic acid via upregulation of fibronectin and integrin α5 in the colonic epithelium. GPR35 signalling promotes mucosal repair by inducing fibronectin and integrin α5 expression, coupling to Gi protein, and activating ERK1/2 in colonic epithelial cells. These findings define GPR35 as a candidate therapeutic target in IBD.

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.

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.

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.

A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4–21) were synthesized and evaluated for their serotonin (5-HT1A/5-HT7) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The study enabled the identification of potent 5-HT1A, 5-HT7 and mixed 5-HT1A/5-HT7 receptor ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed with the determination of lipophilicity and metabolic stability using micellar electrokinetic chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam. Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for antidepressant and/or anxiolytic application.

This work was supported in part by National Institutes of Health Grants AI-110027 and AI-096836 (to S. N. J. M.) and 1DP5OD017892 (to S. L.). The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition. Publisher PDF

Summary Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N-benzyl-N-ethyl-2-(7,8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA-induced neurotoxicity in vitro. The present study aims to examine the role of ZBD-2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD-2 (10 mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of spinal cord. High-dose ZBD-2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B-containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD-2 (10 mg/kg) increased phosphorylated Akt (p-Akt) and decreased the ratio of Bax/Bcl-2. These results demonstrate that ZBD-2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway. This article is protected by copyright. All rights reserved.

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs.

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.

Testosterone (T) plays a permissive role in the development of benign prostatic hyperplasia (BPH), and phosphodiesterase 5 inhibitors (PDE5is) have been found to be effective for BPH and lower urinary tract symptoms (LUTS) in clinical trials. This study investigated the effect of T on smooth muscle (SM) contractile and regulatory signaling pathways, including PDE5 expression and functional activity in prostate in male rats (sham-operated, surgically castrated, and castrated with T supplementation). In vitro organ bath studies, real-time RT-PCR, Western blot analysis, and immunohistochemistry were performed. Castration heavily attenuated contractility, including sensitivity to phenylephrine with SM myosin immunostaining revealing a disrupted SM cell arrangement in the stroma. PDE5 was immunolocalized exclusively in the prostate stroma, and orchiectomy signficantly reduced PDE5 immunopositivity, mRNA, and protein expression, along with nNOS and ROKβ mRNA, whereas it increased eNOS plus α1a and α1b adrenoreceptor expression in castrated animals. The PDE5i zaprinast significantly increased prostate strip relaxation to the nitric oxide donor sodium nitroprusside (SNP) in control but not castrated rats. But SNP alone was more effective on castrated rats, comparable with sham treated with SNP plus zaprinast. T supplementation prevented or restored all above changes, including SNP and zaprinast in vitro responsiveness. In conclusion, our data show that T positively regulates PDE5 expression and functional activities in prostate, and T ablation not only suppresses prostate size but also reduces prostatic SM contractility, with several potential SM contraction/relaxation pathways implicated. Zaprinast findings strongly suggest a major role for PDE5/cGMP in this signaling cascade. PDE5 inhibition may represent a novel mechanism for treatment of BPH.

To develop a PET ligand for imaging TSPO in peripheral organs, we designed three novel oxopurine analogues [11C]3a–c (LogD: 1.81–2.17) by introducing a pyridine ring in place of a benzene ring in the lead compound [11C]2 (LogD: 3.48). The desmethyl precursors 10 for radiosynthesis were synthesized by reacting glycine 7 with picolylamines 4, followed by hydrolysis and by Curtius rearrangement with diphenylphosphoryl azide. Methylation of 10a–c with methyl iodide produced unlabeled compounds 3a–c. The radiosynthesis of [11C]3a–c was performed by reacting 10a–c with [11C]methyl iodide. Compounds 3a–c displayed high or moderate in vitro binding affinities (Ki: 5–40 nM) for TSPO. PET with [11C]3a–c in rats showed high uptake in the lung, heart, and kidney, which are organs with high TSPO expression. Metabolite analysis with [11C]3a showed that radioactivity in these organs mainly corresponded with unchanged [11C]3a. PET with [11C]3a using a rat model of lung inflammation showed a significant signal in the lipopolysaccharide-treated lung.

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.

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.

G-protein coupled receptor 35 (GPR35) is a former "orphan receptor" expressed in brain and activated by either kynurenic acid or zaprinast. While zaprinast has been studied as a phosphodiesterase inhibitor, kynurenic acid (KYNA) is a tryptophan metabolite and has been proposed as the endogenous ligand for this receptor. In the present work, we showed that GPR35 is present in the dorsal root ganglia and in the spinal cord and in order to test the hypothesis that GPR35 activation could cause analgesia, we administered suitable doses of zaprinast or we increased the local concentration of KYNA by administering a precursor (kynurenine) or by inhibiting its disposal from the CNS (with probenecid). We used the "writhing test" induced by acetic acid i.p. injection in mice. KYNA and kynurenine plasma and spinal cord levels were measured with HPLC techniques. Kynurenine (30, 100, 300 mg/kg s.c.) increased plasma and spinal cord levels of KYNA and decreased the number of writhes in a dose dependent manner. Similarly, probenecid was able to increase KYNA levels in plasma and spinal cord, to reduce the number of writes and to amplify kynurenine effects. Furthermore, zaprinast had antinociceptive effects in the writhing test without affecting KYNA levels. In agreement with its affinity for GPR35 receptor (approximately 10 times higher than that of KYNA), zaprinast action occurred at relatively low doses. No additive actions were obtained when kynurenine and zaprinast were administered at maximally active doses. Our results suggest that GPR35 could be an interesting target for innovative pharmacological agents designed to reduce inflammatory pain. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

A methodology enabling investigation of a multicomponent tautomeric and acid-base equilibria by 13 C NMR spectroscopy supported by theoretical calculations has been proposed. The effectiveness of this method has been illustrated in a study of 2-oxopurine, 6-oxopurine (hypoxanthine), 8-oxopurine, and 2,6-dioxopurine (xanthine) in neutral and alkaline aqueous solutions. For each compound a series of 13 C NMR spectra were recorded at pH ranges in which neutral molecules, δ monoanions and/or dianions occurred in dynamic equilibrium. The carbon chemical shifts for these three forms of the investigated compounds were retrieved from the analysis of pH-dependence of the measured, dynamically averaged values of these parameters. The structures of several stable tautomers of the neutral and monoanionic oxopurine forms were predicted from theoretical calculations and nuclear magnetic shielding constants for 13 C nuclei in these tautomers were calculated. At both calculation steps (molecular geometry optimization and calculation of NMR parameters) the PBE1PBE/6-31 1++G(2d,p) level of theory was used. The populations of the most stable tautomers were determined from the experimental data analysis exploiting the fact that they were population-weighted averages of the chemical shifts of particular tautomers. It has been shown that only the oxo forms of the investigated oxopurines are present in aqueous solutions and that the determined populations in most cases remain in a qualitative agreement with the calculated free energies of the appropriate tautomers. The obtained results are in general agreement with other literature reports on oxopurine tautomerism and confirm importance of the hydration phenomena for the investigated systems. The data analysis has shown that the best compliance between theory and experiment is obtained when the hydration phenomenon is modeled by discrete hydration augmented by PCM (polarizable continuum solvation model).

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.

Nitric oxide (NO) signaling in tumors and endothelial cells regulates vascular permeability and blood flow and therefore influences tumor uptake and response to therapeutic compounds. As delivery and efficacy of chemotherapy is impaired in CNS neoplasms due to a partially intact blood–brain barrier (BBB), we studied the effects of NO released by the short-acting NO donor disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate methanolate (PROLI/NO) on BBB integrity and blood flow in C6 gliomas using [14C]-aminoisobutyric acid (AIB) and [14C]-iodoantipyrine quantitative autoradiography. PROLI/NO selectively increased intratumoral uptake of [14C]AIB and [14C]sucrose when given as a 3-minute intracarotid infusion or a 15-minute i.v. infusion (AIB: tumor, K1 = 68.7 ± 3.2 vs 24.9 ± 0.9 µL g−1 min−1, P < .0001; sucrose, K1 = 16.9 ± 0.9 vs 11.5 ± 0.9 µL g−1 min−1, P = .0007). This effect was achieved without significant changes in cerebral and tumor blood flow or arterial blood pressure, which indicates that the effect on vascular permeability is independent of changes in vascular tone induced by NO. This effect was mediated by activation of the NO/3',5'-cyclic guanosine monophosphate (cGMP) pathway, as it was blocked by guanylate cyclase inhibition by LY83583 and reproduced by the delivery of 8-bromoguanosine 5'-monophosphate or inhibition of cGMP degradation by the phosphodiesterase inhibitor zaprinast. Inhibition of inducible NO synthase by aminoguanidine or cyclooxygenase inhibition by indometacin or dexamethasone did not reduce the blood–tumor barrier (BTB) response to PROLI/NO. PROLI/NO, and perhaps other NO-donating compounds, can be used to selectively increase BTB permeability in gliomas through the NO/cGMP pathway at doses that do not cause unwanted vasodilatory changes in blood flow and that do not affect the systemic circulation.

Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, relaxes gastrointestinal smooth muscle. L-type calcium channel currents play an important role in regulating smooth muscle contraction. The effect of DNP on L-type calcium channel currents in gastrointestinal tract is still unclear. This study was designed to investigate the effect of DNP on barium current (I Ba ) through the L-type calcium channel in gastric antral myocytes of guinea pigs and cGMP-pathway mechanism. The whole-cell patch-clamp technique was used to record L-type calcium channel currents. The content of cGMP in guinea pig gastric antral smooth muscle and perfusion solution was measured using radioimmunoassay. DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in perfusion medium. DNP concentration-dependently inhibited I Ba in freshly isolated guinea pig gastric antral circular smooth muscle cells (SMCs) of guinea pigs. DNP-induced inhibition of I Ba was partially blocked by LY83583, an inhibitor of guanylate cyclase. KT5823, a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked DNP-induced inhibition of I Ba . However, DNP-induced inhibition of I Ba was potentiated by zaprinast, an inhibitor of cGMP-sensitive phosphodiesterase. Taken together, DNP inhibits L-type calcium channel currents via pGC–cGMP–PKG-dependent signal pathway in gastric antral myocytes of guinea pigs.