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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.

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.

This study has assessed the expression and functional significance of cGMP-dependent signalling components in BRIN-BD11 cells. RT-PCR analysis revealed the expression of two subunits of soluble guanylate cyclase (sGC) suggesting the presence of an α2/β1 heterodimer. The expression of three particulate guanylate cyclases (pGC) was also detected (GC-A, GC-B and GC-C), as well as two cGMP-selective PDE isoforms (PDE5A and PDE9). Stimulation of BRIN-BD11 cells with agonists selective for sGC (NO, YC-1 and BAY 41-2272), GC-A (atrial natriuretic peptide (ANP) or GC-C (guanylin) caused an elevation in cGMP, and in the case of sGC, this was blocked by the selective inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). The stimulatory effects of each activator on cGMP levels were further potentiated by the PDE5A inhibitor, zaprinast. Treatment of cells with sGC activators induced a loss of viability and increased insulin secretion. However these effects were not attenuated by ODQ suggesting that they were independent of a rise in cGMP. A modest increase in β-cell death and insulin secretion were also observed in guanylin and ANP treated cells, although the latter only reduced cell viability in the presence of a PDE5A inhibitor. Taken together, the data reveal that BRIN-BD11 cells express several functionally active enzymes capable of modulating cGMP levels, and they imply that signalling through these proteins may impact upon β-cell viability. The results further suggest that pGC isozymes can also regulate insulin secretion but that the pool of cGMP controlling insulin release is small relative to the global cGMP concentration in the cell.

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.

Many pancreatic cancers consume excessive glutamine as a preferable source for anaplerosis, maintenance of proper redox state, and synthesis of intermediates for cancer cell growth and proliferation. Abrogating glutamine metabolism is under investigation to inhibit the proliferation/ growth of glutamine-addicted cancers. In addition, inhibition of the mitochondrial pyruvate carrier (MPC1) can potentially induce cancer cells to become more dependent on glutaminolysis. In this regard, Zaprinast, originally developed as a phosphodiesterase 5 (PDE5) inhibitor, has recently been identified as an inhibitor of both glutaminase as well as MPC1. We compared the growth inhibition effect of Zaprinast against BPTES, a targeted glutaminase inhibitor, and UK5099, a targeted MPC1 inhibitor, as well as the combination of BPTES and UK5099. Using pancreatic ductal adenocarcinoma (PDAC) cells, KRAS mutant (Mia Paca2) and KRAS wildtype (Panc03.27), KRAS mutant neuroendocrine (QGP1), and normal pancreatic epithelial cells (HPNE), Zaprinast alone showed more potent growth inhibition in all tested cells lines than single agent treatment with BPTES or UK5099. Further, Zaprinast effectively depleted mitochondrial reserve respiratory capacity similar to UK5099, rendering mitochondria more vulnerable to oxidative stress or pharmacological inhibitors targeting the electron transport chain. In addition, Zaprinast treatment of cancer cells differentially elevated ROS levels greater than 10-fold compared to normal HPNE cells (ROS levels: Mia Paca2 ~ Panc03.27 > QGP1 > HPNE). However, the increase in ROS levels did not correlate with Zaprinast-induced growth inhibition (IC50: Mia Paca2=300 µM, Panc03.27=110 µM, HPNE=650 µM, QGP1 >1 mM). Although Zaprinast-induced growth inhibition was selective toward adenocarcinoma cells but not the neuroendocrine subtype, the selective Zaprinast-induced elevation of ROS levels toward all cancer subtypes renders possibilities for potential synthetic lethality when combined with other metabolic inhibitors. Citation Format: Yi Rao, Seth Gammon, David Piwnica-Worms. Differential metabolic targeting of PDAC cells with Zaprinast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3514.

The aim of this study was to examine the expression of G protein-coupled receptor (GPR)35 in human invariant natural killer T (iNKT) cells and to determine the functional effects induced by selective activation of this receptor. RT-PCR analysis showed that both human iNKT cells and resting PBMC expressed GPR35; GPR35 protein resulted mostly localized in the plasma membrane, while it internalized in punctate intracellular structures following specific receptor activation (Western blot and immunofluorescence/confocal microscopy analysis). The specific activation of GPR35 by selective receptor agonists [l-kynurenic acid (KYNA)] or 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo [4,5-d]pyrimidine-7-one (zaprinast)] functionally correlated with a significant reduction in IL-4 release from alpha-galactosylceramide (alpha-GalCer)-activated human iNKT cells, and this effect resulted mediated by pertussis toxin (PTX)-sensitive Gi/o proteins. In conclusion, our results demonstrate that human iNKT cells express GPR35 functionally active in reducing IL-4 release.

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca(2+) signaling, and Ca(2+) sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca(2+)](i) revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca(2+) oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca(2+) oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca(2+) oscillations. NOC-5 inhibited the increase of [Ca(2+)](i) and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP(3)) in airway SMCs. The effect of NO on the Ca(2+) sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca(2+) by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca(2+)-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP-PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca(2+) via the IP(3) receptor to decrease the frequency of agonist-induced Ca(2+) oscillations.

We report that the asthma drugs cromolyn disodium and nedocromil sodium are potent G-protein-coupled receptor 35 (GPR35) agonists. We utilized calcium flux and inositol phosphate accumulation assays to examine the pharmacology of these asthma drugs on the human, mouse and rat GPR35. The compounds were more potent on the human GPR35 than on mouse and rat receptors. In contrast, zaprinast, a known GPR35 agonist, was more potent on mouse and rat GPR35 than the human ortholog. We show by quantitative PCR that GPR35 is expressed in human mast cells, human basophils and human eosinophils. We also demonstrate that GPR35 mRNA is upregulated upon challenge with IgE antibodies. We show that, unlike zaprinast, a potent phosphodiesterase 5 (PDE5) inhibitor, cromolyn disodium and nedocromil sodium lack inhibitory activity towards PDE5. These findings suggest that GPR35 may play an important role in mast cell biology and be a potential target for the treatment of asthma.

Recent reports suggest that xanthine oxidase (XO), a modified form of the native xanthine dehydrogenase enzyme, plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases, and chronic heart failure. The XO inhibitors allopurinol and its oxidation product oxypurinol held considerable promises in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil, a well-known type-5 phosphodiesterase inhibitor, has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil (pyrimidine and pyrazole rings), and with zaprinast (featuring pyrimidine and triazolo rings), we have investigated the potential effects of these latter compounds on the buttermilk purified XO and on nontumorigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and, to a lesser extent, zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthine-induced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO enzyme and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5.

Background and purpose: 5-HT receptors may have a role in pulmonary hypertension. Their relationship with the activity of BK a T-type voltage-operated calcium channel (VOCC) and cyclic nucleotide-mediated relaxation was examined. Experimental approach: Ring segments of bovine pulmonary arteries were mounted in organ baths in modified Krebs-Henseleit buffer (37 C) under a tension of 20 mN and gassed with 95% O /5% CO . Isometric recordings were made using Chart 5 software. Key results: Contractile responses to 5-HT (10 nM-300 μM) were inhibited similarly by the 5-HT receptor antagonist SB216641 (100 nM) and the T-type VOCC blockers mibefradil (10 μM) and NNC550396 (10 μM) with no additive effect between SB216641 and mibefradil. Inhibition by SB216641 was prevented by the potassium channel blocker, charybdotoxin (100 nM). 5-HT receptor activation and charybdotoxin produced a mibefradil-sensitive potentiation of responses to U46619. Bradykinin (0.1 nM-30 μM), sodium nitroprusside (0.01 nM-3 μM), zaprinast (1 nM-3 μM), isoprenaline (0.1 nM-10 μM) and rolipram (1 nM-3 μM) produced 50% relaxation of arteries constricted with 5-HT (1-3 μM) or U46619 (30-50 nM) in the presence of 5-HT receptor activation, but full relaxation of arteries constricted with U46619, the 5-HT receptor agonist 2,5 dimethoxy-4 iodoamphetamine (1 μM) or 5-HT in the presence of 5-HT receptor antagonism. Enhanced relaxation of 5-HT-constricted arteries by cGMP-dependent pathways, seen in the presence of the 5-HT receptor antagonist, was reversed by charybdotoxin whereas cAMP-dependent relaxation was only partly reversed by charybdotoxin. Conclusions and implications: 5-HT receptors couple to inhibition of BK , thus increasing tissue sensitivity to contractile agonists by activating a T-type VOCC and impairing cGMP-mediated relaxation. Impaired cAMP-mediated relaxation was only partly mediated by inhibition of BK .

The L-arginine/nitric oxide/cyclic GMP pathway has been proposed as the mechanism of action for peripheral antinociception concerning several groups of drugs, including opioids and nonsteroidal analgesics. The aim of the present study was to investigate the involvement of the L-arginine/NO/cGMP pathway on antinociception induced by xylazine, an alpha(2)-adrenoceptor agonist extensively used in veterinary medicine and animal experimentation. The rat paw pressure test was used by inducing hyperalgesia via intraplantar injection of prostaglandin E(2) (2 microg). Xylazine was administered locally into the right hind paw (25, 50 and 100 microg) and either NO synthase inhibitor L-NOarg (12, 18 and 24 microg/paw), soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100 microg/paw) or cGMP-phosphodiesterase inhibitor zaprinast (50 microg/paw) were previously administered to the right hind paw of Wistar rats. Xylazine administration elicited a local antinociceptive effect, since only much higher doses produce a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine (100 microg/paw) was antagonized by L-NOarg and by ODQ; however, zaprinast potentiated the antinociceptive effect of xylazine at 25 microg/paw. The results provide evidence that xylazine probably induces peripheral antinociceptive effect by L-arginine/NO/cGMP pathway activation.

Nitric oxide (NO) is the primary mediator of blood flow in female genital tissues and drugs that enhance the activity of nitric oxide, such as phosphodiesterase type-5 (PDE-5) inhibitors, increase vaginal blood flow in anesthetized rats. The goal of the present study was to test the effects of one PDE-5 inhibitor, zaprinast, on the display of sexual behaviors in gonadectomized, estrogen and progesterone-treated female rats. Experiment 1 demonstrates that zaprinast alters paced mating behavior by lengthening the contact-return latency to ejaculation; there is a significant relationship between dose of zaprinast (range 1.5 – 6 mg/kg) and contact-return latency to ejaculation. Experiment 2 illustrates that zaprinast has no effect on preference for an intact male as measured in a No Contact partner preference test. Rats receiving zaprinast tend to exhibit reduced locomotor activity in both experiments. Collectively, these findings demonstrate that modulation of the NO-cGMP pathway using a PDE-5 inhibitor alters the display of paced mating behaviors in rats.

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.

When secreted from malignant cells, hyaluronan facilitates tumor invasion and metastasis, as inhibition of its export by zaprinast inhibited metastasis formation in mice. However, the precise steps of the metastatic cascade, which were influenced by zaprinast, have not been identified as yet. Here we analyzed the cell biological effects of the inhibitor on three human melanoma cell lines that differed in their hyaluronan production and their metastatic capability when xenografted into SCID mice. We measured the influence of zaprinast on cellular hyaluronan export, surface coat formation, proliferation, random migration, colony formation in soft agar, adhesion, and transepithelial resistance. Concentrations of zaprinast not affecting cell proliferation, adhesion and transepithelial resistance, nevertheless reduced hyaluronan export by 50%, surface coat formation, random migration, and colony formation in soft agar. These results indicate that hyaluronan enhances metastasis formation primarily in those steps of the metastatic cascade, which involves tumor cell migration.

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.

Our previous studies have shown that there is a lower cGMP concentration in the aged brain as well as an alteration in the activity of cGMP-hydrolyzing phosphodiesterases (PDEs) and nitric oxide synthase (NOS). The aim of this study was to investigate the effect of specific inhibitors of selected PDEs on object recognition memory and locomotor activity during aging, and to correlate their action with NOS activity in the following brain regions: hippocampus, striatum, and cerebral cortex. The study was carried out using 3, 12, and 24 month-old rats. Inhibitors of PDE2 and PDE5 (Bayer 60-7550 and zaprinast, respectively) were used. Evaluation of memory and locomotor activity was carried out using an object recognition task and the open field test. NOS activity was determined using a radiochemical method after behavioral analysis in the cytosolic fraction from all brain areas investigated. We have found that the inhibitor of PDE2, Bay60-7550, improves object recognition memory in all age groups investigated and increases basal constitutive NOS activity in the hippocampus and striatum. Moreover, in 3 month-old rats, additional inhibition of PDE5 by zaprinast improves object memory and elevates NOS activity in all brain regions studied. Specific inhibition of nNOS eliminates the effect of Bay60-7550 on memory function and on NOS activity in 24 month-old rats. In summary, our results indicate that inhibition of PDE2 is able to improve cognition and memory function in 3, 12, and 24 month-old rats through the enhancement of nNOS activity in the brain, whereas inhibition of PDE5 is effective only in 3 month-old animals.

In asthma and chronic obstructive pulmonary disease (COPD), the number of eosinophils and neutrophils in the lung is increased. One described mechanism leading to the impaired clearance of these cells from the lung is the delay in their programmed cell death (apoptosis). Selective inhibitors of phosphodiesterases (PDEs) are under development for the treatment of lung diseases because of their anti-inflammatory and bronchodilator activity. The aim of the present study was to establish whether inhibitors of PDE3, PDE4 and PDE5 modulate human eosinophil or neutrophil apoptosis or beta 2-adrenoceptor agonist- or cytokine-afforded survival. We also evaluated whether a PDE4 inhibitor could modulate the effect of a corticosteroid on eosinophil and neutrophil apoptosis. Apoptosis was measured by using the relative DNA fragmentation assay and Annexin-V binding. Inhibitors of PDE4 (rolipram; 0.1-10 microM) and PDE3 (cilostazol; 0.1-10 microM) delayed spontaneous eosinophil apoptosis maximally by 25% and 15%, respectively. A combination of a PDE4 or PDE3 inhibitor (10 microM) with salbutamol (100 nM) further delayed eosinophil apoptosis maximally by 42-49%. In neutrophils, rolipram (10 microM) also decreased apoptosis with a maximal inhibition of 13%. The combination of rolipram (10 microM) and salbutamol (100 nM) produced a 27% inhibition of neutrophil apoptosis. Inhibitor of cGMP-specific PDE5 (zaprinast; 0.1-10 microM) did not affect eosinophil apoptosis and only slightly increased spontaneous neutrophil apoptosis. The effect of budesonide on apoptosis was not significantly modulated by a PDE4 inhibitor in eosinophils or neutrophils. The present results show that selective inhibitors of cAMP-hydrolyzing PDEs (PDE3 and PDE4) delay eosinophil apoptosis and, thus, increase their survival in vitro. Furthermore, beta 2-adrenoceptor agonists enhance the anti-apoptotic effects of PDE3 and PDE4 inhibitors, suggesting that such drug combinations may prolong eosinophil and neutrophil longevity in the lung.

The aim of this study was to assess the role of K(+) and Ca(2+) fluxes in the cerebroarterial vasoactive effects of the phosphodiesterase-5 inhibitor sildenafil. We used isolated rabbit basilar arteries to assess the effects of extracellular K(+) raising on sildenafil-induced vasodilatation, and studied the pharmacological interaction of sildenafil with selective modulators of membrane K(+) and Ca(2+) channels. Expression of Kv1 subunits of K(+) channels was assessed at messenger and protein levels. Parallel experiments were carried out with zaprinast for comparison. Sildenafil (10 nM-0.1 mM) induced concentration-dependent relaxation of endothelin-1 (10 nM)-precontracted arteries, which was partially inhibited by depolarization with KCl (50 mM), 3 mM tetraethylammonium (non-selective K(+) channel blocker) or 1 mM aminopyridine (inhibitor of K(v) channels), but not by 1 microM glibenclamide (inhibitor of K(ATP) channels) or 50 nM iberiotoxin (inhibitor of K(Ca) channels). Arterial smooth muscle expressed messengers for Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6, and proteins of Kv1.1, Kv1.2 and Kv1.4. CaCl(2) (10 microM- 10 mM) induced concentration-dependent contraction in Ca(2+)-free, depolarizing (50 mM KCl) medium. Sildenafil (0.1-100 microM) produced reversible concentration-dependent inhibition of the response to CaCl(2), which was completely abolished by the highest sildenafil concentration. By contrast, only 100 microM zaprinast inhibited the response to CaCl(2). The L-type Ca(2+) channel activator Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent potentiation of the response to CaCl(2) inhibited by 100 microM sildenafil. Moreover, Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent contraction in slightly depolarizing (15 mM) medium, which was inhibited to the same extent and in a concentration-dependent way by sildenafil (0.1-100 microM) and zaprinast (1 or 100 microM). These results show that sildenafil relaxes the rabbit basilar artery by increasing K(+) efflux through K(v) channels, which in turn may affect Ca(2+) signalling. Expression of Kv1 subunits involved in this pharmacological effect occurs at the messenger and, in some cases, at the protein level. In addition to this phosphodiesterase-5-related effect, sildenafil and zaprinast inhibit cerebroarterial vasoconstriction at least in part by directly blocking L-type Ca(2+) channels, although a decrease in the sensitivity of the contractile apparatus to Ca(2+) can not be discarded.

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.

GPR35 is a Gi/o- and G16-coupled receptor abundantly expressed in gastrointestinal tissues and immune cells. Kynurenic acid (a tryptophan metabolite and ionotropic glutamate receptor antagonist) and zaprinast (a phosphodies- terase inhibitor) are GPR35 agonists. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) is also a GPR35 agonist. NPPB activates the GPR35-Gi/o and GPR35-G16 pathways in human embryonic kidney 293 (HEK293) cells and induces intracellular calcium mobilization in a concentration-dependent manner in HEK293 cells coexpressing human, rat or mouse GPR35 and the chimeric G protein Gqi5. These results suggest a novel pharmacological activity of NPPB and will provide useful information to search for more potent and selective GPR35 agonists.

Previous behavioural studies which have administered phosphodiesterase type-5 (PDE5) inhibitors have consistently demonstrated improved retention. However, when young chicks were trained on a strongly reinforced passive avoidance task 100 μM zaprinast caused two periods of transient retention loss. This is opposed to past findings and may suggest an effect on retrieval. It is hypothesised that the level of reinforcement is central to this phenomenon. The molecular corollary of this may be the need to maintain cGMP homeostasis such that strong reinforcement + zaprinast may impair retention through the production of excessive levels of cGMP. This was demonstrated by two challenge studies whereby increasing concentrations of 8-Br-cGMP were administered in the presence of the guanylate cyclase inhibitor ODQ (100 μM; ic) resulting in an inverted “U-shaped” retention curve. These findings suggest a more complex role for PDE5 and cGMP in memory processing than previously described and question the role of PDE5 inhibitors as nootropes under all circumstances.

GPR35 is a G protein-coupled receptor recently "de-orphanized" using high-throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein alpha-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (Ca(V)2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonists, indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of Gbetagamma-mediated modulation. Both agonist-independent and -dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of G(i/o) proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC(50) = 1 microM) than kynurenic acid (58 microM) but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release.

The free radical peroxynitrite (ONOO-) is formed in biological systems from the reaction of nitric oxide (NO) with superoxide (O2-) and can react with protein and nonprotein thiol groups to produce tissue injury. However, these pathologic actions of (ONOO-) may have been overemphasized, in that (ONOO-) has vasorelaxant properties through activation of soluble guanylate cyclase; inhibits leukocyte-endothelial cell interactions; and reduces ischemia-reperfusion injury in the heart, lung, and liver. It has been reported that tolerance develops to the vasodilator actions of (ONOO-) and that (ONOO-) impairs vascular function. However, little, if anything, is known about responses to (ONOO-) in the hindlimb circulation of the cat. To better understand the effects of (ONOO-) on responses to vasoactive agonists and the mechanism by which (ONOO-) induces vasodilation, the effects of short-term exposure to (ONOO-) were investigated under constant-flow conditions in the hindlimb vascular bed of the cat. In these studies, direct intraarterial injections of (ONOO-) produced dose-dependent decreases in hindquarters perfusion pressure. The vasodilator responses to (ONOO-) were rapid in onset, were short in duration, and could be repeated without exhibiting tachyphylaxis. Vasodilator responses to (ONOO-) were not changed in the presence of inhibitors of nitric-oxide synthase, cyclooxygenase, or K+-ATP (adenosine triphosphate-sensitive potassium) channels. Furthermore, responses to (ONOO-) were enhanced in duration by the type 5-cGMP (cyclic guanosine monophosphate) phosphodiesterase inhibitor zaprinast, whereas rolipram, a type 4-cGMP phosphodiesterase inhibitor, was without effect. Repeated administration of (ONOO-) had no significant effect on responses to vasoconstrictor or to vasodilator agents including acetylcholine. These results show that (ONOO-) has significant vasodilator activity in the hindlimb vascular bed of the cat and suggest that the response is mediated by a cGMP- dependent mechanism. The results of experiments with repeated injections of (ONOO-) indicate that (ONOO-) does not impair vasoconstrictor and endothelium-dependent or endothelium-independent vasodilator responses. Furthermore, tolerance did not develop with repeated short-term exposure to (ONOO-). Moreover, the results of experiments with inhibitors suggest that responses to (ONOO-) are not dependent on K-ATP (adenosine triphosphate-sensitive potassium) channel activation, increased NOS activity, or the formation of products in the cyclooxygenase pathway. The results of these studies are consistent with the hypothesis that (ONOO-) is rapidly converted in the hindlimb circulation to a substance that has the properties of an NO donor. These studies suggest that under physiologic conditions, the cytotoxic effects of (ONOO-) on a short-term basis may have been overemphasized.

The release of the inhibitory neurotransmitter GABA is generally enhanced under potentially cell-damaging conditions. The properties and regulation of preloaded [3H]GABA release from mouse hippocampal slices were now studied in free radical-containing medium in a superfusion system. Free radical production was induced by 0.01% of H2O2 in the medium. H2O2 markedly potentiated GABA release, which was further enhanced about 1.5-fold by K+ stimulation (50 mM). In Ca2+-free media this stimulation was not altered, indicating that the release was mostly Ca2+-independent. Moreover, omission of Na+ increased the release, suggesting that it is mediated by Na+-dependent transporters operating outwards, a conception confirmed by the enhancement with GABA homoexchange. Inhibition of the release with the ion channel inhibitors diisothiocyanostilbene-2,2'-disulphonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate indicates that Cl(-) channels also participate in the process. This release was not modified by the adenosine receptor (A1 and A2a) agonists and ionotropic glutamate receptor agonists kainate, N-methy-D: -aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, whereas the agonists of metabotropic glutamate receptors of group I [(S)-3,5-dihydroxyphenylglycine] and of group II [(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate] enhanced it by receptor-mediated mechanisms, the effects being abolished by their respective antagonists. The group III agonist L+-2-amino-4-phosphonobutyrate reduced the evoked GABA release, but this was not affected by the antagonist. Furthermore, the release was reduced by activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate and by inhibition of tyrosine kinase by genistein and of phoshoplipase by quinacrine. On the other hand, increasing cGMP levels with the phosphodiesterase inhibitor zaprinast, selective for PDE5, 6 and 9, and NO production with the NO-generating compounds hydroxylamine, sodium nitroprusside and S-nitroso-N-penicillamine enhanced the release. The regulation of GABA release induced by free radical production proved thus to be rather complex. Under potentially cell-damaging conditions, the potentiation of GABA release may be a mechanism to counteract hyperactivity and reduce the effects of excitatory amino acid release. On the other hand, reduction of GABA release could be harmful and contribute to excitotoxic damage and neuronal degeneration.

Hyaluronan must be exported from its site of synthesis, the inner side of plasma membrane, to the extracellular matrix. Here, we identified the multidrug-associated protein MRP5 as the principle hyaluronan exporter from fibroblasts. The expression of the MRP5 (ABC-C5) transporter was silenced in fibroblasts using RNA interference, and a dose-dependent inhibition of hyaluronan export was observed. Hyaluronan oligosaccharides introduced into the cytosol competed with the export of endogenously labeled hyaluronan and the MRP5 substrate fluorescein. Because cGMP is a physiological substrate of MRP5, the intracellular concentrations of cGMP were modulated by the drugs 3-isobutyl-1-methylxanthin, propentofyllin, L-NAME, zaprinast, and bromo-cGMP, and the effects on hyaluronan export were analyzed. Increasing the cGMP levels inhibited hyaluronan export and decreasing it afforded higher concentrations of zaprinast to inhibit the export. Thus, cGMP may be a physiological regulator of hyaluronan export at the level of the export MRP5.

Inhalation of nitric oxide (NO) and inhibition of phosphodiesterase type 5 (PDE5) selectively dilate the pulmonary circulation in patients with acute lung injury (ALI) associated with pulmonary hypertension. PDE5 inhibitors administered at doses that decrease pulmonary artery pressures have been shown to worsen arterial oxygenation. We investigated the efficacy of doses of PDE5 inhibitors that do not reduce pulmonary artery pressure alone (subthreshold doses) to improve the response to inhaled NO in an animal model of ALI.Adult Sprague-Dawley rats were pre-treated with 0.5 mg/kg Escherichia coli 0111:B4 endotoxin and 16 to 18 h later, their lungs were isolated perfused and ventilated. The thromboxane mimetic U46619 was used to induce pulmonary hypertension. After the determination of subthreshold doses of two different PDE5 inhibitors, either 50 microg zaprinast or 10 ng sildenafil was added to the perfusate and the decrease of pulmonary artery pressure measured in the presence and absence of inhaled NO.In the presence of 4 or 10 ppm NO, zaprinast (-1.6 +/- 0.4 and -2.9 +/- 0.6 mmHg, respectively) and sildenafil (-1.9 +/- 0.4 and -2.4 + 0.3 mmHg, respectively) improved responsiveness to inhaled NO compared to lungs from rats treated with LPS only (0.7 +/- 0.1 and -1.0 +/- 0.1 mmHg, respectively; P0.05). Neither zaprinast nor sildenafil prolonged the pulmonary vasodilatory response to inhaled NO.Subthreshold doses of PDE5 inhibitors improved responsiveness to inhaled NO. Combining inhaled NO with subthreshold doses of PDE5 inhibitors may offer a therapeutic strategy with minimal side-effects in ALI associated with pulmonary hypertension.

In kidneys, stimulation of adenylyl cyclase causes egress of cAMP, conversion of cAMP to AMP by ecto-phosphodiesterase, and metabolism of AMP to adenosine by ecto-5'-nucleotidase. Although much is known about ecto-5'-nucleotidase, the renal ecto-phosphodiesterase remains uncharacterized. We administered cAMP (10 microM in the perfusate) to 12 different groups of perfused kidneys. AMP was measured in perfusate using ion trap mass spectrometry. In control kidneys (n=19), basal renal secretion rate of AMP was 0.49+/-0.08 and increased to 3.0+/-0.2 nmol AMP/g kidney weight/min during administration of cAMP. A broad-spectrum phosphodiesterase (PDE) inhibitor (1,3-isobutyl-1-methylxanthine, 300 microM, n=6) and an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine, 1 mM, n=6) significantly attenuated cAMP-induced AMP secretion by 60 and 74%, respectively. Blockade of PDE1 (8-methoxymethyl-3-isobutyl-1-methylxanthine, 100 microM), PDE2 [erythro-9-(2-hydroxy-3-nonyl)adenine, 30 microM], PDE3 (milrinone, 10 microM; cGMP, 10 microM), PDE4 (Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one], 100 microM), PDE5 and PDE6 (zaprinast, 30 microM), and PDE7 [BRL-50481 (5-nitro-2,N,N-trimethylbenzenesulfonamide), 10 microM] did not alter renal ecto-phosphodiesterase activity. Administration of a concentration (100 microM) of dipyridamole that blocks PDE8 inhibited ecto-phosphodiesterase activity (by 44%). However, a lower concentration of dipyridamole (3 microM) that blocks PDE9, PDE10, and PDE11, but not PDE8, did not inhibit ecto-phosphodiesterase activity. These data support the conclusion that renal ecto-phosphodiesterase activity is not mediated by PDE1, PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE9, PDE10, or PDE11 and is inhibited by high concentrations of dipyridamole. Ecto-phosphodiesterase has some pharmacological characteristics similar to PDE8.

We tested the hypothesis that the negative functional effects of natriuretic peptides would be blunted in thyroxine (T4)-induced hypertrophic cardiac myocytes. We also studied the causes of these changes. Ventricular myocytes were obtained from control (n=8) and T4 (0.5 mg/kg/16 days) treated rabbit hearts (n=7). Cell shortening parameters were studied with a video edge detector. We also determined particulate (pGC) and soluble (sGC) guanylyl cyclase activity and cyclic GMP levels. Myocyte function was examined at baseline and after brain natriuretic peptide (BNP 10(-7,-6) M) or C-type natriuretic peptide (CNP 10(-7,-6) M) or zaprinast (cyclic GMP phosphodiesterase inhibitor 10(-6)M) followed by BNP or CNP. Baseline function was similar in control and T4 myocytes. BNP (5.7 +/- 0.2 to 4.3 +/- 0.1%) and CNP (5.7 +/- 0.4 to 4.2 +/- 0.2%) significantly reduced percent shortening in control myocytes. These reductions were not observed with T4 (BNP, 5.7 +/- 0.6 to 5.6 +/- 0.6; CNP, 5.6 +/- 0.4 to 5.5 +/- 0.5). BNP and CNP responded similarly after zaprinast. Baseline cyclic GMP was similar in control and T4, but BNP only increased cyclic GMP in controls. The activity of pGC was similar at baseline in control and T4, but the stimulated activity was significantly lower in T4 myocytes. Both basal and stimulated sGC activity were similar in control and hypertrophic myocytes. These results demonstrated that the ability of natriuretic peptides to reduce ventricular myocyte function was blunted in T4 hypertrophic myocytes. This blunted response was related to the reduced ability of natriuretic peptides to increase cyclic GMP levels due to a reduced stimulated particulate guanylyl cyclase activity.

Background and purpose: Nitrergic neurons are important for erectile responses in the corpus cavernosum and impaired signalling results in erectile dysfunction, today treated successfully by oral administration of the selective phosphodiesterase 5 (PDE 5) inhibitors sildenafil, tadalafil and vardenafil. Although the importance of nitrergic neurons in urogenital function has become evident, it has not been investigated if the PDE 5 inhibitors affect the nerve-induced release of nitric oxide (NO). In a previous study we found that the soluble guanylate cyclase (sGC)/cyclic guanosine 3’,5’–monophosphate (cGMP) pathway might modulate nerve-induced release of NO in isolated cavernous tissue. Experimental approach: Electrical field stimulation (EFS 5 Hz, 40 V, 0.3 ms pulse duration, 25 pulses at intervals of 2 min) of rabbit isolated cavernous tissue elicited reproducible, nerve-mediated relaxations in the presence of scopolamine (10 � 5 M), guanethidine (10 � 5 M) and phenylephrine (3 � 10 � 6 M). In superfusion experiments, nerve stimulation (20 Hz, 40 V, 1 ms) of the cavernous tissue evoked release of NO/NO2 , measured by chemiluminescence. Key results: Sildenafil, tadalafil and vardenafil decreased the muscular tone and prolonged the relaxations to nerve stimulation. The evoked release of NO decreased to 72711%, 55716% and 61714% of control, respectively after addition of

Introduction Phosphodiesterase-4 (PDE4) enzymes specifically break down the second messenger cAMP, thereby terminating the intracellular signaling cascade that plays an essential role in neurohormonal modulation of many physiological systems. PDE4 activity and expression are regulated by cAMP levels, suggesting that measurement of PDE4 provides an index of intracellular cAMP signaling. Methods Male Sprague–Dawley rats were administered ( R )- or the less active enantiomer ( S )-[ 11 C]rolipram and sacrificed 30 min later with tracer retention measured in various tissues. Co-injections with saturating doses of unlabeled ( R )-rolipram, ( S )-rolipram and Ro 20-1724, as well as subtype-selective PDE inhibitors vinpocetine, Bay 60-7550, cilostazol and zaprinast were used to establish binding selectivity for PDE4 over PDE1, PDE2, PDE3 and PDE5 subtypes, respectively. Autoradiography was performed to substantiate results of biodistribution studies in the myocardium. Results In vivo ( R )-[ 11 C]rolipram retention was dose-dependently reduced by co-injections of ( R )-rolipram and ( S )-rolipram (ED 50 values of 0.03 mg/kg and 0.2 mg/kg, respectively). Vinpocetine, Bay 60-7550, cilostazol and zaprinast had no effect on ( R )-[ 11 C]rolipram binding, while ( R )-rolipram and Ro 20-1724 reduced the tracer uptake to nonspecific levels in PDE4-rich tissues. Conclusions In addition to the brain, ( R )-[ 11 C]rolipram binds selectively to PDE4 across all cardiac regions, skeletal muscle, lungs and pancreas, but not in the adipose tissues. In vivo findings were confirmed by in vitro autoradiography studies, suggesting that ( R )-[ 11 C]rolipram can be applied to evaluate alterations in central and peripheral PDE4 levels and cAMP-mediated signaling.

© 2014 Elsevier Inc. Nitric oxide (NO), a gaseous messenger molecule synthesized by nitric oxide synthase (NOS), plays a pivotal role in integrating dopamine transmission in the basal ganglia and has been implicated in the pathogenesis of Parkinson disease (PD). To study the role of the nitrergic system in l-DOPA-induced dyskinesia (LID), we assessed the effect of the pharmacological manipulation of NO levels and NO/cyclic guanosine monophosphate (cGMP) signaling on LID in the Pitx3-/- aphakia mouse, a genetic model of PD. To evaluate the effect of decreased NO signaling on the development of LID, Pitx3-/- mice were chronically treated with l-DOPA and 7-nitroindazole (7-NI, a neuronal NOS inhibitor). To evaluate its effect on the expression of established LID, 7-NI was administered acutely to dyskinetic mice. The chronic 7-NI treatment attenuated the development of LID in the Pitx3-/- mice, and the sub-acute 7-NI treatment attenuated established dyskinesia without affecting the beneficial therapeutic effect of l-DOPA. Moreover, 7-NI significantly reduced FosB and pAcH3 expression in the acutely and chronically l-DOPA-treated mice. We also examined how increasing NO/cGMP signaling affects LID expression by acutely administering molsidomine (an NO donor) or zaprinast (a cGMP phosphodiesterase 5-PDE5 inhibitor) before l-DOPA in mice with established dyskinesia. Paradoxically, the administration of either of these drugs also significantly diminished the expression of established LID; however, the effect occurred at the expense of the antiparkinsonian l-DOPA properties. We demonstrate that targeting the NO/cGMP signaling pathway reduces dyskinetic behaviors and molecular markers, but only the 7-NI treatment preserved the antiparkinsonian effect of l-DOPA, indicating that NOS inhibitors represent a potential therapy to reduce LID.

Inhibition of phosphodiesterase 5 (PDE) improved recognition memory and counteracted spatial learning impairment induced by nitric oxide synthase (NOS) inhibition in recent studies. Aim of this study was to investigate effects of rolipram, a PDE4 inhibitor and zaprinast, a PDE5 inhibitor, on learning and memory in Morris water maze (MWM) and radial arm maze (RAM) tests in naive mice. Male Balb-c mice were treated subchronically with zaprinast (3 and 10 mg/kg) and rolipram (0.05 and 0.1 mg/kg) for 6 days in the MWM test and acutely before the retention trial of radial arm maze test. Rolipram (0.05 and 0.1 mg/kg) significantly decreased escape latency between 2(nd) and 5(th) sessions, while zaprinast (10 mg/kg) significantly decreased escape latency only in 2(nd) session. Rolipram (0.05 and 0.1 mg/kg) and zaprinast (10 mg/kg) significantly increased time spent in escape platform's quadrant in probe trial of MWM test; only rolipram decreased mean distance to platform, while zaprinast had no effect on mean distance to platform. Zaprinast (3 and 10 mg/kg) significantly decreased number of errors compared to control group, while rolipram (0.05 and 0.1mg/kg) had no effect on number of errors in retention trial of RAM test. Rolipram (0.05 and 0.1 mg/kg) and zaprinast (10 mg/kg) significantly decreased time spent to complete retention trial (latency) compared to control group. Our study revealed that both zaprinast and rolipram enhanced spatial memory in MWM, while zaprinast seems to have more memory enhancing effects compared to rolipram in radial arm maze test.

Adenosine is an important inhibitory neuromodulator that regulates neuronal excitability. Several studies have shown that nitric oxide induces release of adenosine. Here we investigated the mechanism of this release. We studied the effects of nitric oxide on evoked field excitatory postsynaptic potentials (fEPSPs) recorded in the CA1 area of rat hippocampal slices. The nitric oxide donor 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO; 100 microm) depressed the fEPSP by 77.6 +/- 4.1%. This effect was abolished by the adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 400 nm), indicating that the nitric oxide effect was mediated by adenosine accumulation. The DEA/NO effect was unaltered by the 5'-ectonucleotidase inhibitor alpha,beta-methylene-adenosine 5'-diphosphate (AMP-CP; 100 microm), indicating that extracellular adenosine did not derive from ATP or cAMP release. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ; 5 microm) did not affect nitric oxide depression of the fEPSPs, indicating that nitric oxide-mediated adenosine release was not mediated through a cGMP signaling cascade. This conclusion was confirmed by the observation that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP; 1 mm) reversibly depressed the fEPSP by 24.9 +/- 4.5%, but this effect was not blocked by adenosine antagonists. Adenosine kinase inhibitor 5-iodotubercidin (ITU; 7 microm) occluded the nitric oxide effects by 74%, suggesting that inhibition of adenosine kinase activity contributes to adenosine release. In conclusion, exogenous nitric oxide evokes adenosine release by a cGMP-independent pathway. Intracellular cGMP elevation partially inhibits the fEPSP but not through adenosine release. Although a direct block of adenosine kinase by nitric oxide can not be excluded, the depression of adenosine kinase activity may be due to inhibition by its own substrate adenosine.

GABA is the inhibitory neurotransmitter in most brain stem nuclei. The properties of release of preloaded [(3)H]GABA were now investigated with slices from the mouse brain stem under normal and ischemic (oxygen and glucose deprivation) conditions, using a superfusion system. The ischemic GABA release increased about fourfold in comparison with normal conditions. The tyrosine kinase inhibitor genistein had no effect on GABA release, while the phospholipase inhibitor quinacrine reduced both the basal and K(+)-evoked release in normoxia and ischemia. The activator of protein kinase C (PKC) 4beta-phorbol 12-myristate 13-acetate had no effects on the releases, whereas the PKC inhibitor chelerythrine reduced the basal release in ischemia. When the cyclic guanosine monophosphate (cGMP) levels were increased by superfusion with zaprinast and other phosphodiesterase inhibitors, GABA release was reduced under normal conditions. The NO donors S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine (HA) enhanced the basal and K(+)-stimulated release by acting directly on presynaptic terminals. Under ischemic conditions GABA release was enhanced when cGMP levels were increased by zaprinast. This effect was confirmed by inhibition of the release by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The NO-producing agents SNAP, HA, and sodium nitroprusside potentiated GABA release in ischemia. These effects were reduced by the NO synthase inhibitor N(G)-nitro-L: -arginine, but not by ODQ. The results show that particularly NO and cGMP regulate both normal and ischemic GABA release in the brain stem. Their effects are however complex.

In the study of anti-proinflammation by 7-[2-[4-(2-chlorobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3), exposure of rat tracheal smooth muscle cells (TSMCs) to tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase alpha1 (sGCalpha1), soluble guanylate cyclase beta1 (sGCbeta1), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-Br-cGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-alpha-induced increases of iNOS expression and NO levels and reversed TNF-alpha-induced decreases of sGCalpha1, sGCbeta1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-alpha also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E2 and 6-keto-PGF1alpha (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-alpha-induced expression of COX-2 and activated productions PGE2 and PGI2. However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-alpha. It is suggested that TNF-alpha-induced increases of PKA expression and cAMP levels are mediated by releasing PGE2 and PGI2, the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-alpha-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.