YC-1, a novel potential anticancer agent,inhibit multidrug-resistant protein via cGMP-dependent pathway

The aim of the present study was to evaluate the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) on multidrug resistance. Expression of human P-glycoprotein was assessed by realtime quantitative RT-PCR and western blot. The efflux function of P-glycoprotein was evaluated by rhodamine 123 accumulation and calcein-AM uptake models. The mechanisms of action of YC-1 on different signaling pathways were studied using series of antagonists and the kinetics was also assessed. Cytotoxicity was evaluated by MTT assay. The results demonstrated that increased intracellular accumulation of rhodamine 123 and increased fluorescence of calcein were observed after YC-1 treatment. Furthermore, increased YC-1 concentration resulted in significant decrease in Vmax while K_M remained unchanged suggested that YC-1 acted as a noncompetitive inhibitor of P-glycoprotein. Moreover, the inhibition of Pgp efflux function by YC-1 was significantly reversed by NO synthase inhibitor, _L-NAME, the sGC inhibitor, ODQ, the PKG inhibitor, Rp-8-Br-PET-cGMPS, and the PKG inhibitor KT5823. In addition, ERK kinase inhibitor PD98059 also significantly restored YC-1 inhibited Pgp efflux function. These results indicated that YC-1 inhibited Pgp efflux via the NO-cGMP-PKG-ERK signaling pathway through noncompetitive inhibition. The present study revealed that YC-1 could be a good candidate for development as a MDR modulator.     

YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cells

YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-alpha, -iota, -lambda, -zeta and -mu isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-alpha, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-alpha. The MEK inhibitor, PD 98059 (10 - 50 microM), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-alpha activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression.     

Nitric oxide-soluble guanylyl cyclase signaling regulates corticostriatal transmission and short-term synaptic plasticity of striatal projection neurons recorded in vivo

Striatal medium-sized spiny neurons (MSNs) contain the highest levels of soluble guanylyl cyclase (sGC) in the brain. Striatal sGC signaling is activated by nitric oxide (NO) and other neuromodulators. MSNs also express cGMP-dependent protein kinase and other components of the cGMP signaling system which are critically involved in integrating corticostriatal transmission and regulating synaptic plasticity in striatal networks. However, the influence of tonic and phasic activation of this signaling pathway on striatal MSN activity is poorly understood. The present study examined the impact of systemic administration of the selective sGC inhibitor [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) on spike activity evoked using low and high frequency electrical stimulation of the frontal cortex. MSN activity was monitored using single-unit extracellular recordings in urethane-anesthetized rats. ODQ administration significantly decreased spike activity evoked by low frequency cortical stimulation in a stimulus intensity- and time-dependent manner. Additionally, ODQ administered along with the neuronal NO synthase inhibitor 7-nitroindazole (7-NI) potently decreased the incidence of excitatory responses observed during high-frequency train stimulation of the contralateral frontal cortex. The short-term depression of cortically-evoked spike activity induced by train stimulation was enhanced following pretreatment with ODQ in MSNs exhibiting an excitatory response during cortical train stimulation. Unexpectedly, this effect of ODQ was reversed in animals receiving co-administration of ODQ and 7-NI. 7-NI/ODQ co-administration also reversed measures of short-term depression observed in MSNs exhibiting an inhibitory response during cortical train stimulation. These observations extend previous studies showing that tonic and phasic NO-sGC signaling modulates the responsiveness of MSNs to corticostriatal input. Moreover, phasic activation of NO signaling is likely to regulate short-term changes in corticostriatal synaptic plasticity via complex mechanisms involving both sGC-cGMP-dependent and independent pathways.     

Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague-Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or Nomega-propyl-L-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4'-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or L-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.     

Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells

The nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), is commonly manipulated pharmacologically in two ways. Inhibition of activity is achieved using 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) which oxidizes the haem prosthetic group to which NO binds, while the compound 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1) is considered an 'allosteric' activator. Knowledge of how these agents function and interact in a normal cellular environment is limited. These issues were addressed using rat cerebellar cells. Inhibition by ODQ was not simply competitive with NO. The rate of onset was ODQ concentration-dependent and developed in two kinetic phases. Recovery from inhibition occurred with a half-time of approximately 5 min. YC-1 slowed the rate at which sGC deactivated on removal of NO by 45 fold, consistent with YC-1 increasing the potency of NO for sGC. YC-1 also enhanced the maximal response to NO by 2 fold. Furthermore, when added to cells in which sGC was 90% desensitized, YC-1 abruptly enhanced sGC activity to a degree that indicated partial reversal of desensitization. After pre-exposure to YC-1, sGC became resistant to inhibition by ODQ. In addition, YC-1 rapidly reversed inhibition by ODQ in cells and for purified sGC, suggesting that YC-1 either increases the NO affinity of the oxidized sGC haem or reverses haem oxidation. It is concluded that the actions of ODQ and YC-1 on sGC are broadly similar in cells and purified preparations. Additionally, YC-1 transiently reverses sGC desensitization in cells. It is hypothesized that YC-1 has multiple actions on sGC, and thereby both modifies the NO binding site and enhances agonist efficacy.     

Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole

AMP-activated protein kinase (AMPK) activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major antiaggregatory mechanism residing in platelets. Based on the hypothesis that direct activation of AMPK might be a potential strategy to inhibit platelet aggregation, the antiplatelet effect of AMPK activators was investigated. Treatment of isolated platelets with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in AMPK activation and a decrease in aggregation, which was abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A. Such an AMPK-dependent antiaggregatory effect was also observed with other AMPK activators such as A-769662 and PT1. AICAR induced eNOS activation was followed by NO synthesis, cGMP production, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a PKG substrate. All these events were blocked by CC or ara-A pretreatment, and each event was inhibited by the eNOS inhibitor L-NAME, the sGC inhibitor ODQ, and the PKG inhibitor Rp-8-pCPT-cGMPS. Simultaneous treatment of dipyridamole, a phosphodiesterase (PDE) inhibitor, with AICAR potentiated the antiaggregatory effect by enhancing the cGMP elevation. Administration of AICAR increased platelet cGMP and prolonged FeCl₃-induced arterial occlusion time in rats, which further increased in combination with dipyridamole. In conclusion, AMPK activators inhibited platelet aggregation by stimulating the eNOS-NO/sGC-cGMP/PKG signaling pathway. The antiplatelet effect of AMPK activators could be potentiated in combination with a PDE inhibitor through the common mechanism of elevating cGMP. Thus, AMPK may serve as a potential target for antiplatelet therapy.     

不同给药时间间隔对吗啡诱导的条件性位置偏爱的影响

目的:比较两种不同给药时间间隔的实验方案对大鼠吗啡条件性位置偏爱(CPP)的影响。方法:采用三箱式非平衡设计的CPP装置,在实验方案1中,吗啡组大鼠注射吗啡(10 mg.kg-1.d-1,ip)后放入伴药箱50 min,间隔6 h后注射同等剂量的生理盐水后放入非伴药箱50 min,共12 d,检测期检测13 d。实验方案2中,奇数天给吗啡组大鼠注射吗啡(10 mg.kg-1,ip),偶数天注射同等剂量的生理盐水,共8 d,在检测期不定期地把大鼠放入到CPP箱中进行检测。结果:两种实验方案的吗啡组大鼠在伴药箱停留的时间均明显多于生理盐水对照组(P<0.01,P<0.01);自然戒断后,实验方案1和实验方案2中吗啡组大鼠的CPP维持分别为5 d和42 d。结论:两种实验方案均能有效建立大鼠吗啡CPP模型,而实验方案2更经济、省时,可提高实验效率。     

The inhibition of the nitric oxide-cGMP-PKG-JNK signaling pathway avoids the development of tolerance to the local antiallodynic effects produced by morphine during neuropathic pain

Tolerance to the local antiallodynic effects of morphine, DPDPE ([D-Pen(2),D-Pen(5)]-Enkephalin) or JWH-015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) after their repeated administration during neuropathic pain was evaluated. The role of the nitric oxide-cGMP-protein kinase G (PKG)-c-Jun N-terminal kinase (JNK) signaling pathway on the peripheral morphine-induced tolerance after the chronic constriction of sciatic nerve in mice was also assessed. The mechanical and thermal antiallodynic effects produced by a high dose of morphine, DPDPE or JWH-015 subplantarly administered daily from days 10 to 20 after nerve injury were estimated with the von Frey filaments and cold plate tests. The antiallodynic effects of the repeated administration of morphine combined with a sub-analgesic dose of a selective inducible nitric oxide synthase (NOS2) (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) or JNK (anthra[1,9-cd]pyrazol-6(2H)-one; SP600125) inhibitor from days 10 to 20 after injury were also evaluated. The repeated administration of morphine, but not DPDPE or JWH-015, produced a rapid development of tolerance to its mechanical and thermal antiallodynic effects in sciatic nerve-injured mice. The co-administration of morphine with L-NIL, ODQ, Rp-8-pCPT-cGMPs or SP600125 avoided the development of morphine antiallodynic tolerance after nerve injury. These findings reveal that the repeated local administration of DPDPE or JWH-015 did not induce antinociceptive tolerance after sciatic nerve injury-induced neuropathic pain. Our data also indicate that the peripheral nitric oxide-cGMP-PKG-JNK signaling pathway participates in the development of morphine tolerance after nerve injury and propose the inactivation of this pathway as a promising strategy to avoid morphine tolerance during neuropathic pain.     

Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator-induced relaxation of porcine coronary veins

BACKGROUND AND PURPOSE: Coronary venous activity is modulated by endogenous and exogenous nitrovasodilators. The present study was to determine the role of protein kinase G (PKG) in the regulation of the basal tension and nitrovasodilator-induced relaxation of coronary veins. EXPERIMENTAL APPROACH: Effects of a PKG inhibitor on the basal tension and responses induced by nitroglycerin, DETA NONOate, and 8-Br-cGMP in isolated porcine coronary veins were determined. Cyclic cGMP was measured with radioimmunoassay. PKG activity was determined by measuring the incorporation of 32P from gamma-32P-ATP into the specific substrate BPDEtide. KEY RESULTS: Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, increased the basal tension of porcine coronary veins and decreased PKG activity. The increase in tension was 38% of that caused by nitro-L-arginine. Relaxation of the veins induced by nitroglycerin and DETA NONOate was accompanied with increases in cGMP content and PKG activity. These effects were largely eliminated by inhibiting soluble guanylyl cyclase with ODQ. The increase in PKG activity induced by the nitrovasodilators was abolished by Rp-8-Br-PET-cGMPS. The relaxation caused by these dilators and by 8-Br-cGMP at their EC50 was attenuated by the PKG inhibitor by 51-66%. CONCLUSIONS AND IMPLICATIONS: These results suggest that PKG is critically involved in nitric oxide-mediated regulation of the basal tension in porcine coronary veins and that it plays a primary role in relaxation induced by nitrovasodilators. Since nitric oxide plays a key role in modulating coronary venous activity, augmentation of PKG may be a therapeutic target for improving coronary blood flow.     

The Guanylyl Cyclase Activator YC-1 Directly Inhibits the Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We investigated the effects of YC-1, an activator of soluble guanylyl cyclase (sGC), on voltage-dependent K⁺ (Kv) channels in smooth muscle cells from freshly isolated rabbit coronary arteries by using the whole-cell patch clamp technique. YC-1 inhibited the Kv current in a dose-dependent fashion with an apparent K _d of 9.67 μ M. It accelerated the decay rate of Kv channel inactivation without altering the kinetics of current activation. The rate constants of association and dissociation for YC-1 were 0.36 ± 0.01 μ M ^?1·s^?1 and 3.44 ± 0.22 s^?1, respectively. YC-1 did not have a significant effect on the steady-state activation and inactivation curves. The recovery time constant from inactivation was decreased in the presence of YC-1, and application of train pulses (1 or 2 Hz) caused a progressive increase in the YC-1 blockade, indicating that YC-1–induced inhibition of Kv currents is use-dependent. Pretreatment with Bay 41-2272 (also a sGC activator), ODQ (a sGC inhibitor), or Rp-8-Br-PET-cGMPs (a protein kinase G inhibitor) did not affect the basal Kv current and also did not significantly alter the inhibitory effect of YC-1. From these results, we suggest that YC-1 directly inhibits the Kv current independently of sGC activation and in a state-, time-, and use-dependent fashion.     

Mechanisms of the relaxant effect of vardenafil in rat penile arteries

The aim of the present study was to investigate the mechanisms underlying the vasorelaxation induced by the selective phosphodiesterase 5 (PDE5) inhibitor vardenafil in rat penile small arteries. Segments of the rat dorsal penile artery were mounted in microvascular myographs for isometric tension recording. Concentration-response curves for vardenafil (1 nM-3 microM) and other PDE inhibitors (sildenafil, rolipram and milrinone) were constructed by adding cummulative concentrations of the drugs to arteries precontracted with phenylephrine. The effect of mechanical endothelial cell removal and of selective blockers of the nitric oxide (NO)/cGMP pathway and K+ channels were evaluated on the vardenafil relaxant responses. Vardenafil was the most potent of the four PDE inhibitors tested that maximally relaxed penile arteries, pD2 and maximum relaxation being 6.96+/-0.08 and 97+/-1% (n=48), respectively. Blockade of guanylate cyclase with ODQ (5 microM), mechanical removal of the endothelium or inhibition of NO synthase with l-NOARG (100 microM) markedly reduced vardenafil-induced relaxations, without altering maximum response. Inhibitors of both the cGMP-dependent (PKG) and the cAMP-dependent (PKA) protein kinases, Rp-8-Br-PET-cGMPS (5 microM) and Rp-8-CPT-cAMPS (50 microM), respectively, both reduced vardenafil relaxant responses and the later abolished that of rolipram. Vardenafil-elicited relaxation was reduced by the selective inhibitor of the large-conductance Ca2+-activated K+ channels (BK(Ca)), iberiotoxin (30 nM) and also by the ATP-sensitive K+ channel (K(ATP)) inhibitor, glibenclamide (1 microM). Vardenafil induces a potent vasodilatation in rat penile arteries that is partially dependent on the endothelium and the NO/cGMP pathway and involves activation of both BK(Ca) and K(ATP) channels.     

Prolonged exposure to YC-1 induces apoptosis in adrenomedullary endothelial and chromaffin cells through a cGMP-independent mechanism.

YC-1, a benzyl indazole derivative, is an NO-independent direct activator of soluble guanylyl cyclase (sGC), which presents a synergistic action with NO in stimulating cGMP synthesis. These properties have served to suggest YC-1 as an attractive therapeutic agent by permitting the reduction of nitrovasodilator dosage and regulating endogenous cGMP metabolism. Here we studied the effect of prolonged exposure of adrenomedullary endothelial and chromaffin cells to YC-1. We found that YC-1 increased cGMP in the two types of cells and this action was blocked by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, cytoskeletal disorganization and changes in membrane permeability after prolonged incubation with YC-1. Caspase-3-like protease activity and DNA fragments in the cytoplasm were increased in a dose-dependent manner by 16 h YC-1 treatment. The specific and cell permeable caspase-3-like protease inhibitor DEVD-CHO effectively inhibited YC-1-mediated caspase-3-like activation and DNA fragmentation. Moreover, YC-1 also induced cell shape changes accompanied by actin filament disorganization and alterations in membrane permeability. Cells incubated for 24h with YC-1 showed damaged membranes by binding to nucleic acid of a dye excluded by the intact plasma membrane of live cells. YC-1 also induced a decrease in the intracellular non-specific esterase activity, another indication of cell toxicity. Apoptotic phenomena were not prevented by the presence of ODQ although it effectively inhibited the YC-1-elicited cGMP increases. These findings indicate that YC-1 induces apoptosis by activating caspase-3-like protease through a mechanism independent of sGC activation.     

The vasodilator-stimulated phosphoprotein (VASP): target of YC-1 and nitric oxide effects in human and rat platelets

The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser(239) and Ser(157)) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser(157) phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser(239), the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.     

Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP / protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO(2), and intracellular cGMP. Like L-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the L-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca(2+)-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO(2) level was decreased by dantrolene, but not by the other 3 blockers. L-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.     

YC-1 stimulates the expression of gaseous monoxide-generating enzymes in vascular smooth muscle cells

The benzylindazole derivative 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-alpha]quinozalin-1-one (ODQ) or by the protein kinase G inhibitors (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823) and YGRKKRRQRRRPPLRKKKKKH-amide (DT-2) and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator 5-cyclopropyl-2[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b] pyridine-3-yl] pyrimidin-4-ylamine (BAY 41-2272). However, the YC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor (9S,10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)-benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and was mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides and simultaneously stimulate their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified.     

酸枣仁提取物对小鼠吗啡条件性位置偏爱的影响

目的:观察酸枣仁提取物对吗啡(MOR)诱导建立小鼠条件性位置偏爱(CPP)行为的影响。方法:连续给予吗啡(6mg·kg-1,sc)5d,引起小鼠产生显著的CPP效应,观察酸枣仁提取物对CPP效应的影响。结果:吗啡可诱导小鼠对伴药箱产生显著的位置偏爱;训练阶段于每次给予吗啡前30min给予酸枣仁醇提物800mg·kg-1可拮抗小鼠对吗啡产生的CPP效应(与吗啡对照组比较P<0.05),但酸枣仁醇提物200和400mg·kg-1不影响其效应(P>0.05);酸枣仁乙酸乙酯提取物13mg·kg-1、20mg·kg-1,正丁醇提取物160mg·kg-1可拮抗小鼠对吗啡产生的CPP效应(P<0.05),但酸枣仁石油醚提取物、乙醚提取物、水提物均不影响其效应(P>0.05)。结论:酸枣仁醇提物及其乙酸乙酯提取物、正丁醇提取物能在一定程度上抑制吗啡诱导小鼠建立的CPP行为。     

GABA(A) receptors of hippocampal CA1 regions are involved in the acquisition and expression of morphine-induced place preference.

In the present study, the effects of bilateral intra-hippocampal CA1 (intra-CA1) injections of GABA(A) receptor agonist and/or antagonist on the acquisition and expression of morphine-induced place preference in male Wistar rats have been investigated. The conditioning treatments with subcutaneous (s.c.) injections of different doses of morphine (0.5-7.5 mg/kg) induced a conditioned place preference (CPP) for the drug-associated place in a dose-dependent manner. Intra-CA1 administration of the GABA(A) receptor agonist, muscimol (0.25, 0.5 and 1 microg/rat) significantly inhibited the morphine (5 mg/kg, s.c.)-induced CPP. Intra-CA1 injections of different doses of the GABA(A) receptor antagonist, bicuculline (0.25, 0.5 and 1 microg/rat), in combination with an ineffective dose of morphine (0.5 mg/kg, s.c.) elicited a significant CPP. However, muscimol or bicuculline by themselves did not elicit any effect on place conditioning. Furthermore, the muscimol-induced inhibition of morphine response was reversed by bicuculline (1 microg/rat, intra-CA1) administration. On the other hand, the bilateral intra-CA1 injections of muscimol (0.25, 0.5 and 1 microg/rat) or bicuculline (0.5, 1 and 2 microg/rat) significantly decreased the expression of morphine-induced CPP. Intra-CA1 administration of different doses of muscimol or bicuculline had no effect on locomotor activity in the testing phase. Our data indicated that the GABA(A) receptors of the hippocampal CA1 regions may play an important role in the acquisition and expression of morphine-induced place preference.     

Pharmacological evidence for the participation of NO-cyclic GMP-PKG-K+ channel pathway in the antiallodynic action of resveratrol

The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG)-K+ channels pathway in the antiallodynic action of resveratrol and YC-1 in spinal nerve injured rats was assessed. Ligation of L5/L6 spinal nerves produced a clear-cut tactile allodynia in the rats. Intrathecal administration of resveratrol (100-600 microg) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (0.1-2.7 microg, YC-1, a soluble guanylyl cyclase activator) decreased tactile allodynia induced by ligation of L5/L6 spinal nerves. Intrathecal treatment with NG-L-nitro-arginine methyl ester (10-100 microg, L-NAME, a NO synthase inhibitor), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (1-10 microg, ODQ, a soluble guanylyl cyclase inhibitor), KT-5823 (5-500 ng, a PKG inhibitor) and iberiotoxin (5-500 ng, a large-conductance Ca2+ -activated K+ channel blocker), but not NG-D-nitro-arginine methyl ester (100 microg, D-NAME, an inactive isomer of L-NAME), glibenclamide (12.5-50 microg, ATP-sensitive K+ channel blocker) or vehicle, significantly diminished resveratrol (300 microg)- and YC-1 (2.7 microg)-induced spinal antiallodynia. These effects were independent of prostaglandin synthesis inhibition as indomethacin did not affect resveratrol-induced antiallodynia. Results suggest that resveratrol and YC-1 could activate the proteins of the NO-cyclic GMP-PKG spinal pathway or large-conductance Ca2+ -activated, but not ATP-sensitive, K+ channels at the spinal cord in order to produce at least part of their antiallodynic effect in this model of neuropathy.     

NO/cyclic GMP pathway mediates the relaxation of feline lower oesophageal sphincter

1. We examined the role of the NO/cyclic GMP (cyclic GMP) pathway in nitric oxide (NO)- and vasoactive intestinal peptide (VIP)-induced relaxation of feline lower oesophageal sphincter (LES). Furthermore, it was studied whether methylene blue, LY83583 and ODQ, which are soluble guanylate cyclase (sGC) inhibitors, could inhibit NO-induced relaxation. 2. The nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine (L-NNA) had no effect in sodium nitropruside (SNP)-induced relaxation, but 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1)-induced relaxation was decreased by the pretreatment of L-NNA, which showed that SIN-1, not SNP, could activate NOS to cause relaxation. Methylene blue and LY83583 did not inhibit the relaxation by SNP and SIN-1. However, the more specific sGC inhibitor ODQ blocked the relaxation induced by NO donors. 3. To identify the relationship of NOS, sGC and adenylate cyclase in VIP-induced relaxation, tissue were pretreated with L-NNA and ODQ and SQ22536. These inhibitors produced significant inhibition of this response to VIP. The adenylyl cyclase inhibitor SQ 22536 also inhibited relaxation by VIP. 4. In conclusion, our data showed that SNP- and SIN-1-induced relaxation was mediated by sGC. Of sGC inhibitors, methylene blue and LY83583 were not adequate for the examination of NO donor-induced feline LES smooth muscle relaxation. VIP also caused relaxation by the pathway involving NO and cGMP and cAMP.