Advances in PDE5 inhibitors and diseases related to cGMP signaling pathway

Phosphodiesterase 5 （PDE5） inhibitors， as drugs to dilate the corpora cavernosa of the penis through sexual stimulation， are used to treat erectile dysfunction， and are also approved for treating pulmonary arterial hypertension. With the in-depth studies， PDE5 inhibitors have been approved to treat a variety of diseases， such as cardiovascular diseases， Alzheimer disease， etc， but there is currently no overall review on clinical applications of PDE5 inhibitors. As known， the classic pathway of PDE5 inhibitors promotes cyclic guanosine monophosphate （cGMP） production. However， reports on downstream pathways regulated by cGMP are still unclear. This review summarized the research and clinical application progress of PDE5 inhibitors in different diseases， and the pharmacological bases of PDE5 inhibitors through cGMP signaling pathway are discussed.     

Effect of VASP mutant phosphorylation on migration of endothelial cells induced by PDGF-BB

AIM: To investigate the effect of vasodilator-stimulated phosphoprotein (VASP) phosphorylation on the cell migration induced by platelet-derived growth factor BB (PDGF-BB), and to identify which phosphorylation site was more important among the three phosphorylation sites, namely Ser157,Ser239 and Thr278. METHODS: Two phosphorylation mutants of VASP, pcDNA3.1(+)/VASP-S157A and pcDNA3.1(+)/VASP-S239A, were constructed and respectively transfected into the cultured ECV304 cells by means of liposome. The stable expression cells were screened by using antibiotic G418. Protein expression of VASP was measured by Western blotting. The ECV304 cell migration was evaluated using Transwell chamber. RESULTS: Compared to control group, the cell migration was significantly inhibited in ECV304 transfected with VASP-S157A and VASP-S239A (P<0.05), although slight differences existed between VASP-S157A and VASP-S239A transfected cells (P>0.05). CONCLUSION: VASP mutation on the phosphorylation sites of Ser157 and Ser239 inhibits cell migration, and the phosphorylation sites of Ser157 and Ser239 both greatly affect the function of VASP.     

Effect of ERK1/2 phosphorylation on the aggregation of the rat platelets in vitro

AIM: To study the influence of PD098059 on the rat platelet aggregation rate and the phosphorylation of ERK1/2 induced by the different agonists, and to observe the effects of phosphorylation of ERK1/2 on the platelet aggregation. METHODS: The maximal aggregation rate (MAR) was measured by nephelometry. The inhibitory rate of PD098059 and the appearing time of MAR were also observed. ERK1/2 phosphorylation was detected by Western blot. RESULTS: The phosphorylation of ERK1/2 was detected during aggregation induced by thrombin and ADP. PD098059 inhibited the MAR and phosphorylation of ERK1/2. Effects of PD098059 were different on the aggregation induced by thrombin and ADP. CONCLUSIONS: The phosphorylation of ERK1/2 is one of the cellular signal transduction mechanisms of platelets aggregation. Phosphorylation of ERK1/2 plays different roles during the platelet aggregation induced by thrombin and ADP.     

Signal pathway of Cx40/43 regulates vascular contractile reactivity of superior mesenteric artery under hypoxic condition

AIM: To investigate the effect of connexin 40/43(Cx40/43) on cyclic adenosine monophosphate-protein kinase A (cAMP-PKA), cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) and diacylglycerol-protein kinase C (DG-PKC) signal pathways and the regulatory role on the vascular contractile reactivity. METHODS: Rat superior mesenteric arteries (SMA) were isolated. The vascular rings of SMA were prepared and treated with Cx40/43 antisense oligodeoxyribonucleotide (Cx40/43AODN). The changes of the concentration of cAMP, cGMP and DG, the activity of PKA, PKG and PKC, the contractile response of hypoxia treated SMA rings were observed. RESULTS: Cx40AODN decreased the concentrations of cAMP, cGMP and the activities of PKA and PKG, increased the level of DA, the activity of PKC and the endothelium-dependent contractile response in SMA. Cx43AODN increased the concentrations of cAMP, cGMP and the activities of PKA and PKG, decreased the level of DA, the activity of PKC and the endothelium-dependent contractile response in SMA. CONCLUSION: Cx40/43 regulates endothelium-dependent vasoconstrictor reactivity of SMA after hemorrhagic shock, which may be related to cAMP-PKA, cGMP-PKG and DG-PKC signal pathways.     

Effect of fraction F of naja naja atra venom on protein tyrosine phosphorylation in platelet activation

AIM: To observe the mechanism of intracellular signal transduction that fraction F of naja naja atra venom inhibits platelet aggregation. METHODS: Tests were divided into six groups: (1) blank group; (2) control group and (3)-(6) ADP plus fraction F group (doses of fraction F were 100, 30, 10, 3 mg/L, respectively). Protein tyrosine phosphorylation in platelets was assayed by Western blotting and platelet aggregation was assayed by nephelometer. RESULTS: Fraction F significantly inhibited molecular masses (MW) 76, 66 and 37.5 kD protein tyrosine phosphorylation in platelet that induced by ADP in a dose-dependent manner, in which 30 and 100 mg/L dose group showed obviously different effects when compared to control group (P<0.05). Inhibition of platelet aggregation was positive correlated with protein tyrosine phosphorylation in platelets （r=0.9367, P<0.01）. CONCLUSIONS: Fraction F of naja naja atra venom affected intracellular signal transduction pathway in platelets by inhibiting MW 76, 66 and 37.5 kD protein tyrosine phosphorylation. The result suggests that this effect may be one of the anti-thrombus mechanisms of fraction F.     

Role of ERK5 in platelet activation in vitro and arterial thrombosis in vivo

AIM: To investigate the role of extracellular signal-regulated kinase 5 (ERK5) in platelet aggregation in vitro and arterial thrombosis in vivo. METHODS: The expression and phosphorylation levels of ERK5 in human platelet were detected by Western blot. The effects of ERK5 selective inhibitor XMD8-92 on platelet aggregation and dense granule secretion were detected by Chrono-Log aggregometer. The effect of ERK5 on in vivo thrombosis was analyzed using an FeCl₃ artery thrombosis model. The effects of XMD8-92 on protein kinase B (PKB/Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) phosphorylation levels were determined by Western blot. RESULTS: ERK5 was stably expressed in human platelets and its phosphorylation level increased significantly after platelet activation (P<0.05). XMD8-92, a selective inhibitor of ERK5, inhibited platelet aggregation and dense granule secretion in response to several platelet stimulators (P<0.05). The results of Western blot showed that XMD8-92 inhibited Akt phosphorylation level by down-regulating PTEN Ser370 phosphorylation and enhancing PTEN activity. The pathway was further confirmed using platelet specific PTEN deficiency mice. The first occlusion time was obviously extended in the mice intravenously given XMD8-92 in the FeCl₃-induced carotid artery injury model. CONCLUSION: ERK5 plays a role in platelet activation and arterial thrombosis by influencing PTEN and Akt phosphorylation.     

Effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia

AIM: To investigate the effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) from rats exposed to chronic hypoxia. METHODS: (1) Wistar rats were randomly divided into control group (group A) and chronic hypoxia group (group B). Then group B received hypoxia 8 hours per day for 4 consecutive weeks. (2) Single PASMC was obtained via acute enzyme separation method. (3) Conventional whole-cell patch clamp technique was used to record resting membrane potential (Em) and ion currents of voltage-gated potassium channel. The changes of ion currents of voltage-gated potassium channel before and after applying cGMP (1 mmol/L), an agonist of protein kinase G (PKG), and cGMP plus H-8 (1 mmol/L), an inhibitor of PKG were compared between two groups. RESULTS: The Em of group B were significantly lower than that of group A. The ion currents of voltage-gated potassium channel in group A and group B were all significantly inhibited by cGMP [control group: from (118.0±5.0) pA/pF to (89.9±16.5) pA/pF, n=6, P＜0.05;chronic hypoxia group: from (81.0±5.0) pA/pF to (56.8±9.1) pA/pF, n=6, P＜0.05]and these effects were reversed by H-8 [control group: from (119.2±10.3) pA/pF to (117.8±9.1) pA/pF, n=6, P＞0.05;chronic hypoxia group: from (96.8±6.2)　pA/pF to (98.0±2.2)　pA/pF, n=6, P＞0.05]. CONCLUSIONS: The currents of voltage-gated potassium channel was inhibited by chronic hypoxic. The inhibitory effect of cGMP on currents of voltage-gated potassium channel in PASMCs from both normal and chronic hypoxic rats may be probably through the phosphorylation of voltage-gated potassium channel.     

Effects of the juice of Fructus Hippophae (JFH) on thrombocytopenia induced by cyclophosphamide in rats

AIM:To investigate the effects of the juice of fructus hippophae(JFH) on thrombocytopenia in rats.METHODS:Forty-eight wistar rats were divided into control group, model group, Yixuesheng-Jiaonang (YSJN) group and JFH group. Rats were injected intraperitoneally with cyclophosphamide (CTX, 30 mg/kg) or saline once a day for 3 days. And then, Saline, YSJN or JFH was administered (ig) once a day for 11 days. On the second, fouth, sixth and eighth day, the thrombocyte were counted, the mean platelet volume (MPV) and PDW were examined. On the eighth day, the cAMP, cGMP in platelets and platelet aggregation function were also examined.RESULTS:JFH could shorten the time of blood coagulation, increase the count of platelet, and enhance the platelet aggregation function. The experiment also showed that the JFH could decrease cGMP content in platelet.CONCLUSION:JFH could enhance blood coagulation and quality of platelet and prevent thrombocytopenia induced by cyclophosphamide in rats.     

Effect of nitric oxide on adiponectin-induced inhibition of platelet aggregation in hyperlipidemia rats

AIM: To investigate the mechanism that adiponectin inhibits platelet aggregation via nitric oxide (NO) signaling pathway. METHODS: Adult rats were fed with normal or high-fat diet for 14 weeks. Their platelets were immediately isolated and treated with or without recombinant full-length adiponectin (rAPN). The platelet aggregation, NO and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. RESULTS: Treatment with rAPN inhibited platelet aggregation induced by hyperlipidemia (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregate and thrombus formation, was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN significantly decreased superoxide production by 62% (P<0.05) and increased antioxidant capacity by 38% (P<0.05) in hyperlipidemic platelets. Importantly, hyperlipidemia-induced reduction of eNOS phosphorylation and increase in iNOS expression were markedly reversed by rAPN treatment (P<0.05 and P<0.01, respectively). CONCLUSION: Adiponectin is an adipokine that inhibits platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blockage of iNOS expression and superoxide production.     

Effects of cimetidine on platelet function and thrombosis

AIM:To observe the effects of cimetidine(Cim) on platelet function and thrombosis. METHODS:After incubated with Cimin vitro, rat platelets were activated with ADP or thrombin. The platelet aggregation, platelet malondialdehyde(MDA) formation, platelet intracellular free calcium( [Ca²⁺]_i), and thromboxane B₂ (TXB₂) were measured. The effects of Cim on electric-induced thrombosis in rat carotid artery were examined. RESULTS:Cim potentiated ADP induced platelet aggregation, increased the thrombin induced [Ca²⁺]_i and MDA formation, decreased TXB₂. Also, Cim shortened the duration of electric-stimulated occlution time in rat carotid artery. CONCLUSION:Cim increased platelet function and accelerated thrombosis.     

Establishment and assessment of a method for acid elution of platelet HLA class I antigens

AIM: To establish an effective method for acid elution of platelet HLA class I antigens, and to evaluate the optimal condition and the feasibility of clinical application of the acid-elution technique. METHODS: Platelets were treated with citric acid buffer at different pH levels (pH=2, 3, 5, 7). Expression of HLA class I antigens and P-selectin (CD62P) on the platelet surface was analyzed by multicolor flow cytometry. The proportion of early apoptotic platelets was detected by Annexin V staining. The maximum platelet aggregation rate was determined by electrical impedance aggregometry.RESULTS: With the decrease in the pH levels of the citric acid buffer (from pH=7 to pH=2), the expression of HLA class I antigens on the platelet surface was remarkably decreased. However, the rates of platelets activation (CD62P expression) and early apoptosis (Annexin V expression) were both significantly increased. Compared with PBS, treatment of the platelets with citric acid buffer at pH 3.0 remarkably reduced the expression of platelet HLA-class I antigens (P<0.05). Although the rates of the platelet activation and apoptosis were also significantly increased (P<0.05), the aggregation of platelet was not remarkably reduced (P>0.05).CONCLUSION: Acid elution of platelet HLA-class I antigens with citric acid buffer at pH 3.0 at 0 ℃ can be use as an attempt to produce HLA-eluted platelets. This technique of acid-elution needs further improvement and standardization before clinical use.     

Effect of cGMP-dependent protein kinase on endothelial cytoskeleton changes

AIM: To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of burn shock. METHODS: Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with 10% burn serum or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 were used to pretreat the endothelial cells before the administration of burn serum or PKG activator 8-Br-cGMP. RESULTS: Exposures to burn serum and 8-Br-cGMP led to a rapid time-dependent increase in endothelial PKG activity and the polar distribution of intracellular filamentous actin, and preincubation with KT5823 abolished those effects. CONCLUSIONS: The results suggest that burn serum induces PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably contributes to the endothelial hyperpermeability after burn shock.     

Effects of different group B streptococci strains on platelet activation

AIM: To explore the ability of different group B streptococci (GBS) strains on inducing platelet activation. METHODS: Six strains of GBS, separated from the septic patients with thrombocytopenia, were used as the inducers. Light transmission aggregometry was used to measure platelet aggregation. Scanning electron microscopy (SEM) was performed to investigate the interaction of platelets with bacteria. The expression of platelet CD62P, Toll-like receptor 2 (TLR2) and TLR4 was determined by flow cytometry and Western blotting. Furthermore, the activity of platelet TLR2 (or TLR4) was blocked by anti-TLR2 (or anti-TLR4) monoclonal antibody, and the platelet aggregation induced by GBS was detected. RESULTS: Only 3 of 6 GBS strains isolated from the septic patients induced platelet aggregation and up-regulated the expression of CD62P and TLR2 in the platelets (P < 0.05), but not TLR4. Incubation with anti-TLR2 antibody, but not anti-TLR4 antibody, significantly blocked platelet aggregation induced by GBS.CONCLUSION: Some GBS strains from the patients are able to trigger platelet activation in vitro, and platelet TLR2 may play an important role in the interaction between GBS and platelets.     

Vasonatrin peptide attenuates injuries of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium

AIM：To investigate the neuroprotective effects of vasonatrin peptide (VNP) on the injury of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium (MPP+). METHODS：Cultured dopaminergic neurons from the mouse ventral mesencephalon were exposed to MPP+, and the effects of VNP on the neurotoxicity of MPP+ were eva-luated by cell viability analysis and immunofluorescence staining. Various kinds of agonists and antagonists were used to clarify the mechanism underlying the effects of VNP. RESULTS：MPP+ caused injuries in the dopaminergic neurons. VNP significantly increased the viability, axon number and axon length of the dopaminergic neurons. The MPP+-induced depolymerization of β-tubulin Ⅲ was also attenuated by the treatment with VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-Br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 ［the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors (NPR)］, or KT-5823 ［a cGMP-dependent protein kinase (PKG) inhibitor］. CONCLUSION：VNP attenuates the neurotoxicity of MPP+ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neural degeneration of dopaminergic neurons in Parkinson disease.     

rAAV-mediated VLDLR gene transfection reduces tau hyperphosphorylation in hippocampus of type 2 diabetic rats

AIM: Abnormal hyperphosphorylation of tau plays a critical role in the pathogenesis of Alzheimers disease(AD), and tau protein was hyperphosphorylated in type 2 diabetes. The present study was designed to explore the phosphorylation level of tau in hippocampus of type 2 diabetes rats which interrupted by very low density lipoprotein receptor(VLDLR)gene transfection. METHODS: Wistar male rats were randomized into 3 groups. The control group(CTL)was fed with normal food. The T2DM group and T2DM mediated VLDLR gene group were on high sugar, high fat and high protein diet for 3 months. The plasma insulin level was measured by RIA method, and the plasma glucose was determined by glucose-oxidase method. Total tau level, the phosphorylation level of tau at individual phosphorylation sites and the level of VLDLR were analyzed by Western blotting. The activity of glycogen synthase kinase 3β, a key component of insulin signal transduction pathway and a known tau kinase, in the hippocampus of rats was determined by using ［γ-32P］-ATP and the specific peptide substrate. RESULTS: No significant difference of total tau level in hippocampus between T2DM group and T2DM mediated VLDLR gene group was observed. Tau protein in T2DM group was found to be more hyperphosphorylated at several AD-related phosphorylation sites(Ser214, Thr217, Ser396, Ser422 and Ser199/202)than that in CTL, while the immunoreaction at tau-1 site is weaker than that in CTL. VLDLR gene therapy reduced hyperphosphorylation sites of Thr217, Ser396, Ser422 and Ser199/202 of tau to almost the control level, but did not change the phosphorylation of Ser214 or Ser422 on tau. The expression of Ser214 was also observed by immunohistochemical assay. The phosphorylated tau modestly increased in hippocampus in T2DM group compared to CTL, but VLDLR gene treatment did not change the phosphorylation level. The phosphorylation of GSK-3β was decreased dramatically in the hippocampus in T2DM rats, and this phosphorylation was significantly increased after VLDLR gene treatment. CONCLUSION: These findings suggest that Raav mediated VLDLR gene treatment partially reverses tau hyperphosphorylation at several sites in T2DM rat hippocampus, which may mediate by inhibition of GSK-3β activity.     

Scutellaria barbata flavonoids inhibits NFT aggregation and regulatory mechanism in rats induced by composited Aβ

AIM: To investigate the effects of Scutellaria barbata flavonoids (SBF) on neurofibrillary tangle (NFT) aggregation, tau protein phosphorylation and the regulated mechanism of glycogen synthase kinase (GSK) 3β and protein phosphatase (PP) 2A in the rats induced by amyloid β protein 25-35 (Aβ_25-35) in combination with AlCl₃ and recombinant human transforming growth factor (RHTGF)-β1(composited Aβ). METHODS: The male SD rats were used to establish the simulated Alzheimer disease (AD) model by intracerebroventricular injection of composited Aβ. The Morris water maze was applied for screening the successful model rats with learning and memory deficits. The successful model rats were daily and orally administrated with SBF at doses of 35, 70 and 140 mg/kg or positive control drug Ginkgo biloba leaves flavonoids (GLF) at 140 mg/kg for 37 d. The silver nitrate staining was used to determine the cortical NFT. The protein levels of total tau, phosphorylated protein of tau at Ser199 and Ser214 sites, GSK3β and PP2A in hippocampus and cortex were determined by Western blot. The mRNA expression of GSK3β and PP2A in the hippocampus and cortex was detected by RT-PCR. RESULTS: Compared with sham group, the cell number of positive NFT with silver nitrate staining in model rat cerebral cortex was significantly increased. The protein levels of phosphorylated tau protein at Ser199 and Ser214 sites, GSK3β in the hippocampus and cerebral cortex in the model rats dramatically elevated, and PP2A was marked decreased as compared with the sham group rats. Meanwhile, the mRNA expression of GSK-3β significantly increased but PP2A was decreased. However, these above abnormalities were differently attenuated by treating with SBF at different doses or GLF at 140 mg/kg for 37 d. CONCLUSION: SBF suppresses the NFT aggregation by inhibition of the regulatory functions of GSK-3β and PP2A, thus reducing the phosphorylation of tau protein.     

Phosphodiesterase 4 inhibitor rolipram prevents depression- and anxiety-like behaviors in rats exposed to chronic restraint stress

AIM: To discuss the impact of phosphodiesterase 4 (PDE4) inhibitor rolipram on chronic restraint stress-induced depression- and anxiety-like behaviors in rats. METHODS: (1)Forty SD rats were randomly divided into 4 weight-matched groups: unstressed animals injected with vehicle of lithium chloride (LiCl) and rolipram, restraint-stressed animals injected daily with vehicle prior to stress, restraint stress plus 100 mg/kg LiCl group and restraint stress plus 1 mg/kg rolipram group. The open field test was conducted 24 h before the first stress and drug administration,and then the rats received drugs daily 1 h prior to restraint stress (6 h/d) for 25 d. Daily body weight recording, forced swimming test, elevated plus-maze and open field test were conducted to determine the changes of depression- and anxiety-like behaviors. The expression of phosphorylated cAMP response element-binding protein (p-CREB), brain-derived Reurotrophic factor (BDNF), p-Ser21-glycogen synthase kinase (GSK) 3α, p-Ser9-GSK3β, p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β was measured by Western blotting. (2)Thirty SD rats were randomly divided into 6 groups and the cannula was surgically placed above the CA1 region in the hippocampus. Seven days after the surgery, the restraint stress was conducted for 21 d after microinjection of protein kinase A (PKA) antagonist H89 and intraperitoneal injection of LiCl and rolipram everyday. The expression of PDE4D, PKA, p-CREB and p-Ser9-GSK3β was measured by Western blotting. RESULTS: (1)No difference of the locomotor activity among all groups before stress was observed. After repeated stress, the body weight,and the crossing, rearing and grooming in open field test were lower than those in control group, and LiCl and rolipram reversed these effects significantly. In addition, in comparison with control group, the immobility in forced swimming test was increased, the climbing in forced swimmming test and the open-arm exploration in elevated plus-maze were decreased and the expression of p-CREB, BDNF, p-Ser21-GSK3α and p-Ser9-GSK3β was down-regulated. Stress induced depression- and anxiety-like behaviors, and rolipram reversed these changes. The LiCl showed similar effects as rolipram except for the expression of p-CREB and BDNF. No significant difference of the expression of p-Tyr279-GSK3α, p-Tyr216-GSK3β, total GSK3α and total GSK3β among all groups was found. (2)The expression of PDE4D was increased, the expression of PKA, p-CREB and p-Ser9-GSK3β was decreased in the hippocampus induce by restraint stress. However, the effect of rolipram on the expression of PKA, p-CREB and p-Ser9-GSK3β was blocked by PKA inhibitor H89. CONCLUSION: Rolipram significantly reduces the depression- and anxiety-like behaviors, possibly through CREB/BDNF signaling and inhibitory serine-phosphorylation of GSK3-mediated signaling. Importantly, the CREB/BDNF signaling also plays a key role in the down-regulation of serine-phosphorylation of GSK3.     

PKA is required in the induction and early-phase maintenance of LTP of C-fiber evoked field potentials in spinal dorsal horn of adult rats

AIM: The role of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in the induction and maintenance of spinal long-term potentiation (LTP) was evaluated. METHODS: The C-fiber evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement. RESULTS: (1) 8-Br-cAMP induced LTP of C-fiber evoked field potentials and 8-Br-cAMP-induced LTP occludes tetanus-induced LTP. (2) Rp-CPT-cAMPS, an inhibitor of PKA, blocked the induction of spinal LTP and reversed established LTP of C-fiber evoked field potentials in a time-dependent manner. (3) In the presence of anisomycin, a protein synthesis inhibitor, the potentiation induced by 8-Br-cAMP was completely blocked. (4) PD98059, a selective inhibitor of mitogen-activated protein kinase (MAPK),completely blocked the 8-Br-cAMP-induced LTP.CONCLUSION: Activation of PKA signal pathway in spinal dorsal horn may be crucial for the induction and the early-phase maintenance of LTP of C-fiber evoked field potentials.     

In vitro anti-platelet aggregative effect of procyanidins isolated from grape seeds

AIM: To study the possible anti-platelet aggregative mechanisms of procyanidins (PC) isolated from grape seeds in vitro. METHODS: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from the blood of healthy volunteers. PC,diphenylene iodonium(DPI,a nonspecific NADPH oxidase inhibitor) and apocynin (a specific NADPH oxidase inhibitor) were used to observe the effects on collagen-induced platelet maximum aggregation rate using platelet aggregometer. The influences of PC on platelet NADPH oxidase activity, NO content and superoxide anion (O₂^-·) level were evaluated by chemiluminescence spectrometer. The role of PC in the expression of activated platelet markers (PAC-1 and CD62P) was observed by flow cytometry. RESULTS: PC (100 μmol/L), apocynin (10 μmol/L) and DPI (100 μmol/L) significantly inhibited collagen-induced maximum platelet aggregation rate (P<0.01). In collagen-activated platelets, NO content reduced and O₂^-· level increased,both of which were recovered by PC at concentration of 100 μmol/L (P<0.05). PC also obviously inhibited NADPH oxidase activity (P<0.01), and significantly down-regulated PAC-1 and CD62P expression (P< 0.05) in platelets. CONCLUSION: Procyanidins isolated from grape seeds have the anti-platelet aggregation function through inhibiting NADPH oxidase activity, further influencing platelet NO and O₂^-· levels.