Effects of Electroacupuncture at Baihui and Shenting on Learning and Memory and Expression of Purinoceptor P2X7 in Hippocampal CA1 after Cerebral Ischmeia-reperfusion in Rats / 中国康复理论与实践

Objective To observe the effect of electroacupuncture at Baihui (GV20) and Shenting (GV24) on learning and memory in rats after cerebral ischmeia-reperfusion and the possible mechanism. Methods A total of 42 male Sprague-Dawley rats were randomly divided into sham group (n=12) and operation group (n=30). The left middle cerebral arteries of the operation group were occluded with the modified Longa's method for 90 minutes and reperfused, and 24 qualified rats were randomly divided into model group (n=12) and elec-troacupuncture group (n=12), and the latter accepted electroacupuncture at Baihui and Shenting for seven days. They were assessed with Longa's score two hours after modeling, and one, three, seven days after intervention. They were tested with Barnes maze since three days after intervention, once a day for five days. The expression of purinoceptor P2X7 in CA1 of the hippocampus were detected with immunofluorescence seven days after inter-vention, while the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in CA1 were detect-ed with enzyme-linked immunosorbent assay. Results The Longa's score was improved in the electroacupuncture group compared with that in the model group seven days after intervention (P<0.05); while the escape latency and the times entering the wrong hole increased in the model group compared with that in the sham group (P<0.001), and decreased in the electroacupuncture group compared with that in the model group (P<0.001). The expression of P2X7, IL-1β and TNF-α increased in the model group compared with the sham operation group (P<0.001), and decreased in the electroacupuncture group compared with that in the model group (P<0.05). Conclusion Electroacupuncture at Baihui and Shenting can improve the learning and memory in rats after cerebral isch-emia-reperfusion, which may associate with inhibition of P2X7 to alleviate inflammation in hippocampus.

The Impact of Generic Clopidogrel Bisulfate on Platelet Inhibition in Patients with Coronary Artery Stents: Results of the ACCEL-GENERIC Study

BACKGROUND/AIMS: In patients with coronary artery stents, the cost of clopidogrel has been cited as a factor in the premature discontinuation of therapy. Thus, the introduction of lower-cost generic clopidogrel may increase patient compliance. However, platelet inhibition by generic clopidogrel has not been compared to the original clopidogrel formulation in patients with coronary artery stents. METHODS: We prospectively enrolled 20 patients receiving chronic therapy with the original clopidogrel bisulfate (Plavix(R)). After assessing patient compliance with Plavix(R), maintenance therapy was switched to generic clopidogrel bisulfate (Plavitor(R)). Platelet reactivity was assessed at baseline and 30-day after the switch using conventional aggregometry and the VerifyNow P2Y12 assay. RESULTS: All patients completed maintenance therapy with Plavitor(R). Before and after switching therapy maximal (36.5 +/- 7.9% vs. 39.8 +/- 16.2%, p = 0.280) and late platelet aggregation (23.5 +/- 10.9% vs. 29.1 +/- 18.3%, p = 0.156) with 5 micromol/L adenosine diphosphate (ADP) stimulus did not differ. Likewise, 20 micromol/L ADP-induced platelet aggregation and P2Y12 reaction unit in patients on Plavitor(R) therapy was comparable to that in patients on Plavix(R) therapy. However, Bland-Altman analysis showed wide limits of agreement between measured platelet reactivity on Plavix(R) vs. Plavitor(R) therapies. CONCLUSIONS: Among patients on Plavix(R) maintenance therapy with coronary stents, replacement with Plavitor(R) shows a comparable inhibition of ADP-induced platelet aggregation. However, due to poor inter-therapy agreement, between two regimens, physicians may be cautious when introducing generic clopidogrel bisulfate.

Inhibition of P2X7 Receptor by Extracts of Chinese Medicine / 针灸推拿医学（英文版）

Objective: To investigate the influence of Acorus gramineus (Soland), a crude extract, SCP01, and a purified component, SCP02, and of Rosmarinus officinalis L., X0728 on human mast cells (HMC-1 Cell Line). Methods: Current-voltage of P2X7 receptors on human mast cell membrane activated by ATP was recorded by the whole-cell patch clamp technique. Results: The current at-100 mV mediated by P2X7was inhibited by (27.6±2.0) % in the presence of 40 μg/mL SCP01 and by (29.5±2.2) % in the presence of 40 μg/mL SCP02, which was identified as α-asarone. 42 μg/mL of the commercially available α-asarone inhibited the P2X7-mediated current by (52.2±2.0) %. In contrast to SCP01 and SCP02, 40μg/mL X0728 provoked stimulation of the current by (28.6±2.8) %. All effects were voltage-independent. Conclusion: The inhibition of P2X7by α-asarone will inhibit intracellular calcium increase and this may account for the inhibition of reported excitotoxic cell death. The pharmacological function of P2X7stimulation by X0728 needs further investigation.

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia

Recent studies have reported that the "cholinergic anti-inflammatory pathway" regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X7 receptor (P2X7R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fA beta(1-42))-induced ROS production by modulating ATP efflux-mediated Ca2+ influx through P2X7R. Nicotine inhibited ROS generation in fA beta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca2+ influx in fA beta(1-42)-stimulated microglia. Moreover, ATP release from fA beta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca2+ influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X7R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fA beta(1-42)-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X7R.

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta(1-42)) induced ATP release, which in turn activated NADPH oxidase via the P2X(7) receptor (P2X(7)R). Reactive oxygen species (ROS) production in fAbeta(1-42)-treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X(7)R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta(1-42)-induced Ca2+ influx was mediated through P2X(7)R activation. In serial experiments, we found that microglial pretreatment with the P2X(7)R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 micrometer) or oxidized ATP (100 micrometer) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta(1-42)-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.

Effects of different ways of administration on the P2X_1 purinoceptor-mediated vasoconstriction in the rat mesenteric artery / 中国药理学通报

Aim To investigate the effects of ?,?-methylene ATP ( ?,?-MeATP) on the P2X1 purino-ceptor-mediated vasoconstriction by different administration ways in the rat mesenteric artery. Methods Isometric vasoconstrictive responses to ?,?-MeATP,administered in non-accumulative manner or single concentration manner,were recorded in the rat isolated mesenteric arterial rings. Results ?,?-MeATP ( 10 -7 ～ 10 -4 mol ? L -1 ) administered in both of the two manners produced concentration-dependent vasocon-strictive responses in the rat isolated mesenteric artery. The vasoconstrictive responses to ?,?-MeATP in single-concentration administration group were greater than those in non-accumulative administration group when the vasoconstriction was standardized either by tissue wet weight or by maximal response to 120 mmol ?L-1 KCl ( P

HCG attenuates ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) / 대한산부인과학회잡지

OBJECTIVE: To determine the effects of hCG on extracellular ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) and 5 IU hCG to luteinized granulosa cells obtained during in vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Calcium imaging with fura-2 revealed that ATP elevated [Ca2+]i by mobilizing intracellularly stored Ca2+. A patch clamp study showed that ATP exerted its effect by initially binding to the P2Y type purinoceptor, as evidenced by the ATP-evoked outward Ca2+-activated K+ current. Probing mitochondria with JC-1, a mitochondrial transmembrane potential-sensitive dye, revealed that ATP induced mitochondrial depolarization in a concentration-dependent manner. A quantitative flow cytometric analysis with Annexin V showed that apoptotic cells were increased in number in proportion to the concentration of ATP, having 18.57% of apoptotic cell populations in the presence of 0.75 mM ATP compared to 7.88% in the control. Moreover, treatments with human chorionic gonadotropin (hCG) at 5 IU reversed both the ATP-induced mitochondrial depolarization and apoptosis (18.57 vs 6.32%). CONCLUSION: Taken together, these results indicate, first, extracellular ATP recognized by the P2Y type purinoceptor on h-LGCs increases the intracellular Ca2+. Second, the increased intracellular Ca2+ triggers the apoptotic cascade by acting at least, in part, on mitochondria. Third, hCG reverses the ATP-induced apoptosis, raising a possible clinical implication of hCG in the treatment of degeneration of granulosa cells such as follicular atresia.

Apoptosis of human luteinized granulosa cell via change of mitochondria / 대한산부인과학회잡지

OBJECTIVE: To determine the effects of extracellular ATP on mitochondrial function and apoptosis during human luteinized granulosa cell cultures. METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) to luteinized granulosa cells obtained during In vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Measurement by patch clamp of the granulosa cell membrane potential after ATP addition to cultured granulosa cells showed that both the inward and outward currents were expressed. After treatment of the granulosa cells with JC-1, measurement of the mitochondrial activity by confocal microscopy showed that the with increasing concentrations of ATP the relative ratio of undamaged mitochondria (red/green ratio) tended to decrease (P=0.027). After double staining of the cultured granulosa cells with Annexin V and Propidium Iodide, quantitative flow cytometry analysis showed that apoptosis increased with increasing concentrations of ATP (7.88%, 8.44%, 11.40%, 13.52%, 18.57%). CONCLUSION: The results of this study shows that apoptosis of granulosa cells increases with increasing extracellular ATP concentrations in cultured human luteinized granulosa cells. This is observed to be a consequence of cell membrane purinoceptor activity and functional changes in the mitochondria. It is therefore thought that remodelling processes of the ovarian tissue is regulated by neuroendocrine factors of the extracellular ATP.

Effects of exogenous ATP on calcium mobilization and cell proliferation in C6 glioma cell

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ((Ca2+)i) mobilization and cell proliferation were investigated. ATP-induced (Ca2+)i increased in a dose-dependent manner (10-7 M apprx 10-3 M). ATP-induced (Ca2+)i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced (Ca2+)i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced (Ca2+)i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.

Effects of Adenosine 5'-Tetraphosphate on the Cardiac Activity*

BACKGROUND: Adenosine 5'-tetraphosphate(ATPP), an endogenous nucleotide, is stored in cells and released into the extracellular space upon stimulation. Some of the biological responses to ATPP were reported, but characteristics of its receptor were not well known. Present study was conducted to investigate the effects of ATPP on mechanical contractility, resting membrane potential and action potential of rat left atrium. METHODS: Left atrium was isolated from Sprague-Dawley rat. Mechanical contraction induced by electrical field stimulation(EFS) was recorded on polygraph using force transducer. With glass microelectrodes(10 MOmega), potential difference across the membrane was measured and recorded on an oscilloscope and a polygraph. RESULTS: ATPP reduced the left atrial contractility with concentration-dependent manner. ATPP also hyperpolarized the resting membrane potential and decreased the action potential duration of the left atrial cell. Nucleotides other than ATPP, such as ATP, ADP, AMP and adenosine, have the same effect as ATPP. However, there is no difference among the nucleotides. Prior treatment of DPCPX, a P1-purinoceptor blocker, inhibited the ATPP-induced negative inotropism and changes of the membrane potential. But suramin, a nonselective P2-purinoceptor blocker, did not alter the effects of ATPP. alpha, beta methylene ADP and adenosine deaminase, which attenuates hydrolysis of adenine nucleotides and inactivates adenosine respectively, did not influence the effects of adenine nucleotides except for adenosine. CONCLUSION: ATPP reduced the mechanical contractility, hyperpolarized the resting membrance potential and decreased duration of action potential of rat left atrium. These effects were induced by ATPP directly, not by adenosine from hydrolyzed ATPP.

Effects of adenosine tetraphosphate (ATPP) on vascular tone in the isolated rat aorta

Effects of a platelet-released, naturally occurring nucleotide, adenosine 5'-tetraphosphate (ATPP) on vascular tone were analyzed in the isolated rat aorta. Under resting tension ATPP (1 approximately 100 microM) elicited concentration-dependent contractions in endothelium-intact aortic rings in contrast to the concentration-dependent relaxation with ATP. In endothelium-denuded aortic rings, ATPP induced contraction, as ATP did, but with a greater potency. alpha, beta-methylene ATP (APCPP 50 microM), a P2x-purinoceptor antagonist, significantly inhibited ATPP- as well as ATP-induced contractions in the endothelium-denuded preparations suggesting that ATPP acts via P2x-purinoceptors. ATPP (10 approximately 100 microM) relaxed precontracted aortic rings with an intact endothelium in a concentration-dependent manner. This effect of ATPP was 3.7 fold less potent than that of ATP. However, after P2x-purinoceptor blockade, the effect became identical between the two nucleotides. Reactive blue 2, a selective antagonist of P2x-purinoceptors, significantly attenuated the ATPP-induced relaxation with no change in the ATP-induced relaxation. These results indicated that the rat aortic endothelium contains heterogeneous populations of P2-purinoceptors (possibly P2y and nucleotide receptors). Since ATPP shows dual effects depending upon the vascular tension, it may play a significant role in the physiological regulation of vascular tone.

Electrophysiological Effects of Purinergic Receptor Agonists on Atrial Muscle Fiber under Normal and Ischemic Conditions

BACKGROUND: The electrophysiological effects of purinergic receptor agonists, adenosine triphosphate(ATP) and adenosine were examined using conventional microelectrode technique in rat atrial muscle fibers under superfused with a normal or a simulated ischemic(hypoxic, hyperkalemic and acidotic) physiologic salt solution(PSS) in vitro. METHODS: Action potential parameters, such as maximal diastolic potential(MDP), action potential amplitude(APA), rate of phase 0 depolarization(dv/dtmax) and action potential duration(APD90) were measured in electrically paced, physiologic salt solution(Tyrode's) superfused left rat atrium. In the experiment of ischemic simulation in vitro, normal physiologic salt solutions(NPSS0 were modified(MPSS) and superfused in substitute for normal Tyrode's solution. To investigate the effects of purinergic receptor agonists, ATP or adenosine was added to the superfused tyrode's solutions(NPSS or MPSS) in molar concentration. RESULTS: Under superfused with normal PSS, ATP(10(-3), 10(-4)M) elicited slight hyperpolarization in MDP, and both ATP(10(-6)-10(-3)M) and adenosine(10(-6)-10(-3)M) shortened the duration of normal action potential in a dose-dependent manner. The other paramaters were not affected by the drugs. Superfusion with ischemic PSS caused reductions in MDP as well as APA, dv/dtmax and, especially, APD90. The effects produced by the initial 10 minutes of superfusion with ischemic PSS almost completely disappeared during a subsequent period of continued superfusion with normal PSS, but, those by the initial 20 min lasted in some degree. Both ATP(10(-4)M) and adenosine(10(-4)M) attenuated the reduction in the rate of phase 0 depolarization and the amplitude of the action potential amplitude produced by the ischemic PSS. CONCLUSION: Purinergic receptor agonists, ATP and adensoine, caused a concentration-dependent shortening of the action potential duration in rat atrial muscle fibers and they attenuated the reductions in the rate of phase 0 depolarization and action potential amplitude in fibers superfused with ischemic PSS.

The functional binding antibodies to novel purinoceptor selected from phage display library / 中国药理学通报

Aim To screen antibodies of novel purinoceptor as a marker for further study of the purinoceptor. Method BALB/c mice were immunized for 4 times with rat aortic endothelial cell. Then the phage display system was used to construct a single-chain Fv fragment (ScFv) cDNA library from the total RNA of immunized mice. The characteristics of novel purinoceptor not existing on vascular smooth muscle cell but on aortic endothelium were used to enrich the aortic endothelium specific antibodies. Induced with IPTG, these antibodies were secreted into the periplasm of E. coli. The functional experiment of novel purinoceptor named organ bath experiment was used to screen out the positive ScFv from the soluble expressed antibodies. Immunohistochemistry experiment was used for positive ScFv identification. Results The total mouse anti-rat endothelium lgG is 1 ∶16 000. 8?106 mouse anti-rat endothelium ScFv cDNA library was successfully constructed. After 4 times of rat endothelium and rat smooth muscle cells screening, 2 500 ScFv cDNA binding membrane of aortic endothelium was enriched. After 4 times of functional screening, a phage-ScFv named B inhibiting the adenosine induced NO dependent construction by 83.4%?21.6% was selected from the expressed antibodies. Immunohistochemistry experiment showed that ScFv-B combined with aortic endothelium specifically and functional experiment showed that ScFv-B did not have any effect on adenosine induced ileum contraction, indicating that ScFv-B specifically binding to the novel purinoceptor. Conclusions ScFv-B binding specifically to the novel purinoceptor was selected by phage display technique and functional screening experiment which provide a good marker for further study of the novel purinoceptor.

Progress in the studies of P_2 purinoceptors / 中国药理学通报

P 2 purinoceptors were first subdivided into P 2X and P 2Y subtypes, and later this classification was broadened to include P 2T , P 2Z and P 2D subtypes. In the 1990s, a new kind of receptors was found, which respond to UTP, ATP and ATP?S, but not to 2 MeSATP or ?,? MeATP, this finding led to the definition of the so called “P 2U ” or “nucleotide” receptor. Most recent evidence demonstrated the existence of a pyrimidine receptor responding to UTP but not to ATP. For this case, IUPHAR (International Union of Pharmacology) committee recommended that P 2 purinoceptors be subdivided into P2X and P2Y subtypes, any subtypes of intrinsic ion channel be termed P2Xn, and any subtypes of G protein coupled receptor be termed P2Yn purinoceptors. With the development and application of the molecular biologic technique and the cloning and expression of the receptors, the classification was strongly confirmed.