This study is to observe the difference in pharmacological characteristics between circular smooth muscles of rat isolated gastric body and gastric fundus, and to investigate the effects of nucleoside and nucleotide on circular smooth muscle of the rat gastric body and the involved receptors. Circular muscle strips of the rat gastric body and gastric fundus were prepared, and contractile responses to agonists were investigated with a technique of drug-receptor interaction in functional system. There was no significant difference between the circular muscle strips of the gastric body and gastric fundus in the responses to KCl, and no difference in EC50 values of contractile responses for 5-HT and His between the two kinds of preparations (P > 0.05). However, Emax values of contractile responses to 5-HT and His [(0.81 +/- 0.26) and (0.88 +/- 0.27) g] in gastric body were significantly smaller than those in gastric fundus [(2.67 +/- 0.61) and (1.90 +/- 0.68) g, P < 0.01], and EC50 value of CCh produced contractile response [(0.45 +/- 0.15) micromol x L(-1)] in gastric body was significantly higher than that in gastric fundus [(0.20 +/- 0.09) micromol x L(-1), P < 0.01]. In precontracted circular muscle strips of the gastric body, ATP (0.1-3000 micromol x L(-1)) produced only a contractile response concentration-dependently, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus. ATP, UTP, ADP, 2-MeSATP and alpha,beta-MeATP produced contractile responses concentration-dependently in circular muscle strips of the rat gastric body. The EC50 value for 2-MeSATP [(7.2 +/- 5.2) nmol x L(-1)] was about 500 times lower than that for Ach [(3.47 +/- 1.20) micromol x L(-1)]. The rank order of potency for the contraction was 2-MeSATP>ADP>ATP=UTP>alpha,beta-MeATP>adenosine. The contractile responses to ATP and UTP were not significantly affected by phentolamine, propranolol, atropine or tetrodotoxin. In conclusion, there is a significant difference in pharmacological characteristics between the circular smooth muscles of the rat gastric body and gastric fundus and nucleotides might be important mediators responsible for the contraction via a specific P2Y receptor in circular smooth muscle of the rat gastric body.
Adenosine ; pharmacology ; Adenosine Diphosphate ; pharmacology ; Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Animals ; Gastric Fundus ; drug effects ; physiology ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; Purinergic P2 Receptor Agonists ; Rats ; Rats, Wistar ; Receptors, Purinergic P2 ; drug effects ; Stomach ; drug effects ; physiology ; Thionucleotides ; pharmacology ; Uridine Triphosphate ; pharmacology

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.
Adenosine Triphosphate/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Autocrine Communication/*drug effects/physiology ; Cells, Cultured ; Microglia/*drug effects ; Peptide Fragments/*pharmacology ; Pyridoxal Phosphate/analogs & derivatives/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Purinergic P2/physiology

OBJECTIVETo investigate the effects and related mechanisms of 2-methylthio-adenosine-5'-triphosphate (2-MeSATP), an important extracellular agonist that activates receptors for purine nucleotides (P2XR), on ventricular arrhythmias in rabbits with chronic heart failure (CHF).

METHODSThe male New Zealand rabbits were divide into control (n=12), CHF (n=12) and CHF+2-MeSATP groups (2-MeSATP, n=12). CHF was induced by isoproterenol injection (0.3 mg·kg⁻¹·d⁻¹ for 3 weeks) and rabbits were observed 6 months later. The main cardioelectrophysiological parameters and ventricular arrhythmias were tested by recording monophasic action potential (MAP) with burst-pacing (BCL) in rabbits in vivo. The transient outward potassium current (Ito) was recorded via whole-cell patch clamp technique and the fluorescence intensity of intracellular free Ca²⁺ was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbet ventricular myocytes.

RESULTSCHF rabbits developed severely clinical CHF signs and symptoms, reduced left ventricular ejection fraction and fractional shortening as well as enlarged end-diastolic dimension. Compared with CHF group, APA and MaxdV/dt were significantly increased, while APD20, APD50 and APD90 were significantly reduced in 2-MeSATP group (all P<0.01). Moreover, 2-MeSATP could obviously shorten BCL induced ventricular arrhythmias, and decrease deducibility and persistence time of ventricular arrhythmias with burst-pacing in 2-MeSATP group in vivo (all P<0.05). With voltage clamp model, 2-MeSATP could significantly increase the current density of Ito in different command potential in CHF ventricular myocytes (all P<0.01). When holding potential was set at -50 mV and command potential was set at +50 mV, the current densities of Ito increase was more significant in 2-MeSATP group than that in CHF group ((11.79 ± 4.51) pA/pF vs. (7.94 ± 3.53) pA/pF, P<0.01). 2-MeSATP could completely change the I-V curve upward without changing the I-V curve direction in CHF ventricular myocytes. The fluorescence intensities of intracellular free Ca²⁺ increase was more significant in 2-MeSATP group compared to CHF group ((1 291.98 ± 123.31) µmol/L vs. (793.59 ± 114.65) µmol/L, P<0.01).

CONCLUSION2-MeSATP as a potent agonist acting on P2XR could significantly shorten APD, increase heart rate and improve cardiac performance as well as decrease the susceptibility of ventricular arrhythmias in this rabbit CHF model. Our results suggest that Ito increase and sarcoplasmic reticulum uptake Ca²⁺ enhancement as well as dynamic balance of intracellular Ca²⁺ cycling sustenance might linked to the beneficial effects of 2-MeSATP in this CHF model.

Action Potentials ; Adenosine Triphosphate ; analogs & derivatives ; Animals ; Brugada Syndrome ; Cardiac Conduction System Disease ; Chronic Disease ; Heart Failure ; Heart Ventricles ; Isoproterenol ; Male ; Myocytes, Cardiac ; Patch-Clamp Techniques ; Potassium ; Rabbits ; Thionucleotides

In order to investigate the role of non-adrenergic non-cholinergic nerves in regulating mechanical and electrical activity of gastric circular smooth muscle, the effects of ATP and its analogues on gastric motility and electrical activities were observed in guinea-pig. In organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured. Electrical activity of the muscle was recorded by using intracellular microelectrode. Electrical and mechanical activities were recorded by chart recorder. ATP and 2-MeSATP potentiated the mechanical activity but did not affect electrical activity in gastric circular smooth muscle. ATP and 2-MeSATP-induced contraction was effectively blocked by nonselective P2y-purinoceptor antagonist, reactive-blue-2 and suramin, but ATP-induced contraction was not blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. However, alpha,beta-MeATP, P2x-purinoceptor agonist, attenuated slow waves with membrane hyperpolarization and inhibited contraction. The relaxation by beta,gamma-MeATP was blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. ATP-induced contraction was blocked by external calcium-free, but not by nicardipine, a L-type calcium channel blocker. Indomethacin, a nonselective cyclooxygenase inhibitor, did not block ATP-induced contraction. The results suggest that: (1) ATP- and analogues-induced contraction is mediated by P2y-purinoceptor, whereas alpha,beta-MeATP-induced relaxation by P2x-purinoceptor in guinea-pig gastric antral circular smooth muscle. (2) ATP-induced contraction is dependent on extracellular calcium, but Ca2+ entry is not mediated by L-type calcium channel. (3) Prostaglandins are not involved in ATP- and analogue-induced contraction of gastric circular smooth muscle in guinea-pigs, and alpha,beta-MeATP-induced relaxation is related to membrane hyperpolarization.
Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Animals ; Electrophysiology ; Guinea Pigs ; In Vitro Techniques ; Microelectrodes ; Muscle Contraction ; drug effects ; physiology ; Muscle, Smooth ; drug effects ; physiology ; Purinergic Agonists ; Pyloric Antrum ; drug effects ; physiology ; Thionucleotides ; pharmacology

The adenosine triphosphate-based chemotherapy response assay (ATP-CRA) is a chemosensitivity test that offers the potential of selecting cancer treatments based on the responsiveness of individual tumors. We report a case of 47-yr-old male, presented with sigmoid colon cancer with multiple liver and peritoneal metastases, in which there was a complete response for the primary colon cancer after administration of preoperative chemotherapy selected by ATP-CRA. Oxaliplatin was the most sensitive drug based on the ATP-CRA where the specimen obtained by ultrasound- guided percutaneous liver biopsy was used. After twelve cycles of oxaliplatincapecitabine chemotherapy, abdominopelvic computed tomography revealed marked shrinkage of the liver metastases and positron emission tomography showed no uptake of (18)F-fluoro-deoxy-glucose (FDG) either in the liver or peritoneum except localized uptake in the sigmoid colon. The patient underwent an anterior resection and radiofrequency ablation of the liver metastases, which resulted in a macroscopic curative resection of the cancer cells. Histological examination revealed no residual cancer cells in the resected specimen of the sigmoid colon. This result suggested that preoperative chemotherapy chosen by ATP-CRA may be useful for treating advanced colon cancer with unresectable liver and peritoneal metastases.
Adenosine Triphosphate/*metabolism ; Antineoplastic Agents/*pharmacology ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Colonic Neoplasms/*diagnosis/*drug therapy ; Deoxycytidine/administration & dosage/analogs & derivatives ; Fluorouracil/administration & dosage/analogs & derivatives ; Humans ; Liver Neoplasms/drug therapy/secondary ; Male ; Medical Oncology/methods ; Middle Aged ; Neoplasm Metastasis ; Organoplatinum Compounds/administration & dosage ; Positron-Emission Tomography ; Remission Induction ; Treatment Outcome

The aim of this study is to investigate the effect of isosorbide-5-mononitrate (ISMN) on the electric field stimulation induced sympathetic purinergic vasoconstriction of the rabbit saphenous arterial rings. Isometric vasoconstrictive responses to electric field stimulation and to exogenous noradrenaline and adenosine triphosphate were recorded. We found that the vasoconstrictive responses to electric field stimulation (15 V, 1 ms pulse duration, 2 - 16 Hz) were frequency-dependant in the rabbit saphenous arterial rings, and abolished by tetrodotoxin (0.1 micromol x L(-1)). The alpha1-adrenoceptor antagonist prazosin (1 micromol x L(-1)) did not affect the vascular responses to the electric field stimulation (2 -8 Hz). After a combination treatment with both alpha,beta-meATP (3 micromol x L(-1), desensitizing P2X1 receptors) and prazosin (1 micromol x L(-1)), the vasoconstrictive responses to electric field stimulation were abolished. When the arterial preparation was treated with ISMN (one preparation was exposed to only one concentration of ISMN), ISMN at 0.1 mmol x L(-1) significantly inhibited the vasoconstriction induced by electric stimulation at 8 Hz, 0.3 and 1.0 mmol x L(-1) significantly inhibited the vasoconstrictive responses to electric stimulation at 2 - 16 Hz. The highest concentration of ISMN (1.0 mmol x L(-1)) reduced the vasoconstrictive responses by 46% (2 Hz), 47% (4 Hz), 34% (8 Hz) and 22% (16 Hz), separately. ISMN (0.3 and 1.0 mmol x L(-1)) did not affect the vascular responses to exogenous noradrenaline (0.01-100 micromol x L(-1)) and adenosine triphosphate (1 mmol x L(-1)). It is reasonable to suggest that ISMN inhibits the purinergic vasoconstriction induced by sympathetic nerve stimulation via a prejunctional mechanism.
Adenosine Triphosphate ; analogs & derivatives ; pharmacology ; Adrenergic alpha-Antagonists ; pharmacology ; Animals ; Arteries ; drug effects ; Delayed-Action Preparations ; Electric Stimulation ; Isosorbide Dinitrate ; administration & dosage ; analogs & derivatives ; pharmacology ; Male ; Norepinephrine ; pharmacology ; Prazosin ; pharmacology ; Purinergic P2 Receptor Agonists ; Rabbits ; Receptors, Purinergic P2X ; Vasoconstriction ; drug effects

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 micrometer) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-beta S that showed maximal IL-10 release were 100, 10, 100, and 100 micrometer respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down- regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 micrometer) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertusis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.
Adenosine Diphosphate/analogs & derivatives/pharmacology ; Adenosine Triphosphate/analogs & derivatives/*pharmacology ; Adenylate Cyclase/antagonists & inhibitors ; Animals ; Calcium/metabolism ; Chelating Agents/pharmacology ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors ; Enzyme Inhibitors/pharmacology ; Extracellular Space/drug effects/*metabolism ; Gene Expression Regulation/drug effects ; Interleukin-10/*biosynthesis ; Microglia/*drug effects/enzymology/*metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor Cross-Talk/*drug effects ; Receptors, Purinergic P2/agonists/antagonists & inhibitors/genetics/*metabolism ; Thionucleotides/pharmacology

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.
Adenosine Triphosphate/analogs & derivatives/metabolism/pharmacology ; Amyloid/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Calcium/metabolism ; Enzyme Activation/drug effects ; Microglia/cytology/*drug effects/enzymology/*metabolism ; NADPH Oxidase/metabolism ; Nicotine/pharmacology ; Nicotinic Antagonists/pharmacology ; Peptide Fragments/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Nicotinic/*metabolism ; Receptors, Purinergic P2/metabolism

We sought to know whether a free radical spin trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) influences brain cell membrane function and energy metabolism during and after transient global hypoxia-ischemia (HI) in the newborn piglets. Cerebral HI was induced by temporary complete occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min, followed by release of carotid occlusion and normoxic ventilation for 1 hr (reoxygenationreperfusion, RR). PBN (100 mg/kg) or vehicle was administered intravenously just before the induction of HI or RR. Brain cortex was harvested for the biochemical analyses at the end of HI or RR. The level of conjugated dienes significantly increased and the activity of Na+, K+-ATPase significantly decreased during HI, and they did not recover during RR. The levels of ATP and phosphocreatine (PCr) significantly decreased during HI, and recovered during RR. PBN significantly decreased the level of conjugated dienes both during HI and RR, but did not influence the activity of Na+, K+-ATPase and the levels of ATP and PCr. We demonstrated that PBN effectively reduced brain cell membrane lipid peroxidation, but did not reverse ongoing brain cell membrane dysfunction nor did restore brain cellular energy depletion, in our piglet model of global hypoxic-ischemic brain injury.
Adenosine Triphosphate/metabolism ; Animals ; Animals, Newborn ; *Anoxia ; Brain/*drug effects/metabolism/pathology ; Cell Membrane/*metabolism ; Cerebral Cortex ; *Ischemia ; Lipid Peroxidation ; Na(+)-K(+)-Exchanging ATPase/metabolism ; Neuroprotective Agents/*pharmacology ; Nitrogen Oxides/*pharmacology ; Phosphocreatine/*analogs & derivatives/metabolism ; Reperfusion Injury/*drug therapy ; Support, Non-U.S. Gov't ; Swine ; Time Factors

OBJECTIVETo explore the expression of purinergic p2X4 receptor (P2X4R) in trigeminal ganglion of rats after occlusal interference. Investigation of peripheral receptor mechanism of occlusal interference-induced masticatory muscle pain will aid the development of drug intervention against this condition.

METHODSExperimental occlusal interference was established by application of 0.4 mm metal crown to the upper right first molar of male Sprague-Dawley rats. Real-time PCR assay was used to investigate P2X4R mRNA level in trigeminal ganglion in rats with occlusal interference for 3, 7, 10 and 14 days and in control rats without occlusal interference (n=5 in each). Retrograde labelling combining immunofluorescence was performed to evaluate the percentage of P2X4R-positive cells in masseter afferent neurons (n=5 in each group). Graded concentrations of P2XR antagonist TNP-ATP (0.1, 10, 125, 250, 500 μmol/L) or saline (n=5 in each group) was administrated in right masseter and the mechanical sensitivity of bilateral masseters was measured before occlusal interference application, before the injection, and 30 min as well as 60 min after the injection.

RESULTSCompared with control rats (P2X4R mRNA: right side: 1.00±0.26, left side: 0.94± 0.21; percentage of P2X4R-positive masseter afferents: right side: [64.3±6.3]%, left side: [67.7±5.8]%), the level of P2X4R mRNA in bilateral trigeminal ganglia (right side: 5.98±3.56; left side: 5.06±2.88) of rats with occlusal interference for 7 days up-regulated (P<0.01) and the percentage of P2X4R-positive masseter afferent neurons(right side: [81.7±1.5]%; left side: [82.9±2.3]%) increased (P<0.05). Local administration of 10, 125, 250, 500 μmol/L TNP-ATP increased the mechanical withdrawal threshold in masseter 30 min after injection, compared with those before injection (P<0.05).

CONCLUSIONSIncreased expression of trigeminal P2X4R involves in the development of occlusal interference-induced masseter hyperalgesia.

Adenosine Triphosphate ; administration & dosage ; analogs & derivatives ; pharmacology ; Animals ; Dental Occlusion ; Hyperalgesia ; etiology ; Male ; Masseter Muscle ; drug effects ; Masticatory Muscles ; Purinergic P2X Receptor Antagonists ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Purinergic P2X4 ; genetics ; metabolism ; Time Factors ; Trigeminal Ganglion ; metabolism