AIMTo further explore the roles of endogenous nitric oxide (NO) or NO derivatives in complex partial seizures and generalized convulsions.

METHODSThe effect of pretreatment with L-nitroarginine (L-NNA), an inhibitor of nitric oxide synthase (NOS), or L arginine (L-Arg), a precursor of NO on kainic acid (KA)-induced seizure in rats and the changes in the concentration of NO2 -/NO- in the hippocampus were determined.

RESULTSThe rats appeared with wet dog shakes (WDS) at 15 min and then occurred generalized convulsions during 1 h to 3 h after administration of KA (10 mg/kg i.p.). However, the pretreatment of L-NNA (50 mg/kg) so dramatically promoted and enhanced KA-induced behavioral seizures that the latency of generalized convulsion was shorten dramatically, and the mortality was greatly high. In contrast, the pretreatment with L-Arg (40 mg/kg) markedly delayed or weakened KA-induced behavioral changes, such as increasing latency of WDS and generalized convulsion, shortening time o f seizure and none of animal died during observed time. The concentration of NO2- /NO3- in the hippocampus increased immediately at 30 min and remained to 7 d after the administration of KA. Compared with control group (pretreatment with NS), the concentration of NO2- / NO3- in the hippocampus apparently increased at 3 h and 3 d after the administration of KA in the rats with L-Arg pretreatment.

CONCLUSIONThe endogenous NO (NO or NO derivatives) mediators may play an important role against excitotoxin induced seizures in rats.

Animals ; Arginine ; pharmacology ; Kainic Acid ; adverse effects ; Male ; Nitric Oxide ; metabolism ; Nitroarginine ; pharmacology ; Rats ; Rats, Wistar ; Seizures ; chemically induced ; metabolism

This study was designed to clarify the dependency of hypoxic coronary vasodilation (HCD) on the endothelium and the role of the K+ channels on HCD in the rabbit coronary artery. HCD was investigated in an isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit (KH) solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and was maintained for 15 approximately 25 min. Hypoxia elicited a vasodilation in the precontracted coronary artery with and without endothelium. There was no difference between the amplitude of the HCD induced by two consecutive hypoxic challenges and the effects of 20% O2 + 5% CO2 + 75% N2 and 95% O2 + 5% CO2 control K-H solution of subsequent responses to hypoxia. Inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HCD. Blockades of the tetraethylammonium chloride-sensitive K+ channel abolished HCD. Apamin, a blocker of the small conductance Ca(2+)-activated K+ (KCa) channel, and iberiotoxin, a blocker of the large conductance KCa channel had no effect on HCD, respectively. Glibenclamide, a blocker of the ATP-sensitive K+ (K+ATP) channel, reduced HCD. Cromakalim, an opener of the K+ATP channel, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia while glibenclamide reduced both hypoxia- and cromakalim-induced vasodilatations. In conclusion, these results suggest that HCD is independent on endothelium and HCD is considered to be induced by activation of K+ATP channel.
Animal ; Anoxia/physiopathology* ; Coronary Vessels/physiopathology* ; Coronary Vessels/drug effects ; Cyclooxygenase Inhibitors/pharmacology ; Enzyme Inhibitors/pharmacology ; Female ; Indomethacin/pharmacology ; Male ; Nitroarginine/pharmacology ; Rabbits ; Tetraethylammonium/pharmacology ; Vasodilation/physiology*

OBJECTIVETo investigate the effect of emodin on the contraction of jejunum smooth muscle and its underlying mechanisms.

METHODSRats were randomly divided into 7 groups (n = 6): control group, emodin group (1, 5, 10, 20 micromol/L), propranolol (PRO) plus emodin group, glibenclamide (GLI) plus emodin group, NG-Nitro-L-arginine Methyl Ester (L-NAME) plus emodin group, calcium free control group and calcium free emodin group. The rats were sacrificed by cervical dislocation and the small intestine was isolated. The jejunum segment specimens were mounted on an Organ Bath System with a tension transducer. The effect of emodin on contraction of jejunum smooth muscle was measured by BL-420E+ biological signal processing system and the amplitude (AM), tension (TE) and frequency (FR) of contraction were determined.

RESULTS(1) Emodin inhibited the tension and amplitude of jejunum smooth muscle contraction in a dose-dependent manner (P < 0.05, P < 0.01) while the frequency was not obviously influenced. (2) PRO (P < 0.05) or GLI (P < 0.01) partly abolished the inhibitory effect of emodin on jejunum smooth muscle. (3) L-NAME had no obvious effect on the inhibitory effect of emodin. (4) Emodin attenuated the contraction of jejunum smooth muscle induced by calcium chloride application into calcium free K-H solution (P < 0.01).

CONCLUSIONEmodin obviously inhibits the amplitude and tension, while has no influence on the frequency of jejunum smooth muscle contraction in rats. Activation of beta adrenergic receptor, open of ATP sensitive potassium channels, and inhibition of the extracellular calcium influx through calcium channels of smooth muscle cell membrane might be involved in the process.

Animals ; Calcium Signaling ; Emodin ; pharmacology ; Glyburide ; pharmacology ; Jejunum ; drug effects ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; NG-Nitroarginine Methyl Ester ; pharmacology ; Propranolol ; pharmacology ; Rats

AIMIn order to discover new inhibitors and enhancers of nitric oxide synthase (NOS), an in vitro assay to determine NOS activity was established for high throughput screening.

METHODSThe activity of NOS was detected based on the change of nicotinamide-adenine dinucleotide phosphate (NADPH) concentration in the reaction system by the fluorescence density. The enzyme was prepared from bovine brain by gradient centrifugation. The reaction performed in black 96 well micro-plate with a final volume of 90 microL. Every factor which would affect the results such as the concentration of NADPH, L-arginine (L-Arg, used as substrate) and enzyme protein was optimized in different conditions. At last, 5,600 samples (compounds and extracts) were screened by the method.

RESULTSThe test signal (fluorescence density) in the reaction system was influenced by many different factors such as temperature and concentration of substrates. The ideal system contains protein 1.50 mg.mL-1, L-Arg 1 mmol.L-1, NADPH 0.1 mmol.L-1 at 37 degrees C. In this method, there were about 2% samples which emit fluorescence, and about 0.5% samples which quench the fluorescence. So these samples were deleted from the sample library. The effects of these samples on activity of NOS were distributed in a normal manner. About 2% samples had potential effects on the NOS activity (including inhibitors and enhancers).

CONCLUSIONThe method can be performed by high throughput screening and gives the stable data, not only for inhibitors, but also for enhancers of NOS activity.

Animals ; Arginine ; pharmacology ; Cattle ; Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; isolation & purification ; pharmacology ; In Vitro Techniques ; NADP ; pharmacology ; Nitric Oxide Synthase ; antagonists & inhibitors ; metabolism ; Nitroarginine ; pharmacology

OBJECTIVETo investigate the involvement of immunoreactive-dynorphin A in the inhibitory effect of N-nitro-L-arginine on the morphine physical dependence in rats.

METHODSThe rats were rendered dependent on morphine by subcutaneous administration of morphine solution three times daily in a manner of dose increment of 5 mg.kg(-1) for 6 days. The degree of morphine physical dependence was monitored by scoring the abstinence syndromes precipitated by 5 mg.kg(-1) naloxone of the rats. The expression levels of immunoreactive dynorphin A in tissues were determined using a radioimmunoassay.

RESULTSIntraperitoneal injection of 5 mg.kg(-1) N-nitro-L-arginine suppresses most of the withdrawal symptoms of morphine dependent rats. N-nitro-L-arginine can elevate the expression of immunoreactive dynorphin.

CONCLUSIONSChronic N-nitro-L-arginine administration can inhibit the development of morphine physical dependence in a manner of dose-dependence, which is significantly related to its role of regulating the endogeneous dynorphin system.

Animals ; Dose-Response Relationship, Drug ; Dynorphins ; physiology ; Male ; Morphine Dependence ; prevention & control ; Nitroarginine ; pharmacokinetics ; pharmacology ; Rats ; Rats, Sprague-Dawley

Although hypoxic pulmonary vasoconstriction (HPV) has been recognized by many researchers, the precise mechanism remains unknown. As isolated pulmonary arteries will constrict in vitro in the response to hypoxia, the oxygen sensor/transduction mechanism must reside in the pulmonary arterial smooth muscle or in the endothelium, or in both. Unfortunately, much of the current evidence is conflicting, especially as to the dependency of HPV on the endothelium and the role of a K+ channel. Therefore, this experiment was attempted to clarify the dependency of HPV on the endothelium and the role of a K+ channel on HPV in rat pulmonary artery. The effects of hypoxia were investigated in isolated main pulmonary arteries precontracted with norepinephrine. Vascular rings were suspended for isometric tension recording in an organ chamber filled with a Krebs-Henseleit solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and this was maintained for 20 min. Hypoxia elicited a vasoconstriction in arteries with endothelium. Mechanical disruption of the endothelium abolished HPV. There was no difference between the amplitude of the HPV induced by two consecutive hypoxic challenges and the effect of normoxic and hyperoxic control Krebs-Henseleit solution on a subsequent response to hypoxia. Inhibition of NO synthesis by treatment with N(omega)-nitro-L-arginine reduced HPV, but inhibition of a cyclooxygenase pathway by treatment with indomethacin had no effect on HPV. Blockades of a tetraetylammonium chloride-sensitive K+ channel abolished HPV. Verapamil, a Ca2+ entry blocker reduced HPV. In conclusion, these results suggest that HPV was dependent on the endothelium and that HPV can be considered to be induced by inhibition of the mechanisms of NO-dependent vasodilation such as the opening of a K+ channels.
Animal ; Anoxia/physiopathology* ; Blood Vessels/physiopathology ; Calcium Channel Blockers/pharmacology ; Cyclooxygenase Inhibitors/pharmacology ; Enzyme Inhibitors/pharmacology ; Indomethacin/pharmacology ; Nitroarginine/pharmacology ; Pulmonary Circulation/physiology* ; Pulmonary Circulation/drug effects ; Rats ; Tetraethylammonium/pharmacology ; Vasoconstriction/physiology* ; Vasoconstriction/drug effects ; Verapamil/pharmacology

OBJECTIVETo observe the effects of nitric oxide(NO) on the DNA ploidy of germ cells and to evaluate the role of NO in modulating spermatogenesis by using SNP, a donor of NO and N-nitro-l-arginine-mythel-ester(L-NAME), an inhibitor of nitric oxide synthese(NOS) in rats physically in vivo.

METHODSForty adult male, Sprague-Dawley rats (60-70 days) were divided into four groups, and injected ultraperitoneally with one of the following agents (once a day, for 12 days): SNP, L-NAME and SNP + L-NAME with normal saline. Two hours after the last injection the rats were sacrificed. The sera were collected and stored at -70 degrees C for subsequent hormone assay. The concentration of serum testosterone was measured by radioimmunoassay. Serum NOx- (nitrite/nitrate) concentration was measured by Greiss method. DNA of spermatogenic cells was detected by flow cytometry(FCM), and the percentage of 1c, 2c and 4c germ cells calculated.

RESULTSIn the SNP treatment group, the serum concentration of NOx- was higher, testosterone concentration was lower and the number of 1c cells was smaller compared with the control group. However, in rats treated with L-NAME, the concentration of NOx- was significantly lower, testosterone concentration was higher and the number of 1c cells was larger compared with the control group(P < 0.01). No changes were observed in the SNP + L-NAME group.

CONCLUSIONEnhancing ectogenous NO will suppress spermatogenesis while inhibiting NO productive pathway will promote it.

Animals ; DNA ; analysis ; Flow Cytometry ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; physiology ; Nitroprusside ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis ; drug effects

The role of nitric oxide during neonatal sepsis is complex. We tested the hypothesis that nonselective inhibition of nitric oxide synthase with N(w) -nitro-L-arginine methyl ester (L-NAME) is detrimental during the early phase of experimental sepsis in the newborn piglet. Newborn piglets were divided into four groups: 6 in the control group, 6 in the L-NAME control group, 12 in the sepsis group (SG), and 11 in the sepsis with L-NAME group (NS). Sepsis was induced by intravenous injection of 10(8) colony forming units of Escherichia coli. L-NAME 10 mg/kg was given intravenously 60 min before the induction of sepsis. The survival rate of piglets after 4 hr was 27% in NS, while it was 100% in other groups. Systemic hypotension, observed in both SG and NS, were more profound in NS. Leukopenia was observed in both SG and NS. Thrombocytopenia, prolongation of prothrombin time and activated partial thromboplastin time, and increase in thrombin-antithrombin complexes were observed only in NS. Decreased PaO2 /FiO2 ratio, arterial pH and base excess, and increased blood lactate levels observed in both SG and NS, but were more profound in NS. These findings suggest that nonselective inhibition of nitric oxide synthase with L-NAME is detrimental during the early phase of experimental neonatal sepsis.
Animals ; Animals, Newborn ; Enzyme Inhibitors/pharmacology ; Escherichia coli/*metabolism ; NG-Nitroarginine Methyl Ester/*pharmacology ; Sepsis/*drug therapy ; Support, Non-U.S. Gov't ; Swine ; Time Factors

The aim of the present study is to explore the interaction of nitric oxide (NO) and nicotinic acetylcholine receptor (nAChR) on learning and memory of rats. Rats were intracerebroventricularly (i.c.v.) injected with L-arginine (L-Arg, the NO precursor) (L-Arg group) or choline chloride (CC, an agonist of α7nAChR) (CC group), and with combined injection of L-Arg and CC (L-Arg+CC group), and methyllycaconitine (MLA, α7nAChR antagonist) or N(ω)-nitro-L-arginine methylester (L-NAME, nitric oxide synthase inhibitor) i.c.v. injected first and followed by administration of L-Arg combined with CC (MLA+L-Arg+CC group or L-NAME+L-Arg+CC group), respectively, and normal saline was used as control (NS group). The learning and memory ability of rats was tested with Y-maze; the level of NO and the expressions of neuronal nitric oxide synthase (nNOS) or α7nAChR in hippocampus were measured by NO assay kit, immunohistochemistry or Western blot. The results showed that compared with L-Arg group or CC group, the rats' learning and memory behavioral ability in Y-maze was observably enhanced and the level of NO, the optical density of nNOS-like immunoreactivity (LI) or α7nAChR-LI in hippocampus were significantly increased in L-Arg+CC group; Compared with L-Arg+CC group, the ability of learning and memory and the level of NO as well as the expressions of nNOS-LI or α7nAChR-LI were obviously decreased in MLA+L-Arg+CC group or in L-NAME+L-Arg+CC group. In conclusion, i.c.v. administration of L-Arg combined with CC significantly improved the action of the L-Arg or CC on the content of NO and the nNOS or α7nAChR expressions in hippocampus along with the learning and memory behavior of rats; when nNOS or α7nAChR was interrupted in advance, the effects of L-Arg combined with CC were also suppressed. The results suggest that there are probably synergistic effects between NO and nAChR on learning and memory.
Animals ; Enzyme Inhibitors ; pharmacology ; Hippocampus ; physiology ; Learning ; Memory ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; physiology ; Nitric Oxide Synthase Type I ; physiology ; Rats ; alpha7 Nicotinic Acetylcholine Receptor ; physiology

Mastoparan is an amphiphilic tetradecapeptide derived from wasp venom which activates G-proteins. Several secondary effects have been attributed to this peptide, including activation of phospholipase and phosphatidylinositol kinase. The aim of the present study was to investigate the effects of mastoparan on vascular contractility. Rabbit aortic rings were cut and mounted on a force transducer to record isometric tension on a polygraph. The effects of mastoparan were then investigated on the contractile responses in the isolated rabbit aorta with or without endothelium. The results were summarized as follows; 1. Mastoparan caused biphasic response, a transient relaxation followed by a further contraction, in norepinephrine (NE)-precontracted ring with endothelium. These effects were not observed in the aorta in the absence of endothelium. 2. Mastoparan-induced transient relaxation was significantly inhibited by treatment with a N-omega-nitro-L-arginine or methylene blue. 3. When an inhibitor of phospholipase C, neomycin was added to the precontracted aortic ring with NE, the transient relaxation induced by mastoparan was inhibited, but sustained contraction was not inhibited. 4. When an inhibitor of phospholipase A2, quinacrine and inhibitor of the cyclooxygenase pathway, indomethacin, were added to a precontracted ring with NE, the transient relaxation induced by mastoparan was not inhibited, but sustained contraction was inhibited. 5. Mastoparan induced a contraction of the aorta either with or without endothelium. Indomethacin and nifedipine inhibited mastoparan-induced contraction. From the above results, we concluded that mastoparan acts on the endothelium and modifies the release of endothelium-derived relaxing factors such as nitric oxide and also endothelium-derived contracting factors such as metabolites of arachidonic acid.
Animal ; Aorta/*drug effects/physiology ; Arginine/analogs & derivatives/pharmacology ; Calcium/metabolism ; In Vitro ; Indomethacin/pharmacology ; Neomycin/pharmacology ; Nitroarginine ; Quinacrine/pharmacology ; Rabbits ; Support, Non-U.S. Gov't ; Vasoconstriction/*drug effects ; Wasp Venoms/*pharmacology