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BACKGROUND/AIMS: Myenteric plexus interstitial cells of Cajal (ICC-MY) are involved in the generation of gut pacemaker activity and neuronal communication. We performed patch clamp on ICC-MY in situ to observe the changes of pacemaker activity in response to neural modulations. METHODS: A fresh longitudinal muscle with myenteric plexus (LMMP) from mouse jejunum was prepared. ICC-MY and ganglion neurons embedded in the layer of longitudinal muscles were targeted by patch clamping in whole-cell configuration in a model of current or voltage clamp. Neurogenic modulators were applied to evaluate their effects on ICC pacemaker activity. RESULTS: In situ ICC-MY showed spontaneous and rhythmical voltage oscillations with a frequency of 27.2 ± 3.9 cycles/min, amplitude of 32.6 ± 6.3 mV, and resting membrane potential of −62.2 ± 2.8 mV. In situ neurons showed electrically evocable action potential in single or multiple spikes. Pacemaker activity was modulated by neuronal activators through receiving a neuronal input. Application of tetrodotoxin depolarized pacemaker potentials in a dose dependent manner, and decreased the amplitude at tetrodotoxin 0.3 μM for about 40 ± 10%; capsaicin (1 μM) ameliorated ICC-MY K+ current for about 49 ± 14.8%; and, nitric oxide hyperpolarized pacemaker potential and decreased the amplitude and frequency. CONCLUSIONS: The in situ preparation patch clamp study further demonstrates that the pacemaker activity is an intrinsic property of ICC. The neurogenic activators change and shape pacemaker potential and activity in situ. LMMP preparation in situ patch clamp provides an ideal platform to study the functional innervation of the ICC and the enteric neural system, thereby, for evaluating the neural regulation of pacemaker activity, especially in disorder models.
Subject(s)
Animals , Mice , Action Potentials , Capsaicin , Constriction , Enteric Nervous System , Ganglion Cysts , Interstitial Cells of Cajal , Jejunum , Membrane Potentials , Muscles , Myenteric Plexus , Neurons , Nitric Oxide , TetrodotoxinABSTRACT
Objective To investigate the relationship of the beneficial effect of CsA on vascular reactivity to Rho-kinase,protein kinase C ( PKC) and mitochondrial permeability transition pore ( MPTP) in traumatic hemorrhagic shock rats. Methods With traumatic hemor-rhagic shock rats and hypoxia-treated vascular smooth muscle cells ( VSMCs) ,the role of Rho-kinase and PKC in CsA-regulating vascular re-activity following shock and their relationship to MPTP was observed. Meanwhile,the effects of CsA on inflammatory mediators including TNF-α, IL-1β and IL-6 were also studied. Results CsA significantly improved the vascular reactivity of superior mesenteric artery following hemor-rhagic shock. Rho-kinase inhibitor Y27632 significantly antagonized CsA-induced increase of vascular reactivity,while PKC inhibitor stauros-porine had no significant influences on the effects of CsA. Further studies showed that CsA and Rho-kinase agonist U46619 inhibited the o-pening of MPTP in hypoxia-treated VSMCs. In addition,shock induced a significant increase of TNF-αand IL-1β,but CsA did not show a sig-nificant inhibitory effect on their level. Conclusion CsA-induced restoration of vascular reactivity following traumatic hemorrhagic shock via inhibiting MPTP opening,and Rho-kinase participate in this process.
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Objective To detect the mutagenicity of sulfur mustard in the tk gene of L5178Y3.7.2c tk +/- mouse lymphoma cells. Methods The plating efficiency and the mutant frequency were detected by the microtiter procedure. Results Sulfur mustard induced tk locus mutation with mutation frequency 2-15 times higher than that of spontaneous mutation frequency of L5178Y3.7.2c tk +/- cells. There were two different phenotypes of mutation colonies induced, which were large and small colonies, but most of them were small colonies. Conclusion Sulfur mustard is characterized by marked mutagenic activity and cytotoxicity. The compound may result in large range of DNA damage.
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Objective To investigate the effects of anticholinergic antidote and rhodosin on the brain injury induced by soman intoxication combined with hypobaric hypoxia in rats. Methods A total of 72 Wistar rats were divided into 4 groups: hypoxia control (HC), hypoxia plus soman (HS), hypoxia plus soman plus anticholinergic antidote (HSAA), and hypoxia plus soman plus anticholinergic antidote plus rhodosin (HSAAR). The animals after soman intoxication (72 ?g/kg) were placed in a hypobaric (62 kPa) apparatus for hypoxic exposure for 48 h. Rats were sacrificed for brain tissue detachment at the time points of 12, 24, and 48 h. Evans blue (EB) content and PLA 2 activity were detected biochemically. CaM concentration was determined by radioimmuno assay. Results Compared with the rats in HC, soman induced significant increases of brain EB, PLA 2, and CaM at 12, 24, and 48 h in HS. Elevated EB, PLA 2, and CaM induced by hypoxia and soman intoxication in rats in group HSAA were obviously attenuated by anticholinergic antidote. More significant decreases of brain EB, PLA 2, and CaM were found in rats in group HSAA. Conclusion Both anticholinergic antidote and anticholinergic antidote plus rhodosin have the preventive effect on rat brain damage induced by soman intoxication combined with hypoxia.
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Objective To investigate the roles of nitric oxide and caspase 3 in PC12 cell apoptosis induced by rotenone. Methods The acute injury effect of rotenone on PC12 cells and viability of the PC12 cells were detected by MTT assay. Nitrite (NO 2 -) was quantified by Greiss reaction. The activity of caspase 3 and the cell apoptosis were detected by fluoremetry and agarose gel electrophoresis, respectively. Results The MTT colorimetry indicated that PC12 cell viability reached a peak at 7 d after passage. L NAME at the concentration of 100 ?mol/L showed the strongest protective effect for PC12 cell in the four different doses. Rotenone also induced the formation of DNA ladder in PC12 cells. Additionally, the nitric oxide synthase inhibitor L NAME inhibited the activity of caspase 3 protease. L NAME also inhibited the production of nitric oxide induced by rotenone. Conclusion Nitric oxide and caspase 3 are involved in the process of PC12 cell apoptosis induced by rotenone.
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Objective To investigate the effect of soman on the Janus kinases (JAKs) expression in cell line PC 12 . Methods The PC 12 cell was used in these experiments and treated with soman at a concentration of 20 ?mol/L . RT PCR and Western blotting were employed to detect the mRNA and protein expressions of JAK1, JAK2 and JAK3 at the time points of 0, 6, 12 and 24 h. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of JAK1, JAK2 and JAK3 mRNAs and proteins increased at 2 h, reached the highest at 12 h and decreased at 24 h, but they were still higher than those of the control. It was shown that the sequences of amplification products by RT PCR were the same to corresponding ones in GenBank. Conclusion Soman intoxication enhances the expression of Janus kinases in PC 12 cells. JAKs genes may play an important role in brain injury due to soman intoxication.
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Objective To illustrate the features of soman induced signal transducers and activators of transcription (STATs) gene and protein expressions in cell line PC 12 . Methods The expression levels of STAT1, 3 and 5 mRNAs and protein in PC 12 cells were detected by semi quantitative RT PCR and Western blotting. PC 12 cells at 5～8 passages were randomly divided into 5 groups: control, intoxication groups for 2, 6, 12 and 24 h respectively. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of STAT1, 3 and 5 mRNAs and proteins increased in PC 12 cell at 2 h and reached the highest at 12 h, then decreased at 24 h, but they were still higher than those of the control. The sequences of amplification products by RT PCR were the same to those in GenBank. Conclusion Soman intoxication can enhance the expression of STATs in PC 12 cells. STAT genes may possibly play an important role in brain injury.
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Objective To investigate the changes of caspase 3 activity and the interventional effect of tetrapeptide AC DEVD CHO on sulfur mustard induced apoptosis of lymphocyte. Methods The activity of caspase 3 was detected by fluorospectrophotometry. DNA ladder and apoptosis were detected by DNA agarose gel electrophoresis and flow cytometry. Results Sulfur mustard enhanced the activity of caspase 3 and its polypeptide inhibitor (AC DEVD CHO) partially blocked apoptosis, which was mainly presented as an obscure phenomenon of DNA ladder and a decrease in percentage of apoptosis. Conclusion Sulfur mustard can induce the apoptosis of lymphocyte by means of activating caspase 3. The inhibitor may have an interventional effect on lymphocyte apoptosis induced by sulfur mustard.
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Objective To investigate the manner and molecular mechanism of lymphocyte cytotoxic action induced by sulfur mustard. Methods Lymphocytes were separated from the spleen of Wistar rats and were cultured. DNA strand breaks were detected by fluorospectrophotometry. RT PCR and Western blotting were employed to detect the gene and protein expressions of caspase 3 and its activation. Results Sulfur mustard induced DNA strand breaks in the lymphocytes. Gene and protein expressions of caspase 3 were increased in a time dependent manner. Conclusion Sulfur mustard induces lymphocyte cytotoxic action by means of depending on the expression and activation of caspase 3.
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Objective To observe the rotenone induced changes of nitric oxide (NO) concentration and nitric oxide synthase (NOS) activity in rat caudate nuclei and blood plasma. Methods Wistar rats were daily administrated with rotenone at the doses of 0.335, 0.670 and 1.005 mg/kg(sc). Animals were sacrificed on days 30, 60 and 90. The NO concentration and the NOS activity in the caudate nuclei and blood plasma were determined by biochemical methods. Results At each time point of days 30, 60 and 90, rotenone induced significant increases in NO concentration and NOS activity in both the caudate nuclei and blood plasma in dose and time dependent manners. Conclusion Long term exposure to rotenone leads to the increase of nitric oxide and nitric oxide synthase in brain tissues and blood plasma in rat.
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Objective To investigate the effects of hypoxia and sodium cyanide(NaCN)on oxidative stress in rabbit arterial blood.Methods An artificial hypobaric hypoxia chamber was used to simulate 4 000-meter high altitude.Twenty rabbits were randomly divided into 4 groups:hypoxia group with high-or low-dose,non-hypoxia with high-or low-dose.The animals in the non-hypoxia groups were operated under normal circumstances while those in hypoxia groups were subjected to chamber in low pressure for 72 h before receiving the hypoxia experiments.Femoral arterial cannulation was performed on all animals under anesthetization with pentobarbital sodium(30 mg/kg,iv)and NaCN(ip)at the doses of 1.5 mg/kg and 2 mg/kg.Blood samples were collected at 10 min before intoxication,and 5,10,15,20,30,60,120 and 180 min after intoxication and blood seperation was conducted.The activity of superoxide dismutase(SOD),contents of reduced glutathione(GSH)and malondialdehyde(MDA)were determined.Results The MDA content was significantly increased(P
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Objective To study the toxic effects of Rotenone on glutamate transporter and glutamine synthetase in rat brain.Methods The glutamate levels in the striatum of SD rats were detected by high performance liquid chromatography(HPLC),the expression of glutamate tansporter mRNA and proteins were detected by RT-PCR and Western blotting and the activity of glutamine synthetase was determined by using GS detect kit.Results Rotenone was shown to increase the release of glutamate in rat striatum,the expression of glutamate/aspartate transporter(GLAST)mRNA and protein decreased significantly in 0.6 mg/kg and 1.2 mg/kg Rotenone intoxicated groups,but the expression of GLT-1 and the activity of GS enhanced obviously.Conclusion Down-regulation of GLAST may be responsible for increased Glu level in rat brain induced by Rotenone,but the increased expression of GLT-1 and GS activity may represent a protective mechanism of the brain cells by limiting the neurotoxicity of Glu.
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Objective To study the changes of IL 2 and IL 6 levels and lymphocyte DNA damage in peripheral blood of dog intoxicated by sulfur mustard. Methods Chongqing dogs were subcutaneously injected with sulfur mustard at the dose of 16 mg/kg. Lymphocytes and serum were isolated from dog peripheral blood at different time points as designed. The IL 2 and IL 6 levels in blood serum were measured by radioimmunoassay and lymphocyte DNA damage represented as comet tail length was determined by single cell gel electrophoresis (SCGE). Results Four hours after administration of sulfur mustard, the serum IL 2 and IL 6 levels dogs injected with sulfur mustard decreased and reached the lowest level at 72 h. A significant recovery was found at the time point of 120 h. SCG revealed sulfur mustard induced DNA fragmentation (comet) in dog peripheral lymphocytes. The rate of the damaged lymphocytes and the degree of DNA fragment migration began to increase at 4 h and progressively increased from 24 h to 72 h after sulfur mustard treatment. Conclusion Sulfur mustard intoxication induces early and long lasting decreases in serum IL 2 and IL 6 levels and lymphocyte DNA fragmentation in dog peripheral blood.
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Objective To observe the changes of the concentration of Ca2+, contents of cAMP, CaM and activity of Ca2+/CaM-PK II in pheochromocytoma PC12 cells after combined soman and hypoxia injury. Methods The changes of ［Ca2+］, and activity of CaM, cAMP and Ca2+/CaM-PK II in PC12 cells were studied after combined soman and hypoxia injury with radioimmunoassay. Results The changes of ［Ca2+］, the contents of CaM, cAMP were significantly higher in hypoxic and soman intoxicated group than in soman intoxicated group and control group under hypoxia; but the activity of Ca2+/CaM-PK Ⅱ were significantly decreased. Conclusion ［Ca2+］, CaM, cAMP and Ca2+/CaM-PK Ⅱ exert important role in the damage of PC12 after combined soman and hypoxia injury.
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Objective To investigate the apoptotic effect induced by sulfur mustard on the lymphocytes of the rat spleen,and define the role of caspase-3 during the process.Methods Sulfur mustard was given by intraperitoneal injection in a dose of 3.5mg/kg.The animals were anesthetized and the spleens were harvested at different timepoints after intoxication.The histopathogy of spleen was studied with hematoxylin-eosin staining.Caspase-3 mRNA was detected with RT-PCR.Protein expression of caspase-3 was assayed with Western blotting,lymphocytes of rat spleen were isolated and cultured in vitro and they were challenged with sulfur mustard(100?mol/L).The effect of Ac-DEVD-CHO(a specific inhibitor of caspase-3)on sulfur mustard-induced apoptosis of the lymphocytes cultured in vitro was evaluated.Fluorescent probe labeled with Rhodamine 123 was used to study mitochondrial potential.Results The histology of rat spleen was affected after sulfur mustard intoxication,as evidenced by apoptosis of a part of lymphocytes.Protein and mRNA expressions of caspase-3 were increased significantly in the spleens of intoxicated rats as compared with that in control group.DNA ladder and 'sub-G1' peak of lymphocytes which were treated with sulfur mustard in vitro were partially improved by Ac-DEVD-CHO(the specific inhibitor of caspase-3).In addition,mitochondrial potential decreased in a time-dependent manner in the lymphocytes intoxicated by sulfur mustard in vitro compared with that in the control group.Conclusions The spleen is injured in the rat which is intoxicated by sulfur mustard.Lymphocyte apoptosis is one of the mechanisms splenic injury.Caspase-3 may be involved in the process of lymphocyte apoptosis induced by sulfur mustard.
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Objective To study the toxic effects of rotenone on substantia nigra of rats. Methods Rotenone was given subcutaneously once a day during 28 days. Substantia nigra was dissected. Morphology of dopamine neuron, synapse and myelin sheath were observed. SOD and GSH-Px activity were assayed; MDA and GSH contents were measured. Results Autonomic activities of rats decreased, similar to Parkinson's Symptoms. Morphology of dopamine neuron changed, synapse structures were abnormal, synapse vesicals increased and myelin sheath degenerated. MDA content increased (P
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Effects of soman on lymphocyte proliferation are investigated in mice. 1 d after soman(154 ?g/kg,sc) poisoning B-lymphocyte reaction to lipopolysaccharide(LPS) is inhibited significantly and is persistent.T-cell proliferation response stimulated by concanavalin(Con A)has a 2-phase changeson the 1st day an increase followed by a dramatical reduction. Both the decreases of T-and B-lymphocyte mitogenic response and the primary increase of T-cell response show a close correlation with the doses of soman in this experiment. It has no relation to the change of the whole blood cholinesterase activity.
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Objective To investigate the effects of sodium cyanide (NaCN) and hemorrhagic shock on the level of serum oxidative stress in rabbits. Methods A total of 15 Japanese white rabbits were randomly divided into 5 groups: sham operation, hemorrhagic shock (HS), NaCN intoxation, HS+NaCN and HS+NaCN+4-dimethy laminophenol (4-DMAP). The rabbits were anesthetized for left arteria carotis communis cannulation, and connected with three-way tube and RM6240 electronic physiological signal recording system. The models of hemorrhagic shock and/or sodium cyanide intoxation (blood pressure was controlled at 5.3 kPa) were established. At 0.5, 1 and 2 h after the intraperitoneal injection of 2.5 mg/kg NaCN and/or 3.2 mg/kg 4-DMAP, the blood were drawn. The serum superoxide dismutases (SODs), content of malonaldehyde (MDA) and active oxygen (ROS) were detected by spectrophotometric method. Results After HS, NaCN intoxation, HS+NaCN, the SODs decreased significantly (P
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Objective To summarize our experience on the food safety for residents and disaster relief workers in Li county after Wenchuan earthquake in Sichuan province.Methods A comprehensive survey was conducted to assess the current food hygiene status.According to the survey results,we had integrated the local forces and strengthened the food hygiene surveillance and quality detection focusing on the crucial procedures.Besides,effective health educations were applied to advocate the rational dietary after earthquake.Results There was no any food safety incident in the county,and the hygiene awareness of local residents has been improved.Conclusion Powerful organization,focused management and multi-collaboration are the important elements to accomplish the food safety control after earthquake.
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Objective To observe the effects of rotenone on the mitochondrial membrane potential and cell cycles in pheochromocytoma (PC12) cells and to explore the injury mechanism of rotenone on dopaminergic neuron. Methods The mitochondrial membrane potential and cell cycles were determined by flow cytometry. Results After treatment with 5.0 ?mol/L rotenone for 12 and 24 h, the percentage of G 0/G 1 phase and S phase in cultured PC12 cells decreased gradually (P