Glutamate release through connexin 43 by cultured astrocytes in a stimulated hypertonicity model.

We investigated the role of connexin 43 (Cx43) hemichannels in the release of glutamate by astrocytes after hypertonic stimulus. Mechanical, osmotic and oxidative stress, and changes in the extracellular or intracellular Ca(2+) levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as glutamate, ATP, etc. In our past studies, we primarily found that acute hypertonic stimulus induced the release of glutamate. Since glutamate release was involved with several routes, we studied its release routes by astrocytes incubated in a hypertonic media for various periods. The glutamate release was increased after hypertonic stimulus. Glutamate release in hypertonic stimulus was inhibited by gap junction or Cx43 hemichannel blockers, but not by antagonists of purinergic receptor (P2XnR), glutamate transport inhibitors, intracellular Ca(2+) blockers, and pannexin 1(Panx1) hemichannel. The results suggest that glutamate release by the Cx43 hemichannels is likely to feature in the response of cultured astrocytes to hypertonic stimulus.

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells.

OBJECTIVE: To investigate whether hypertonic saline (HS) can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. METHODS: Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat (1 day). The astrocytes were randomly divided into five groups: isotonic (IS) and HS groups, astrocytes were incubated by IS and HS (320 mosM NaCl) medium, respectively, for 1, 3, 5, 10 or 15 min; carbenoxolone (CBX)+IS and CBX+HS groups, astrocytes were pre-treated with CBX (100 mmol/L) for 1 h at 37 degrees C in a 5% CO(2) / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca(2+)+HS group, astrocytes were pre-incubated with Ca Ca(2+) (1,000 micromol/L) for 1 h at 37 degrees C in a 5% CO(2) / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein (GFAP) and anti-glutamate. The C6 cells were divided into four groups: IS, HS, CBX+IS and CBX+HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry (FCM). RESULTS: (1) Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. (2) The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 min (P < 0.01 vs the 5 min of HS group). In CBX+HS group, the glutamate intensity was higher than that in CBX+IS and HS groups. (3) The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min (P< 0.01 vs 1 min and 3 min). On the contrary, the medium glutamate concentrations in the CBX+HS or Ca(2+)+HS group were significant lower than that in the HS group (P < 0.01). (4) FCM showed HS and CBX+HS induced glutamate increase in C6 cells. CONCLUSION: HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

[Changes in plasticity of rat hypothalamic neurons and astrocytes in humid and hot environment].

OBJECTIVE: To study the changes in the plasticity of the neurons and astrocytes in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus of rats exposed to a humid and hot environment. METHODS: The rats were subjected to stimulation with a humid and hot environment for 120 min in a climate chamber (dry bulb temperature of 40.0-/+0.5 degrees C with relative humidity of 60-/+5%). During the exposure, the behavioral responses of the rats were observed, and the changes in the expressions of Fos and GFAP in the PVN and SON in response to the exposure evaluated using immunohistochemical ABC methods. RESULTS: Exposure to a humid and hot environment caused restlessness and agitation in the rats, which showed increased respiratory frequency and scratching of the face with the forelimbs. Two rats died after the 120-min exposure. Significantly increased expressions of Fos and GFAP were detected in the PVN and SON following the exposure as compared with the control group. CONCLUSION: The neurons and astrocytes in the PVN and SON both participate in the regulation of responses to exposure to a humid and hot environment.

Electro-acupuncture of Tsusanli and Shangchuhsu regulates gastric activity possibly through mediation of the vagus-solotary complex.

BACKGROUND/AIMS: Acupuncture has been reportedly used to treat gastrointestinal diseases, however, its precise mechanism remains unknown. METHODOLOGY: In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on regulation of gastric activity were observed. RESULTS: EA at Tsusanli showed that gastric electric change was the most obvious, with significantly higher frequency and wave amplitude compared with that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated that the change of gastric electric level was much higher than that of the non-acupoint group and control group. After bilateral vagotomy, Tsusanlis was electro-acupunctured, the changes of electro-gastric graph (EGG) weren't significant with the control group. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in the Tsusanli group was markedly increased compared with that in the other group. Fos and GFAP expression in NTS and DMV in the Tsusanli group was significantly higher than that in other groups and control group. The results have indicated that EA at Tsusanli and Shangchuhsu cannot only regulate gastric activity, but also activate neurons and astrocytes in NTS and DMV. The effects on regulation and activation of EA at Tsusanli were very obvious. CONCLUSIONS: Our study suggests that this electroacupuncture regulation of gastric activity may partially depend upon integrated nerve pathway and related central neurons and astrocytes in the vagus-solitary complex.

[Influence of intrathecal injection of fluorocitrate on the protective effect of electroacupuncture on gastric mucosa in high humid heat stress rats].

OBJECTIVE: To study if the spinal glial cells involve in the protective effect of acupuncture on gastric mucosa in high humid heat stress rats. METHODS: Thirty-six male SD rats were randomized into 6 groups: control, stress model, electroacupuncture (EA), Fluorocitrate(FCA, intrathecal injection of FCA. 1 microL, 60 min before humid heat stress), EA+ normal saline (NS, intrathecal injection of NS) and EA+ FCA groups. Stress model was established by putting the rats in a container with higher temperature and higher humidity [(40.0 +/- 0.5) degrees C, relative humidity (60 +/- 5)%] for 60 min. EA (50 Hz, intermittent waves, 2-5 V) was applied to bilateral "Zusanli" (ST 36) for 60 min. Using immunofluorescent methods, we observed glial fibrillary acidic protein (GFAP) and microglia OX42 immunoreactivity (OD value) in the tissue of the lumbar enlargement segment of the spinal cord. We evaluated and recorded the damage index (DI) of gastric mucosa of rats according to Guth's method. RESULTS: There were clear dot-line-like hemorrhage foci with formation of ulcer in the gastric mucosa 60 minutes after high humid heat stimulation. Compared with model group, DI of gastric mucosa in EA and EA+ NS groups decreased significantly (P < 0.05, suggesting a protective effect of EA on the gastric mucosa under stress), OD values of EA and EA + NS groups increased considerably (P < 0.05). Comparison between EA and EA+ FCA groups showed that DI of EA + FCA group was higher than that of EA group, while the OD values of GFAP and OX42 in EA+ FCA group was markedly lower than those in EA group (P < 0.05), suggesting an inhibitory effect of FCA on the effects of EA in improving stress-induced damage of gastric mucosa and upregulation of GFAP and OX42 expression. CONCLUSION: EA at "Zusanli" (ST 36) can prevent the gastric mucosa from injury caused by high humid heat stimulation. The lumbar spinal glial cells may play a role in EA's protective function.

Electro-acupuncture of Foot YangMing regulates gastric activity possibly through mediation of the dorsal vagal complex.

Acupuncture at some specific acupoints of Foot Yangming can regulate gastric activity. However, its precise mechanism remains unknown. In our study, the effects and mechanism of electro-acupuncture (EA) at Tsusanli (ST 36), Shangchuhsu (ST 37) on the regulation of gastric activity were observed. EA at Tsusanli showed that gastric electric change had a significantly higher frequency and wave amplitude as compared to that of the Shangchuhsu group and other groups. EA at Shangchuhsu demonstrated the change of gastric electric was greater than that of the non-acupoint group and the control group. After bilateral vagotomy, the change of electro gastric graph (EGG) of EA at Tsusanlis was not significant compared to the control group. In the mean time, we have observed the electric discharge of the neurons in NTS and DMV. The frequency of electro-physiological activity in nucleus of solitary tract (NTS) and dorsal motor nucleus of the vagus nerve (DMV) in Tsusanli group and Shangchuhsu group were markedly increased compared with that in other groups. The results have indicated that EA at Tsusanli and Shangchuhsu not only regulate gastric activity, but also activate neurons in NTS and DMV significantly. Our study suggests that the effect of EA at Tsusanli and Shangchuhsu on the gastric activity may partially depend upon integrated nerve pathway and related central neurons in dorsal vagal complex.

[Influences of repeated lower +Gz exposures on high +Gz exposure induced brain injury in rats].

Objective. To explore the influence of repeated lower +Gz exposures on high +Gz exposure induced brain injury in rats. Method. Forty SD male rats were randomly divided into control group (5 rats), +10 Gz/5 min group (5 rats) +4 Gz exposure one time, three times and five times group (10 rats each group). After 1 d or 6 d of +4 Gz exposure each group were exposed to +10 Gz again. Three days after +10 Gz exposure the neuron damage was observed by light microscope in HE stained section. Result. There was no brain damage after repeated +4 Gz/3 min exposure 5 times so it was reasonable to use this exposure intensity as ischemia stimulation. +10 Gz/5 min exposure could result in irreversible neuron damage such as neuron degeneration and coagulation necrosis. The experiment results suggested that after +10 Gz/5 min exposure there were degenerated neurons in cortex, hippocampus and thalamus. The number of degenerated neurons were obviously decreased in cortex, hippocampus and thalamus when exposed to +10 Gz/5 min again after repeated +4 Gz/3 min 3-5 times. Conclusion. The degree of neuron damage was obviously slight at the time of exposure to +10 Gz/5 min again after repeated +4 Gz/3 min 3-5 times. The ischemia tolerance at the time of exposure to +Gz was similar to other brain ischemia.