Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP3 pathway.

Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca²âº concentration ([Ca²âº](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10⁻7 to 10⁻5 M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10⁻5 M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca²âº](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10⁻6 M) or CdCl2. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP3 receptor antagonist 2-APB (10⁻4 M) or the PLC inhibitor U73122 (10⁻5 M). OT (10⁻6 M) also increased the IP3 concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP3-Ca²âº signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.

Brain-derived neurotrophic factor exerts antinociceptive effects by reducing excitability of colon-projecting dorsal root ganglion neurons in the colorectal distention-evoked visceral pain model.

The mechanism underlying visceral pain is still largely unclear. Recently, much attention has focused on a potential modulatory role of brain-derived neurotrophic factor (BDNF) in visceral pain. In the present study, we investigated the expression of BDNF in dorsal root ganglia (DRG) primary sensory neurons and its role in a colorectal distention (CRD)-induced model of visceral pain. Results obtained from enzyme-linked immunosorbant assay (ELISA) revealed that BDNF protein was upregulated after CRD. An abdominal withdrawal reflex (AWR) assay confirmed that BDNF played an antinociceptive role in this model. Application of BDNF directly to DRG neurons decreased their hypersensitivity when evoked by CRD. Pretreatment with k252a partially blocked the effect of BDNF. These findings suggest that BDNF might be a novel analgesic agent for the treatment of visceral pain.

CCK mediated the inhibitory effect of oxytocin on the contraction of longitudinal muscle strips of duodenum in male rats.

The aim of the present study was to investigate the effect of oxytocin (OT) on duodenum motility in rats and the possibility that cholecystokinin (CCK) was involved in this process. The isometric contraction of longitudinal muscle strips of duodenum was monitored by polygraph. ELISA was used to measure the concentration of CCK and OT in duodenum. CCK mRNA was assayed by RT-PCR. Oxytocin receptor (OTR) and CCK in duodenum were located by immunohistochemistry and immunofluorescence staining. OT (10⁻5 and 10⁻6 M) inhibited the spontaneous contraction of the muscle strips. On the contrary, atosiban (OT receptor antagonist), lorglumide (CCK1 receptor antagonist), and tetrodotoxin (TTX, blocker of voltage-dependent Na(+) channel on nerve fiber) excited the contraction. The inhibitory effect of OT on duodenal motility was reversed by pretreatment of atosiban, lorglumide, or TTX. Exogenous OT did not influence the expression of OT mRNA in duodenum but increased the concentration of CCK in the culture medium of the cells isolated from longitudinal muscle myenteric plexus. The OTR and CCK were co-expressed in the neurons of the myenteric plexus in duodenum. We concluded that OT inhibited the contraction of the LD spontaneous contraction of rats in vitro. This effect was mediated by the CCK released from the neurons of the myenteric plexus in duodenum.

Blocking TRAIL-DR5 signaling with soluble DR5 reduces delayed neuronal damage after transient global cerebral ischemia.

Mechanisms underlying delayed selective neuronal death after global cerebral ischemia remain to be clarified. Here, we report a critical role for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in the pathogenesis of cerebral ischemia. C57BL/6j mice were subjected to transient global brain ischemia. RT-PCR and immunohistochemistry showed that the expression of TRAIL and DR5 was upregulated following transient ischemia-reperfusion. Dual immunofluorescence analysis indicated that TRAIL expression was significantly more pronounced in astrocytes and activated microglia/macrophages, whereas DR5 expression was more pronounced in neurons, which had a good correlation with the distribution of apoptotic cells. Treatment with soluble DR5 reduced ischemic cell death after transient global ischemia through blocking the interaction of endogenous TRAIL with DR5. These results indicate that TRAIL plays a deleterious role in the pathogenesis of delayed neuronal damage after global cerebral ischemia and inhibition of TRAIL function in the brain may represent a novel neuroprotective strategy to treat ischemic stroke.

Protection by 3'-methoxypuerarin of rat hippocampal neurons against ischemia/reperfusion injury.

3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified four-vessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P < 0.001) and markedly reduced the number of apoptotic pyramidal neurons (P < 0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.

Diazoxide reverses the enhanced expression of KATP subunits in cholinergic neurons caused by exposure to Aß1₋42.

The ATP-sensitive potassium channel (KATP) play a crucial role in coupling metabolic energy to the cell membrane potential, ß-amyloid peptide (Aß) neurotoxicity has been associated with cellular oxidative stress and metabolic impairment. Whether there is an interaction between KATP and Aß or not? The expression of KATP subunits was to be investigated after the cultured primary rat basal forebrain cholinergic neurons being exposed to Aß1₋42. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing change was observed by double Immunofluorescence and immunoblotting in cultured cholinergic neurons from different groups: treatment with Aß1₋42 (group Aß1₋42), pretreatment with diazoxide and then exposure to Aß1₋42 (group diazoxide + Aß1₋42), and the control (group control). The results showed that in response to the treatment with Aß1₋42 (2 µmol/L) for 24 h, the expression of Kir6.1 and SUR2 were significantly up-regulated, while this change can be partly reversed by pretreatment with diazoxide (1 mmol/L) for 1 h. There were significant increases in all KATP subunits expression levels after exposure to Aß1₋42 for 72 h. However, the up-regulation of Kir6.1, Kir6.2 and SUR2 except SUR1 can be partly reversed by pretreatment with diazoxide (1 mmol/L) for 1 h. It is concluded that exposure to Aß1₋42 for different time (24 and 72 h) induced differential regulation of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to the dysfunction of KATP and membrane excitability disturbance. The effect of diazoxide on KATP subunits expression may explain, in part, the resistance of diazoxide to the toxicity of Aß1₋42.

Inhibition of caspase-9 activation and apoptosis is involved in ischemic preconditioning-induced neuroprotection in rat brain.

OBJECTIVES: Cerebral ischemic pre-conditioning (IPC) is capable of protecting hippocampal neurons from ischemia/reperfusion (I/R) injury. In the current study, we investigated the role of activated caspase-9 in the protective process induced by IPC and related it to cytochrome c release and apoptosis. METHODS: I/R injury was induced by a four-vessel occlusion model in Wistar rats which were randomly divided into ischemia/reperfusion group (I/R), ischemic pre-conditioning + I/R group (IPC + I/R) and control group. Histologic changes in the pyramidal layer of the hippocampal CA1 region were determined by hematoxylin and eosin (H&E) staining. The relative proportion of apoptotic neurons in this area was assessed with TUNEL staining. The redistribution of cytochrome c and activation of caspase-9 were detected in the same area with immunohistochemistry and Western blotting respectively. RESULTS: Compared to the I/R group, IPC increased the number of surviving neurons in the hippocampal CA1 region (p<0.001), markedly reduced the number of apoptotic pyramidal neurons (p<0.001), inhibited the release of cytochrome c from mitochondria to cytoplasm (p<0.001 for positively stained neurons) and decreased the amount of activated caspase-9 (p<0.001). DISCUSSION: These findings confirm that IPC is capable of protecting neurons from injury by apoptosis. The release of cytochrome c to the cytosol demonstrates that the mitochondrial pathway was involved, and the reduction in this release caused by IPC was clearly associated with reduced caspase-9 activation. Together, these results suggest that IPC protects neurons via action on the mitochondrial/caspase-9 pathway of apoptosis.

Ghrelin reduces injury of hippocampal neurons in a rat model of cerebral ischemia/reperfusion.

Ghrelin, an acyl-peptide gastric hormone and an endogenous ligand for growth hormone secretagogue (GHS) receptor 1a (GHS-R 1a) exerts multiple functions. It has been reported that synthetic GHS-hexarelin reduces injury of cerebral cortex and hippocampus after brain hypoxia-ischemia in neonatal rats. However, the effect of ghrelin in tolerance of the brain tissues to cerebral ischemia/reperfusion (I/R) injury has not been studied. The aim of the present study was to examine whether ghrelin have potential protective effect on hippocampal neurons of rats against I/R injury. I/R injury was induced by a modified four-vessel occlusion model. Ghrelin was administered intraperitoneally after the insult. Histological damage of the neurons was determined with hematoxylin-eosin (H&E) staining and assay of the neuronal apoptosis was performed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). The results showed that I/R decreased the number of surviving neurons and induced apoptosis of the neurons in CA1 area of the hippocampus in rats. In contrast, administration of ghrelin significantly increased the number of surviving neurons and reduced the number of TUNEL-positive apoptotic neurons in the equivalent areas after I/R. In conclusion, the present data provide evidence for the first time that ghrelin can exert a neuroprotective role in vivo in the tolerance of hippocampal neurons to I/R injury, and that the mechanism underlying this effect involves an anti-apoptotic property of ghrelin.

Sexual differences of the inhibitory effect of ethanol on gastrointestinal motility: in vivo and in vitro studies.

Female brain is more sensitive to the acute exposure of ethanol. This study aimed to investigate the sexual difference of the ethanol-induced inhibition of gastrointestinal motility. Wistar rats were fasted and allowed drinking water only 12 - 18 h before the experiments. In the in vivo experiments, by using an oral radiochromium motility marker, the liquid gastric emptying and intestinal transit were [corrected] measured 30 min after ethanol treatment. In the in vitro study, strips of stomach and duodenum smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were also examined. Systemic administration of ethanol (2 g/kg, i.p.) significantly inhibited the gastric emptying and intestinal transit, and the effect on female rats turned out to be greater than that on the male rats (P < 0.05). In an in vitro study, ethanol (0.38 x 10(-3) M - 1.34 x 10(-3) M) inhibited the motility of gastric antrum and duodenum in rats of both sexes, but there was no sexual difference in the inhibitory effect of ethanol on muscle strips. We concluded that sexual difference of the ethanol-induced inhibition of gastrointestinal motility was not resulted from the smooth muscle itself.

The antinociception of oxytocin on colonic hypersensitivity in rats was mediated by inhibition of mast cell degranulation via Ca(2+)-NOS pathway.

This study was conducted to investigate the effects of oxytocin (OT) on visceral hypersensitivity/pain and mast cell degranulation and the underlying mechanisms. We found that oxytocin receptor (OTR) was expressed in colonic mast cells in humans and rats, as well as in human mast cell line-1 (HMC-1), rat basophilic leukemia cell line (RBL-2H3) and mouse mastocytoma cell line (P815). OT decreased 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, colonic mast cell degranulation and histamine release after mast cell degranulation in rats. Also, OT attenuated the compound 48/80 (C48/80)-evoked histamine release in P815 cells and inward currents, responsible for the mast cell degranulation, in HMC-1, RBL-2H3 and P815 cells. Moreover, these protective effects of OT against visceral hypersensitivity and mast cell degranulation were eliminated by coadministration of OTR antagonist atosiban or a nonselective inhibitor of nitric oxide synthase (NOS), NG-Methyl-L-arginine acetate salt (L-NMMA). Notably, OT evoked a concentration-dependent increase of intracellular Ca(2+) in HMC-1, RBL-2H3 and P815 cells, which was responsible for the activation of neuronal NOS (NOS1) and endothelial NOS (NOS3). Our findings strongly suggest that OT might exert the antinociception on colonic hypersensitivity through inhibition of mast cell degranulation via Ca(2+)-NOS pathway.

[Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine].

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.

Effect of oxytocin on contraction of rabbit proximal colon in vitro.

AIM: To investigate the effects of oxytocin (OT) on isolated rabbit proximal colon and its mechanism. METHODS: Both longitudinal muscle (LM) and circular muscle (CM) were suspended in a tissue chamber containing 5 mL Krebs solution (37 degrees ), bubbled continuously with 950 mL x L(-1) O(2) and 50 mL x L(-1) CO(2). Isometric spontaneous contractile responses to oxytocin or other drugs were recorded in circular and longitudinal muscle strips. RESULTS: OT (0.1 U x L(-1)) failed to elicit significant effects on the contractile activity of proximal colonic smooth muscle strips (P>0.05). OT (1 to 10 U x L(-1)) decreased the mean contractile amplitude and the contractile frequency of CM and LM. Hexamethonium (10 micromol x L(-1)) partly blocked the inhibition of oxytocin (1 U x L(-1)) on the contractile frenquency of CM. N(omega))-nitro-L-arginine-methylester (L-NAME, 1 micromol x L (-1)), progesterone (32 micromol x L(-1)) and estrogen (2.6 micromol x L(-1)) had no effects on OT-induced responses. CONCLUSION: OT inhibits the motility of proximal colon in rabbits. The action is partly relevant with N receptor, but irrelevant with that of NO, progesterone or estrogen.

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits.

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.

Chloroquine impairs visual transduction via modulation of acid sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are extracellular pH sensors activated by protons, which influence retinal activity and phototransduction. Among all ASICs, ASIC1a is abundantly expressed in the retina and involved in normal retinal activity. Chloroquine, which has been used in the treatment of malaria, rheumatoid arthritis and systemic lupus erythematosus, has been shown to be toxic to the retina. However, the underlying mechanisms remain unclear. In this study, we investigated the role of chloroquine in phototransduction by measuring the electroretinogram (ERG). The effect of chloroquine on acid-evoked currents in either isolated rat retinal ganglion neurons (RGNs) or Chinese hamster ovary (CHO) cells transfected with ASIC1a were assessed using a whole-cell patch-clamp technique. Chloroquine reduced the b-wave of scotopic 0.01 and photopic 3.0 and amplitudes of oscillatory potentials (OPs), an effect which was almost completely reversed by PcTx1, an ASIC1a-specific channel blocker. Further, patch-clamp experiments demonstrated that chloroquine reduced the peak current amplitude and prolonged the activation and desensitization of ASIC1a currents. These chloroquine-induced effects on the kinetics of ASIC 1a were dose-, pH- and Ca(2+)-dependent. Taken together, these results demonstrate that chloroquine affects vision conduction by directly modifying the kinetics of ASIC1a. Such a mechanism, may, in part, explain the retinal toxicity of chloroquine.

H2 S modulates duodenal motility in male rats via activating TRPV1 and K(ATP) channels.

BACKGROUND AND PURPOSE: H2 S induces vasodilatation by opening KATP channels but it may also affect other ion channels. The aim of this study was to investigate the effect of H2 S on intestinal motility in rats and its underlying mechanism. EXPERIMENTAL APPROACH: The tension of intestinal muscle strips, afferent firing of intestinal mesenteric nerves, length of duodenal smooth muscle cells and whole-cell membrane potential of dorsal root ganglion (DRG) neurons were monitored. H2 S-producing enzymes were located by immunofluorescence staining. KEY RESULTS: NaHS exerted early transient excitation and late long-lasting inhibition on the intestinal contraction. The excitation was attenuated by TRPV1 antagonists capsazepine, A784168, SB-366791 and NK1 receptor antagonist L703606, while the inhibition was attenuated by glibenclamide. NaHS increased duodenal afferent nerve firing and depolarized DRG neurons. These effects were reduced by capsazepine and A784168. NaHS relaxed isolated duodenal smooth muscle cells. The KATP channels were expressed in smooth muscle cells. Cystathionine ß-synthase and cystathionine γ-lyase were expressed in rat duodenal myenteric neurons. L-cysteine and S-adenosyl-L-methionine increased the contraction of duodenal muscle strips, an effect attenuated by capsazepine and L703606. CONCLUSIONS AND IMPLICATIONS: NaHS induces biphasic effects on intestinal motility in rats while endogenous H2 S only exerts an excitatory effect. This transient excitatory effect might be mediated by activation of TRPV1 channels in sensory nerve terminals with the consequent release of substance P. The long-lasting inhibitory effect might be mediated by activation of KATP channels in the smooth muscle cells. These findings reveal a novel mechanism for the excitatory effect of H2 S on gastrointestinal motility.

Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats.

Enhanced neurogenesis in the dentate gyrus of the hippocampus following seizure activity, especially status epilepticus, is associated with ectopic residence and aberrant integration of newborn granule cells. Hilar ectopic granule cells may be detrimental to the stability of dentate circuitry by means of their electrophysiological properties and synaptic connectivity. We hypothesized that status epilepticus also increases ectopic granule cells in the molecular layer. Status epilepticus was induced in male Sprague-Dawley rats by intraperitoneal injection of pilocarpine. Immunostaining showed that many doublecortin-positive cells were present in the molecular layer and the hilus 7 days after the induction of status epilepticus. At least 10 weeks after status epilepticus, the estimated number of cells positive for both prospero homeobox protein 1 and neuron-specific nuclear protein in the hilus was significantly increased. A similar trend was also found in the molecular layer. These findings indicate that status epilepticus can increase the numbers of mature and ectopic newborn granule cells in the molecular layer.

Diazoxide and cyclosporin A protect primary cholinergic neurons against beta-amyloid (1-42)-induced cytotoxicity.

OBJECTIVE: Activation of mitochondrial (MitoKATP) channels was found to protect against anoxic and chemical stress in brain. This present study sought to investigate the ability of diazoxide and cyclosporin A to antagonize cytotoxicity induced by beta-amyloid peptide (A-beta1-42) in cultured rat primary basal forebrain cholinergic neurons. METHODS: Cytotoxicity was induced by A-beta1-42 (2 µM) in the presence of either diazoxide (500 µM), a selective opener of the mitochondrial ATP-sensitive potassium channel (MitoKATP), or cyclosporin A (20 µM), an inhibitor of the mitochondrial permeability transition pore (MTP), or the combination of both the reagents. We determined cell morphology and cell viability using MTT assay and expression levels of anti-apoptotic protein (Bcl-2), pro-apoptotic proteins (Bax, cytochrome C, caspase-3 and cleaved caspase-3) using Western blotting at 24 hours and 72 hours. RESULTS: Cell viability decreased markedly after exposure to A-beta1-42 for 72 hours with a decrease in the expression of Bcl-2 protein and cytochrome C and an increase in the caspase-3 and cleaved caspase-3 levels. Both diazoxide and cyclosporin A exerted significant protective effects on cell viability by ameliorating the decrease in Bcl-2 and the increase in cytochrome c and caspase-3 activity induced by A-beta1-42. The combination of both the reagents had a greater protective effect than either one alone. CONCLUSIONS: The present research demonstrates that activation of MitoKATP channels independently or in combination with inhibitors of the MTP can elicit a protective effect against primary cholinergic neuron cytotoxicity induced by A-beta1-42. These findings suggest new mitochondrial targets for the development of therapeutic agents against A-beta-induced cytotoxicity.

Long-term downregulation of protease-activated receptor-2 expression in distal colon in rats following bacillary dysentery.

The aim of this study was to determine changes of PAR-2 expression in distal colon and the sensitivity of colonic muscle to SLIGRL-NH2, the PAR-2-activating peptide (PAR-2-AP) following bacillary dysentery. Shigella flexneri was administrated intragastrically in healthy male rats to induce bacillary dysentery. The effect of SLIGRL-NH2 on the motility of colonic muscle strips were examined. The expression of PAR-2 was determined by immunohistochemistry and Western blotting. Intragastric administration of S.flexneri induced acute inflammation at the mucosa of the distal colon at 4-11 days, and the inflammation disappeared 18 days later. PAR-2-AP-induced TTX insensitive relaxation of the colonic muscle strips. This inhibitory effect on colonic circular muscle strips was reduced on days 11-35, but the carbachol-induced contraction did not change. PAR-2 was located at the colon smooth muscles cells and myenteric nerve plexus. The amount of PAR-2 expression in distal colon was down regulated on days 11-35. These data indicated that bacillary dysentery exerted a long-term downregulation on the expression of PAR-2 in distal colon. This might be the reason of the low sensitivity of the colon circular muscle strips to the PAR-2-AP-induced relaxation following intragastric administration of S.flexneri.

V1 receptor in ENS mediates the excitatory effect of vasopressin on circular muscle strips of gastric body in vitro in rats.

The purpose of this study was to localize vasopressin (VP) V(1a) receptor in stomach and to characterize the role of VP in the regulation of gastric motility in rats. Double staining was used to locate the V(1a) receptor in the gastric body of the rat. The contraction of the circular muscle strips of gastric body was monitored by a polygraph. V(1a) receptor was expressed on the neurons of myenteric plexus of the gastric body. VP (10(-10)-10(-6) M) caused a concentration-dependent contractile effect on the circular muscle strips of gastric body in vitro. V-1880 ([deamino-Pen(1), O-Me-Tyr(2), Arg(8)]-Vasopressin, 10(-7) M), a V(1) receptor antagonist, inhibited the spontaneous contraction of the strips. Tetradotoxin (TTX, 10(-6) M) and V-1880 (10(-7) M) abolished the excitatory effect of VP. Atropine (10(-6) M) partially inhibited VP-induced excitatory effect on the muscle strips but hexamethonium (10(-4) M) did not influence it. These results suggest that V(1a) receptor was expressed on the neurons of myenteric nerves. The cholinergic nerve was involved in the excitatory effect of VP on the contraction of gastric body.

Effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain neurons.

ATP-sensitive potassium channels (KATP) play a crucial role in coupling metabolic energy to the membrane potential of cells, thereby functioning as cellular "metabolic sensors." Recent evidence has showed a connection between the amyloid neurotoxic cascade and metabolic impairment. With regard to their neuroprotection in other neuronal preparations, KATP channels may mediate a potential neuroprotective role in Alzheimer's disease (AD). To investigate the effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain cholinergic neurons, primary rat basal forebrain neurons were cultured and evaluated. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing changes were observed by double immunofluorescence and immunoblotting when the neurons were exposed to Abeta1-42(2 microM) for different time (0, 24, 72 h). We found a significant increase in the expression of Kir6.1 and SUR2 in the cultured neurons being exposed to Abeta1-42 for 24 h, while Kir6.2 and SUR1 showed no significant change. However, after being treated with Abeta1-42 for 72 h, the expression of the four subunits was all increased significantly compared with the control. These findings suggest that being exposed to Abeta1-42 for different time (24 and 72 h) induces differential regulations of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to resist the toxicity of Abeta1-42.