Carbachol Research Articles

The effects of carbachol on early oral fluid resuscitation of burn shock in dogs

Objective To investigate the effect of carbachol on early oral fluid resuscitation of a 50% total body surface area (TBSA) burn in dogs. Methods Seventeen Beagle dogs were surgically pre-pared for cannulation of carotid artery,jngular vein ,gastric and bladder,and 24 h later they were subjected to a 50% TBSA full-thickness flame injury. The dogs were randomized to three groups in the first 24 h after burn:non-resuscitation (NR group), gastric resuscitation with glucose-electrolyte solution (GES group), and gastric resuscitation with GES containing 20 μg/kg of carbachol (GES/CAR group). The delivery rate of GES was 4 ml/kg of fluid per 1% TBSA bum during the first 24 h. In the second 24 h all dogs received IV fluid resuscitation. At the end of 72 h-period experiment,the mortality was recorded. Gastric emptying rote in the first 8 h was determined in two gastric resuscitation groups. Changes in homodynamics, cardiac output (CO) urinary output,plasma level of tumor necrosis factor-alpha (TNF-α) during the period of 72 h, and activity of nitric oxide synthase (NOS) of organ tissues at 72 h after bum were also determined. Results The levels of mean arterial pressure (MAP) ,CO,urinary output and the rate of gastric emptying were markedly rdeuced, and the contents of TNF-α obviously elevated in all three groups compared with those before burn injury. The levels of MAP and CO in two gastric infusion groups were significantly in-creased as compared with those in NR group. The levels of CO and the rate of gastric emptying were signifi-cantly increased in GES/CAR group as compared with those in GES group from 4 h after burn (P<0.01). Urinary output was increased in GES/CAR compared to that in GES group from 24 h post burn (P< 0.01). GES/CAR group showed lower TNF-α at 2 h,4 h ,8 h and NOS levels at 72 h than CES group (P< 0.05 or P<0.01) after injury. Conclusion Carbachol efficiently improved the resuscitative effects of oral resuscitation of 50% TBSA bum,which may be related to their effects of promoting gastric emptying and of attenuating systematic inflammation. Key words: Bum; Fluid therapy; Carbachol

Study of the Intestinal Motility Effects of Chaenomelis Sinensis Fructus and Chaenomelis Lagenariae Fructus

Objectives : This study was conducted to compare Chaenomelis Sinensis Fructus with Chaenomelis Lagenariae Fructus, and to examine Fructus chaenomelis having an influence on Intestinal Motility. Methods : We compared Chaenomelis Sinensis Fructus with Chaenomelis Lagenariae Fructus, by observing their effects on Intestinal Motility. Oral administration of water extracts of Chaenomelis Sinensis Fructus and Chaenomelis Lagenariae Fructus into albino rats was followed by dealing with carbachol or loperamide, injecting charcoal meal and measuring the moving length in the intestine. Results : By Chaenomelis Sinensis Fructus and Chaenomelis Lagenariae Fructus, the Intestinal Motility of normal albino rats did not change significantly. Chaenomelis Sinensis Fructus and Chaenomelis Lagenariae Fructus controled the accelerated Intestinal Motility of albino rats. Chaenomelis Sinensis Fructus was not different from the extracting method, Chaenomelis Lagenariae Fructus had effects of control on extracting by methanol significantly. When Chaenomelis Sinensis Fructus was compared with Chaenomelis Lagenariae Fructus, the former was proved to have control effects on Intestinal Motility in decoction extracts and extracts by ethyl ether higher than the latter. By both Chaenomelis Sinensis Fructus and Chaenomelis Lagenariae Fructus, the declined Intestinal Motility was not changed significantly. Conclusion : Chaenomelis Fructus suppressed the exasperated Intestinal Motility by carbachol, but did not influence the dropped Intestinal Motility by loperamide. In addition, Chaenomelis Sinensis Fructus was more excellent than Chaenomelis Lagenariae Fructus in its control effects of Intestinal Motility. inhibitory effects by DKT represent a potential therapeutic approach to the treatment of inflammatory diseases.

Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells

Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions. The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNgamma) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-kappaB) were studied by Western blot. CARB increased NO synthesis by 57 +/- 5%, while a 150 +/- 10% increase in NO liberation was triggered by LPS plus IFNgamma treatment. CARB added to LPS plus IFNgamma potentiated NO synthesis by 227 +/- 19%. CARB also upregulated NOS1 protein expression via NF-kappaB activation. In addition CARB and LPS plus IFNgamma stimulated PGE(2) synthesis by 72 +/- 9 and 42 +/- 4%, respectively, while CARB added to LPS plus IFNgamma treated cells produced a synergism in PGE(2) liberation (130 +/- 12%) via COX-2. Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.

The protective effects of carbachol on visceral ischemia-induced lipid peroxidation injury in rats with sepsis

To investigate the effect of carbachol (CAR) on visceral perfusion and lipid oxidation injury in rats with sepsis. Sixty-four Sprague-Dawley (SD) rats received cecal ligation and puncture (CLP) surgery, and they were divided randomly into two groups: septic model group (CLP group, n=32) and septic model with CAR-treatment group (CAR group, n=32). CAR (10 microg/kg, CAR group) or normal saline (CLP group) was immediately injected into penial vein. Sixteen animals in each group were used to observe the mortality rates 12 hours and 24 hours after CLP, and the remaining rats for measurement of variables of blood and tissue. At the 18 hours after CLP, the mean arterial pressure (MAP), the blood flow (BF) of liver, kidney and jejunum, the plasma levels of alanine aminotransferase (ALT) and creatinine (Cr) were measured. Animals were sacrificed after the aforementioned determinations, and specimens of liver, kidney and jejunum were harvested for evaluation of malondialdehyde (MDA), xanthine oxidase (XOD), and assessment of tissue water content (ratio of dry to wet weight) of those organs . The activity of diamine oxidase (DAO) in jejunal tissue was detected. The mortality rates of 12 hours and 24 hours of CAR group were 25.0% (4/16)and 50.0% (8/16) respectively, all significantly lower than those of CLP group [37.5% (6/16) and 75%(12/16), both P<0.05]. CAR treatment did not result in significant statistical difference in the levels of MAP compared with CLP group at 18 hours after CLP (P>0.05), but led to significant increases in BF of CAR group in liver, kidney and jejunum compared with those of CLP group (all P<0.05). The levels of XOD and MDA, as well as the tissue water content were significantly lower in CAR group than CLP group in kidney and jejunum (all P<0.05). The parameters of organ function were significantly different in CAR group compared with CLP group [ALT: (64.3+/- 8.3) U/L vs. (81.5+/-7.9) U/L, Cr: (96.4+/-7.0) micromol/L vs. (117.1+/-6.7) micromol/L, DAO: (0.20+/- 0.04) U/L vs. (0.12+/-0.03) U/L, all P<0.05]. The results indicate that CAR promotes visceral perfusion, inhibits lipid peroxidation production and alleviates visceral edema and dysfunction in rats with sepsis.

Cholinergic modulation of GABAergic and glutamatergic transmission in the dorsal subcoeruleus: mechanisms for REM sleep control.

Dorsal subcoeruleus (SubCD) neurons are thought to promote PGO waves and to be modulated by cholinergic afferents during REM sleep. We examined the differential effect of the cholinergic agonist carbachol (CAR) on excitatory and inhibitory postsynaptic currents (PSCs), and investigated the effects of CAR on SubCD neurons during the developmental decrease in REM sleep. Whole-cell patch clamp recordings were conducted on brainstem slices of 7- to 20-day-old rats. CAR acted directly on 50% of SubCD neurons by inducing an inward current, via both nicotinic and muscarinic M1 receptors. CAR induced a potassium mediated outward current via activation of M2 muscarinic receptors in 43% of SubCD cells. Evoked stimulation established the presence of NMDA, AMPA, GABA, and glycinergic PSCs in the SubCD. CAR was found to decrease the amplitude of evoked EPSCs in 31 of 34 SubCD cells, but decreased the amplitude of evoked IPSCs in only 1 of 13 SubCD cells tested. Spontaneous EPSCs were decreased by CAR in 55% of cells recorded, while spontaneous IPSCs were increased in 27% of SubCD cells. These findings indicate that CAR exerts a predominantly inhibitory role on fast synaptic glutamatergic activity and a predominantly excitatory role on fast synaptic GABAergic/glycinergic activity in the SubCD. We hypothesize that during REM sleep, cholinergic "REM-on" neurons that project to the SubCD induce an excitation of inhibitory interneurons and inhibition of excitatory events leading to the production of coordinated activity in SubCD projection neurons. The coordination of these projection neurons may be essential for the production of REM sleep signs such as PGO waves.

3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Effect of carbachol on the expression of la on monocyte and lymphocyte apoptosis rate of the burned rats with delayed resuscitation

Objective To explore the changing trend of Ia on monocyte, lymphocyte apoptosis rate, TNF-α and IL-6 in abdominal aorta of burned rats with delayed resuscitation and the influence of application of carbachol on them. Methods Adult male Wistar rats were randomly divided into normal control group(n =8), scald group(n =48) and scald with carbachol treatment group(n =48). In latter two groups, rats were inflicted with 30% total body surface area (TBSA) full-thickness scald and delayed fluid resuscitation. All scald rats were sacrificed at the 6th hours or 1st, 2nd, 3rd, 7th, 14th day after scald, with 8 rats at each time point. Expression of Ia antigen on monocyte and lymphocyte apoptosis rate were determined by direct immunofluorescence on a flow cytometer, and TNF-α and IL-6 was measured by ELISA. Results Expression of la on monocyte was obviously lower than that of controls. The lowest levels were recorded on the 6th hours and 1st day after scald. Subsequently, Ia was elevated gradually, but still lower than that of normal rats(P <0. 01). After administration of carbachol, Ia expression was obviously promoted, compared with the simple scald group (P <0. 01). Lymphocyte apoptosis rate, TNF-α and IL-6 was higher than that of controls(P <0. 01). After administration of cavachol, , lymphocyte apoptosis rate and TNF-α and IL-6 was obviously down-regulated on the 6th hours, 1st day, 2nd day and 3rd day after scald injury, compared with the simple scald group (P < 0. 01 or 0. 05). Conclusion After severe burn with delayed fluid resuscitation, there is a low la expression, high lymphocyte apoptosis rate and increased releasing of proinflammatory cytokine. Immune function was suppressed. Carbacho] could improve the immune function of scald rats with delayed fluid resuscitation. Key words: CarbachoL/PD ; Bums/DT/IM

Cholinergic Responses and Intrinsic Membrane Properties of Developing Thalamic Parafascicular Neurons

Parafascicular (Pf) neurons receive cholinergic input from the pedunculopontine nucleus (PPN), which is active during waking and REM sleep. There is a developmental decrease in REM sleep in humans between birth and puberty and 10-30 days in rat. Previous studies have established an increase in muscarinic and 5-HT1 serotonergic receptor-mediated inhibition and a transition from excitatory to inhibitory GABA(A) responses in the PPN during the developmental decrease in REM sleep. However, no studies have been conducted on the responses of Pf cells to the cholinergic input from the PPN during development, which is a major target of ascending cholinergic projections and may be an important mechanism for the generation of rhythmic oscillations in the cortex. Whole cell patch-clamp recordings were performed in 9- to 20-day-old rat Pf neurons in parasagittal slices, and responses to the cholinergic agonist carbachol (CAR) were determined. Three types of responses were identified: inhibitory (55.3%), excitatory (31.1%), and biphasic (fast inhibitory followed by slow excitatory, 6.8%), whereas 6.8% of cells showed no response. The proportion of CAR-inhibited Pf neurons increased with development. Experiments using cholinergic antagonists showed that M2 receptors mediated the inhibitory response, whereas excitatory modulation involved M1, nicotinic, and probably M3 or M5 receptors, and the biphasic response was caused by the activation of multiple types of muscarinic receptors. Compared with CAR-inhibited cells, CAR-excited Pf cells showed 1) a decreased membrane time constant, 2) higher density of hyperpolarization-activated channels (I(h)), 3) lower input resistance (R(in)), 4) lower action potential threshold, and 5) shorter half-width duration of action potentials. Some Pf cells exhibited spikelets, and all were excited by CAR. During development, we observed decreases in I(h) density, R(in), time constant, and action potential half-width. These results suggest that cholinergic modulation of Pf differentially affects separate populations, perhaps including electrically coupled cells. Pf cells tend to show decreased excitability and cholinergic activation during the developmental decrease in REM sleep.

Food allergy alters jejunal circular muscle contractility and induces local inflammatory cytokine expression in a mouse model

BackgroundWe hypothesized that food allergy causes a state of non-specific jejunal dysmotility. This was tested in a mouse model.MethodsBalb/c mice were epicutaneously sensitized with ovalbumin and challenged with 10 intragastric ovalbumin administrations every second day. Smooth muscle contractility of isolated circular jejunal sections was studied in organ bath with increasing concentrations of carbamylcholine chloride (carbachol). Smooth muscle layer thickness and mast cell protease-1 (MMCP-1) positive cell density were assayed histologically. Serum MMCP-1 and immunoglobulins were quantified by ELISA, and mRNA expressions of IFN-γ, IL-4, IL-6 and TGFβ-1 from jejunal and ileal tissue segments were analyzed with quantitative real-time PCR.ResultsOvalbumin-specific serum IgE correlated with jejunal MMCP-1+ cell density. In the allergic mice, higher concentrations of carbachol were required to reach submaximal muscular stimulation, particularly in preparations derived from mice with diarrhoea. Decreased sensitivity to carbachol was associated with increased expression of IL-4 and IL-6 mRNA in jejunum. Smooth muscle layer thickness, as well as mRNA of IFN-γ and TGF-β1 remained unchanged.ConclusionIn this mouse model of food allergy, we demonstrated a decreased response to a muscarinic agonist, and increased levels of proinflammatory IL-6 and Th2-related IL-4, but not Th1-related IFN-γ mRNAs in jejunum. IgE levels in serum correlated with the number of jejunal MMCP-1+ cells, and predicted diarrhoea. Overall, these changes may reflect a protective mechanism of the gut in food allergy.

The effect of carbachol on pulmonary vascular permeability and lung water content during oral fluid resuscitation of burn shock induced by a 50% total body surface area full-thickness flame injury in dogs

To investigate the effects of carbachol (CAR) on pulmonary vascular permeability and pulmonary water content during oral fluid resuscitation of burn shock. Twelve male Beagle dogs with intubation of carotid artery and jugular vein for 24 hours were subjected to a 50% total body surface area (TBSA) full-thickness burn, then they were equally divided into oral resuscitation (OR) and OR plus CAR groups (OR+CAR). Dogs were given either a glucose-electrolyte solution (GES) in OR group or GES containing CAR (20 microg/kg) in OR+CAR group by gavage within 24 hours after burn. Dogs in each group were given intravenous fluid resuscitation after 24 post burn hour (PBH). The delivery rate and volume of GES was in accordance with that of Parkland formula. Respiratory rate (RR), arterial partial pressure of oxygen (PaO(2)), extravascular lung water index (ELWI) and pulmonary vascular permeability index (PVPI) were determined before burn (0 hour), and at 2, 4, 8, 24, 48 and 72 PBH. At 72 PBH or before death, dogs were sacrificed to collect lung tissue for evaluation of myeloperoxidase (MPO), malondialdehyde (MDA), and assessment of the tissue water content by dry to wet weight. Compared with those before burn, RR, ELWI and PVPI were greatly increased, and PaO(2) obviously decreased in two groups after burn (all P<0.01). At 72 PBH, PaO(2) returned to pre-burn level, while RR, ELWI and PVPI were still higher than pre-burn levels. RR, ELWI and PVPI at 4, 8 and 24 PBH, and PaO(2) at 8, 24, 48 PBH in OR+CAR group were respectively lower or higher than those in OR group (P<0.05 or P<0.01), but those measurements showed no statistical differences between two groups at 72 PBH (all P>0.05). MPO, MDA and lung water contents in OR+CAR group were significantly lower than those in OR group at 72 PBH [(2.64+/-0.38) U/mg vs.(4.12+/-0.46) U/mg, P<0.01; (3.60+/-0.54) micromol/mg vs.(5.14+/-0.62) micromol/mg, P<0.01; (77.40+/-0.56)% vs. (78.30+/-0.54)%, P<0.01]. The results indicate that CAR inhibits inflammatory response and oxidative damage in lung tissue, and alleviates pulmonary vascular permeability and lung edema during oral fluid resuscitation of burn shock.

Lateral Hypothalamic-Induced Alpha-Adrenoceptor Modulation Occurs in a Model of Inflammatory Pain in Rats

Previous work from our lab showed that stimulation of the lateral hypothalamus (LH) produces analgesia (antinociception) in a model of thermal nociceptive pain. This antinociceptive effect is mediated by alpha2-adrenoceptors in the spinal cord dorsal horn. However, a concomitant, opposing hyperalgesic (pro-nociceptive) response also occurs, which is mediated by alpha1-adrenoceptors in the dorsal horn. Antinociception predominates but is attenuated by the pronociceptive response. To determine whether such an effect occurs in a model of inflammatory pain, we applied mustard oil (allyl isothiocyanate; 20 microl) to the left ankle of female Sprague-Dawley rats. We then stimulated the LH using carbamylcholine chloride (carbachol; 125 nmol). The foot withdrawal latencies were measured. Some rats received intrathecal alpha-adrenoceptor antagonists to determine whether the opposing alpha-adrenoceptor response was present. Mustard oil application produced hyperalgesia in the affected paw, while the LH stimulation increased the foot withdrawal latencies for the mustard oil paw as compared to the control group. Following carbachol microinjection in the LH, WB4101, an alpha1-adrenoceptor antagonist, produced significantly longer foot withdrawal latencies compared to saline controls, while yohimbine, an alpha2-antagonist, decreased the foot withdrawal latencies from 10 min postinjection (p < .05). These findings support the hypothesis that the LH-induced nociceptive modulation is mediated through an alpha-adrenoceptor opposing response in a model of inflammatory pain.

Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis

Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the beta2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Galpha(s) and Galpha(q), localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Galpha(s) and Galpha(q) remained associated with the PM and these endosomal membranes, whereas beta2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.

Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M1, methoctramine for M2, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M3, and tropicamide for M4 receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC50), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 microM), activator of adenylate cyclase (AC), was unidirectional with beta-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC- or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M3 acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M3 acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M3-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.

Effect of carbachol on expression of TNF-α and aquaporin-1 in small intestine during enteral resuscitation of scald injury in rats

目的：研究拟胆碱药卡巴胆碱对烫伤大鼠口服补液时小肠TNF-α及水通道蛋白-1（AQP-1）表达的影响． 方法：♂Wistar大鼠50只，随机分为假烫（N）、单纯烫伤（S）、肠内葡萄糖-电解质液（GES）、肠内卡巴胆碱组（CAR）和肠内葡萄糖-电解质液＋卡巴胆碱组（GES／CAR）5组（n=10）．大鼠背部用沸水造成35％TBSA烫伤．N，GES和GES／CAR组于伤后30min开始补液．免疫组化法测定肠组织AQP-1的表达，ELISA法检测肠组织TNF-α含量，酚红法测定大鼠小肠对水的吸收率． 结果：S组大鼠小肠AQP-1与N组比明显降低（90．3±18．4 vs 4851．6±654．5，P〈0．01）：CAR，GES和GES／CAR组AQP-1与S组相比均显著增加（1806．1±110．1，2272．3±113．8，3322．0±595．9 vs 90．3±18．4，均P〈0．01）．给予卡巴胆碱组（CAR，GES／CAR）与未给予卡巴胆碱组（S，GES）相比肠组织TNF-α含量明显下降（0．9±0．3，1．0±0．47 vs 1．8±0．3，1．9±0．2，P〈0．05）．GES／CAR．CARTLS组AQP-1表达量与TNF-α含量成负相关（r=0．9030，-0．9602，-0．9866，均P〈0．05）．GES／CAR组水吸收率较GES组明显升高（21．0％±0．1％ vs 12．7％±0．1％，P〈0．05）． 结论：卡巴胆碱可抑制促炎因子TNF-α的释放，上调小肠AQP-1表达，改善大鼠烫伤早期肠道对水的吸收．

Effects of carbachol on intestinal inflammation and mucosal blood flow in rats with gut ischemia-reperfusion: P-084.

Objective To investigate the effects of carbachol on intestinal inflammation and mucosal blood flow after gut ischemia-repedusion(I/R) in rat. Method A jejunal sac was formed in Wistar rats. The superior mesenteric artery (SMA) was occluded for 45 mi-nutes followed by 240 minutes of reperfusion. Animals were random divided into three groups: sham operation, L/R + saline injection (I/R + NS) and I/R + carbachol injection (0.1mg/kg, I/R + Ca). Immediately after occluded of SAM blood flow, either 0.1mg/kg of carba-chol or same account of 0.9% saline was injected into the jejunal sac. The pathological injury was observed with HE staining. The activity of DAO and content of TNF-α in intestinal mucosa tissue were determined. Mucosal blood flow was measured by laser Doppler. All measure-ments were done at 0 min, 30 min, 60 min, 120 min, and 240 min after reperfusion. Result In I/R group the activity of DAO in intestinal mucosa and mucosal blood flow deceased, meanwhile the content of TNF-α gut tissue was dramatically increased than those in sham operation (P<0.01). Severe pathological changes were observed in intestinal mucosa. After injection of carbachol, the activity of DAO and mucosal blood flow increased (P<0.01), but the content of TNF-α in intestinal mucosa were dramatically decreased (P<0.01), compared with those in I/R group. Conclusion Administration of carbachol protects intestinal ischemia-reperfusion injury by attenuating intestinal mucosa inflammation and increasing gut mueosal blood flow. Key words: Carbachol/PD; Reperfusion injury; Inflammation; Tumor necrosis factor-alpha; Blood volume

Activity-dependent release of transforming growth factor-beta in a neuronal network in vitro

For neurotrophins and also for members of the transforming growth factor beta (TGF-β) family an activity-dependent regulation of synthesis and release has been proposed. Together with the observation that the secretion of neurotransmitters is initiated by neurotrophic factors, it is reasonable to assume that they might act as retrograde modulators enhancing the efficacy and stabilization of synapses. In the present study, we have tested this hypothesis and studied the release and regulation of TGF-β in vitro using mouse primary hippocampal neurons at embryonic day E16.5 as model. We show that neuronal activity regulates TGF-β release and TGF-β expression in vitro. Treatment of the cultures with KCl, 3-veratroylveracevine (veratridine), glutamate or carbamylcholine chloride (carbachol) increased the levels of secreted TGF-β, as assessed by the MLEC/plasminogen activator inhibitor (PAI)–luciferase-assay, whereas TGF-β release stimulated by KCl or veratridine was reduced in the presence of tetrodotoxin or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). In addition, application of glutamate significantly upregulated expression of TGF-β2 and TGF-β3 in the culture. Notably, KCl stimulation caused Smad (composite term from SMA ( C. elegans) and MAD=mothers against dpp ( Drosophila)) translocation into the nucleus and upregulated TGF-β inducible early gene (Tieg1) expression, demonstrating that activity-dependent released TGF-β may exert autocrine actions and thereby activate the TGF-β-dependent signaling pathway. Together, these results suggest an activity-dependent release and gene transcription of TGF-β from mouse hippocampal neurons in vitro as well as subsequent autocrine functions of the released TGF-β within the hippocampal network.

Activation of muscarinic cholinergic receptors induces MCF-7 cells proliferation and angiogenesis by stimulating nitric oxide synthase activity

Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10-9 M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that non-neuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.

Autonomous contractile activity in the isolated rat bladder is modulated by a TRPV1 dependent mechanism

Resiniferatoxin (RTX), a vanilloid compound and agonist of the transient receptor potential channel 1 (TRPV1), is known for its beneficial effects on neurogenic detrusor overactivity. The mainstream rationale for its use is the desensitization of TRPV1 on sensory bladder afferents. However, recent findings showed that TRPV1 is present in other cell types in the bladder. To eliminate the effects of RTX on spinal and central neural circuits, we investigated autonomous contractility in normal and neurogenic rat bladders after treatment with RTX. Female Wistar rats were made paraplegic at vertebral level T8-T9. Animals were intravesically pre-treated with vehicle (ethanol 5%) or RTX (100 nM) and sacrificed after 72 hr. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. Effects on contractile parameters of intravesical volume load, the non-selective muscarinic receptor agonist carbachol (CA) and electrical stimulation (ES) of nerves were studied in both groups. In RTX-treated normal bladders we found shorter contractions with higher amplitude than in control bladders (P < 0.05). In RTX-treated neurogenic bladders the amplitude and duration of autonomous contractions were increased compared with controls (P < 0.05). Furthermore RTX induced an increased response to CA and to ES (P < 0.05). RTX significantly affected the properties of autonomous bladder contractile activity. This provides evidence for local effects of RTX on bladder contractile activity, which are not mediated by afferent neural pathways and which may contribute to the beneficial effects on detrusor overactivity. TRPV1 and TRPV1(+) cells seem to play an important role in (autonomous) bladder contractility.