[Study on correlation between serum 25-hydroxyvitamin D3 level and esophageal variceal bleeding in cirrhotic patients].

Objective: To explore the correlation between serum 25-hydroxyvitamin D3 (25[OH]D(3)) levels and esophageal variceal bleeding (EVB) in cirrhotic patients. Methods: Eighty-three cases with liver cirrhosis hospitalized from November 2016 to January 2017 were collected. The patients were divided into bleeding group (51 cases) and non-bleeding group (32 cases) depending on the presence or absence of bleeding under gastroscopy. Serological tests were performed on both groups, including hemoglobin (Hb), albumin (ALB), alkaline phosphatase (ALP),γ-glutamyltransferase (GGT), interleukin-6 (IL-6), and 25-hydroxyvitamin D3 (25[OH]D(3)). Both groups were analyzed by univariate analysis. The differences between both groups were compared by t-test, after normality test. The other variables were compared by Mann-Whitney U test. The correlation between the relevant variables and EVB were analyzed by Spearman's rank correlation and a multivariate analysis. Cases with primary biliary cirrhosis were relatively low in number (four cases in bleeding group, accounting for 8%, 10 cases in non-bleeding group, accounting for 31%). The effects of ALP and GGT on serum 25(OH)D(3) level were analyzed by stratified analysis. Moreover, ALP and GGT levels were divided into two and three groups: < 140 U/L and >140 U/L and < 30 U/L, > 30 U/L, and ~≤60 U/L. Results: Bleeding group had low levels of hemoglobin (t= -2.827,P= 0.005), alkaline phosphatase (t= -3.097,P= 0.002), gamma-glutamyltransferase (t= -2.292,P= 0.022), and 25(OH)D(3) (t= -3.134,P= 0.002) than non-bleeding group. Both groups (P> 0.05) had similar levels of albumin, interleukin-6, AAR, and FIB-4. Logistic regression analysis showed that 25(OH)D(3), alkaline phosphatase and hemoglobin were independent risk factors for EVB. Spearman's correlation coefficient analysis showed that 25(OH)D(3)was significantly positively and negatively correlated with interleukin-6 (r= 0.306,P= 0.005) and albumin (r= -0.327,P= 0.003). Stratified analysis showed that serum 25(OH)D(3) level was lower in ALP≤140U/L group and the bleeding group, and the difference was statistically significant than non-bleeding group (P= 0.007), while the serum level of 25(OH)D(3)was decreased in both groups for alkaline phosphatase > 140 U/L group, and the difference was not statistically significant (P= 0.051). Furthermore, in the GGT > 60 U/L group, the serum level of 25(OH)D(3)was significantly lower in the bleeding group, and the difference was statistically significant in non-bleeding group (P= 0.003), while the difference between the two groups was not statistically significant (P> 0.05) in GGT≤30 U/ L, > 30 U/L, and ~≤60 U/L group. Conclusion: Serum 25(OH)D(3)level was significantly lower in EVB cirrhotic patients, and it was an independent risk factor for EVB. Serum 25(OH)D(3) low levels was more apparent with ALP normalization or GGT level > 60 U/L.

Epigallocatechin gallate inhibits the growth of salivary adenoid cystic carcinoma cells via the EGFR/Erk signal transduction pathway and the mitochondria apoptosis pathway.

ACC is one of the most malignant tumors in salivary gland, and of poor prognosis. A critical role in ACC development and progression is played by EGFR family members including EGFR. EGCG, a low molecular weight polyphenol contained in green tea, has broad anticancer properties, but whether EGCG regulates activity of ACC is unknown. In the present study, the effects of EGCG were investigated in vitro with particular attention to the pathway of EGFR/Erk and mitochondria apoptosis in SACC-83 cell lines. The results of MTS assay and flow cytometry demonstrated that EGCG (20-80 µM) could inhibit proliferation and promote apoptosis of SACC-83 cells. Furthermore, by Western blotting with antibodies specific for EGFR, Erk 1/2 (p-Erk 1/2), Mek (p-Mek), Bcl-2, and Bax, it was demonstrated that EGCG could reduce the expression of EGFR, inhibit phosphorylation of Erk 1/2 and Mek, downregulate Bcl-2, and upregulate Bax. In addition, it was also shown that EGCG could inhibit mRNA expression of P90 RSK by RT-PCR. In conclusion, the results suggest that EGCG might be a potential therapeutic or adjuvant strategy for the treatment of patients with ACC, by inhibiting proliferation and inducing the apoptosis of the tumor cells.

[Changes in peripheral blood 25 - hydroxyvitamin D3, Th17 cells, and CD4+ regulatory T cells and their clinical significance in patients with primary biliary cirrhosis].

Objective: To investigate the changes in peripheral blood 25-hydroxyvitamin D3[25-(OH)D3], CD4+regulatory T (Treg) cells, and Th17 cells in patients with primary biliary cirrhosis (PBC) and their mechanism of action in PBC. Methods: A total of 22 patients with PBC were enrolled and the male/female ratio was 1:21, with a mean age of 61±12 years. There were 7 healthy volunteers matched for age in the normal control group. Electrochemiluminescence immunoassay was used to measure the peripheral blood 25-(OH)D3level in the PBC group and normal control group, and flow cytometry was used to analyze the changes in Th17 cells and CD4+Treg cells. The t-test, rank sum test, Pearson correlation analysis, or Spearman's rank correlation analysis was used for statistical analysis according to the type of the data. Results: The PBC group had a significantly lower serum 25-(OH)D3level than the normal control group (9.49±3.65 vs 27.35±2.35 ng/ml,P< 0.01). Compared with the normal control group, the PBC group had a significantly higher percentage of Th17 cells (2.05%±1.17% vs 0.99%±0.12%,P< 0.01) and a significantly lower percentage of CD4+Treg cells (2.54%±1.14% vs 3.78%±0.51%,P< 0.05); there was a significant difference in Th17/Treg ratio between the PBC group and the normal control group (1.00±0.63 vs 0.26±0.02,P< 0.01). In the PBC group, peripheral blood 25-(OH)D3 was not correlated with Th17 cells or Th17/Treg ratio (r= -0.062 and -0.328,P> 0.05), while it was positively correlated with the percentage of CD4+Treg cells (r= 0.468,P< 0.05). Conclusion: Patients with PBC have significant reductions in peripheral blood 25-(OH)D3and percentage of CD4+Treg cells, a significant increase in the percentage of Th17 cells, and immune unbalance of Th17 cells and CD4+Treg cells. 25-(OH)D3can upregulate the percentage of CD4+Treg cells and thus affect the development and progression of PBC, and exogenous vitamin D may improve immune function in PBC patients.

Collapse and stability of single- and multi-wall carbon nanotubes.

The collapse and stability of carbon nanotubes (CNTs) have important implications for their synthesis and applications. While nanotube collapse has been observed experimentally, the conditions for the collapse, especially its dependence on tube structures, are not clear. We have studied the energetics of the collapse of single- and multi-wall CNTs via atomistic simulations. The collapse is governed by the number of walls and the radius of the inner-most wall. The collapsed structure is energetically favored about a certain diameter, which is 4.12, 4.96 and 5.76 nm for single-, double- and triple-wall CNTs, respectively. The CNT chirality also has a strong influence on the collapsed structure, leading to flat, warped and twisted CNTs, depending on the chiral angle.

Intrinsic energy loss mechanisms in a cantilevered carbon nanotube beam oscillator.

Classical molecular dynamics is applied to study the energy dissipation (the Q factor) of the cantilever-type beam oscillators of single wall and double-walled carbon nanotubes (CNTs). The study finds that the Q factor of the CNT beam oscillator varies with the temperature T following the 1/T(0.36) dependence. For single wall CNT, the Q factor drops from 2 x 10(5) at 0.05 K to 1.5 x 10(3) at 293 K. The study further reveals that the weak interlayer binding strength and the interlayer commensurance significantly increases the energy dissipation in the double-walled CNT oscillator.

Amphetamine induces differential changes in the gene expression of metabotropic glutamate receptor 5 in cultured cortical and hippocampal neurons.

Mechanisms underlying the short-term effects of amphetamine (AMPH) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) in cultured rat neurons. The cortical and hippocampal neurons were incubated with 0.1-100 microM of AMPH for 1 h or 1 microM of AMPH for 10 min to 3 h. Immunocytochemical and in situ hybridization (ISH) analyses revealed that the levels of mGluR5 immunoreactivity and mRNA in the cortical neurons were initially increased with the treatment time and dosage, to reach maximal elevations of 34 and 53% from control values following 1 h incubation of 1 microM, and then returned toward the controls. When the cortical neurons were preincubated with the antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) to mGluRs, before treated with 1 M of AMPH for 1 h, the levels of mGluR5 protein and mRNA became 120 and 116% of control values. In hippocampal neurons, the AMPH treatment persistently upregulated the mGluR5 protein by 50-62%; however, the mRNA responded with the bell-shaped pattern to the treatment times and doses, with 20-43% increases from controls. These modifications of the receptor were reversible, since removal of AMPH resulted in regular levels of the receptor. Notably, the AMPH-generated increases in mGluR5 protein and mRNA were completely blocked by the pretreatment with cycloheximide and actinomycin D, respectively. The data indicate differential responsive patterns of mGluR5 in the cortical and hippocampal neurons to the drug perturbation. The action of AMPH may involve regulation to transcriptional and translational events in the neurons, and the activation of the MCPG-sensitive receptors.

Effect of amphetamine on the expression of the metabotropic glutamate receptor 5 mRNA in developing rat brain.

Mechanisms underlying the acute effects of amphetamine (AMP) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) and specific 3H-glutamate binding in the developing rat brain. Each of the postnatal day (P) 4, P21 and P60 rats received one intraperitoneal injection of AMP, 5 mg/kg or saline and were sacrificed one hour later. In situ hybridization analysis revealed that the AMP treatment raised the levels of the mGluR5 mRNA by 9-28% in the neurons of the layer 5 of motor and somatosensory cortices, whereas reduced the levels by 12-28% in the layer 5 of perirhinal cortex and the ventromedial part of caudate-putamen of the 3 ages. In the layer 2/3 neurons of cingular cortex, an 18% higher and 14% and 22% lower than control levels of the mRNA were detected in the P4 and in the P21 and P60 rats injected with AMP. Moreover, the levels of mGluR5 mRNA in the hippocampi and dentate gyri were elevated by AMP to 110-151% of controls in the rats of 3 ages. Reversible 3H-glutamate binding assay showed an increase of 25% and a 12% decrease in the binding levels in the cortices of AMP-treated P4 and P21 rats. The AMP administration also produced a 27% reduction and 62% elevation in the binding of the hippocampi of P4 and P60 rats. The results reveal age- and region-dependent changes in the expression of the glutamate receptors induced by AMP and may indicate differential plastic capability of the neurons to the drug perturbation.

Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent.

In an effort to further understand the processes underlying hypoxic pulmonary vasoconstriction, we examined the mechanism by which sodium hydrosulfite (Na2S2O4), a potent reducing agent and oxygen scavenger, induces smooth muscle contraction. In rat pulmonary arterial strips, sodium hydrosulfite (10 mM) induced contractions that were 65.9 +/- 12.8% of the response to 60 mM KCl (n = 9 segments). Contractions were not inhibited by nisoldipine (5 microM) or by repeated stimulation with caffeine (10 mM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (10 microM), or cyclopiazonic acid (10 microM), all of which eliminated responses to contractile agonists. Maximum force generation after exposure to sodium hydrosulfite was 0.123 +/- 0.013 mN in the presence of 1.8 mM calcium and 0.127 +/- 0.015 mN in the absence of calcium. Sodium hydrosulfite contractions in pulmonary arterial segments were not due to the generation of H2O2 and occurred in the presence of chelerythrine (10 microM), which blocked phorbol ester contractions, and solution hyperoxygenation. Similar contractile responses were obtained in rat aortic and tracheal smooth muscles. Finally, contractions occurred in the complete absence of an increase in myosin light chain phosphorylation. Therefore sodium hydrosulfite-induced smooth muscle contraction is not specific to pulmonary arterial smooth muscle, is independent of calcium and myosin light chain phosphorylation, and is not mediated by either hypoxia or protein kinase C.

Differential development of the enhanced metabolic response during amphetamine sensitization.

Behavioral sensitization elicited by repeated administration of amphetamine does not fully develop until a period after discontinuation of amphetamine, but then persists undiminished for a long time. This experiment investigated the regional metabolic changes in rats pretreated with amphetamine and challenged after different abstinence periods (2, 7 and 28 days), using the 2-[14C]deoxyglucose method. The results demonstrated that chronic amphetamine administration enhanced rates of local cerebral glucose utilization in specific cerebral regions. The magnitude and distribution of effects varied with the abstinence period. A challenge dose of d-amphetamine 2 days after pretreatment was found to have no more, or only mildly elevated, local cerebral glucose utilization compared with that following a single acute dose. In rats challenged at the 7th and 28th day, a supersensitive metabolic response was found in dopaminergic and non-dopaminergic areas. This finding suggested regional differences in the development of sensitization and underscored the importance of an abstinence period in the study of sensitization and amphetamine psychosis.

Gene transfer by adenovirus in smooth muscle cells.

We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (13-21 days). In two experiments in which the mucosa and serosa were removed, nearly all cells expressed beta-galactosidase, whereas in a third experiment in which the tissue was not dissected, about 40% of cells were stained. Viral infection had no effect on tension development of strips following organ culture. In vitro gene transfer may provide a useful method to alter protein expression and examine the effect of this alteration on excitation/contraction coupling in smooth muscle.

Effects of hydrogen peroxide on isolated trachealis muscle of horses.

During acute bouts of recurrent airway obstruction (heaves) in horses, neutrophils that are capable of increased production of reactive oxygen species accumulate in the airways. In the study reported here, the effect of hydrogen peroxide (H2O2; 1 microM to 0.1M), one of these reactive oxygen species products, on the responses of isolated trachealis muscle of horses was determined. Before and after incubation with H2O2, contractile responses to acetylcholine, electrical field stimulation (EFS), 127 mM KCl, and relaxation responses to isoproterenol and activation of the nonadrenergic noncholinergic inhibitory response (iNANC) were evaluated. Beginning at 1 mM, H2O2 contracted trachealis muscle in a concentration-dependent manner. This contraction was unaffected by atropine (1 microM), tetrodotoxin (1 microM), or 1 microM meclofenamate. Contraction of trachealis muscle in response to H2O2 is, therefore, not attributable to release of prostaglandins, acetylcholine, or other neurotransmitters. Above a concentration of 0.1 mM, H2O2 depressed the responses to EFS, acetylcholine, and KCl in a concentration-dependent manner. At 0.1M, H2O2 decreased the maximal responses to EFS, acetylcholine, and KCl by 62.7 +/- 7.2, 60.58 +/- 6.12, and 37.8 +/- 9.54%, respectively. In the presence of meclofenamate (1 microM), partial but significant protection against 1 to 100 mM H2O2 was observed. In tracheal strips contracted with 0.3 microM methacholine, H2O2 had no effect on the isoproterenol concentration-response curve. Up to a concentration of 100 mM, H2O2 had no effect on iNANC response. However, in the presence of 100 mM H2O2, this response was abolished in 2 of 4 horses. We conclude that high concentrations of H2O2 affected the responses of airway smooth muscle by actions on neurotransmission, muscarinic receptors, and downstream from receptors; some of the H2O2 effects were in part mediated by cyclooxygenase products; and H2O2 had no effect on beta-adrenergic- or iNANC-induced relaxation.

Metabolic and behavioral response in rats treated with amphetamine chronically with and without challenge.

Chronic amphetamine (AMPH) treatment may cause behavioral sensitization in animals and can be used as an animal model of psychosis. The aim of the study was to check the behavioral and metabolic response in this animal model. In rats pretreated with normal saline (NS) or AMPH, with or without AMPH challenge, the [14C]deoxyglucose method was employed to check the metabolic changes in 42 regions. Behavioral testing was performed in rats with the same treatment. The results showed that after challenge with AMPH, glucose utilization was enhanced in most of the regions investigated. However, metabolic enhancement of the AMPH-pretreated group was lower in the caudate nucleus when compared with that of the NS-pretreated group though the stereotypy rating was higher in the former. Dissociation between the metabolic enhancement and behavioral response was noted. Furthermore, more significant differences between the two pretreated conditions of glucose utilization were found with challenge than without challenge. Further evaluation using procedures which include advanced techniques can be applied in further investigation.

Acetylcholine release from airway cholinergic nerves in horses with heaves, an airway obstructive disease.

The present study was conducted to determine if acetylcholine (ACh) release from airway cholinergic nerves is increased and if modulation of ACh release by prejunctional receptors is altered in horses with heaves, an obstructive airway disease characterized by airway inflammation and bronchospasm. Trachealis strips and bronchial segments of normal horses and horses affected with heaves were suspended in 2-ml tissue baths. ACh release was induced by electrical field stimulation and the bath ACh content was measured by high performance liquid chromatography (HPLC) with electrochemical detection. In response to electrical field stimulation, the rate of ACh release from cholinergic nerves innervating the trachea was not significantly different between the two groups of horses. The nonselective muscarinic receptor antagonist atropine augmented ACh release to the same extent in both groups, indicating that there is no dysfunction of muscarinic autoreceptors on cholinergic nerves of large airways from horses with heaves. Compared with the data from normal horses, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine on ACh release was lacking in the bronchi and less potent in the trachea, suggesting that the prejunctional alpha 2-adrenoceptors are dysfunctional in horses with heaves. Neither exogenous prostaglandin E2 (PGE2) nor the cyclooxygenase inhibitor indomethacin influenced ACh release from the bronchi, suggesting that a decrease in airway mucosal PGE2 production reported previously in horses with heaves does not alter ACh release.

Prejunctional muscarinic autoreceptors on horse airway cholinergic nerves.

Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M1-selective), AF-DX 116 (M2-selective), and hexahydrosiladifenidol (M3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence on the measured amount of ACh. Hexahydrosiladifenidol was more potent in inhibiting ACh-induced muscle contraction, which is known to be mediated by M3 receptors, than in augmenting ACh release. The maximal ACh release rate in response to the selective antagonists was much less than that following atropine. Furthermore, the concentrations of the selective antagonists required to augment ACh release far exceeded their KdS for M1, M2, or M3 receptors. These observations suggest that the muscarinic autoreceptors on horse airway cholinergic nerves may belong to a novel subtype.

Dose-dependent effects of chronic amphetamine administration in local cerebral glucose utilization in rat.

The daily dose of amphetamine pretreatment may affect the development of behavioral-sensitized patterns in rodents and amphetamine psychosis in humans. This experiment investigated the regional cerebral metabolic changes in rats after pretreatment with different doses of amphetamine by the 2-[14C]deoxyglucose method. Rates of local cerebral glucose utilization were examined in 37 regions of the rat brain. The result showed generally maximal metabolic augmentation in the 5.0 mg/kg group instead of in the 1.0 or the 10.0 mg/kg groups. Behavioral testing using motor activity cages in rats with the same regimen found no difference among groups. The findings demonstrate that there might be a window effect by daily amphetamine dose on the development of drug dependence and amphetamine psychosis. It was suggested that the 2(-)[14C]deoxyglucose method could be used for further study of animal models of amphetamine psychosis.

PGE2 inhibits acetylcholine release from cholinergic nerves in canine but not equine airways.

The effects of exogenous prostaglandin E2 (PGE2) and endogenous prostanoids on cholinergic neurotransmission were determined by measurement of acetylcholine (ACh) release from canine and equine airway tissues. Trachealis strips and bronchial segments were suspended in 2 ml tissue baths. ACh release was induced by electrical field stimulation (EFS), and its content in tissue bath liquid was measured by high pressure liquid chromatography (HPLC) with electrochemical detection. In canine airways, exogenous PGE2 (10(-9) to 10(-7) M) inhibited ACh release concentration-dependently, whereas inhibition of endogenous prostanoid production by indomethacin (3 x 10(-6) M) augmented ACh release. By contrast, in equine airways, exogenous PGE2 had no effect on ACh release in bronchi but at 10(-7) M augmented ACh release in the trachea. Cyclooxygenase inhibition by either indomethacin or meclofenamate (10(-6) M) did not influence ACh release. We conclude that exogenous PGE2 and endogenous prostanoids inhibit ACh release from cholinergic nerves in canine but not equine airways.

Modulation of bronchial smooth muscle function in horses with heaves.

Four mechanisms that modulate airway smooth muscle function in normal horses were studied in the bronchi of horses affected by the airway obstructive disease heaves. Results were compared with data from historical controls studied by the same personnel in the same laboratory. Rings from the left cranial lobar bronchus (LB1) and small bronchi (5 mm OD) were suspended in muscle baths, and the isometric tension were measured. The inhibitory nonadrenergic noncholinergic (iNANC) function was studied in LB1. After the LB1 segments were pretreated with atropine and contracted with histamine, electrical field stimulation (EFS) induced little or no relaxation, indicating iNANC dysfunction in horses with heaves. Bronchi from animals with heaves were hyporesponsive to EFS and acetylcholine. Epithelial removal augmented the contractile response of small bronchi to acetylcholine more in animals with heaves than in control animals, indicating an enhanced function of epithelial-derived relaxing factor. In contrast, cyclooxygenase inhibition with meclofenamate (10(-6) M) increased the EFS-induced contraction of small bronchi less in affected horses than in control horses, suggesting a change in prostaglandin production in favor of excitatory prostanoids. We conclude that in the bronchi of horses with heaves; the iNANC function is defective, the response of smooth muscle to cholinergic activation is diminished, the production of epithelial-derived relaxing factor is enhanced, and the inhibitory function of prostanoids is reduced.

Evidence for the absence of a functional role for muscarinic M2 inhibitory receptors in cat trachea in vivo: contrast with in vitro results.

1. The effect of the selective muscarinic M2 receptor antagonist, gallamine and the selective M2 receptor agonist, pilocarpine, on airway constriction induced by vagal stimulation was studied in anaesthetized cats. In addition, the effect of gallamine on contraction of cat isolated tracheal and bronchi preparations induced by electrical field stimulation was also investigated. 2. In in vivo experiments, extrathoracic airway constriction was measured with an electromechanical caliper that was attached to the outer surface of tracheal ring 4. Intrathoracic airway constriction was determined by measuring the changes in total lung resistance and dynamic compliance during vagal stimulation. 3. Intravenous gallamine (0.1, 1, and 10 mg kg-1) augmented the rise in total lung resistance induced by vagal stimulation in a dose- and frequency-dependent manner. At stimulation frequencies of 8 and 12 Hz the fall in dynamic compliance provoked by vagal stimulation was also significantly increased by gallamine (10 mg kg-1). Gallamine was without effect on airway constriction induced by acetylcholine. 4. Vagal stimulation at 4 Hz produced significant tracheal constriction, but the amount of constriction did not change following injection of increasing doses of gallamine. Similarly, there was no difference in tracheal constriction at any frequency of stimulation (0.5-16 Hz) when frequency-response curves before and after gallamine injection (10 mg kg-1) were compared. 5. Pilocarpine (0.01-10 micrograms kg-1, i.v.) diminished changes in total lung resistance and dynamic compliance induced by vagal stimulation, an effect that was reversed by gallamine (10 mg kg-1, i.v.). Vagally-induced tracheal constriction was not significantly affected by any dose of pilocarpine, nor was it modified by gallamine (10mg kg- ') given subsequently.6. Atropine (0.5 mgkg-') completely blocked tracheal constriction induced by vagal stimulation, indicating that the changes in tracheal ring diameter provoked by stimulation were mediated by muscarinic receptors and that intravenous drugs could reach the cervical trachealis muscle.7. In vitro tissue bath studies demonstrated a significant leftward shift of the frequency-response curve to electrical field stimulation in both tracheal strips and bronchial rings following gallamine (10-4M) administration.8. Although the functional presence of muscarinic M2 autoreceptors was demonstrated in feline isolated tracheal and bronchial preparations, a corresponding functional role was not detected in cat trachea in vivo. This was despite repeated demonstration of muscarinic M2 receptor-mediated limitation of airway constriction of intrathoracic airways in vivo.