Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.(AU)

Research progress on mechanism of atropine in controlling myopia / 中华实验眼科杂志

The prevalence of myopia and high myopia has gradually increased,increased myopia is associated with an enhanced risk of pathological ocular complications and may lead to blinding disorders.The optometrists and parents initially pay more attention to juvenile myopia prevention and control.Atropine is the previous drug used to control myopia,and the evidence-based medicine has corroborated its clinical effect.However,its mechanism has been undetermined yet.It has been reported that the effective locations of atropine in inhibiting experimental myopia may be not related to the accommodation of ciliary muscle or the ciliary muscle is not the primary target,more and more attention is currently focused on the retina and sclera.Recently,the application of the highly selective muscarinic receptor antagonists and the combination of other drugs have been found that atropine exerts its myopia-controlling effect may be linked to multiple M receptors,and with the secretion and expression of dopamine,nitric oxide (NO),early grow response (EGR)-1,the protein change of the sclera and γ-aminobutyric acid (GABA) ergic signaling pathway.This paper reviewed the atropine on controlling experimental myopia in effective locations and its relationship with the cholinergic pathway,the dopamine pathway,the GABA pathway,the secretion of NO,the expression of EGR-1,and the process of scleral remodeling,hence offers the prospects for the study of atropine in myopia control.

Research progress on mechanism of atropine in controlling myopia / 中华实验眼科杂志

The prevalence of myopia and high myopia has gradually increased,increased myopia is associated with an enhanced risk of pathological ocular complications and may lead to blinding disorders. The optometrists and parents initially pay more attention to juvenile myopia prevention and control. Atropine is the previous drug used to control myopia,and the evidence.based medicine has corroborated its clinical effect. However,its mechanism has been undetermined yet. It has been reported that the effective locations of atropine in inhibiting experimental myopia may be not related to the accommodation of ciliary muscle or the ciliary muscle is not the primary target, more and more attention is currently focused on the retina and sclera. Recently, the application of the highly selective muscarinic receptor antagonists and the combination of other drugs have been found that atropine exerts its myopia.controlling effect may be linked to multiple M receptors,and with the secretion and expression of dopamine,nitric oxide ( NO) , early grow response (EGR).1,the protein change of the sclera and γ.aminobutyric acid (GABA)ergic signaling pathway. This paper reviewed the atropine on controlling experimental myopia in effective locations and its relationship with the cholinergic pathway, the dopamine pathway, the GABA pathway, the secretion of NO, the expression of EGR.1,and the process of scleral remodeling,hence offers the prospects for the study of atropine in myopia control.

GABA-enriched fermented Laminaria japonica improves cognitive impairment and neuroplasticity in scopolamine- and ethanol-induced dementia model mice

BACKGROUND/OBJECTIVES: Fermented Laminaria japonica (FL), a type sea tangle used as a functional food ingredient, has been reported to possess cognitive improving properties that may aid in the treatment of common neurodegenerative disorders, such as dementia. MATERIALS/METHODS: We examined the effects of FL on scopolamine (Sco)- and ethanol (EtOH)-induced hippocampus-dependent memory impairment, using the Passive avoidance (PA) and Morris water maze (MWM) tests. To examine the underlying mechanisms associated with neuroprotective effects, we analyzed acetylcholine (ACh) and acetylcholinesterase (AChE) activity, brain tissue expression of muscarinic acetylcholine receptor (mAChR), cAMP response element binding protein (CREB) and extracellular signal-regulated kinases 1/2 (ERK1/2), and immunohistochemical analysis, in the hippocampus of mice, compared to current drug therapy intervention. Biochemical blood analysis was carried out to determine the effects of FL on alanine transaminase (ALT), aspartate transaminase (AST), and triglyceride (TG) and total cholesterol (TC) levels. 7 groups (n = 10) consisted of a control (CON), 3 Sco-induced dementia and 3 EtOH-induced dementia groups, with both dementia group types containing an untreated group (Sco and EtOH); a positive control, orally administered donepezil (Dpz) (4mg/kg) (Sco + Dpz and EtOH + Dpz); and an FL (50 mg/kg) treatment group (Sco + FL50 and EtOH + FL50), orally administered over the 4-week experimental period. RESULTS: FL50 significantly reduced EtOH-induced increase in AST and ALT levels. FL50 treatment reduced EtOH-impaired step-through latency time in the PA test, and Sco- and EtOH-induced dementia escape latency times in the MWM test. Moreover, anticholinergic effects of Sco and EtOH on the brain were reversed by FL50, through the attenuation of AChE activity and elevation of ACh concentration. FL50 elevated ERK1/2 protein expression and increased p-CREB (ser133) in hippocampus brain tissue, according to Western blot and immunohistochemistry analysis, respectively. CONCLUSION: Overall, these results suggest that FL may be considered an efficacious intervention for Sco- and EtOH-induced dementia, in terms of reversing cognitive impairment and neuroplastic dysfunction.

Dipyrone in association with atropine inhibits the effect on gastric emptying induced by hypoglycemia in rats

Atropine (AT) and dipyrone (Dp) induce a delay of gastric emptying (GE) of liquids in rats by inhibiting muscarinic receptors and activating β2-adrenergic receptors, respectively. The objective of the present study was to determine the effects of pretreatment with AT and Dp, given alone or in combination, on the effect of hypoglycemia in the liquid GE in rats. Male Wistar adult rats (280-310 g) were pretreated intravenously with AT, Dp, AT plus Dp or their vehicle and then treated 30 min later with iv insulin or its vehicle (n=8-10 animals/group). Thirty min after treatment, GE was evaluated by determining, in awake rats, the percent gastric retention (%GR) of a saline meal labeled with phenol red administered by gavage. The results indicated that insulin induced hypoglycemia in a dose-dependent manner resulting in a significant reduction in %GR of liquid only at the highest dose tested (1 U/kg). Pretreatment with AT significantly increased %GR in the rats treated with 1 U/kg insulin. Surprisingly, after pretreatment with AT, the group treated with the lowest dose of insulin (0.25 U/kg) displayed significantly lower %GR compared to its control (vehicle-treated group), which was not seen in the non-pretreated animals. Pretreatment with Dp alone at the dose of 40 mg/kg induced an increase in %GR in both vehicle and 0.25 U/kg-treated rats. A higher dose of Dp alone (80 mg/kg) significantly reduced the effect of a marked hypoglycemia induced by 1 U/kg of insulin on GE while in combination with AT the effect was completely abolished. The results with AT suggest that moderate hypoglycemia may render the inhibitory mechanisms of GE ineffective while Dp alone and in combination with AT significantly overcame the effect of hypoglycemia on GE.

Atropine is more efficient than ZM241385 to reduce a drastic level of fade caused by cisatracurium at 50 Hz / Atropina é mais eficiente que ZM241385 para reduzir o intenso nível de fadiga causada por cisatracurium a 50 Hz

The effects of atropine (non selective muscarinic antagonist) and ZM241385 (A2A receptors antagonist) in the cisatracurium-induced drastic (100%) level of fade at 50 Hz (10 s) (100% Fade) were compared in the phrenic nerve-diaphragm muscle preparations of rats indirectly stimulated at a physiological tetanic frequency (50 Hz). The lowest dose and the instant cisatracurium caused 100% Fade were investigated. Cisatracurium induced 100% Fade 15 min after being administered. Atropine reduced the cisatracurium-induced 100% Fade, but the administration of ZM241385 separately, or combined with atropine, did not cause any effect in the cisatracurium-induced 100% Fade. Data indicate that the simultaneous blockage of M1 and M2 muscarinic receptors on motor nerve terminal by atropine is more efficient than the blockage of presynaptic A2A receptors for a safer recovery of patients from neuromuscular blockade caused by cisatracurium.

Os efeitos de atropina e ZM241385 (antagonistas de receptores A2A) no drástico (100%) nível de fadiga (100% Fadiga) produzido pelo cisatracúrio a 50 Hz (10 s) foram comparativamente investigados em preparações nervo frênico músculo diafragma isolado de ratos indiretamente estimuladas com a frequência fisiológica tetanizante de 50 Hz. A menor dose e o instante no qual cisatracúrio causou 100% Fadiga foram pesquisados. O cisatracúrio induziu 100% Fadiga 15 min depois de ser administrado. A atropina reduziu a fadiga de 100% causada pelo cisatracúrio, mas ZM241385, ou a administração combinada de atropina com ZM241385, não causou qualquer efeito na 100% Fadiga produzida pelo cisatracúrio. Os dados indicam que o bloqueio simultâneo dos receptores muscarínicos M1 e M2 no terminal nervoso motor pela atropina é mais eficiente do que o bloqueio dos receptores pré-sinápticos A2A no auxilio da recuperação mais segura do bloqueio da transmissão neuromuscular causada por cisatracúrio.

Possible Mechanisms for Functional Antagonistic Effect of Ferula assafoetida on Muscarinic Receptors in Tracheal Smooth Muscle

Background: The contribution of histamine (H1) receptors inhibitory and/or β-adrenoceptors stimulatory mechanisms in the relaxant property of Ferula assa-foetida. (F. asafoetida) was examined in the present study. Methods: We evaluated the effect of three concentrations of F. asafoetida extract (2.5, 5, and 10 mg/mL), a muscarinic receptors antagonist, and saline on methacholine concentration-response curve in tracheal smooth muscles incubated with β-adrenergic and histamine (H1) (group 1), and only β-adrenergic (group 2) receptors antagonists. Results: EC50 values in the presence of atropine, extract (5 and 10 mg/mL) and maximum responses to methacholine due to the 10 mg/mL extract in both groups and 5 mg/mL extract in group 1 were higher than saline (P < 0.0001, P = 0.0477, and P = 0.0008 in group 1 and P < 0.0001, P = 0.0438, and P = 0.0107 in group 2 for atropine, 5 and 10 mg/mL extract, respectively). Values of concentration ratio minus one (CR-1), in the presence of extracts were lower than atropine in both groups (P = 0.0339 for high extract concentration in group 1 and P < 0.0001 for other extract concentrations in both groups). Conclusion: Histamine (H1) receptor blockade affects muscarinic receptors inhibitory property of F. asafoetida in tracheal smooth muscle

The Effect of Urocortin 1 on Motility in Isolated, Vascularly Perfused Rat Colon / 대한소화기학회지

BACKGROUND/AIMS: Urocortin 1, a corticotropin-releasing factor related peptide, increases colonic motility under stressful conditions. We investigated the effect of urocortin 1 on colonic motility using an experimental model with isolated rat colon in which the blood flow and intestinal nerves were preserved. Furthermore, we assessed whether this effect was mediated by adrenergic or cholinergic nerves. METHODS: Colonic motility was measured in the proximal and distal parts of resected rat colon. The colon resected from the peritoneum was stabilized, and then urocortin 1 (13.8, 138, 277, and 1,388 pM) was administered via a blood vessel. Motility index was measured in the last 5 min of the 15 min administration of urocortin 1 and expressed as percentage change from baseline. Subsequently, the change in motility was measured by perfusing urocortin 1 in colons pretreated with phentolamine, propranolol, hexamethonium, atropine, or tetrodotoxin. RESULTS: At concentrations of 13.8, 138, 277, and 1,388 pM, urocortin 1 increased the motility of proximal colon (20.4+/-7.2%, 48.4+/-20.9%, 67.0+/-25.8%, and 64.2+/-20.9%, respectively) and the motility of distal colon (3.3+/-3.3%, 7.8+/-7.8%, 71.1+/-28.6%, and 87.4+/-32.5%, respectively). The motility induced by urocortin 1 was significantly decreased by atropine to 2.4+/-2.4% in proximal colon and 3.4+/-3.4% in distal colon (p<0.05). However, tetrodotoxin, propranolol, phentolamine, and hexamethonium did not inhibit motility. CONCLUSIONS: Urocortin 1 increased colonic motility and it is considered that this effect was directly mediated by local muscarinic cholinergic receptors.

Uterine Relaxation Potential of Ethanol Leaf Extract of Moringa oleifera Lam. VIA the Muscarinic Receptor Pathway.

Aim: The effect of Ethanol Leaf Extract of Moringa oleifera (ELMO) on uterine smooth muscles of non- pregnant female rats was studied in vitro with a view to finding out the mechanism(s) for observed effects. Experiential Design: In vitro studies using isolated rat’s uteri. Methods: Female albino rats (140-180g) pretreated 24 hours before experiments with diethylstilbestrol were sacrificed and the uterine horns carefully harvested into a beaker of De Jalon solution bubbled with 95% oxygen and 5% carbon dioxide. Each horn was mounted in an organ bath and allowed to equilibrate for 30 minutes, then the effects of graded doses of Acetylcholine, oxytocin and ELMO were established, using a physiograph and its accessories. The drugs were re-administered in the presence of their respective antagonists (Atropine for Acetylcholine and Atosiban for Oxytocin) and also in the presence of ELMO. Results: Results obtained showed that while Acetylcholine and oxytocin induced uterine contractions, ELMO caused relaxation. ELMO significantly (P<0.05) blocked the uterine contractile effect of Acetylcholine but had no effect on Oxytocin induced uterine contractions. The experiments therefore indicate that ELMO contain active ingredients capable of inducing uterine relaxation via the muscarinic receptor pathway. Conclusion: The extract therefore, may not be a valuable tocolytic agent in cases of Oxytocin induced uterine contractions, particularly in pregnancy but its observed strong anticholinergic activity may be exploited in the treatment of diseases associated with hyper activity of the parasympathetic arm of the autonomic nervous system.

Preliminary pharmacological investigation of the ischuretic property and safety of a hydro-ethanolic extract of Amaranthus spinosis (Fam: Amaranthaceae).

Background: Ischuria is a health and social problem, having a negative impact on sufferers. This study therefore was a preliminary investigation of the ischuretic property and safety for use of a hydro-ethanolic extract of Amaranthus spinosus used traditionally in managing ischuria. Methods: Phytochemical screening, thin layer chromatography and high performance liquid chromatography were performed on the extract to establish fingerprints for identification. Acetylcholine, Nicotine, and the extract were applied to an isolated rat urinary bladder to ascertain contractile response. The possible receptor site(s) of action was also investigated using isolated rabbit jejunum, and guinea-pig ileum preparations. In-house observation, hematological analysis, and liver and kidney function tests were performed on Sprague-Dawley rats, in acute and sub-acute toxicity studies. Results: The extract had contractile effects on the rat urinary bladder (similar to acetylcholine and nicotine) and rabbit jejunum. Its contractile effect of the guinea-pig ileum was significantly inhibited by hexamethonium (77.50 ± 8.50 %; P ≤ 0.001) and to a lesser extent by mepyramine (49.2 ± 6.80 %; P ≤ 0.001) and Atropine (22.45 ± 5.22 %; P ≤ 0.01). The extract (80-800 mg kg-1) was not lethal and a 160 and 240 mg kg-1 dose had no adverse effect on blood, liver, kidney metabolic function. Conclusions: The hydro-ethanolic extract of Amaranthus spinosus has ischuretic activity possibly mediated via nicotinic, histaminic and muscarinic receptor stimulation and is safety to use in ischuria.

Muscarinic antibodies and heart rate responses to dynamic exercise and to the Valsalva maneuver in chronic chagasic patients / Anticorpos muscarínicos e resposta da frequência cardíaca ao exercício dinâmico e a manobra de Valsalva na doença de Chagas crônica

We have studied the cardiac chronotropic responses to the Valsalva maneuver and to dynamic exercise of twenty chronic chagasic patients with normal left ventricular function and no segmental wall abnormalities by two-dimensional echocardiogram. The absolute increase in heart rate of the patients (Δ = 21.5 ± 10 bpm, M±SD) during the maneuver was significantly diminished when compared to controls (Δ = 31.30 ± 70, M±SD, p = 0.03). The minimum heart rate (58.24 ± 8.90 vs. 62.80 ± 10, p = 0.68) and the absolute decrease in heart rate at the end of the maneuver (Δ = 38.30 ± 13 vs. Δ = 31.47 ± 17, p = 0.10) were not different from controls. The initial heart rate acceleration during dynamic exercise (Δ = 12 ± 7.55 vs. Δ = 19 ± 7.27, M±SD, p = 0.01) was also diminished, but the heart rate recovery during the first ten seconds was more prominent in the sero-positive patients (Median: 14, Interquartile range: (9.75-17.50 vs. 5(0-8.75, p = 0.001). The serum levels of muscarinic cardiac auto-antibodies were significantly higher in the chagasic patients (Median: 34.58, Interquartile Range: 17-46.5, Optical Density) than in controls (Median: 0, Interquartile Range: 0-22.25, p = 0.001) and correlated significantly and directly (r = 0.68, p = 0.002) with early heart rate recovery during dynamic exercise. The results of this investigation indirectly suggest that, the cardiac muscarinic auto-antibodies may have positive agonist effects on parasympathetic heart rate control of chagasic patients.

Foram estudadas as respostas cronotrópicas cardíacas à manobra de Valsalva e ao exercício dinâmico de vinte pacientes chagásicos com função ventricular esquerda normal e sem alterações da contractilidade segmentar por ecocardiografia bidimensional. O aumento absoluto da frequência cardíaca dos pacientes (Δ = 21,5 ± 10 bpm, M ± DP) durante a manobra de Valsalva foi significativamente menor quando se comparava ao grupo controle (Δ = 31,30 ± 70, p = 0,03). A frequência cardíaca mínima (58,24 ± 8,90 vs 62,80 ± 10, p = 0,68) e a diminuição da frequência cardíaca absoluta no final da manobra (Δ = 38,30 ± 13 vs Δ = 31,47 ± 17, p = 0,10) não foram diferentes em comparação com o grupo controle. A aceleração inicial da frequência cardíaca durante o exercício dinâmico (Δ = 12 ± 7,55 vs Δ = 19 ± 7,27, p = 0,01) também foi menor, mas a recuperação da frequência cardíaca, durante os primeiros dez segundos, foi maior no grupo sero-positivos [mediana:14 (intervalo interquartil: 9,75-17,50) vs 5 (0 - 8,75), p = 0,001]. Os níveis séricos de auto-anticorpos muscarínicos cardíacos foram significativamente maiores nos pacientes chagásicos do que no grupo controle [(mediana: 34,58 densidade óptica (intervalo interquartil 17 - 46,5) vs (mediana: 0, intervalo interquartil 0 - 22,25) p = 0,001] e a correlação é significativa e direta (r = 0,68, p = 0,002) com o início da recuperação da frequência cardíaca durante o exercício dinâmico. Os resultados desta investigação sugerem que indiretamente, os auto-anticorpos muscarínicos cardíacos, podem ter ação agonista positiva sobre o controle parassimpático da frequência cardíaca dos pacientes chagásicos.

Rationale for the Use of Anticholinergic Agents in Overactive Bladder With Regard to Central Nervous System and Cardiovascular System Side Effects

PURPOSE: Central nervous system (CNS) and cardiovascular system (CVS) side effects of anticholinergic agents used to treat overactive bladder (OAB) are underreported. Hence, this review aimed to focus on the mechanisms of CNS and CVS side effects of anticholinergic drugs used in OAB treatment, which may help urologists in planning the rationale for OAB treatment. MATERIALS AND METHODS: PubMed/MEDLINE was searched for the key words "OAB," "anticholinergics," "muscarinic receptor selectivity," "blood-brain barrier," "CNS," and "CVS side effects." Additional relevant literature was determined by examining the reference lists of articles identified through the search. RESULTS: CNS and CVS side effects, pharmacodynamic and pharmacokinetic properties, the metabolism of these drugs, and the clinical implications for their use in OAB are presented and discussed in this review. CONCLUSIONS: Trospium, 5-hydroxymethyl tolterodine, darifenacin, and solifenacin seem to have favorable pharmacodynamic and pharmacokinetic properties with regard to CNS side effects, whereas the pharmacodynamic features of darifenacin, solifenacin, and oxybutynin appear to have an advantage over the other anticholinergic agents (tolterodine, fesoterodine, propiverine, and trospium) with regard to CVS side effects. To determine the real-life situation, head-to-head studies focusing especially on CNS and CVS side effects of OAB anticholinergic agents are urgently needed.

Muscarinic Receptors Distributed in the Lower Urinary Tract

Acetylcholine (Ach) acting on the muscarinic receptors is the major stimulus for the detrusor inducing contraction if the bladder is distended and the bladder volume is at the threshold. There are 5 types muscarinic receptor subtypes cloned (M1-M5). Among them, the M2 subtype distributed dominantly in amount in the detrusor and its role seems to be primarily by blocking stimuli from cAMP-coupled receptors that induce relaxation. In contrast, through M3 receptors on the detrusor the strong contractile stimulus evoked even if its distribution in secondarily common. The excitatory M1 and inhibitory M2 and M4 subtypes are also expresswed prejunctionally. Like detrusor, urothelium also expressed all subtypes of muscarinic receptors, and on stimulation it releases factors affecting bladder afferents and smooth muscle. During inflammation, the expression of muscarinic M5 receptors is increased, particularly in the urothelium, together with a cholinergic-induced production of nitric oxide in the mucosa. This review will be discussed expression and functional effects of muscarinic receptors in the lower urinary tract. Clinical aspects of the different muscarinic receptors and roles in the pathologic conditions included in this discussions.

The Effects of Estrogen on Detrusor Contraction and the Expression of Muscarinic Receptors in Ovariectomized Rats / 대한비뇨기과학회지

PURPOSE: The purpose of this study was to evaluate the effects of estrogen on detrusor contraction and the expression of muscarinic receptors in ovariectomized rats. MATERIALS AND METHODS: 24 Sprague-Dawley female virgin rats(12 weeks old) were separated into three groups of 8 rats each. Group I served as a control group, group II was the ovariectomized only rats(Ovx group) and Group III was given estradiol benzoate(0.8mg/kg/day) subcutaneously for 7 consecutive days, beginning 1 week after ovariectomy(Ovx+E group). At the end of the experimental period, each rat was sacrificed and the urinary bladder was removed for contractile studies. The expressions of M2 and M3 receptors in the bladder epithelium and the muscle layer were investigated by performing immunofluorescent staining. RESULTS: The Ovx group showed a significantly decreased bladder contractile function on the KCl and carbachol-induced contractile tests, whereas the Ovx+E group showed increased contractility(p<0.05). The Ovx+E group showed an increase of smooth muscle compared to the other groups. Ovariectomy induced a significant increase in the M3 receptors density in the bladder body, as compared to the control group(p<0.05) but there was no significant difference between the Ovx group and the Ovx+E group. CONCLUSIONS: Bladder dysfunction of menopausal women is thought not to be related with the changes of muscarinic receptors. Our results suggest that the detrusor contractility of menopausal women might be improved after estrogen replacement therapy.

Pacientes chagásicos crônicos portadores de disfunção do nódulo sinusal: a presença de anticorpos IgG com ação agonista muscarínica independe da disfunção ventricular esquerda? / Chronic Chagas disease patients with sinus node dysfunction: is the presence of IgG antibodies with muscarinic agonist action independent of left ventricular dysfunction?

Estudos mostram que anticorpos IgG agonistas muscarínicos, de pacientes chagásicos, alteram a atividade elétrica de células cardíacas in vitro. Outros consideram sua presença, e a da síndrome do nódulo sinusal, conseqüências da lesão cardíaca progressiva. Objetivou-se avaliar a relação entre os anticorpos e as disfunções nodal e ventricular esquerda, em 65 pacientes chagásicos crônicos divididos em grupo I, composto de 31 pacientes portadores da síndrome do nódulo sinusal, e grupo II, de não portadores. A análise dos dados, pelo modelo log linear, mostrou uma interdependência entre a disfunção do nódulo sinusal e os anticorpos (p=0,0021) e entre a disfunção nodal e a ventricular (p=0,0005), mas não houve relação entre esta última e os anticorpos. Idade e sexo não tiveram influência sobre as outras variáveis. Chagásicos crônicos com a síndrome do nódulo sinusal têm maior prevalência de anticorpos agonistas muscarínicos, independentemente da presença de disfunção miocárdica.

Studies have shown that muscarinic agonist IgG antibodies from Chagas disease patients alter the electrical activity of cardiac cells in vitro. Others have considered their presence, along with sinus node dysfunction, to be consequences of progressive cardiac lesions. The aim of this study was to evaluate the relationship between these antibodies and sinus node and left ventricular dysfunction in 65 chronic Chagas disease patients. These patients were divided into group I, composed of 31 patients with sinus node dysfunction, and group II, composed of the patients without this syndrome. Data analysis using the log linear model showed interdependence between sinus node dysfunction and the antibodies (p = 0.0021) and between nodal and ventricular dysfunction (p = 0.0005). However, no relationship was found between the antibodies and ventricular function. Age and sex did not influence any other variables. The chronic Chagas disease patients with sinus node dysfunction had higher prevalence of muscarinic agonist antibodies, independent of the presence of myocardial dysfunction.

The influences of morpholin ring and perizine ring compounds on relaxation of rat aortic rings / 中国药理学通报

AIM Our investigation is to screen bioactive novel compounds using the isolated rat aortic rings and depending on the similar and distinct characteristics between the endothelial target for acetylcholine(ETA) and muscarinic receptors and to investigate the mechanisms of vasodilatory effects of candidate compounds. METHODSIn isolated rat aorta precontracted with NE, the vasodilatory effects of novel structure compounds were investigated. We Compared the maximal relaxation of endothelium denuded aorta with that of the endothelium intact aorta elicited by the 8 candidate compounds respectively. The aortas were precubated with L NAME, indomethacine and atropine before using NE, and measured the changes of the maximum vasodilatory rate of candidate compound. RESULTS AND CONCLUSION Among 81 compounds, we found 8 novel compounds which induced relaxation. Their maximal relaxation rates ranged of from 50 percent to 85 percent. The endothelium dependent relaxation induced by DMHPPP and PPVP was blocked by indomethacin and L NAME, but not by atropine. DMHPPP and PPVP also enhanced the maximal endothelium dependent relaxation induced by acetylcholine. These suggest that novel compounds may regulate functions of endothelial cell target for acetylcholine(ETA) to induce relaxation of isolated rat aortic rings, which may involve the prostacycline and nitric oxide pathways.

Function of the neuronal M2 muscarinic receptor in asthmatic patients / 결핵

BACKGROUND: The dominant innervation of airway smooth muscle is parasympathetic fibers which are carried in the vagus nerve. Activation of these cholinergic nerves releases acetylcholine which binds to M3 muscarinic receptors on the smooth muscle causing bronchocontraction. Acetylcholine also feeds back onto neuronal M2 muscarinic receptors located on the postganglionic cholinergic nerves. Stimulation of these receptors further inhibits acetylcholine release, so these M2 muscarinic receptors act as autoreceptors. Loss of function of these M2 receptors, as it occres in animal models of hyperresponsiveness, leads to an increase in vagally mediated hyperresponsiveness. However, there are limited data pertaining to whether there are dysfunctions of these receptors in patients with asthma. The aim of this study is to determine whether there are dysfunction of M2 muscarinic receptors in asthmatic patients and difference of function of these receptors according to severity of asthma. METHODS: We studied twenty-seven patients with asthma who were registered at Pulmonology Division of Korea University Hospital. They all met asthma criteria of ATS. Of these patients, eleven patients were categorized as having mild asthma, eight patients moderate asthma and eight patients severe asthma according to severity by NAEPP Expert Panel Report 2(1997). All subjects were free of recent upper respiratory tract infection within 2 weeks and showed positive methacholine challenge test(PC 20<16mg/ml). Methacholine provocation tests performed twice on separate days allowing for an interval of one week. In the second test, pre-treatment with the M2 muscarinic receptor agonist pilocarpine(180µg) through inhalation was performed before the routine procedures. RESULTS: Eleven subjects with mild asthma and eight aubjects with moderate asthma showed significant increase of PC20 from 5.30±5.23mg/ml(mean±SD) to 20.82±22.56mg/ml(p=0.004) and from 2.79±1.5mg/ml to 4.67±3.53mg/ml(p=0.012) after pilocarpine inhalation, respectively. However, in the eight subjects with severe asthma significant increase of PC20 from 1.76±1.50mg/ml to 3.18±4.03mg/ml(p=0.161) after pilocarpine inhalation was not found. CONCLUSION: In subjects with mild and moderate asthma, function of M2 muscarinic receptors was normal, but there was a dysfunction of these receptors in subjects with severe asthma. These results suggest that function of M2 muscarinic receptors is different according to severity of asthma.

Protective effects of penehyclidine hydrochloride on transient forebrain ischemia reperfusion injury in gerbils / 中国临床药理学与治疗学

AIM:To study the protective effects of penehyclidine hydrochloride(PHC) on transient forebrain ischemia reperfusion injury in gerbils.METHODS:The model of transient forebrain ischemia reperfusion was established in gerbils by bilateral carotid artery clamping.The effects of PHC on neurological function scores and the morphous of hippocampal pyramidal neuron of gerbils were observed after receiving transient forebrain ischemia reperfusion.SOD activities and contents of MDA in the hippocampus and cortex of gerbils were measured.RESULTS:In the groups of PHC(0.08),(0.24)(mg?kg~(-1)) and atropine,the stroke index was decreased,compared with the ischemia-reperfusion group after the gerbils received transient forebrain ischemia reperfusion for six hours.PHC could reduce the degree of injury in hippocampal pyramidal neuron after ischemia reperfusion for three days.CONCLUSION: PHC has protective effects on transient forebrain ischemia reperfusion injury in gerbils.

Enhanced coupling of M-1 muscarinic receptors to activation of phospholipase C upon mutation of a transposed amino acid triplet repeat

The C-terminus ends of the second putative transmembrane domains of both M-1 and M-2 Muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in M-1 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-LYT) in the sequence Of M-2 receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of M-1 receptors to the corresponding M-2 receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT M-1 receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant M-1 receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant M-1 receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in M-1 muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of M-1 receptors to the stimulation of phospholipase C.

Primary role of posterior hypothalamic cholinergic receptors in central regulation of blood pressure and heart rate in rats

The purpose of the present study is to determine the role of muscarinic cholinergic receptors of posterior hypothalamus in the central blood pressure regulation when respiration is controlled. In anesthetized and artificially ventilated rats, vasodepressor response was evoked by injection of L-glutamate (10 nmol) neuroexcitatory amino acid into the posterior hypothalamic area. The injection of carbachol (0.5 ~ 8 nmol) into the same area induced dose-dependent vasodepressor and bradycardic responses. Pretreatment with atropine (4 nmol) completely blocked the vasodepressor response to carbachol (2 nmol). In contrast, in spontaneously breathing rats, the injection of carbachol (8 nmol) into the posterior hypothalamic area induced the vasopressor and tachycardic responses. These results suyggest that the muscarinic cholinergic receptors in the posterior hypothalamic area primarily play an inhibitory role in the central regulation of blood pressure and heart rate.