Effects of JO 1784, A selective sigma ligand, on the autoradiographic localization of M2 and M2 muscarinic receptor subtypes in trimethyltin treated rats

The distribution patterns of M₁ and M₂ muscarinic receptor subtypes following TMT and JO 1784 administration in the male Sprague-Dawley rat were investigated. In the present study, JO 1784 was injected in doses of 1, 4 and 16 mg/kg i.p. for one week prior to the single injection of TMT (8 mg/kg i.p.) and subsequently for 33 days. The effects of JO 1784 on the density of muscarinic receptor sub-types (M₁ and M₂) in the control and trimethyltin (TMT) treated rats were then evaluated. The topographic distribution and changes in muscarinic (M₁ and M₂) receptor densities were determined by means of autoradiography using [³H]quinuclidinylbenzilate (QNB). Both sub-types of muscarinic receptors contributed to the observed decrease in total muscarinic receptor binding in TMT-treated rats. In control rats, JO 1784 alone decreased M₁ receptor density in the amygdaloid nuclei, basal ganglia, cortex and hippocampus and decreased M₂ receptor density in the amygdaloid nuclei, basal ganglia, cortex, hippocampus, hypothalamus and septal regions. In TMT treated rats, chronic JO 1784 administration has a “neuroprotective effect” on both M₁ and M₂ receptors subtypes. Thus, following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M₁ receptor density were observed relative to TMT animals. A significant increase in M₁ receptor density was found in the cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. In the hippocampus (CA₂ and CA₃), a significant decrease in M₁ receptor density was observed. In TMT-treated rats, JO 1784 produced a significant increase in M₂ receptor density in several brain regions with the most marked effects occurring in the amygdaloid nuclei, basal ganglia, cortex, hippocampus and hypothalamus. The ability of the selective sigma ligand, JO 1784, to attenuate the loss of muscarinic receptors in TMT treated rats could be of importance in the development of novel neuroprotective drugs.     

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes

A set of novel heterocyclic ligands (6–27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1–5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M₁, M₂, and M₃ tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC₅₀: 7.40 (M₁), 8.18 (M₂), and 8.14 (M₃)], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M₁ subtype [pK_B: 6.88 (M₁), 5.95 (M₂), 5.53 (M₃)]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M₁ antagonist/M₂ partial agonist/M₃ full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M_1–3 receptors, with an appreciable selectivity for the cardiac M₂ receptors.     

The neuroprotective effect of tacrine on trimethyltin induced memory and muscarinic receptor dysfunction in the rat

In this study chronic (39 days) tacrine (3 mg/kg i.p.) treatment significantly improved trimethyltin (8 mg/kg i.p.) induced deficits in spatial navigation. Tacrine also reduced trimethyltin induced hyperactivity and passive avoidance deficits but these effects did not reach statistical significance. The effect of trimethyltin on muscarinic (M₁ and M₂) receptor sites was determined by means of quantitative autoradiography using [³H]quinuclidinyl benzilate. A selective pattern of M₁ and M₂ receptor loss was observed mainly affecting the hippocampus and other limbic structures while leaving other brain regions intact. Tacrine successfully prevented the M₁ and M₂ receptor loss in the CA₁ and CA₁ hippocampal subfields. The improvement in trimethyltin behavioural toxicity following tacrine treatment may be related to the protective effect of this compound on muscarinic receptor density in the hippocampal formation and lends support to the hypothesis that cholinergic system dysfunction may be primarily responsible for trimethyltin induced deficits in cognitive function.     

Diphenidol-related diamines as novel muscarinic M4 receptor antagonists

A series of hydrochloride derivatives 2a–9a and quaternary ammonium derivatives 3b–9b of diphenidol have been synthesized and characterized in receptor binding and cellular functional assays versus human muscarinic M₁–M₅ receptors expressed in CHO cells. Compound 8b, a methiodide derivative with a bipiperidinyl moiety and a second diphenidol framework, showed a potent and selective M₄ activity as competitive antagonist. Moreover 8b, acting as an allosteric modulator, was able to retard the dissociation rate of [³H]-N-methylscopolamine from CHO-M₄ cell membranes exposed to atropine. Taken together, these data suggest that 8b might open new avenues to the discovery of novel multivalent antagonists for the muscarinic receptors.     

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo

One of the hallmarks of type 2 diabetes is that pancreatic β cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic β cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M₃ muscarinic acetylcholine receptor subtype in pancreatic β cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M₃ receptors in pancreatic β cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that β cell M₃ muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.     

Synthesis, NMR spectroscopy study, and antimuscarinic activity of a series of 2-(Acyloxymethyl)-1,3-dioxolanes

A series of 1,3-dioxolane-based ligands, bearing hydroxymethyl or ester functionalities, was synthesized and tested as potential muscarinic antagonists. The compounds display moderate to low affinity for the three receptor subtypes M₁-M₃, with some of them showing a significant selectivity for the M₃ subtype. The configurational and conformational properties were studied using NOE experiments and vicinal coupling constants. The ¹H and ¹³C NMR chemical shifts show stereochemically dependent trends. Quantitative analysis of conformer populations showed that the exocyclic CH₂N⁺(CH₃)₃ group is prevalently in a pseudo-axial orientation in the cis isomers and in a pseudo-equatorial orientation in the trans isomers.     

6-(4-Phenyl-benzyloxy-methyl) guvacine. Synthesis, GABA uptake inhibitor and muscarinic properties

6-(4-Phenyl-benzyloxy-methyl) guvacine was synthesized. Surprisingly the compound was devoid of the γ-aminobutyric acid (GABA) uptake inhibitory activity of its parent compound guvacine, but instead showed affinities for the muscarinic M₁ and M₂ receptors.     

Muscarinic thioligands with cyclopentane nucleus

Some thio- and the benzoyl-derivatives of deoxamuscarine were synthesized and tested as muscarinic agonists using radioligand binding assays and functional tests. In comparison with deoxamuscarine, used as reference compound, no dimension/distance modification is tolerated for correct lipophilic pocket recognition. The substitution of the ammonium group with a sulphonium group significantly decreased muscarinic potency. The so-called ‘muscarinic sub-site’ accepts relatively bulky functions as long as it is bound to the cyclopentane carrier by an oxygen bridge. Esterification of this moiety increases the M₂ subtype selectivity, while etherification heightens that of M₃.     

小家鼠(Mus musculus)种群动态预测及机制的探讨

我国学者夏武平(1966)用9月铗日捕获率预测东北棕背(鼠平)(Clethrionomys rufocanus)数量以来,严志堂等(1983)应用种群年龄结构的回归方程预测新疆天山北麓农区塔西河地区的小家鼠(Mus musculus)种群密度获初步成功,并根据该文提供的一个改进公式M₁₀=5.60x+5.80(x表示种群年龄的成亚比,M₁₀表示小家鼠10月数量,即年内最高数量),提前11个月预测该地的小家鼠种群最高数量。     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Interaction of amitriptyline with muscarinic receptor subtypes in the rat brain

The affinity of amitriptyline for muscarinic receptors in rat brain areas was studied using autoradiographic techniques including image analysis. As shown by competitive inhibition of [³H]-l-quinuclidinyl benzilate binding, amitriptyline was found to be a potent inhibitor of muscarinic receptors throughout the rat brain. Muscarinic receptors in the external layers of the cortex displayed a high affinity for amitriptyline (IC₅₀ = 65.8 ± 2.1 nM), while the hippocampal regions had somewhat lower affinities (e.g. IC₅₀ = 96.3 ± 3.4 nM). Amitriptyline bound with lower affinity in the thalamus and various midbrain regions, such as the paraventricular nucleus of the thalamus and the superior colliculus, which had IC₅₀ values of 112 ± 6.8 and 117 ± 32.6 nM, respectively. Other midbrain regions displayed higher affinities, for example, the substantia nigra had an IC₅₀ value of 62.8 ± 0.9 nM. The data show that amitriptyline binds with high affinity to muscarinic receptors with a modest subtype selectivity that is unlike that of either pirenzepine or AF-DX 116. In addition, amitriptyline at concentrations of 10 nM-1 μM antagonized the oxotremorine-induced inhibition of acetylcholine release in cortical nerve endings, demonstrating activity at M₂ autoreceptors.     

有机肥氮投入比例对双季稻田根际土壤微生物生物量碳、氮和微生物熵的影响

为探明不同有机肥氮素占总氮投入的百分比对双季稻区早、晚稻各生育时期稻田根际土壤微生物的影响,本研究以大田定位试验为平台,应用氯仿熏蒸-K₂SO₄提取法和化学分析法系统分析了施用化肥N(M₁)、30%有机肥N(M₂)、50%有机肥N(M₃)、100%有机肥N(M₄)和无N对照(M₀)5个不同施肥处理双季稻田根际土壤微生物生物量碳(MBC)、微生物生物量氮(MBN)和微生物熵的差异.结果表明: 在早稻和晚稻各主要生育时期,施肥措施均能提高稻田根际土壤MBC、MBN和微生物熵,各施肥处理根际土壤MBC、MBN和微生物熵均随水稻生育期推进呈先增加后降低的变化趋势,均于齐穗期达到最大值,成熟期为最低值;其中,各处理双季稻田根际土壤MBC、MBN、MBC/MBN值和微生物熵一般均表现为M₄＞M₃＞M₂＞M₁＞M₀,M₂、M₃和M₄处理间均无显著差异,但均显著高于M₀处理.可见,单独施用化肥措施对提高根际土壤微生物生物量碳、氮和微生物熵效果有限,施用有机肥或有机无机肥配施提高根际土壤微生物生物量碳、氮和微生物熵的效果较好.     

Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain

Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [³H]QNB reversibly with a K _d of 260 PM and B_max of 1 pmol/g of heads from direct binding measurements. The K_d calculated from the ratio of the dissociation rate constant (2 × 10⁻⁴ sec⁻¹) to the association rate constant (2.5 × 10⁶ M⁻¹ Sec⁻¹) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.     

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K-depolarized synaptosomes

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked γ-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 μM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca²⁺-dependent and was under GABA_B autoreceptor control. Under these experimental conditions, acetylcholine (0.1–10 μM) and physostigmine (30 μM) decreased the electrically evoked GABA release while the M₂ receptor antagonist AFDX-116 (0.01–0.1 μM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K⁺ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M₂ receptors located on cortical GABAergic terminals.     

有机物沟埋还田模式与花后灌水量对玉米光合特性和产量的影响

有机物沟埋还田与花后灌水配合对增加玉米田保水供水能力,提高玉米花后光合性能、实现节水增产有重要意义.本试验以郑单958为供试材料,设置有机物沟埋还田和花后灌水量两个因素,有机物沟埋还田包括不还田(M₀)、秸秆单还田(M₁)和牛粪秸秆混合还田(M₂)3个还田类型,花后灌水量包括450 mm(W₁)和325 mm(W₂)2个水平,研究了其对玉米穗位叶光合性能、光系统Ⅱ(PSⅡ)效率和产量等的影响.结果表明:与秸秆单还田比较,牛粪秸秆混合还田有效提高了玉米花后光合能力和各器官的干物质积累量;与节水灌溉相比,正常灌溉加强了有机物还田对玉米光合能力的促进作用.牛粪秸秆混合还田与正常灌溉结合可显著提高玉米花后叶片的光合速率(P_n)、气孔导度(g_s)和蒸腾速率(T_r),降低胞间CO₂浓度(C_i);提高玉米花后叶片PSⅡ的最大光化学效率(φ_po)和捕获的激子将电子传递到电子传递链中Q_A下游的电子受体的概率(Ψ_o);改善花后叶片光能利用率,维持花后叶片较高的光合性能;同时增加花后玉米各器官干物质的量,提高干物质总积累量和转运能力,有利于花后同化物对籽粒的分配,最终获得高产.节水灌溉降低了叶片的光合性能,造成产量的下降;但配合牛粪秸秆混合还田与不还田处理相比,水分利用效率、籽粒增长速率和增产效果均优于正常灌水.这表明牛粪秸秆混合还田与正常灌溉结合可有效提高玉米花后光合性能,增加干物质积累量,促进玉米增产;牛粪秸秆混合还田与节水灌溉结合一定程度上降低了因减少灌溉量造成的减产.     

长期不同施肥对玉米根茬生物量及养分累积量的影响

以黄土高原南部两个长期定位试验（分别开始于1990和2003年）为研究对象,探讨了不同肥料处理对玉米根茬生物产量和养分累积的影响.于2011年10月玉米收获后采集0～20 cm土层不同施肥处理玉米根茬.结果表明：与不施肥及偏施N、NK、PK化肥相比,氮磷配施（NP）、氮磷钾平衡施肥（NPK）、有机无机配施（M₁NPK、M₂NPK）及化肥配合秸秆（SNPK）处理均显著提高了玉米根茬干质量.根茬固碳量及氮、磷、钾养分累积量在NP、NPK、M₁NPK、M₂NPK、SNPK处理显著高于不施肥和偏施N、NK、PK化肥处理,其中以有机无机配施处理效果最好.与不施氮肥（N₀）相比,施氮120 kg N·hm^-2（N₁₂₀）和240 kg N·hm^-2（N₂₄₀）处理根茬干质量分别提高38%和45%,高量氮肥对根茬增量效果不显著.施用氮肥也显著提高了根茬碳、氮、磷、钾累积量.根茬可溶性有机碳、可溶性总氮含量在NP、NPK、M₁NPK、M₂NPK、SNPK及N₁₂₀和N240处理中较高.氮磷钾平衡施肥、有机无机配施以及秸秆还田处理降低了根茬的纤维素、木质素含量.根茬C/N、木质素/N在CK、PK、N₀处理间显著高于其他施肥处理.因此,氮磷配施、氮磷钾平衡施肥、有机无机配施及秸秆还田处理能够促进玉米根生长,提高营养成分含量,有利于土壤培肥和固碳.     

Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder

Caveolin-1 (Cav1), a structural protein of caveolae, plays cell- and context-dependent roles in signal transduction pathway regulation. We have generated a knockout mouse homozygous for a null mutation of the Cav1 gene. Cav1 knockout mice exhibited impaired urinary bladder contractions in vivo during cystometry. Contractions of male bladder strips were evoked with electric and pharmacologic stimulation (5–40 Hz, 1–10 μM carbachol, 10 mM ,β-methylene ATP, 100 mM KCl). Acetylcholine (ACh) and norepinephrine (NE) release from bladder strips were measured with a radiochemical method by incubating the strips with ¹⁴C-choline and ³H-NE prior to electric stimulation, whereas ATP release was measured using the luciferin-luciferase assay with a luminometer. A 60–75% decline in contractility was observed when Cav1 knockout muscle strips were stimulated with electric current or carbachol, compared to wildtype muscle strips. No difference in contractility was noted when contractions were evoked either by the purinergic agonist ,β-methylene ATP, or by extracellular potassium. To investigate the relative contribution of non-cholinergic activity to bladder contractility, the amplitude of the electric stimulation-evoked contractions was compared in the presence of the muscarinic antagonist atropine (1 μM). While the non-muscarinic (purinergic) response was unaltered, muscarinic cholinergic response was principally disrupted in Cav1 knockout mice. The loss of Cav1 gene expression was also associated with a 70% reduction in ACh release. NE and ATP release was not altered. It is concluded that the loss of caveolin-1 is associated with disruption of M₃ muscarinic cholinergic activity in the bladder. Both pre-junctional (acetylcholine neurotransmitter release from neuromuscular junctions) and post-junctional (M₃ receptor-mediated signal transduction in bladder smooth muscles) mechanisms are disrupted, resulting in impaired bladder contraction.     

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 2: Non-isothermal conditions

Lactose hydrolysis by β-galactosidase immobilized on two nylon membranes, differently grafted, has been studied in a bioreactor operating under isothermal and non-isothermal conditions. One membrane (M₁) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M₂) by a double chemical grafting: styrene (Sty) and MAA. Hexamethylenediamine was used as a spacer between the grafted membranes and the enzyme. Both membranes have been physically characterized studying their permeabilities in presence of pressure or temperature gradients. Under non-isothermal conditions, the increase in activity of membrane M₂ was higher than that of membrane M₁. The and β coefficients, giving the percentage of activity increase when a temperature difference of 1°C is applied across the catalytic membranes, have been calculated. Results have been discussed with reference to the greater hydrophobicity of membrane M₂ with respect to membrane M₁, the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.