Cholinomimetic teratogens. II. Interaction with inorganic ions.

Treatment of chicken embryos after 96 h of incubation showed that the teratogenic activity of the cholinomimetic compounds carbachol, neostigmine, decamethonium, and tetramethylammonium is reduced in the presence of calcium acetate. Similarly, supplementation with potassium acetate reduced the teratogenicity of carbachol and tetramethylammonium.     

Carbachol effect on kinetics of the alpha1-adrenergic contractile response of the rat vas deferens

Analysis of the control kinetics of the rat vas deferens contractile response to phenylephrine showed that alpha 1-adrenoceptors mediating the contraction could be in different functional states. In some organs these receptors represented a single pool with Hill coefficient n = 1 (the linear mode of the Scatchard plot), in others--not a single pool, n > 1 (not linear mode of the Scatchard plot). Activation of muscarinic cholinergic receptors by carbachol exerted a stimulating effect on the alpha 1-adrenergic contractile response especially to low adrenomimetic concentrations and the maximum response was increased. The action of cholinomimetic was accompanied by a decrease of Hill coefficient. When the control represented a single pool of alpha 1-adrenoceptors in the presence of carbachol Scatchard plot became biphasic with Hill coefficient n < 1, in addition to the low affinity pool the high affinity appeared. In case of not homogeneous control pool, in the presence of carbachol a single pool was revealed and n was close to 1. These findings suggest that the stimulatory effect of carbachol is caused by its modulator action on the alpha 1-adrenoceptors states and activating influence on the intracellular effector's system.     

Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos

The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca(2+)](i)) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca(2+)](i) levels in the unfertilised sea urchin egg, or on the transient [Ca(2+)](i) increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca(2+)](i), which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca(2+)-dependent) modulation of the nuclear status during the first cell cycles.     

Effect of cerebro-ventricular administration of noradrenaline and carbachol on behavioural and autonomic thermoregulation in the monitor lizard Varanus albigularis albigularis

1. 1. When exposed to an ambient temperature of 45°C V. a. albigularis began to pant whne core temperature was between 35.4–38.6°C. The skin temperature, however, appeared to be more critical than the core temperature in determining onset of panting.

2. 2. Cerebro-ventricula injections of saline and moradrenaline had no effect on panting but carbachol inhibited pnting and disrupted normal thermoregulatory behaviour in a photothermal gradient. The significance of this is discussed in relation to comparable studies on mammals.

The ionic mechanisms associated with the excitatory response of kainate, L-glutamate, quisqualate, ibotenate, AMPA and methyltetrahydrofolate on leech Retzius cells

Intracellular recordings were made from Retzius cells from segmental ganglia of the leech, Hirudo medicinalis. The ionic mechanisms of the following compounds were examined: L-glutamate, ibotenate, quisqualate, AMPA, kainate, methyltetrahydrofolate and carbachol. All these compounds depolarise and excite Retzius cells. In sodium-free Ringer, the responses to L-glutamate, kainate, ibotenate and AMPA were greatly reduced, the response to quisqualate was reduced, the response to methyltetrahydrofolate was normal while the response to carbachol was abolished. In sodium-free high calcium Ringer the responses to L-glutamate, ibotenate and carbachol were absent, the responses to quisqualate and AMPA greatly reduced, the responses to methyltetrahydrofolate and kainate were normal. The methyltetrahydrofolate and kainate responses in sodium-free high calcium Ringer were greatly reduced on addition of cobalt. All the responses are associated with an increase in conductance, the increase being the largest in the case of kainate. It is concluded that the response to L-glutamate, ibotenate and carbachol are dependent on sodium, the responses to quisqualate and AMPA are mainly sodium dependent, possibly with a small calcium component. The kainate response in normal Ringer is largely sodium dependent but in sodium-free Ringer calcium can completely substitute for sodium. The methyltetrahydrofolate response appears to be sodium independent but at least partly calcium dependent. These studies provide further evidence that L-glutamate and ibotenate act on a common receptor on leech Retzius cells while kainate acts on a separate receptor which can activate a calcium ionophore. It is probable that methyltetrahydrofolate acts on a different ionophore system to kainate. N-Methyl-D-aspartate has no agonist activity on any of these receptors.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

The Na+, K(+)-ATPase inhibitor ouabain and the Na+ ionophore monensin have opposite effects upon carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms.

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K+. Enhancement of the carbachol response at 6 mM, but not at 18 mM K+, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Ouabain did not affect [3H] pirenzepine binding at any of the concentrations tested (6-600 muM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.     

A neurochemical approach for studying response to acetylcholine in Alzheimer's disease

The proposal of cholinomimetic treatment as a rational basis for the therapy of Alzheimer's disease has been prematurely dismissed by some workers on the hypothesis of impaired coupling/signal transduction of postsynaptic cholinergic receptors. Disparity of reports studying such impairment may be due to inappropriate extrapolation of experimental systems to the physiological stituation, as well as inadequate consideration of disease epiphenoma. In the present study we have used samples with short duration of terminal coma, collected using techniques to minimise postmortem autolysis, and samples obtained during neurosurgery to examine carbachol stimulated hydrolysis of [³H]phosphatidylinositol (PI) as a marker for receptor/signal transduction integrity. The influence of postmortem delay was also studied using another series of samples and a rat model. While a significant correlation of postmortem delay and carbachol stimulated [³H]PI hydrolysis was found, comparison of pooled neurosurgical and postmortem controls with AD samples revealed no significant reduction. Thus this study concurs with a similar one previously reported here, using [³H]phosphatidylinositol 4,5-bisphosphate (1). They provide evidence for competent receptor-signal transduction events in AD, supporting the use of cholinomimetic therapy for disease treatment.     

Comparative affinity of several standard anti-secretory agents for the intestinal cholinergic receptors of of rats and dogs]

Anticholinergic potentialities of four standard anti-secretory drugs (N-methyl-scopolammonium methylsulfate, atropine, diphemanil and prifinium) have been investigated with the help of molecular pharmacology techniques. The results gained with two different agonists (acetylcholine and carbachol) on rat and dog intestinal cholinergic receptors-jejunum and duodenum respectively-show : 1) That relative potentialities of the anticholinergic drugs (pA2) as well as those of the cholinomimetic agonists (pD2) are markedly modified according to which effector is used. 2) The N-methyl scopolammonium methylsulfate remains in any event the most potent anticholinergic drug investigated.     

Method to determine stability and recovery of carboprost and misoprostol in infusion preparations

The two synthetic prostaglandin analogues, carboprost and misoprostol, are used extensively in obstetric and gynaecological practice. Our recent research of these compounds' use for intra-umbilical injection to treat adherent placenta necessitated their storage in solution for 3-4 days. This raised concerns over the stability and applied dosage in the in-house infusion preparations. It requires various pharmacological preparations before administration in clinical practice. We used LCMS to develop a simultaneous, valid, fast and simple method to assess the stability and recovery of their in-house preparations in different conditions. The linearity between 0-40 microg/ml was above 0.995. The reproducibility (CV) was within 5.2%. The limit of quantitation of the method for both compounds is about 2 microg/ml. The accuracy of both compounds from 0.4-40 microg/ml is 96.4-104.3% while the precision is 0.4-7.4%. The recoveries of carboprost in the infusion were from 100.3+/-4.0 to 102.4+/-1.6% and that of misoprostol in Cytotec tablet was from 44.9+/-3.5 to 50.0+/-5.0% in water and saline at 4 degrees C and room temperature. No interference was found from the matrix and between the tested compounds. The compounds were basically stable for 6 days in water and in saline, whether they were stored at 4 degrees C or at room temperature. However, only half of the dosage of misoprostol was recovered in the solution. Therefore, misoprostol dosage should be adjusted before clinical application.     

Gastric acid secretion in streptozotocin-diabetic rats--different responses to various secretagogues.

We compared gastric acid secretion in response to various stimuli in normal and streptozotocin (STZ)-induced diabetic rats, in an attempt to characterize the alteration of acid secretory response in diabetic conditions. Animals were injected STZ (70 mg x kg(-1), i.p.) and used after 5 weeks of diabetes with blood glucose > 350 mg x dL(-1). Under urethane anesthesia, a rat stomach was mounted on an ex vivo chamber, perfused with saline and acid secretion was measured at pH 7.0 using a pH-stat method and by adding 100 mM NaOH. The acid secretion was stimulated by i.v. infusion of either histamine (4 mg x kg(-1) x h(-1)), pentagastrin (60 microg x kg(-1) x h(-1)) or carbachol (20 microg x kg(-1) x h(-1)) or i.v. injection of YM-14673 (0.3 mg x kg(-1)), an analog of thyrotropin-releasing hormone, or vagal electrical stimulation (2 ms, 3 Hz, 0.5 mA). In normal rats, gastric acid secretion was increased in response to either histamine, pentagastrin, carbachol, YM-14673 or electrical vagal stimulation. In STZ diabetic rats, however, changes in acid secretion varied depending on the stimuli; the acid secretory responses to histamine remained unchanged, those to YM-14673 and vagal electrical stimulation significantly decreased, but the responses to both pentagastrin and carbachol were significantly enhanced as compared to normal rats. Luminal release of histamine in response to both pentagastrin and carbachol was increased in STZ-diabetic rats as compared to normal animals. The altered acid secretory responses in STZ diabetic rats were partially reversed by daily injection of insulin with amelioration of high blood glucose levels. These results suggest that STZ-diabetic rats showed different changes in gastric acid secretory responses to various stimuli; no change in response to histamine, a decrease to both YM-14673 and vagal electrical stimulation and an increase to both pentagastrin and carbachol. The increased acid secretory response may be associated with an enhanced release of mucosal histamine, while the decreased response may be due to vagal neuropathy.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.     

Measuring the toxic effects of high gene dosage on yeast cells

 A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed. Received: 18 March 1996 / Accepted: 23 August 1996     

Regulation and function of p38 protein kinase in isolated canine gastric parietal cells

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 microM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca(2+) chelator BAPTA-AM, but incubation of cells in Ca(2+)-free medium led to a 50% inhibition of carbachol induction of p38 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [(14)C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 microM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca(2+). p38 kinase appears to inhibit gastric acid secretion.     

Effects of calcium ionophore A23187 and calcium antagonists on 32Pi incorporation into polyphosphoinositides of rat cortical synaptosomes

The role of Ca2+ on 32Pi incorporation into polyphosphoinositides (PPI) of rat cortical synaptosomes was studied. Stimulation of muscarinic receptor by carbachol (1 mM) resulted in a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphophaphate (TPI) and phosphatidylinositol-4-phosphate (DPI), and an increase in 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA), whereas no significant effect on other membrane phospholipids was found. This response could be blocked by atropine (1 microM). The stimulatory effect of carbachol required Ca2+ in the medium; the presence of 0.5 mM EGTA blocked the effect of carbachol on PPI turnover completely. Calcium ionophore A23187, at 1 microM, had a similar effect on PPI turnover by carbachol (1 mM). At higher concentrations (10-100 microM) of A23187, the PPI turnover rate was much enhanced. Depolarization of the membrane by high potassium (60 mM) in the presence of calcium resulted in an enhanced PPI turnover, which was similar to the results of the carbachol (1 mM) effect but to a lesser extent. Calcium antagonists, diltiazem and trifluoperazine, at 10 microM could block the carbachol effect on 32Pi incorporation into PPI in this preparation. Our results suggest that the enhancement of PPI turnover in rat cortical synaptosomes by carbachol, calcium ionophore or high potassium requires Ca2+, and it can be blocked by compounds which interfere with the availability of this ion, such as EGTA or calcium antagonists.     

Microwave facilitation of methylatropine antagonism of central cholinomimetic drug effects

Pilocarpine-induced hypothermia and oxotremorine-induced tremors in mice are central cholinomimetic drug effects that are readily blocked by the muscarinic antagonist atropine. However, the quaternary ammonium derivative of atropine, methylatropine, is unable to block these cholinomimetic drug effects by virture of its inability to penetrate the blood-brain barrier (BBB) and blood-cerebral spinal fluid barrier (B-CSFB). Dose-response curves for pilocarpine and oxotremorine effects are not appreciably affected either by pretreatment with methylatropine (1.0 mg/kg) or by exposure to moderate-level microwave irradiation (2.45 GHz, 23.7 W/kg, CW, 10-min exposure). However, in mice receiving both the methylatropine pretreatment and microwave irradiation, the dose-response curves for both pilocarpine and oxotremorine effects were significantly shifted to the right, signifying a central anticholinergic action by methylatropine. These data indicate that a single acute exposure to a thermogenic level of microwave irradiation facilitates methylatropine antagonism of centrally mediated cholinomimetic drug effects. One possible explanation for this observation is that microwave radiation may enhance passage of quaternary ammonium compounds like methylatropine across the BBB and B-CSFB.     

萜类化合物对小菜蛾幼虫的拒食活性

采用叶碟浸液法测试从松节油合成的26个不同结构萜类化合物样品对小菜蛾Plutella xylostella(L.)4龄幼虫的拒食活性。结果显示,编号为2,4,8,13,27,31的6个化合物,即诺卜醇、诺卜丙基醚、内型异莰烷基甲醇丙酸酯、4-(1-甲基乙烯基)-1-环己烯-1-乙醇丙酸酯、羟基香茅醛丙酸酯、羟基香茅醛1,2-丙二醇缩醛具有较高的拒食活性。处理有效成分为0.01g/mL时,这6个化合物24h拒食率为70%～100%。其中2,4,8号3个化合物拒食活性最高,处理有效成分为0.01g/mL和0.001g/mL时,24h拒食率分别为98.33%,99.80%,100.00%和85.60%,83.90%,66.97%;并且持效性较好,72h拒食率分别为92.67%,89.97%,98.73%和63.20%,63.30%,45.93%。     

The reaction of the N-cholinoreceptors of the isolated neuron of the mollusk Planorbarius corneus to polymethylene-bis-trimethylammonium compounds

Experiments have been made on isolated giant neurones of the mollusc Planorbarius corneus using clamp technique at temperatures 10 and 20 degrees C. The effect of polymethylene-bis-trimethylammonium compounds with 7-18 methylene groups in the molecule (C7...C18) on N-cholinoreceptors with chloride ionic channels was investigated. All these drugs were found to be agonists. Their cholinomimetic activity depends on the number of methylene groups (up to a certain extent) in their structure. This finding stands true also for skeletal muscles of frog and chick, as it had been shown in our earlier experiments. Analysis of membrane current fluctuations showed that the elementary current, the channel opened time, temperature coefficient (Q10) of the neuronal response to application of an agonist and the calculated Q10 of the reaction rate of the agonist with cholinoreceptor did not significantly differ for C8...C18 from the reaction rate of the agonist with cholinoreceptor. As compared with C8, C12...C18 exhibited 30 ... 40 times higher cholinomimetic activity, all other parameters in them being similar. Presumably, this difference is explained by concentrating capacity of C12...C18 at the membrane site because of their higher hydrophobic properties.     

Effects of immunological sensitization on the responses and sensitivity of guinea pig airways to bronchoconstrictors. Modulation by selective inhibition of arachidonic acid metabolism

The force generated by tracheal spirals and lung parenchymal strips from normal and ovalbumin-sensitized guinea pigs was measured in vitro, after challenge with histamine, carbachol, leukotriene (LT) C4, LTD4, or a prostaglandin endoperoxide analog (U-44069). The responses and sensitivity of airway tissues to the above agonists were identical in normal and sensitized animals. Treatment of tracheal spirals with indomethacin (8.5 microM), phenidone (185 microM), and nordihydroguaiaretic acid (NDGA: 30 microM) reduced resting tension (tone) equally in both normal and sensitized trachea, but did not affect lung parenchymal strips from either group. The responses of tracheal spirals from normal and sensitized animals to low concentrations of histamine, carbachol, LTC4, and LTD4 were reduced or abolished by treatment with the above inhibitors. Responses to higher concentrations of the same agonists were significantly enhanced. In contrast, treatment of normal and sensitized trachea with indomethacin (2.8 and 8.5 microM) did not abolish or reduce the effects of low concentrations of U-44069. However, an enhancement of the effect of high concentrations occurred only on normal tracheal spirals, even though the control tissues from each group responded identically with U-44069 in the absence of any inhibitor. Parenchymal strips increased in sensitivity to histamine, but not carbachol, as a result of time, vehicle, or prior exposure to the drug. Inhibitor treatment did not affect sensitivity or responsiveness of parenchyma to histamine, carbachol, and U-44069, but the contractile activity of LTD4 on both normal and sensitized lung parenchymal strips was reduced by indomethacin, NDGA, and phenidone. We conclude that ovalbumin sensitization does not induce hyperreactivity of guinea pig airways.     

植物气味化合物与斜纹夜蛾性信息素的协同作用

为提高现有性信息素对雄蛾的引诱活性, 本研究通过大量的田间试验探索植物气味化合物与斜纹夜蛾Spodoptera litura性信息素（顺9, 反11-十四碳二烯乙酸酯∶顺9, 反12-十四碳二烯乙酸酯=10∶1）的协同作用机制。从斜纹夜蛾寄主植物和花的气味化合物中, 选择9种有代表性的化合物, 并以一定剂量分别加入到斜纹夜蛾性信息素诱芯中, 在田间测试对雄蛾的引诱活性。结果表明: 在测试的9种植源性化合物中, 发现一定剂量(每个诱芯加入0.4 mg)的苯乙醛(PAA), 具有显著提高斜纹夜蛾性信息素的引诱作用, 而高剂量的苯乙醛则强烈抑制性信息素的引诱活性; 此外, 其他各种浓度的测试化合物或混合物对性信息素则没有统计上显著的增效作用。不同剂量的苯乙醛单个化合物及各种植物气味化合物组成的混合物对斜纹夜蛾也有微弱的引诱作用。苯乙醛必须要与性信息素的完整组分(以10∶1比例混合的顺9, 反11-十四碳二烯乙酸酯和顺9反, 12-十四碳二烯乙酸酯)混合才能起作用, 缺少顺9, 反12-十四碳二烯乙酸酯则没有引诱活性。本研究证明, 苯乙醛作为理想的性信息素诱芯增效剂, 可应用于建立更理想的斜纹夜蛾性信息素诱杀技术, 对性诱害虫防治和测报具有应用价值。