Multi-target bioactive compound screening from the infructescence of Platycarya strobilacea Sieb. et Zucc. by affinity chromatography using immobilized ß2 -adrenoceptor and muscarinic-3 acetylcholine receptor as the stationary phase.

As a main source for the recognition and identification of lead compounds, traditional Chinese medicine plays a pivotal role in preventing diseases for years. However, screening bioactive compounds from traditional Chinese medicine remains challenging because of the complexity of the systems and the occurrence of the synergic effect of the compounds. The infructescence of Platycarya strobilacea Sieb. et Zucc is prescribed for allergic rhinitis treatment with unknown bioactive compounds and unclear mechanisms. Herein, we immobilized the ß2 -adrenoceptor and muscarine-3 acetylcholine receptor onto the silica gel surface to prepare the stationary phase in a covalent bond through one step. The feasibility of the columns was investigated by the chromatographic method. Ellagic acid and catechin were identified as the bioactive compounds targeting the receptors. The binding constants of ellagic acid were calculated to be (1.56 ± 0.23)×107  M-1 for muscarine-3 acetylcholine receptor and (2.93 ± 0.15)×107  M-1 for ß2 -adrenoceptor by frontal analysis. While catechin can bind with muscarine-3 acetylcholine receptor with an affinity of (3.21 ± 0.05)×105  M-1 . Hydrogen bonds and van der Waals' force were the main driving forces for the two compounds with the receptors. The established method provides an alternative for multi-target bioactive compound screening in complex matrices.

[Comparison of effects of electroacupuncture at "Tianshu" (ST25) and "Dachangshu" (BL25) on intestinal sensitivity and expression of muscarinic M3R and 5-HT3AR in irritable bowel syndrome rats].

OBJECTIVE: To observe and compare the effect of electroacupuncture (EA) of "Tianshu" (ST25) and "Dachangshu" (BL25) on intestinal sensitivity and the expression of M3 muscarinic acetylcholine receptor (M3R) and 5-hydroxytryptamine 3A receptor (5-HT3AR) in the colon tissue in irritable bowel syndrome (IBS) rats, so as to explore its mechanism underlying improvement of IBS. METHODS: Forty newborn Wistar rats were randomly divided into blank control, model, ST25 and BL25 groups (n＝10 rats per group). The IBS model was established by joint application of maternal separation, acetic acid enema plus colorectal distension from day 8 to 21 after birth. At the age of 9 weeks, EA (2 Hz/100 Hz, 0.1－0.3 mA) was applied to ST25 and BL25 for 20 min, once every other day for 5 times. The modified method was used to record the abdominal withdrawal reflex (AWR), so as to evaluate the visceral sensitivity by referring to the modified Al-Chaer's and colleagues' methods. The threshold (initial wave), the number and peak-peak value of contraction waves (AWR) within 90 s were recorded. The immunoactivity of M3R and 5-HT3AR in the colon tissue was detected by immunohistochemistry. RESULTS: After modeling, the latency was obviously shortened, the number and peak-peak values of contraction waves of intestine were significantly increased, and the immunoactivity of M3R and 5-HT3AR was notably up-regulated in the model group compared with the blank control group (P<0.01). After the EA intervention, the latency was obviously prolonged, the peak-peak value in both ST25 and BL25 groups, and the number of contraction waves in the ST25 group were significantly decreased (P<0.01), and the expression levels of M3 and 5-HT3A R were considerably down-regulated in both ST25 and BL25 groups relevant to the model group (P<0.01). The therapeutic effects were significantly better in the ST25 group than in the BL25 group in prolonging the latency and reducing the contraction wave number, and in down-regulating the expression of colonic M3R and 5-HT3AR (P<0.05, P<0.01). CONCLUSION: EA at ST25 and BL25 can reduce the intestinal sensitivity in IBS rats, which is probably associated with its effects in down-regulating the expression of M3R and 5-HT3AR in the colon. The therapeutic effects of ST25 were obviously better than those of BL25 in suppressing intestinal sensitivity and expression of colonic M3R and 5-HT3AR.

Amanita muscaria (fly agaric): from a shamanistic hallucinogen to the search for acetylcholine.

The mushroom Amanita muscaria (fly agaric) is widely distributed throughout continental Europe and the UK. Its common name suggests that it had been used to kill flies, until superseded by arsenic. The bioactive compounds occurring in the mushroom remained a mystery for long periods of time, but eventually four hallucinogens were isolated from the fungus: muscarine, muscimol, muscazone and ibotenic acid. The shamans of Eastern Siberia used the mushroom as an inebriant and a hallucinogen. In 1912, Henry Dale suggested that muscarine (or a closely related substance) was the transmitter at the parasympathetic nerve endings, where it would produce lacrimation, salivation, sweating, bronchoconstriction and increased intestinal motility. He and Otto Loewi eventually isolated the transmitter and showed that it was not muscarine but acetylcholine. The receptor is now known variously as cholinergic or muscarinic. From this basic knowledge, drugs such as pilocarpine (cholinergic) and ipratropium (anticholinergic) have been shown to be of value in glaucoma and diseases of the lungs, respectively.

Recovery of hippocampal functions and modulation of muscarinic response by electroacupuncture in young diabetic rats.

The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer's disease.

[Outline of JRS COPD guideline Ed. 4].

JRS COPD guideline Edition 4 updated current concept for clinical management of COPD in Japan. Minor but substantial changes were added to the definition and subtype of the disease. In pharmacological treatment, single use of LAMA or LABA is equally the first choice. If it is not enough, those combination are recommended. A-D classification that was presented in GOLD report 2011 was not adopted. In non-pharmacological treatment, pulmonary rehabilitation is steadily effective and essential. In terms of survival prognosis, at least, the concept of physical activity is important. Those improvement and maintenance were newly added to the objectives of the clinical management of COPD.

Type and treatment of toxic mushroom poisoning in Korea

To eat unidentified or misidentified mushrooms taken from the wild can be very dangerous. In the vast majority of toxic mushroom ingestions in Korea, the mushroom was incorrectly identified. In general, poisoning of toxic mushrooms can be classified into seven types according to the toxins that they contain; amatoxin, gyromitrin, coprine, muscarine, ibotenic acid-muscimol, psilocybin-psilocin and gastrointestinal irritants. When clinicians care for a patient who ingested a toxic mushroom, it is very important to identify what kind of mushroom may have caused a patient's illness. But, in clinical practice, accurate botanical identification of the mushroom can be very difficult. Therefore, for estimating the caused mushroom and adequate treatment of poisoning, clinicians should know the type and treatment of toxic mushroom poisoning.

Argyreia nervosa (Burm. f.): receptor profiling of lysergic acid amide and other potential psychedelic LSD-like compounds by computational and binding assay approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: The convolvulacea Argyreia nervosa (Burm. f.) is well known as an important medical plant in the traditional Ayurvedic system of medicine and it is used in numerous diseases (e.g. nervousness, bronchitis, tuberculosis, arthritis, and diabetes). Additionally, in the Indian state of Assam and in other regions Argyreia nervosa is part of the traditional tribal medicine (e.g. the Santali people, the Lodhas, and others). In the western hemisphere, Argyreia nervosa has been brought in attention as so called "legal high". In this context, the seeds are used as source of the psychoactive ergotalkaloid lysergic acid amide (LSA), which is considered as the main active ingredient. AIM OF THE STUDY: As the chemical structure of LSA is very similar to that of lysergic acid diethylamide (LSD), the seeds of Argyreia nervosa (Burm. f.) are often considered as natural substitute of LSD. In the present study, LSA and LSD have been compared concerning their potential pharmacological profiles based on the receptor binding affinities since our recent human study with four volunteers on p.o. application of Argyreia nervosa seeds has led to some ambiguous effects. MATERIAL AND METHODS: In an initial step computer-aided in silico prediction models on receptor binding were employed to screen for serotonin, norepinephrine, dopamine, muscarine, and histamine receptor subtypes as potential targets for LSA. In addition, this screening was extended to accompany ergotalkaloids of Argyreia nervosa (Burm. f.). In a verification step, selected LSA screening results were confirmed by in vitro binding assays with some extensions to LSD. RESULTS: In the in silico model LSA exhibited the highest affinity with a pKi of about 8.0 at α1A, and α1B. Clear affinity with pKi>7 was predicted for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT6, 5-HT7, and D2. From these receptors the 5-HT1D subtype exhibited the highest pKi with 7.98 in the prediction model. From the other ergotalkaloids, agroclavine and festuclavine also seemed to be highly affine to the 5-HT1D-receptor with pKi>8. In general, the ergotalkaloids of Argyreia nervosa seem to prefer serotonin and dopamine receptors (pKi>7). However, with exception of ergometrine/ergometrinine only for 5-HT3A, and histamine H2 and H4 no affinities were predicted. Compared to LSD, LSA exhibited lower binding affinities in the in vitro binding assays for all tested receptor subtypes. However, with a pKi of 7.99, 7.56, and 7.21 a clear affinity for 5-HT1A, 5-HT2, and α2 could be demonstrated. For DA receptor subtypes and the α1-receptor the pKi ranged from 6.05 to 6.85. CONCLUSION: Since the psychedelic activity of LSA in the recent human study was weak and although LSA from Argyreia nervosa is often considered as natural exchange for LSD, LSA should not be regarded as LSD-like psychedelic drug. However, vegetative side effects and psychotropic effects may be triggered by serotonin or dopamine receptor subtypes.

The contributions of muscarinic receptors and changes in plasma aldosterone levels to the anti-hypertensive effect of Tulbaghia violacea.

BACKGROUND: Tulbaghia violacea Harv. (Alliaceae) is used to treat various ailments, including hypertension (HTN) in South Africa. This study aims to evaluate the contributions of muscarinic receptors and changes in plasma aldosterone levels to its anti-hypertensive effect. METHODS: In the acute experiments, methanol leaf extracts (MLE) of T. violacea (30-120 mg/kg), muscarine (0.16 -10 µg/kg), and atropine (0.02 - 20.48 mg/kg), and/or the vehicle (dimethylsulfoxide (DMSO) and normal saline (NS)) were respectively and randomly administered intravenously in a group of spontaneously hypertensive (SHR) weighing 300 to 350 g and aged less than 5 months. Subsequently, T. violacea (60 mg/kg) or muscarine (2.5 µg/kg) was infused into eight SHRs, 20 min after atropine (5.12 mg/kg) pre-treatment. In the chronic (21 days) experiments, the SHRs were randomly divided into three groups, and given the vehicle (0.2 ml/day of DMSO and NS), T. violacea (60 mg/kg/day) and captopril (10 mg/kg/day) respectively into the peritoneum, to investigate their effects on blood pressure (BP), heart rate (HR), and plasma aldosterone levels. Systolic BP and HR were measured using tail-cuff plethysmography during the intervention. BP and HR were measured via a pressure transducer connecting the femoral artery and the Powerlab at the end of each intervention in the acute experiment; and on day 22 in the chronic experiment. RESULTS: In the acute experiments, T. violacea, muscarine, and atropine significantly (p < 0.05) reduced BP dose-dependently. T. violacea and muscarine produced dose-dependent decreases in HR, while the effect of atropine on HR varied. After atropine pre-treatment, dose-dependent increases in BP and HR were observed with T. violacea; while the BP and HR effects of muscarine were nullified. In the chronic experiments, the T. violacea-treated and captropril-treated groups had signicantly lower levels of aldosterone in plasma when compared to vehicle-treated group. Compared to the vehicle-treated group, significant reduction in BP was only seen in the captopril-treated group; while no difference in HR was observed among the groups. CONCLUSION: The results obtained in this study suggest that stimulation of the muscarinic receptors and a reduction in plasma aldosterone levels contribute to the anti-hypertesive effect of T. violacea.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

Cholinergic activation of M2 receptors leads to context-dependent modulation of feedforward inhibition in the visual thalamus.

In many brain regions, inhibition is mediated by numerous classes of specialized interneurons, but within the rodent dorsal lateral geniculate nucleus (dLGN), a single class of interneuron is present. dLGN interneurons inhibit thalamocortical (TC) neurons and regulate the activity of TC neurons evoked by retinal ganglion cells (RGCs), thereby controlling the visually evoked signals reaching the cortex. It is not known whether neuromodulation can regulate interneuron firing mode and the resulting inhibition. Here, we examine this in brain slices. We find that cholinergic modulation regulates the output mode of these interneurons and controls the resulting inhibition in a manner that is dependent on the level of afferent activity. When few RGCs are activated, acetylcholine suppresses synaptically evoked interneuron spiking, and strongly reduces disynaptic inhibition. In contrast, when many RGCs are coincidently activated, single stimuli promote the generation of a calcium spike, and stimulation with a brief train evokes prolonged plateau potentials lasting for many seconds that in turn lead to sustained inhibition. These findings indicate that cholinergic modulation regulates feedforward inhibition in a context-dependent manner.

Gq/11-induced and spontaneous waves of coordinated network activation in developing frontal cortex.

Repeated episodes of spontaneous large-scale neuronal bursting and calcium influx in the developing brain can potentially affect such fundamental processes as circuit formation and gene expression. Between postnatal day 3 (P3) and P7, the immature cortex can express one such form of activation whereby a wave of neuronal activity propagates through cortical networks, generating massive calcium influx. We previously showed that this activity could be triggered by brief stimulation of muscarinic receptors. Here, we show, by monitoring large cortical areas at low magnification, that although all areas respond to muscarinic agonists to some extent, only some areas are likely to generate the coordinated wave-like activation. The waves can be triggered repeatedly in frontal areas where, as we also show, waves occur spontaneously at a low frequency. In parietal and occipital areas, no such waves are seen. This selectivity may be related in part to differences in the cortical distribution of dopaminergic signaling, because we find that activation of dopamine receptors enables the response. Because M1 muscarinic receptors are typically coupled with G-alpha(q)/11, we investigated whether other receptors known to couple with this G-protein (group I glutamate metabotropic receptors, neurotensin type 1) could similarly elicit wave-like activation in responsive cortical areas. Our results suggest that multiple neurotransmitter systems can enable this form of activation in the frontal cortex. The findings suggest that a poorly recognized, developmentally regulated form of strong network activation found predominantly in the frontal cortex could potentially exert a profound influence on brain development.

Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection.

Neurons that synthesize melanin-concentrating hormone (MCH) may modulate arousal and energy homeostasis. The scattered MCH neurons have been difficult to study, as they have no defining morphological characteristics. We have developed a viral approach with AAV for selective long-term reporter gene (GFP) expression in MCH neurons, allowing the study of their cellular physiology in hypothalamic slices. MCH neurons showed distinct membrane properties compared to other neurons infected with the same virus with a cytomegalovirus promoter. Transmitters of extrahypothalamic arousal systems, including norepinephrine, serotonin, and the acetylcholine agonist muscarine, evoked direct inhibitory actions. Orexigenic neuropeptide Y was inhibitory by pre- and postsynaptic mechanisms; an anorexigenic melanocortin agonist had no effect. In contrast, the hypothalamic arousal peptide hypocretin/orexin evoked a direct inward current and increased excitatory synaptic activity and spike frequency in the normally silent MCH neurons. Together, these data support the view that MCH neurons may integrate information within the arousal system in favor of energy conservation.

Cholinergic modulation of purinergic and GABAergic co-transmission at in vitro hypothalamic synapses.

The lateral hypothalamus (LH) is an important center for the integration of autonomic and limbic information and is implicated in the modulation of visceral motor and sensory pathways, including those underlying feeding and arousal behaviors. LH neurons in vitro release both ATP and GABA. The control of ATP and GABA co-transmission in LH may underlie the participation of LH in basic aspects of arousal and reinforcement. LH neurons receive cholinergic input from the pedunculopontine and laterodorsal tegmental nuclei as well as from cholinergic interneurons within the LH per se. This study presents evidence for nicotinic acetylcholine receptor (nAChR)-mediated enhancement of GABAergic, but not of purinergic, transmission despite the co-transmission of ATP and GABA at LH synapses in vitro. Facilitation of GABAergic transmission by nicotine is inhibited by antagonists of (alphabeta)*-containing nAChRs, but is unaffected by an alpha7-selective antagonist, consistent with a nAChR-mediated enhancement of GABA release mediated by non-alpha7-containing nAChRs. Activation of muscarinic ACh receptors enhances the release of ATP while concomitantly depressing GABAergic transmission. The independent modulation of ATP/GABAergic transmission may provide a new level of synaptic flexibility in which individual neurons utilize more than one neurotransmitter but retain independent control over their synaptic activity.

Muscarine, imidazole, oxazole, thiazole, Amaryllidaceae and Sceletium alkaloids.

A review with 136 references covers the literature from July 2000 to June 2001 on the isolation, bioactivities, and synthetic highlights of complex natural products including muscarine, imidazole, oxazole, thiazole, Amaryllidaceae and Sceletium alkaloids.

Slow synchronized bursts of inhibitory postsynaptic currents (0.1-0.3 Hz) by cholinergic stimulation in the rat frontal cortex in vitro.

Cholinergic inputs from the basal forebrain to cortex exert profound effects on cortical activities, such as a rhythmic synchronization. For these modulatory effects inhibitory interneurons could play crucial roles within the cortical circuitry. To study cholinergic modulation of GABA-mediated inhibition, we recorded inhibitory postsynaptic current (IPSC) during application of cholinergic agonists in the rat frontal cortex in vitro. Both carbachol and muscarine caused two temporally different patterns of IPSC modulation in both pyramidal cells and inhibitory interneurons: tonic or periodic increase of GABA-A receptor-mediated inhibition. The tonic pattern showed a continuous increase of IPSC frequency, while the periodic increase manifested itself as rhythmic (0.1-0.3 Hz, mean 0.2 Hz) bursts of IPSC (frequency: 6-69 Hz, mean 24 Hz; burst duration: 1.2-4.3 s, mean 2.2 s). Both types of increase were suppressed by atropine or pirenzepine, muscarinic-receptor antagonists. The periodical IPSC bursts were not affected by antagonists for ionotropic glutamate receptors. Following cholinergic stimulation, periodic IPSC bursts in nearby cells were synchronized as a whole, but individual inhibitory events within the bursts were not always temporally correlated, suggesting synchronized depolarizations of several presynaptic interneurons. It has been revealed that slow rhythmic depolarizations accompanying spike firing can be generated within the cortex. In addition to this periodic excitation of cortical circuits, these results indicate that cortical inhibitory interneurons have their own acetylcholine-dependent mechanism generating the slow rhythm independent of the excitatory circuits.

Coordinated transitions in neurotransmitter systems for the initiation and propagation of spontaneous retinal waves.

Spontaneous waves of excitation in the developing mammalian retina are mediated, to a large extent, by neurotransmission. However, it is unclear how the underlying neurotransmitter systems interact with each other to play specific roles in the formation of retinal waves at various developmental stages. In particular, it is puzzling why the waves maintain a similar propagation pattern even after underlying neurotransmitter systems have undergone drastic developmental changes. Using Ca(2+) imaging and patch clamp in a whole-mount preparation of the developing rabbit retina, we discovered two dramatic and coordinated transitions in the excitatory drive for retinal waves: one from a nicotinic to a muscarinic system, and the other from a fast cholinergic to a fast glutamatergic input. Retinal waves before the age of postnatal day 1 (P1) were blocked by nicotinic antagonists, but not by muscarinic or glutamatergic antagonists. After P3, however, the spontaneous wave, whose basic spatiotemporal pattern remained similar, was completely inhibited by muscarinic or glutamate antagonists, but not by nicotinic antagonists. We also found that the muscarinic drive, mediated primarily by M1 and M3 receptors, was particularly important for wave propagation, whereas the glutamatergic drive seemed more important for local excitation. Our results suggest (1) a novel mechanism by which a neurotransmitter system changes its functional role via a switch between two completely different classes of receptors for the same transmitter, (2) the cholinergic system plays a critical role in not only early but also late spontaneous waves, and (3) the continued participation of the cholinergic system may provide a network basis for the consistency in the overall propagation pattern of spontaneous retinal waves.

Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons.

The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ channel modulation. The heterologously expressed toxin retained specificity for G alpha(i/o)-dependent pathways as VIP-mediated modulation of N-type Ca2+ channels and muscarine-mediated inhibition of M-type K+ channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.

Effects of ginseng saponins on responses induced by various receptor stimuli.

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

G protein specificity of the muscarine-induced increase in an inward rectifier potassium current in AtT-20 cells.

Muscarine and somatostatin enhance an inward rectifier K+ conductance in the AtT-20 pituitary cell line. Both effects are abolished by pertussis toxin (PTX). To determine which PTX-sensitive G protein mediates these agonist effects, we made cDNAs encoding mutant PTX-insensitive Gi alpha subtypes, in which the cysteine residue fourth from the C terminus was replaced with serine. The mutated cDNA was transfected into AtT-20 cells, resulting in stable cell lines overexpressing a Gi alpha subtype. As controls, wild-type Gi alpha cDNA was transfected into AtT-20 cells. The agonist-induced increase of the inward rectifier K+ conductance in the transfectants was examined with the whole-cell clamp method. Only in the cell lines into which the mutated (PTX-insensitive) Gi2 alpha cDNA was transfected, did the muscarine response become PTX-insensitive, suggesting that Gi2 couples to the muscarinic receptor and enhances the activity of the inward rectifier K+ channel. However, PTX-insensitive somatostatin responses were not obtained in any of the cell lines transfected with a mutated Gi alpha cDNA, suggesting either that none of the Gi subtypes is a transducer for the somatostatin effect or that the mutation prevents the coupling of the Gi alpha to the somatostatin receptor.

Muscarine induces an anomalous inhibition of synaptic transmission in rat auditory thalamic neurons in vitro.

Muscarinic receptor-mediated inhibition of central synaptic transmission was studied in a monosynaptic pathway connecting the inferior colliculus and the auditory thalamus in in vitro rat brain explants. Extra- and intracellular synaptic responses were recorded by sharp electrode and whole-cell patch clamp techniques in the ventral nucleus of the medial geniculate body after electrical stimulation of the brachium of the inferior colliculus. Stimulation of tectal afferents evoked either a high-frequency burst or a single-spike synaptic response in ventral geniculate neurons. Bath application of muscarinic receptor agonists abolished responses consisting of a high-frequency burst, but not responses consisting of a single spike. In the majority of single-spike cells muscarinic agonists often induced a synaptic facilitation. The burst blocking effect was mimicked by a moderate elevation of extracellular potassium. Intracellular recordings showed that the burst synaptic responses similar to that recorded extracellularly were induced by an excitatory postsynaptic potential. This synaptic potential, by first activating a low-threshold spike, was able to evoke a burst of sodium spike discharges. Muscarinic agonists caused a slow membrane depolarization that inactivated the low-threshold spike, leading to a blockade of the burst response. This mechanism is tentatively termed here as EPSP-LTS decoupling. Our results therefore support the hypothesis that part of the muscarinic receptor-mediated synaptic inhibition previously reported in anesthetized animal preparations in vivo represents a membrane depolarization rather than pre- or postsynaptic inhibition.