Effect of postsynaptic blockade of neuromuscualar transmission on properties of the frog fast muscle fiber membrane

Sensitivity of the postsynpatic membrane to acetylcholine, the resting membrane potential, input resistance, and membrane time constant of fast muscle fibers were measured in experiments on frogs. Complete immobilization of the animals with D-tubocurarine or local immobilization of a muscle with α-bungarotoxin was found not to affect these parameters of the muscle membrane, whereas denervation of the muscle widens the zone of postsynaptic sensitivity to acetylcholine, lowers the resting membrane potential, and increases the input resistance and time constant of the muscle membrane. These results are evidence that neurotrophic control of the frog fast muscle fiber membrane is achieved mainly by substances reaching the muscle via axoplasmic transport and not by the character of the neuronal discharge and motor activity or by synaptic acetylcholine.     

Effect of piracetam on the habituation of the neuronal cholinoreceptor membrane of the edible snail

Piracetam is shown to modulate the habituation pattern of cholinoreceptor neuronal membrane in Helix lucorum. Piracetam (10(-2) M) intensified the reversible decrease of depolarization and spike discharge induced by the local microionophoretic rhythmical application of acetylcholine to the neuron soma. The ability of piracetam to intensify the habituation of neuronal cholinoreceptor membrane may serve as a model of its facilitating effect on the development of habituation of behavioural reactions. Piracetam (5.10(-2) M) was shown to induce a shift in the membrane potential towards depolarization in the majority of identified neurons studied.     

The role of intracellular processes in regulating neuronal sensitivity to acetylcholine

Intracellular regulatory mechanisms of neuronal response to acetylcholine were studied on intracellularly perfused isolated neurones of Lymnaea stagnalis using voltage-clamp technique. It was found that at the change of concentration of free intracellular Ca2+ from 0.06 to 0.7 microM the inhibitory effect of intracellularly added serotonin depends on Ca2+, and the modulation of acetylcholine responses by intracellular serotonin is unchanged. The blockers of calmoduline trifluoperazine and W-7 inhibit inward acetylcholine current at both intra- and extracellular introduction. Possible mechanisms mediating the effect of intracellularly added serotonin on the membrane cholinoreceptors of neurones are discussed.     

Cholinergic modulation of neuronal spike reactions to dendritic and somatic application of excitatory acids

Acetylcholine effects on neuronal firing responses evoked by somatic or dendritic applications of excitatory amino acids were studied in slices of guinea-pig parietal cortex. Excitatory reactions initiated by dendritic activation were enhanced by acetylcholine wherever it was iontophoretically applied: either to soma or dendrites. The effect consisted in shortening spike response latencies and increasing response intensity and duration. The modified responses were recorded within 1-min interval after acetylcholine microinjections at a distance within 300 microns of the soma. Parameters of responses to somatic applications of excitatory amino acids were not significantly changed by acetylcholine. The results suggest that acetylcholine improves dendritic propagation rather than membrane excitability.     

The effect of laser puncture on the functional indices of the mammalian body

Biologically active points (acupuncture points) and reflexogenic zones of Wistar rats of different age were exposed to He-Ne laser radiation with the subsequent control of acetylcholine esterase activity, electric parameters of skeletal muscles and heart, and physical activity. Laser puncture was shown to increase the sympathetic nervous system tonus which was manifested by the decreased acetylcholine esterase activity and increased heart rate. Laser puncture stimulated the growth of membrane potential and increased considerably the performance capability of animals, particularly of three-month-olds.     

Effect of ionizing radiation on the functional state of snail neurons. Cyclic nucleotide levels and phosphorylation of membrane proteins

A study was made of the effect of ionizing radiation, phospholipase A2, and low concentrations of mediators (for instance, acetylcholine and gamma-aminobutyric acid) on the content of cyclic nucleotides and phosphorylation of membrane proteins of Helix pomatia nervous ganglia. Ionizing radiation was shown to decrease considerably the levels of cAMP and cGMP which correlated with the diminution of membrane phosphorylation. Phospholipase A2 and low doses of mediators produced a modifying effect on the cyclic nucleotide content.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.     

The non-neuronal cholinergic system in humans: expression,function and pathophysiology

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

The modulatory action of superlow doses of mediators on the functional activity of the neuronal membrane]

It was shown that acetylcholine and gamma-aminobutyric acid at extremely low concentrations, which do not activate receptor-binding ionic channels, modulate the activity of NA-K-pump and Na:Ca exchange, the intracellular level of cAMP and chemosensitivity of the neuronal membrane. These data provide a new evidence in support for Koshtoiants's enzymochemical hypothesis in which synaptic transmitters are considered to be metabolic modulators for postsynaptic neurones.     

Calcium ion modulation of short-term plasticity of the cholinoreceptor membrane of the mollusk neuron

Repeated iontophoretic acetylcholine applications to the external surface of the plasma membrane of identified Helix neurones elicit a gradual reversible reduction of cholinoreceptors (ChR) sensitivity. Influence on Ca-conductivity of neuronal membrane modifies the dynamics of lowering of ChR sensitivity. Administration of cadmium ions--blockader of Ca-conductivity, slows down and weakens ChR habituation, while Ca-conductivity activation by raising of extracellular Ca2+ content accelerates and deepens the habituation. It is suggested that chemo-controlled Ca-conductivity takes part in regulation of short-term ChR plasticity.     

Acetylcholine stimulates cyclic ADP-ribose formation via M1 muscarinic receptors in rat superior cervical ganglion

The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250-300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue.     

Immunocytochemical localization of α-bungarotoxin receptors in the chick ciliary ganglion: Synaptic and extrasynaptic sites?

The immunocytochemical localization of nicotinic acetylcholine receptors in the chick ciliary ganglion was investigated at the ultrastructural level with a procedure utilizing binding of α-bungarotoxin visualized by immunoperoxidase histology. Both ganglionic cell populations, i.e. the ciliary and choroid neurons, showed specific immunoreactivity for receptors. In both types of neurons evident stain was found on the postsynaptic membrane. Often the reaction product filled discrete regions of the synaptic cleft. Furthermore, specific staining was also observed on large areas of the neuronal surface devoid of presynaptic nerve endings. These data probably indicate the occurrence of both synaptic and extrasynaptic nicotinic receptors on the neuronal plasma membrane. Immunoreactivity was also observed on the membrane of the presynaptic nerve endings, and on the part of the satellite plasma membrane which is adjacent to the neuron. These last results are discussed in relation to the occurrence of possible artifacts or, alternatively, to the presence of presynaptic and glial receptors. Immunoreactivity at all sites was prevented almost completely by ganglion incubation in 1 mM d-tubocurarine prior to and during treatment with toxin.     

Identifying nicotinic and muscarinic cholinoreceptors at the soma of neuron RPa4 inHelix lucorum

Cholinoreceptors were identified at the somatic membrane of theHelix lucorum RPa4 neuron using intracellular recording techniques. Application of specific agonists of nicotinic (nicotine, cytisine) and muscarinic (muscarine, arecoline) cholinoreceptors to the soma produced neuronal depolarization. The depolarization produced by applying acetylcholine to the cell was of short duration and was often replaced by hyperpolarization. Both selective desensitization of receptors by nicotine and muscarine as well as receptor occupancy by cytisine and arecoline reduced acetylchloline-induced response. The nicotinic cholinoblocker d-tubocurarine substantially inhibited responses to nicotinic cholinomimetics, while atropine, a muscarinic cholinoblocker, depressed response to muscarinic cholinomimetics. Acetylcholine-induced response was inhibited by both cholinoblockers more or less equally. The presence at the RPa4 neuronal somatic membrane is postulated of standard nicotinic and muscarinic cholinoreceptors similar to those found in vertebrates.M. V. Lomonsov State University, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 203–212, March–April, 1988.     

Regulation of cholinergic development by target contact

Avian ciliary ganglion neurons in cell culture have been used as a model neuronal system to examine the developmental role of direct contact with an appropriate target tissue. Live myotubes, which are immediately innervated, and their membrane remnants, on which neurons form terminal structures, were both found to stimulate or accelerate the acquisition or retention of important parameters of neuronal function. Contact with the target membrane supports survival, aids retention of active nicotinic receptors to acetylcholine, and accelerates the acquisition of adult transmitter synthetic capacity. These results emphasize the multifaceted nature of neurodevelopment, with soluble protein factors, membrane-bound elements, and other functional events all acting in concert in the embryonic nervous system.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

Extrasynaptic localization of α-bungarotoxin receptors in the rat superior cervical ganglion

Binding of [¹²⁵I]α-bungarotoxin to nicotinic cholinergic receptors (α-bungarotoxin receptors) was investigated in the rat superior cervical ganglion by light and electron microscope autoradiography. Both techniques indicated that labelling, which was inhibited by d-tubocurarine, occurred around and/or over neuronal perikarya. In particular, ultrastructural autoradiography showed that the synapses were devoid of radioactivity, suggesting that α-bungarotoxin receptors in the rat superior cervical ganglion are molecules distinct from the nicotinic (postsynaptic) receptors normally involved in ganglionic transmission. By contrast, specific labelling was found in extrasynaptic areas of the neuronal membrane in contact with satellite cells (neuron-satellite cell boundary). Quantitative analysis indicated that at that level silver grains were present on both the neuronal membrane and satellite cells. Furthermore, beside neuronal perikarya, radioactivity was also found around nerve fibres, probably in relation to both the axonal and interstitial sides of the ensheathing Schwann cells. Only a few grains were clearly accumulated inside nerve fibres. Finally, significant amounts of specific radioactivity were detected in the neuronal cytoplasm, especially at the level of rough endoplasmic reticulum and Golgi apparatus. However, parallel diffusion experiments with [¹²⁵I]α-bungarotoxin and [³H]inulin (a marker for the extracellular space) provided no evidence that the toxin enters the neuronal cytoplasm. Thus, the intraneuronal (specific) labeling was probably a reflection of α-bungarotoxin binding to membrane receptors and the subsequent internalization of the toxin-receptor complex in the neurons. We conclude that in the rat superior cervical ganglion extrasynaptic nicotinic acetylcholine receptors (α-bungarotoxin receptors) may be widely located on the neuronal membrane as well as on the plasma membrane of satellite and Schwann cells. The physiological significance of this molecular architecture is discussed.     

Regulation of posttetanic increase in choline sensitivity in Helix neurons by Na,K-pump: the role of Na/Ca-exchange and of mobilized calcium

We studied the role of Na/Ca-exchange and intracellular mobilized calcium in ouabain-mediated suppression of potentiation of cholinosensitivity of somatic membrane in Helix LPa3 and RPa3 command neurons of defensive behaviour after electrical orthodromic tetanisation of n. intestinalis. Cholinosensitivity of neurons was assessed by the amplitude of the inward current evoked by acetylcholine. Inhibitor of a Na/Ca-exchange benzamil and specific inhibitor of Ca-ATPase in endoplasmic reticulum thapsigargin prevented the development of the posttetanic potentiation (PTP). PTP did not arise and at joint action of ouabain with benzamil or thapsigargin. It was concluded that Na/Ca-exchange and mobilized calcium are involved in development of PTP of cholinosensitivity in somatic neuronal membrane and its regulation by Na,K-pump.