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.     

Pharmacological parameters of muscarinic cholinoreceptors in skeletal muscles

It has been shown that bath application of muscarine delayed the early post-denervation depolarization in the muscle fibers incubated for 3 h in culture medium. The greatest reduction of the post-devervation depolarization was observed with 50 nmol/l muscarine. Atropine, a muscarinic antagonist, clozapine, a specific inhibitor of M1/M5-cholinergic receptors, and nitrocaramiphen, a M1-antagonist, completely removed the hyperpolarizing effect of muscarine. 4-DAMP, a specific inhibitor of M3-cholinergic receptors, himbacine, an antagonist of M2-cholinergic receptors, and tropicamide, a specific inhibitor of M2/M4-cholinergic receptors, failed to prevent the effect of muscarine. A M1/M2 muscarine agonists propargyl and but-2-ynyl esters of arecaidine had apparent muscarine-like effect. Nitrocaramiphen, and not himbacine, prevented the hyperpolarizing effect of these cholinomimetics. It is concluded that muscarine and esters of arecaidine delay the development of early postdenervation depolarization in M1-cholinergic receptors of skeletal muscle.     

The effect of plastic restructuring of the local chemoexcitable zone in the somatic membrane of the PPa4 neuron of Helix lucorum on the excitability of neighboring chemo- and electrically excited zones

Rhythmic application of acetylcholine or serotonin to the local zone of somatic membrane was used to study the effect of extinction of RPa4 neuron depolarization in Helix lucorum on the excitability of adjacent chemo- and electroexcitable zone. It has been found that the extinction of response to iontophoretic application of acetylcholine to one somatic zone decreases the sensitivity of serotonin and cholinoreceptors in adjacent zones, as well as the excitability of electroexcitable membrane. The effect on the excitability of adjacent zones does not depend on the type of receptors activated rhythmically, as the extinction of RPa4 response to the repeated application of serotonin also reduces the sensitivity of adjacent cholinoreceptor zones. A cause of this effect may lie in modification of chemoreceptors and ionic channels, by intracellular regulatory systems that become activated by repeated stimulation.     

Participation of muscarinic and nicotinic cholinoreceptors of hypothalamus preoptic area in control of wakefulness and sleep states and of thermoregulation in pigeons Columbia livia

By using electrophysiological methods, it has been established that muscarinic (M-) cholinergic mechanisms of the ventrolateral preoptic area (VLPA) of pigeon hypothalamus participate in maintenance of wakefulness, whereas nicotinic (N-) mechanisms--in maintenance of the slow-wave sleep. Activation of the VLPA M-cholinergic receptors has been found to be accompanied by an elevation of the brain temperature, by development of peripheral vasoconstriction, and by an increase in the muscle contractive activity. Activation of N-cholinoreceptors leads to a decrease in the brain temperature and development of peripheral vasoconstriction. It is suggested that the VLPA M- and N-cholinergic receptors are involved in different mechanisms of regulation of wakefulness and sleep states and brain temperature in pigeons.     

Localization of putative nicotinic cholinoreceptors in the early development of Paracentrotus lividus

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.     

The role of muscarinic cholinoreceptors in the retrieval of the instrumental food conditioned reflex in cats

It was demonstrated in cat experiments that impairment of the retrieval of appetitive instrumental conditioning observed after systemic administration of antagonists of muscarinic central cholinoreceptors scopolamine (a nonselective M1 antagonist) and trihexyphenidyl (relatively selective M1 antagonist) could be connected with central and peripheral side effects of these antagonists. It was established that in case of the absence of side effects (low doses of trihexyphenidyl, 1 mg/kg) the blockade of M1-cholinoreceptors led either to selective fall-off of the motor instrumental reaction with in the presence of contextual behavior and other conditioned reactions or the appearance of errors that seemingly was indicative of the disturbance of triggering and realization of the motor program as the most important component of conditioning performance. The systemic injection of trihexyphenidyl (10 mg/kg), scopolamine (0.03 and 0.06 mg/kg) and nonselective peripheral antagonist methylscopolamine (0.03 mg/kg) led to changes in the general functional state (disorders in the emotional and motivational sphere), the expression of which depended on the individual sensitivity to anticholinergic drugs. The disturbance of CR retrieval observed in parallel with side reactions was characterized by a complete cessation of conditioning and apparently was not associated with memory deterioration.     

The calcium current of Helix neuron

Calcium current, Ica, was studied in isolated nerve cell bodies of Helix aspersa after suppression of Na+ and K+ currents. The suction pipette method described in the preceding paper was used. Ica rises to a peak value and then subsides exponentially and has a null potential of 150 mV or more and a relationship with [Ca2+]o that is hyperbolic over a small range of [Ca2+]o's. When [Ca2+]i is increased, Ica is reduced disproportionately, but the effect is not hyperbolic. Ica is blocked by extracellular Ni2+, La3+, Cd2+, and Co2+ and is greater when Ba2+ and Sr2+ carry the current. Saturation and blockage are described by a Langmuir adsorption relationship similar to that found in Balanus. Thus, the calcium conductance probably contains a site which binds the ions referred to. The site also appears to be voltage-dependent. Activation and inactivation of Ica are described by first order kinetics, and there is evidence that the processes are coupled. For example, inactivation is delayed slightly in its onset and tau inactivation depends upon the method of study. However, the currents are described equally well by either a noncoupled Hodgkin-Huxley mh scheme or a coupled reaction. Facilitation of Ica by prepulses was not observed. For times up to 50 ms, currents even at small depolarizations were accounted for by suitable adjustment of the activation and inactivation rate constants.     

Mechanisms of tubocurarine action on nicotinic cholinoreceptors of rat sympathetic ganglia neurons

The effects of tubocurarine (TC) on current induced by acetylcholine (ACh) in neurons of rat upper cervical ganglia were investigated using techniques for voltage-clamping at the membrane. Reinforcement of TC-induced blockade was achieved by paired application of ACh following prior activation of nicotinic cholinoreceptors, indicating that TC blocked the channels opened by ACh. On average, the TC-open channel complex persisted for 9.8±0.5 sec (n=7) at –50 mV and 20–24°C. It was found that increases exponentially with hyperpolarization at the membrane (a shift in membrane potential of 61 mV corresponds to an e-fold change). Suppression of ACh-induced current (ACh current) was eliminated completely under the effects of 3–30 M with depolarization of up to 80–100 mV at the membrane. Suppression of ACh current produced by membrane potential at negative levels is intensified with increasing doses of ACh. Findings would indicate that blockade of ionic channels opened by ACh is the only mechanism of TC action on nicotinic cholinoreceptors in rat sympathetic ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 672–680, September–October, 1988.     

4-Bromoacetamidoprocaine: An affinity ligand for brain muscarinic and nicotinic cholinergic receptors

This study describes the synthesis, receptor binding characteristics, and some behavioral effects of p-bromoacetamidoprocaine (BAP), a new affinity ligand for brain muscarinic and nicotinic cholinergic receptors. The reversible binding of [³H]QNB to rat brain membranes was inhibited in a concentration dependent and saturable manner by both procaine and BAP, with Ki values of 4×10^–6 and 3×10^–7 M, respectively, and complete inhibition at 1×10^–5 M. Both procaine and BAP, although at much concentrations, inhibited the binding of [³H]methylcarbamylcholine in a concentration dependent manner, with Ki values of 5×10^–5 and 1×10^–5 M, respectively, and complete inhibition for both at 1×10^–3 M. Plots of the % irreversible inhibition of [³H]QNB, [³H]nicotine, and [³H]MCC vs [BAP] yielded Ki values of 7×10^–8, 1×10^–4, and 6×10^–5 M, respectively. In behavioral studies BAP was able to antagonize the QNB-induced hyperactivity in mice; however, BAP did not appear to alter nicotine-induced seizure activity or other behavioral effects in mice. A plot of the time course of inhibition by BAP for [³H]QNB binding revealed that the inhibition was almost complete within 10 min exposure at 37°. The findings indicate that BAP is a useful affinity ligand for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nM concentration range.     

Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium

Acetylcholine (ACh) and its receptors play a crucial role in bladder physiology. Here, we investigated the presence of muscarinic receptor subtypes (MR) and nicotinic acetylcholine receptor (nAChR) alpha-subunits in the mouse urothelium by RT-PCR and immunohistochemistry. With RT-PCR, we detected mRNAs coding for all of the five different MR subtypes and for the nicotinic receptor subunits alpha2, alpha4, alpha5, alpha6, alpha7, alpha9 and alpha10, whereas the alpha3-subunit was not expressed. Using immunohistochemistry, we localised a panel of acetylcholine receptors in the different layers of the murine bladder urothelium, with predominant appearance in the basal plasma membrane of the basal cell layer and in the apical membrane of the umbrella cells. M2R and subunit alpha9 were observed exclusively in the umbrella cells, whereas the MR subtypes 3-5 and the nAChR subunits alpha4, alpha7 and alpha10 were also detected in the intermediate and basal cell layers. The subunit alpha5 was localised only in the basal cell layer. In conclusion, the murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since these receptors have different electrophysiological and pharmacological properties, and therefore are considered to be responsible for different cellular responses to ACh, this differential distribution is expected to confer cell type-specificity of cholinergic regulation in the bladder urothelium.     

Kinetics of the work of the ion channels controlled by nicotinic cholinoreceptors in the membrane of mollusk neurons

Currents entering through single channels with conductivity 10 pS were produced on the membrane of an isolated neuron of the fresh-water molluskPlanorbarius corneus in the presence of suberyldicholine (5 µM) by the patch-clamp technique (cell-attached configuration). The times of stay of the channels in the open and closed states, as well as the durations of pulse bursts and clusters, were measured. The distributions of the time intervals obtained experimentally were approximated for open states by one exponential function: t_o=27±3 msec (n=21), and for closed states by a sum of three exponentials: t_c1=9.5±1.0 msec (n=21); t_c2=171±33 msec (n=19); t_c3=5.2±1.0 sec (n=21). The burst durations are characterized by the presence of two exponential functions in the distribution: t_b2=20±14 msec (n=10), t_b2=203±23 msec (n=10), and the clusters by three exponential functions: t_k1=33±11 msec (n=8), t_k2=274±84 msec (n=8), and t_k3=1.5±0.5 sec (n=9). Thus, for work of a chemoactivated channel associated with nicotinic-type cholinoreceptors in a mollusk neuron we can suggest a kinetic scheme with one open and three nonconducting states: C O D₁A₂ D₂A₂. The two "long-lived" closed states of the channel may be associated with desensitization of the integral response of the neurons to the application of suberyldicholine. Values were obtained for the rate constants of these proposed reactions. It is suggested that this model may be useful in analyzing the action of cholinomimetics and blockers on the molluskan neuronal membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 588–595, September–October, 1991.     

Reaction of cholinoreceptors from an isolated molluskan neuron with analogs of acetylcholine and tetramethylammonium

The effect of 13 acetylcholine and tetramethylammonium derivatives on cholinoreceptors of isolated neurone on the pond snail has been investigated by the analysis of membrane current fluctuations at 10 and 20 degrees C. The elementary current was independent from the agonist structure. The channel open time and its Q10 coefficient were found to be maximum for acetylcholine. At low concentrations, acetylcholine derivative with four groups in its methylene chain exhibited the weakest activity. Agonist activity at higher concentrations was evaluated by maximum increase in the membrane current. With respect to this character, the derivative with two ethyl radicals in its cation group was found to be the weakest one. Possible causes of low activities of the agonists studied in comparison with that of acetylcholine are discussed.     

Independence of the process of habituation from protein synthesis in the mollusk neuron

The influence of protein synthesis blocators--puromicine and gougerotine--on the dynamics of habituation to repeated tactile stimuli in mollusc's neurones was studied microionophoretically. Dynamics of habituation was judged by reduction of impulses number in neurone's response, increase of thresholds and latency of the first synaptic action potential in the response to repeated stimuli. Introduction of the substances produced the greatest effect on the dynamics of thresholds. Puromicine introduction suppressed the habituation only if it was accompanied by generation of epileptiform activity. Gougerotine improved the habituation in the animals with initially weak process. The obtained data testify to the hypothesis that protein synthesis blockade does not impede the process of habituation.