The similarity of the primary structure and homology of rhodopsin, beta-adrenoreceptor and muscarinic cholinoceptor

Computer analysis has been made of the primary structure of 6 different types of receptor proteins: rhodopsin, adrenoreceptor, muscarinic acetylcholine receptor, insulin receptor, nicotinic cholinoreceptor, and bacteriorhodopsin. The aim of the present investigation was to elucidate, at least partially, to what extent insignificant similarity in the primary structure of rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is due to divergent, but not convergent, evolution. Nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor were chosen for comparison with rhodopsin, adrenoreceptor and muscarinic cholinoreceptor since each of these proteins exhibits this or that structural or functional property which is common for rhodopsin, adrenoreceptor or muscarinic cholinoreceptor; on the other hand, nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor differ from other receptor proteins by their molecular mechanisms. Comparison of the primary structure of rhodopsin, adrenoreceptor and muscarinic cholinoreceptor on the one hand, and insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin on the other indicates that only the former exhibit similar primary structure, whereas insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin show no similarity neither in their primary structure, nor in the primary structure of rhodopsin and other receptor proteins which are similar to the latter with respect to their mode of action. The data obtained indicate that similarity in the primary structure between rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is a consequence of divergent, not convergent, evolution; in other words, these receptor proteins are homologous.     

Factors affecting the cholinomimetic activity of alkyltrimethylammonium compounds in experiments on isolated neurons of the mollusk Planorbarius corneus

It has been shown that the elementary current is independent whereas the duration of channel opening is slightly dependent on the number of methylene groups (from 1 to 9) in the molecule of alkyltrimethylammonium compounds. However, substances with more than 4 methylene groups exhibit lower cholinomimetic activity (i.e. the ability to increase the membrane current) and higher values of Q10 for the reaction with cholinoreceptor. It is suggested that lower activity of these compounds is due to a low rate of formation of a complex with cholinoreceptor because of the higher potential energy barrier.     

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.     

Possible approaches to an analysis of the heterogeneous response of the transcription complex to cholinergic actions

The accumulation of tyrosine-aminotransferase (TAT) as a marker of the individual gene activation was studied in the rat tissue after the administration of cholinomimetics and cholinolytics in order to elucidate the relations between cholinoreceptor functional state and the genetic apparatus. The regulation of TAT synthesis was found to be controlled by both cholinomimetic concentration and the density of cholinoreceptors in hepatocytes. Transsynaptic regulation of TAT activity was shown to be different in the brain and liver. It is suggested that the approaches discussed might be useful for the analysis of the relationship between cholinoreceptor state and the regulation of biochemical functions of target cells.     

The cholinomimetic activity of alkyltrimethylammonium compounds in experiments on the isolated mollusk neuron and on frog and chicken skeletal muscle

As revealed by contractile reaction of frog and chick muscles and by changes in the membrane current of isolated molluscan neurone, cholinomimetic activity of alkyltrimethylammonium compounds (ATMC) in the highest in drugs with 4 and 5 methylene groups in a molecule. The decrease in the activity with the decrease in the number of methylene groups was more evident in chick muscle; the decrease in the activity due to the increase in the number of these groups was most significant in experiments on molluscan neurone. Analysis of membrane current fluctuations showed that elementary current does not depend, whereas channel open time only slightly depends on the number of methylene groups in ATMC. However, with the increase of the number of methylene groups above 4, gradual decrease was observed in the ability of ATMC to increase at low (threshold) concentrations the membrane current (response) in the neurone. This decrease in the potency of ATMC correlated with the increase in Q10 value for neuronal response and calculated Q10 value for the reaction rate of ATMC with cholinoreceptor. The decrease in the activity of these ATMC is presumably due to a longer duration of complex formation with cholinoreceptor because of the higher energy barrier. ATMC with 8 and 9 methylene groups at high (saturating) concentrations elicited significantly smaller neuronal response with higher Q10 value. It is suggested that this phenomenon is due to a longer duration of complex formation with cholinoreceptor because of a higher energy cost.     

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.     

Calmodulin blockaders weaken the short-term plasticity of the neuronal cholinoreceptors in the edible snail

By means of recording transmembrane ion currents of identified snail neurones PPa3 and LPa3 a reversible weakening was shown of the speed and depth of extinction of neuronal cholinoreceptor membrane reactions to repeated iontophoretic applications of acetylcholine to the soma by a number of calmodulin blockaders: R24571 (20-50 mmol/l), trifluoperazine (50-200 mmol/l), chlorpromazine (20-60 mmol/l) and prenylamine lactate (30-400 mmol/l). The obtained results testify to a positive control by calmodulin of short-term cholinoreceptors plasticity of the studied neurons.     

Desensitisation of postsynaptic membrane in the neuro-muscular junction due to an increase in spontaneous quantal release of a mediator

Dependence of the amplitude of miniature end-plate currents on frequency of spontaneous quantal release modulated by the elevation of K+ concentration was studied in the frog voltage clamped neuromuscular junctions. A sharp increase of mEPC frequency (not less than approximately 50 per sec) was followed by an obvious fall in both their amplitude and acceleration of decay only in the presence of 3 microM prostigmine (acetylcholinesterase inhibitor) and 5 microM proadiphene, these agents promoting a desensitization of cholinergic postsynaptic membrane. Probable depletion of transmitter store is not involved in the phenomenon observed which is mainly due to the repetitive activation of the postsynaptic zones and the increase of the desensitized cholinoreceptor number.     

Regulation of neuron cholinoreceptor plasticity of Helix lucorum by second messengers and opioids

The rhythmical local ionophoretic applications of acetylcholine (ACh) to the somatic membrane of Helix lucorum identified neurons evokes the reversible depression of the ACh-induced response which shows cholinoreceptor (ChR) desensitization. ChR desensitization is regulated not by one but by several known second messengers and G-proteins. The endogenous opioids perform the excitation or inhibitory tonic control of the membrane potential in some neurons constantly activating the ionotropic opiate receptors. The direction of neuron ChR desensitization modulation by opioids depends on the type of the activated modulatory opiate receptors (mu or kappa) on the neuron membrane. Second messengers are involved in intracellular mechanism of modulation of the ChR desensitization by opiate kappa-agonist bremazocine.     

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.     

Effect of armin on synaptic transmission in frog neuromuscular preparations

The action of armin, an organophosphorus inhibitor of cholinesterases, on synaptic transmission parameters was studied by means of intracellular registration of end plate potentials and currents (EPP and EPC) in the frog. On 10-minute exposure the increase in the temporary parameters became manifest provided the drug was administered at a concentration of 10(-6) g/ml and over. EPC reversal potential and cholinoreceptor sensitivity to armin did not change substantially. At a concentration of 10-(-8) g/ml armin exerted a potentiating effect on the frequency of miniature EPP and quantum composition of EPP, while that effect was not related to armin anticholinesterase activity. The presynaptic acetylcholine release was suppressed by high concentration of armin (10(-5) g/ml). Under the conditions cited there was a decrease in the depot of the available transmitter quanta in nerve terminals.     

An ESR study of the postsynaptic membrane acetylcholinesterase of torpedo marmorata electric organ

Summary The effect of the cholinergic activator, phenyltrimethylammonium, on the ESR spectra of spinlabeled membrane bound acetylcholinesterase was studied; a reduction of maximal hyperfine splitting of the anisotropic ESR spectrum by 2 G was observed. The influence of phenyltrimethylammonium was prevented by the two cholinergic blocking agents d-tubocurarine and-cobratoxine. The present results indicate that the conformation change of the esteratic site of membrane acetylcholinesterase is triggered by the binding of phenyltrimethylammonium to the cholinoreceptor site.entjurcet al.: An ESR study of the postsynaptic membrane Acetylcholinesterase of torpedo marmorata electric organ.     

Effect of α-Bungarotoxin on Acetylcholinesterase bound to Mouse Diaphragm Endplates

IT has been shown that α-bungarotoxin (α-Bgt) irreversibly blocks cholinoreceptors^1–10 and that D-(+)tubocurarine (TC) protects these receptors from the toxin^4–7. On the other hand, it has been emphasized that α-Bgt has no effect on the catalytic activity of acetylcholinesterase (AChE)^4,6,9 and it was concluded that the cholinoreceptor and AChE must be two different macromolecules^8,9. Because typical cholinolytics, including TC and gallamine, characteristically influence the kinetics of a membrane-bound AChE^11,12, it seemed justified to reinvestigate whether α-Bgt really is an exception in this respect.     

Hexamethonium, nicotinic receptor blocker, changes the neuronal reactions on glutamate in the medial septal area in vitro

Despite the great interest in studying the medial septal area, the interactions of its neurochemical systems are not yet clearly understood. The aim of this study was to elucidate the role of nicotinic receptors in the interaction of glutamatergic and cholinergic systems of the medial septal area. The effect of L-glutamate (1 microM) on septal neurons was studied under the application of hexamethonium, nicotinic cholinoreceptor blocker by using the method of extracellular recording of neuronal activity in brain slices of ground squirrels. The response of septal neurons to glutamate depended on the type of their initial activity and on the presence of pacemaker properties. For the first time, the ability of septal neurons to respond to glutamate with an increase in burst frequency was shown. The influence of hexamethonium on the neuronal activity was similar to that of glutamate. After a preliminary application of hexamethonium, the reactions of neurons to glutamate changed. The excitatory reactions were masked, while the inhibitory reactions became stronger. It was found that nicotinic cholinergic receptors modulated the reactions of MS-DB cells to glutamate and the expression of the oscillatory properties of the septal neuronal network.     

Solubilization by various detergents, of the muscarinic cholinoreceptor and its complex with quinuclidinyl benzilate

The possibilities to solubilize the rat brain cortex muscarinic acetylcholine receptor and its complex with [3H]-L-quinuclidinyl benzilate (QNB) were studied, using 14 detergents. It was shown that the native muscarinic cholinoreceptor was solubilized in addition to digitonin, also by CHAPS, with a 6% yield. Besides, the receptor-QNB complex was solubilized with the detergents Triton X-100, -102, -114, -165 (with 30% and 50% yields) and within a narrow concentration range with sodium dodecyl sulfate (50% yield). Some detergents of the Tween series, e.g., Triton X-45 and -305, as well as sodium deoxycholate and sodium oxycholate, did not solubilize the native receptor and its complex with QNB. It was found that yield of receptor solubilization did not exceed half of the total number of the receptor sites in the membranes, despite the fact that different concentrations of detergents were applied. The solubilization yield did not increase, when different mixtures of detergents were used. It was assumed that incomplete solubilization of the receptor protein reflects its heterogeneity in the membrane structure.     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

Repetitive firing in motor nerve endings: Divalent cation,rhythmic activity,and cholinergic agent modulation

Repetitive firing (RF) as two or more end plate currents (EPC) induced by a single stimulus of the motor nerve was studied in sartorius frog muscle under voltage clamp conditions. In the presence of 4-aminopyridine (1.10^–4 M), RF as two EPC with 3–8 msec intervals was found in half of the cells (n=35). When calcium ion concentration was increased from 1.8 to 5.4 mM, magnesium to 5–9 mM, and rhythmic activity beginning with 0.05 Hz and above, RF stopped, but when strontium or barium was substituted for calcium, RF intensified. In the presence of barium, the repeated EPC quantity reached six-eight per one nerve impulse. Slow currents arose in some of the cells when disruption of the EPC series was stimulated. Proserine, an anticholinesterase agent, enhanced RF, whereas -bungarotoxin, a cholinoreceptor blocker, had no effect. The role that calcium and calcium-activated potassium currents play in generating and terminating mechanisms of RF, is discussed.S. V. Kurashov Medical Institute, Russian Federation Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 387–395, July–August, 1992.     

Fluorescence of plant microspores as biosensors

Possibilities of fluorescent microscopic single-cell analysis on plant microspores as biosensors for study of chemosignaling involving neurotransmitters and mechanisms of action of fluorescent medicinal compounds—antagonists of neurotransmitters were studied on some examples. By methods of luminescence microscopy, microspectrofluorimetry and laser scanning confocal microscopy using as an example field horsetail Equisetum arvense microspores, the penetration of these compounds into the cell and associate it with individual compartments (estimated as the changes in their autofluorescence) has been analyzed. Fluorescent in blue antagonists of neurotransmitters d-tubocurarine, yohimbine and azulene (blockers of cholinoreceptor, adrenoreceptor, and histamine receptor, respectively) decreased the number of the E. arvense cells with red fluorescence. Tubocurarine and yohimbine bound to the cellular surface and did not penetrate into the cells. Azulene was found both on the cell surface and inside cells, demonstrating blue (excitation 360–380 nm) or green (excitation 420 nm) fluorescence of DNA-containing organelles. The effects of lipophilic (lecithin and amphotericin B) and proteinous (albumin, enzyme cholinesterase, cytoskeleton proteins actin, myosin, and titin) compounds on the manifestation of the effects of the neurotransmitters and antagonist d-tubocurarine have been shown. The intensity of the red light at 680 nm, has evolved in many variants. Most notable was the decline of the emission in the presence of albumin and cholinesterase as compared with the action of dopamine itself. After the addition of the cytoskeleton proteins and cholinesterase to the medium, the decrease of red fluorescence intensity, usually induced by d-tubocurarine, was not observed.     

Novel dimeric acetylcholinesterase inhibitor bis7-tacrine, but not donepezil, prevents glutamate-induced neuronal apoptosis by blocking N-methyl-D-aspartate receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.