Muscarinic Receptor Sites Research Articles

Effect of axotomy on the cyclic GMP increase induced by preganglionic stimulation and high extracellular K + concentration in superior cervical sympathetic ganglion of the rat

Cyclic GMP generation, induced by preganglionic nerve stimulation or by high extracellular potassium ion concentration (70 mM) in the medium, was studied during aerobic incubation of the excised superior cervical sympathetic ganglion of the rat with and without axotomy, and the results were compared with that of preganglionic denervation. Both axotomy and denervation of the ganglion for a week caused complete loss of increase of cyclic GMP content in the ganglion in response to the preganglionic nerve stimulation. However, the increase of the ganglionic cyclic GMP content evoked by raising the extracellular potassium ion concentration was maintained at a level about two-thirds of the control after axotomy for 1–4 days, while it was abolished within a day after denervation. Ganglionic choline acetyltransferase activity was maintained for several days after axotomy, but it was decreased rapidly by denervation. Acetylcholinesterase, dopamine-β-hydroxylase and muscarinic cholinergic receptor sites had been lost almost completely a week after axotomy. These results suggest that the increase of ganglionic cyclic GMP content caused by depolarization with high extracellular K + is associated with the preganglionic nerve terminals rather than with postganglionic receptor.

Biochemical evidence for cholinergic innervation of intracerebral blood vessels

Muscarinic cholinergic receptor sites were detected with [ 3H]quinuclidinylbenzilate (QNB) binding techniques in two fractions of bovine intracerebral vessels; one of the fractions contained primarily small arteries and veins with some attached capillaries, and the other one was highly enriched in capillaries. The amounts of binding were similar in equivalent vascular fractions isolated from cerebral cortex, caudate nucleus and cerebellar cortex in spite of large differences among the 3 regions in [ 3H]QNB binding to brain tissue. The different distribution of muscarinic receptors in brain tissue and blood vessels argues against the possibility that the receptors represent a contamination of the vascular fractions by brain parenchyma. Cerebral endothelial cells, which were isolated treating capillaries with collagenase, bound [ 3H]QNB to the same extent as did cerebral capillaries. This is consistent with an endothelial localization of capillary muscarinic receptors. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, also was present in the vascular preparations. Within each brain region, ChAT activities in capillaries and larger vessels were similar, but significant regional differences were found for vascular ChAT activity, with the highest values in the caudate. Isolated endothelial cells contained significantly lower levels of ChAT activity than intact capillaries, suggesting a periendothelial location of the enzyme, as would also be the case for attached nerve terminals. The presence of [ 3H]QNB binding sites and ChAT activity in intracerebral blood vessels is consistent with an innervation of the cerebral vasculature by a cholinergic system that may regulate cerebral blood flow and capillary permeability.

Evidence for muscarinic cholinergic receptors in dog portal vein: Binding of [ 3H] quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [ 3H] quinuclidinyl benzilate (QNB) as a ligand. Specific [ 3H] QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (K D = 15.5 ± 2.8 pM) with a Bmax of 110 ± 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 × 10 9 M −1 min −1 and 0.016 min −1, respectively, were calculated. The dissociation constant calculated from the equation K D = K −1/K +1 was 8.3 pM, such being in good agreement with the Scatchard estimate of K D (15.5 pM). Specific binding of [ 3H] QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, α-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [ 3H] QNB binding at 10 μM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [ 3H]quinuclidinyl benzilate to membranes and the binding of [ 3H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [ 125I]α-bungarotoxin or [ 3H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.

STUDIES ON THE SITE OF ACTION OF NEUROTROPIN IN EXPERIMENTS ON DENERVATION-SUPERSENSITIVITY OF THE DUODENUM AND VAS DEFERENS OF RATS AND MICE

In order to clarify to a greater extent its action on peripheral nerves, various experiments were conducted using denervated animals to determine the effects of the nervous sedative, Neurotropin (NSP, containing many types of polysaccharides), on the site considered to be the periphery of autonomic nerves and the central nervous system. The increase in response to ACh due to bilateral cardiac vagotomy was significantly inhibited by a daily administration of NSP, but the increase in the response to noradrenaline (NA) due to celiac sympathectomy was hardly affected by this administration. The supersensitivity to the muscarinic action of ACh or methacholine of a denervated rat vas deferens, owing to ablation of the serous membrane, was significantly inhibited by the daily administrations of NSP. However, supersensitivity to NA was hardly affected. NSP never had any effect on the increase in the NA response of the duodenum and vas deferens isolated from mice given 6-hydroxydopamine, an adrenergic degenerator. Thus, an inhibitory action of NSP on denervation-supersensitivity is conceivably exerted on the parasympathetic nerves, rather than on the sympathetic nerves, and on a muscarinic receptor site, instead of a nicotinic site.

Studies on the site of action of neurotropin in experiments on denervation-supersensitivity of the duodenum and vas deferens of rats and mice.

In order to clarify to a greater extent its action on peripheral nerves, various experiments were conducted using denervated animals to determine the effects of the nervous sedative, Neurotropin (NSP, containing many types of polysaccharides), on the site considered to be the periphery of autonomic nerves and the central nervous system. The increase in response to ACh due to bilateral cardiac vagotomy was significantly inhibited by a daily administration of NSP, but the increase in the response to noradrenaline (NA) due to celiac sympathectomy was hardly affected by this administration. The supersensitivity to the muscarinic action of ACh or methacholine of a denervated rat vas deferens, owing to ablation of the serous membrane, was significantly inhibited by the daily administrations of NSP. However, supersensitivity to NA was hardly affected. NSP never had any effect on the increase in the NA response of the duodenum and vas deferens isolated from mice given 6-hydroxydopamine, an adrenergic degenerator. Thus, an inhibitory action of NSP on denervation-supersensitivity is conceivably exerted on the parasympathetic nerves, rather than on the sympathetic nerves, and on a muscarinic receptor site, instead of a nicotinic site.

Cholinergic receptor site binding, choline acetyltransferase, and acetylcholinesterase activity in the forebrain and brainstem of the Dahl rat model of essential hypertension.

Muscarinic and nicotinic receptor site binding and the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the forebrain and brainstem of Dahl salt-sensitive (DS) and salt-resistant (DR) rats was investigated. The DS line had a greater density of muscarinic sites in the cortex, hypothalamus, and medulla. Hypertensive DS rats had a greater density of sites than normotensive DS rats. ChAT activity was also higher in the cortex and hypothalamus of the DS line than the DR line. No significant differences were found in the activity of AChE or the concentration of nicotinic sites. These results suggest that the central muscarinic cholinergic system may participate in the pathogenesis of hypertension in the DS rat. The data indicate that central cholinergic activity is possibly greater in the DS than the DR rat and that this may help to explain the enhanced pressor response in the DS line after pharmacological activation of the central cholinergic system.

Agonist regulation of the muscarinic cholinergic receptor in embryonic chick heart.

J. B. Galper, L. C. Dziekan and T. W. Smith. The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic agonist (3H)methyl-scopolamine ((3H)MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 days in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist car-bamylcholine were the responses studied. In cells labelled to equilibrium with 42K+, exposure to 10–3 M carbamycholine caused a 33% increase in the rate of 42K+ efflux from the cell with a half-maximal effect at a carbamylcholine concentration of 8×10–5 M. Steady state Intracellular K+ content remained at control levels. A 15% decrease in beating rate occurred in cells exposed to 10–3 M carbamylcholine. Since muscarinic receptors on the surface of the Intact cell are presumed to mediate the physiologic response to the agonist, an assay for binding of (3H)MS to Intact cells was developed. (3H)MS bound specifically to intact heart cells (l85fmol/mg protein) with a Kd of 0.48 nM.KeywordsMuscarinic ReceptorHeart CellReceptor NumberMuscarinic AgonistBeat RateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Presence and axonal transport of cholinoceptor, but not adrenoceptor sites on a cat noradrenergic neurone.

1. Noradrenaline release and radioligand binding studies were carried out in the cat hypogastric nerve ligated in vito 2 cm distal to the inferior mesenteric ganglion for different time periods, and in different effector organs.2. Large quantities of noradrenaline and dopamine beta-hydroxylase (DBH) accumulated in the segments of nerve immediately proximal (P(1)) and distal (D(1)) to the ligation, with rates of about 100 and 25 mm/24 hr for the orthograde and retrograde transport, respectively.3. Nicotine evoked the release of noradrenaline from P(1) and atrial slices; the secretory response to nicotine was completely antagonized by mecamylamine. [(3)H]alpha-bungarotoxin biding to membranes from P(1) allowed the estimation of a K(D) of 2.97 nm and a B(max) of 1639 f-mole/mg protein.4. Acetylcholine inhibited the release of endogenous noradrenaline evoked by high K(+) stimulation in atrial slices, but not in P(1) segments. Similarly, carbachol decreased [(3)H]noradrenaline release induced by electrical stimulation (twenty-six shocks, 2 Hz, 5 msec) in the atrium but not in P(1).5. [(3)H]Quinuclydinilbenzylate ([(3)H]QNB) specifically binds to membranes from P(1) and vas deferens, following a saturation curve. In the case of P(1) segments taken 48 hr after ligation a K(D) of 0.35 nm and a B(max) of 129 f-mole/mg protein were found.6. The fact that the B(max) in P(1) and D(1) increased with the time of ligation suggests that orthograde and retrograde axonal transports of muscarinic binding sites exist in this nerve, with approximate rates of transport of 15 and 8 mm/24 hr, respectively.7. As far as adrenoceptors are concerned, we observed that yohimbine or phentholamine did not modify transmitter release from P(1), evoked by high K(+) or electrical stimulation. However, yohimbine enhanced the release of [(3)H]noradrenaline induced by electrical stimulation from splenic slices of the same animals.8. [(3)H]Clonidine, [(3)H]dihydroergocryptine or [(3)H]dihydroalprenolol ([(3)H]DHA) did not specifically bind to membranes from P(1), in spite of the fact that they showed typical saturation curves for specific binding in cortex and atrial membranes from the same cats.9. In conclusion, these data (a) further show that the ligated hypogastric nerve is a good model of noradrenergic nerve terminal free of effector cell; (b) provide direct evidence for the neural location of nicotinic receptors whose activation trigger noradrenaline release from noradrenergic neurones; (c) demonstrate the neural location and axonal transport of muscarinic receptor sites, but leave certain doubts about its functional role in this noradrenergic neurone; and (d) do not support the hypothesis that alpha and beta-adrenoceptors which modulate noradrenaline release from peripheral noradrenergic nerve terminals are neurally (or prejunctionally) located.

Autoradiography of dendritic acetylcholine receptors: A method for study of isolated neurons of the adult central nervous system in the turtle

Adult neurons were isolated from turtle retina in order to study the subcellular distribution of muscarinic acetylcholine receptors. Retinas were perfused in vitro with [ 3H]quinuclidinylbenzilate [ 3H]QNB) to label muscarinic receptors, and individual neurons with extensive dendritic arborization were obtained for autoradiography by use of papain and trituration. Papain caused no change in the number of muscarinic receptor sites nor in their affinity for [ 3H]QNB. Receptors, indicated by the positions of silver grains, were localized to dendritic processes.

Agonist-induced changes in the modulation of K+ permeability and beating rate by muscarinic agonists in cultured heart cells.

The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic antagonist [3H]methylscopolamine ([3H]MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 d in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist carbamylcholine were the responses studied. Exposure to 10(-3) M carbamylcholine caused a 15% decrease in beating rate and a 33% increase in the rate of 42K+ efflux from cells labeled to equilibrium. An assay for binding of [3H]MS to intact cells was developed. [3H]MS bound specifically to intact heart cells (185 fmol/mg protein) with a Kd of 0.48 nM. Exposure of cells for various times to 10(-3) M carbamylcholine followed by binding of [3H]MS to intact cells demonstrated that a gradual loss of 70% of [3H]MS binding sites took place over the next 6 h with a T 1/2 of 30 min. A decrease in the ability of carbamylcholine to stimulate K+ efflux and to decrease beating rate was observed after pre-exposure of cells to muscarinic agonists. A close correlation was found between the loss of the subclass of muscarinic receptors subject to agonist control and the loss of physiologic responsiveness after agonist exposure. The data suggest the absence of significant numbers of "spare" receptors within this group.

Tolerance, physical dependence and changes in muscarinic receptor binding sites after chronic ethanol treatment in the rat

Rats were treated with ethanol in the drinking water for 73–75 weeks. The daily drinking periods were restricted to only 2×1 h to get significant ethanol blood concentrations. After the end of the ethanol treatment (last day of treatment denoted by 0 of the abstinence) tolerance to sedatives were recorded with a hexobarbital threshold method, convulsions recorded with jiggle cages, and muscarinic receptor binding sites determined with 3H-quinuclidinyl benzilate. Tolerance to hexobarbital was found to have a maximum on day 8 in the abstinence while convulsions recorded from the rats participating in the hexobarbital threshold determinations had a maximum on day 14. Atropine administered as a single dose on day 8 decreased the tolerance to hexobarbital on that day and seemed to reduce the number of convulsions on the following days. Muscarinic receptor sites were significantly increased in the striatum on day 8 of the abstinence at the time of maximal tolerance. This increase in muscarinic receptors was less in rats which had had convulsions during the abstinence before sacrifice. Thus the results indicate that cholinergic mechanisms are involved in the changes seen during the abstinence after chronic ethanol exposure.

Differential down-regulation by carbamylcholine of putative nicotinic and muscarinic acetylcholine receptor sites in chick retina cultures

The effect of chronic exposure to the cholinergic agonist carbamycholine on the number of nicotinic and muscarinic cholinergic ligand binding sites in primary cultures of chick embryo retina has been investigated. Within 24 h, 1 mM carbamylcholine led to a 60–70% loss of specific binding sites for the muscarinic antagonist [ 3H]quinuclidinylbenzilate, whereas the binding of the nicotinic cholinergic ligand [ 125I]α-bungarotoxin remained almost unchanged under the same conditions. The reduction in muscarinic cholinergic ligand binding sites by carbamylcholine was blocked by atropine and scopolamine, but not affected by the inhibition of protein synthesis or the removal of extracellular calcium. These data indicate a different metabolic regulation by agonist occupancy of putative nicotinic and muscarinic acetylcholine receptors in the chick central nervous system.

Regulation of neocortical muscarinic receptors: effects of drug treatment and lesions

Regulation of neocortical muscarinic receptors: effects of drug treatment and lesions

Lowering temperature increases cardiac muscarinic receptor site affinity towards agonists and antagonists

1. 1. The effect of temperature on the binding of agonists and antagonists to cardiac muscarinic receptor sites was investigated. 2. 2. Compared to 37°C, carbachol-[ 3H]QNB and atropine-[ 3H]QNB competition curves were shifted to the left at lower temperatures. This was also observed with other agonists and antagonists. 3. 3. Such an effect of temperature persisted in the N-ethylmaleimide (NEM)- and dithiothreitol (DTT)-treated membranes. 4. 4. Dissociation of the membrane-bound [ 3H]QNB was higher at 37°C (∼40% at 60 min) compared to 18°C (∼20%). Both carbachol and atropine increased the dissociation at 37°C to a similar maximum (to about 60%). 5. 5. The results show marked temperature-dependent alterations in the cardiac muscarinic receptor sites and suggest that these occur either by direct action of the temperature on receptor conformation or its indirect action via membrane fluidity. The likely significance of these results is discussed.

Glutamic acid binding in goldfish brain and denervated optic tectum

The binding of L-glutamic acid to goldfish brain membranes and changes in tectal binding following optic nerve denervation and regeneration were investigated. Saturable, reversible, and specific binding occurred to sodium-free washed membranes from goldfish brain at a single population of sites having an apparent Kd of 3.4 microM and a capacity of 10 pM/mg original tissue. Binding was enriched in crude synaptosomal (P2) subcellular fractions. There was a 10-fold regional variation in the concentration of binding sites. In pharmacological studies protection constants (Kp) (the concentration which resulted in a 50% inhibition of binding) ranged from 4 microM for glutamate to greater than 10 mM for GABA. Following eye removal, the total number of tectal glutamic acid binding sites was stable for 4 days, followed by a rapid loss in binding, reaching 40% of control at 24 days. After optic nerve crush and optic nerve regeneration, the number and concentration of binding sites was not different from control. The relationship between glutamate, nicotinic, and muscarinic receptor sites in the retinotectal pathway is discussed.

Solubilization of muscarinic acetylcholine receptors from mammalian brain: An analytical approach

Muscarinic receptor sites were extracted from microsomal membranes of mammalian brain using digitonin. These detergent preparations were submitted to the control of unambiguous solubilization criteria, and were found to correspond to molecularly dispersed material. In contrast, the corresponding salt preparations did not fulfill these criteria. After comparing various assay methods, we describe a simple and rapid charcoal adsorption binding technique, which was selected for measuring digitonin-extracted muscarinic receptors. Under these conditions the digitonin preparations, from four different species revealed identical biochemical properties in a stereospecific labeling system using [ 3H]dexetimide and [ 3H]levetimide.

Imprinting. A study of cholinergic receptor sites in parts of the chick brain.

The distribution of cholinergic receptor binding sites was studied in certain areas of the chick brain, especially that part (IMHV) of hyperstriatum ventrale which has previously been implicated in the imprinting process. There was relatively little binding of [125I]-alpha-bungarotoxin in this region, as shown using an autoradiographic technique. In contrast muscarinic receptor sites, with an affinity for [3H]-quinuclidinyl benzilate (QNB), are present in relatively high concentration in a medial forebrain sample containing IMHV and also in a visual projection area, the hyperstriatum accessorium (HA). To assess the effects of imprinting on the concentration of QNB binding sites 26 young chicks were exposed to an imprinting stimulus (a pulsing red light) for 20 min. Thirteen of the chicks were returned to the dark and the other 13 were trained for a further 100 min. There was no effect of further training on specific QNB binding in HA or in the medial forebrain sample. In undertrained chicks there was a positive correlation between QNB binding in the medial forebrain sample of the left hemisphere and that in the right; there was no such relationship in overtrained birds. Mean QNB binding in the right medial forebrain sample was greater than in the left by 21%. There was no such hemispheric asymmetry in HA.

Requirement for sulfhydryl groups in the differential effects of magnesium ion and GTP on agonist binding of muscarinic cholinergic receptor sites in rat atrial membrane fraction.

1. The binding of agonist (carbachol) and antagonist (atropine) to the rat heart atrial muscarinic cholinergic receptor sites was investigated. 2. Divalent cations (Mg2+ or Ca2+), in low concentrations, modestly increased the agonist binding affinity of the receptor site without any effect on the antagonist binding affinity. 3. Guanine nucleotides (e.g. GTP), on the other hand, decreased the agonist binding affinity (but not the antagonist binding affinity), and the extent of GTP effect depended on the absence or presence of divalent cation (Mg2+) in the binding assay. 4. Pretreatment of atrial membranes with N-ethylmaleimide (NEM) altered the agonist binding curve (obtained with varying concentrations of carbachol) such that the Hill coefficient (nH) became very close to 1.0, whereas the corresponding nH values for control (untreated) or dithiothreitol (DTT)-treated membranes were much less than 1.0; NEM or DTT treatments failed to show any effect of antagonist binding curve. 5. NEM treatment abolished both divalent cation-induced and guanine nucleotide-induced alterations in the agonist binding affinity of the receptor site. 6. Monovalent cations in low concentrations did not mimic the effects of Mg2+ or Ca2+ on agonist binding. Instead, concentration dependent decreases in both agonist and antagonist binding affinities and abilities were observed. Neither NEM nor DTT treatments failed to alter the monovalent cation effects on carbachol and atropine binding. 7. These observations indicate a likely involvement of -SH groups in the opposing effects of Mg2+ and guanine nucleotides (GTP) on cardiac muscarinic receptor-agonist interaction. The results further suggest some subtle in vitro differences in the brain and heart muscarinic receptor sites with regard to the influence by divalent cations and guanine nucleotides on the receptor-agonist interaction.

The effect of kainic acid on cholinergic enzymes and receptors in the amygdala complex of the rat

Kainic acid (KA) was injected into the amygdala (AM) complex of the rat and its effect on the cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (AChE), and the binding of the muscarinic ligand, [3H]quinuclidinyl-benzilate (QNB) and the nicotinic ligand [125I]alpha-bungarotoxin (aBuTX) was investigated. Ka produced a loss of approximately 35% of the CAT activity in the AM. However, no effect on AChE activity was observed. A 30--50% decrease in the number of muscarinic and nicotinic receptor sites was also found. CAT, AChE and QNB binding in the AM contralateral to the injection did not change. However, the binding of aBuTX was found to decrease by approximately 40%. The present results suggest that the loss of CAT activity in the AM after treatment with KA is due to the destruction of cholinergic neurons within the AM. The lack of effect on AChE suggests that the major cholinergic input to the AM is not affected by KA. In addition, the loss of nicotinic receptors in the contralateral AM may reflect anterograde degeneration of terminals that have nicotinic sites located on them, or may be secondary to the elicitation of intense seizure activity evoked by the KA.