Muscarinic cholinergic receptor binding in rat mast cells

Specific [³H]-QNB binding was present in isolated, purified, intact rat mast cells and in crude membrane preparations. The binding is saturable, time- and temperature-dependent. Cholinergic agents inhibit selectively the binding: atropine is the most effective of the antagonists while oxotremorine is the most potent of the muscarinic agonists. It is concluded that rat mast cells are provided with muscarinic cholinergic receptors.     

Muscarinic cholinergic receptor binding in bovine retina

Using [³H] quinuclidinyl benzylate ([³H] QNB) muscarinic cholinergic receptors have been demonstrated in crude membrane fractions of bovine retina. Specific [³H] QNB binding is saturable with a K_D of 0.5 nM and a maximal number of muscarinic agonists and antagonists for displacing specific [³H] QNB binding closely parallel the affinities for muscarinic receptors in rat brain and guinea pig ileum. The findings may explain atropine sensitive effects of muscarinic agonists on the electroretinogram and on retinal cells in vitro.     

Mast cell receptors controlling histamine release: Influences on the mode of action of drugs used in the treatment of adverse drug reactions

Summary In drug-induced allergic diseases of the immediate type (anaphylactic and anaphylactoid reactions), the primary target cells are tissue mast cells, which discharge their granular content upon interaction with different secretagogues (immunological releasers; histamine liberators) on specific plasma membrane receptors.Experiments are reviewed here which report that IgE-mediated histamine release from mast cells, and the secretion of histamine induced by non-immunological secretagogues (dextran; compound 48/80; acetylcholine) are blocked by beta-adrenoceptor and H₂-receptor agonists, their inhibiting effect being surmountable by beta-adrenoceptor blocking drugs and by anti-H₂-antihistamines. Specific radioligands ([³H]-dihydroalprenolol; [³H]-cimetidine) binding to rat mast cell membranes points to the possibility that inhibition of histamine release is brought about by the activation of mast cell beta-adrenoceptors and H₂-receptors.Drugs used in therapy of anaphylactic or anaphylactoid reactions may act either on tissue receptors, competing with released mediators, or by inhibiting the release of allergic mediators from mast cells, on activation of specific receptors located in mast cell plasma membranes.     

Evidence for H2-receptor-mediated inhibition of histamine release from isolated rat mast cells

Impromidine, an H₂-receptor agonist, inhibited the release of histamine from isolated purified rat mast cells evoked by compound 48/80 and acetylcholine. Pyridilethyl-amine (PEA), an H₁-receptor agonist, on the other hand, was only slightly effective at very high concentrations. The inhibitory effect of impromidine was blocked by preincubating the cells with cimetidine, but not by chlorpheniramine.The existence of an H₂-mediated inhibitory feed-back regulation of histamine release was also suggested by the demonstration of specific binding sites for [³H]-cimetidine in rat mast cell membranes.     

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.     

Selective, unilateral cortical infarction increases striatal muscarinic receptor binding: potential evidence for cortical modulation of intrastriatal cholinergic transmission

The effects of unilateral cortical infarction on subcortical (striatal) muscarinic receptors in rat brain were studied by means of in vitro receptor autoradiography using [3H]quinuclidinyl benzylate ([3H]QNB) and [3H]pirenzepine. The cortical lesions could be produced without compromising subcortical structures. A dramatic (20 to 59 per cent) increase in striatal [3H]QNB binding was observed ipsilateral to the damaged cortex. The increase in binding was greatest in the caudate-putamen, but was also noted in the nucleus accumbens. [3H]Pirenzepine binding (labeling M1 receptors) was also increased but to a lesser degree, as was [3H]QNB binding in the presence of 100 nM (unlabeled) pirenzepine (an indirect means of labeling M2 receptors). The results show that unilateral cortical infarction results in an upregulation of striatal muscarinic receptors, and suggest that both the M1 and M2 subtypes contribute to this effect. These findings provide evidence for cortical modulation of intrastriatal cholinergic transmission.     

Influence of lipid peroxidation and hydrogen peroxide on muscarinic cholinergic receptors and ATP level in rat myocytes and lymphocytes

Isolated rat neonatal cardiac myocytes and rat lymph-node lymphocytes were treated with Fe2+ cumene hydroperoxide or hydrogen peroxide. The intensity of lipid peroxidation was estimated by measuring production of malondialdehyde (MDA). The level of muscarinic cholinergic receptors was determined by [3H]-QNB binding. Cumene hydroperoxide treatment was found to induce lipid peroxidation in both myocytes and lymphocytes, the process being more pronounced in myocytes. An important decrease of muscarinic cholinergic receptor level and a significant drop of intracellular ATP level in these cells were simultaneously observed. In both cell types, hydrogen peroxide treatment decreased the ATP level while lipid peroxidation and muscarinic cholinergic receptors were unaffected. The different effect of hydrogen peroxide on lipid peroxidation and the level of muscarinic cholinergic receptors on the one hand and the ATP level on the other suggests that this substance acts mainly in the intracellular space.     

Effects of aging and cholinergic deafferentation on putative muscarinic cholinergic receptor subtypes in rat cerebral cortex

Despite a 34% decrease in the activity of choline acetyltransferase (ChAT) in the rat cerebral cortex following lesions of the nucleus basalis, there were no changes in the Bmax of the antagonist ligands [3H]quinuclidinyl benzilate ((-)-[3H]QNB) or (-)-[3H]N-methylscopolamine ((-)-[3H]NMS). Furthermore, this treatment produced no significant change in the proportions or affinities of muscarinic receptors having high and low affinity for pirenzepine or (-)-NMS. These data indicate that putative M2 muscarinic receptors are not restricted to ChAT-containing neurons in rat cerebral cortex. In senescent compared to mature rats there was no significant loss of ChAT activity although a significant reduction in the Bmax of both (-)-[3H]QNB and (-)-[3H]NMS binding was observed. However, no changes in the competition of pirenzepine or (-)-NMS for the remaining (-)-[3H]QNB binding sites were observed. Therefore, there is no evidence for any differential regulation of either putative muscarinic receptor subtype in response to cholinergic deafferentation or as a function of the natural aging process.     

Muscarinic cholinergic receptors in the rat deep cerebellar nuclei: A quantitative autoradiographic study

The distribution of muscarinic cholinergic receptors within the rat deep cerebellar nuclei was analyzed using in vitro receptor binding of [³H]quinuclidinylbenzilate (QNB) in conjunction with autoradiography. The highest density of QNB binding sites occurred in the lateral cerebellar (dentate) nucleus. Interpositus nuclei displayed an intermediate density of muscarinic cholinergic binding sites with the posterior interpositus nucleus demonstrating higher binding than the anterior nucleus. The fastigial (medial) cerebellar nucleus exhibited the lowest levels of QNB binding among the four cerebellar nuclei. These results indicate that muscarinic cholinergic receptors are present in the deep cerebellar nuclei and that differences in receptor density occur among the four nuclear groups.     

Immunological modulation of cholinergic histamine release in isolated rat mast cells

Isolated purified rat mast cells release histamine when exposed to acetylcholine according to different patterns of sensitivity.The degree of histamine release is correlated with the levels of reaginic antibodies presumably bound to the mast cell membrane. In fact, mast cells passively sensitized with mouse myeloma IgE against egg albumin or DNP₂-lysine, react to acetylcholine with a release of histamine, which is proportional to the IgE concentration in the incubation medium.The histamine release induced by acetylcholine is due to the stimulation of a musearinic receptor. Accordingly, acetylthiocholine is unable to evoke histamine release and preincubation of sensitized cells with atropine fully inhibits the cholinergic histamine release. The histamine release evoked by acetylcholine is potentiated by the exposure of sensitized cells to the specific antigen.The present results suggest that sensitization of mast cells is a crucial factor in modulating their sensitivity to acetylcholine.     

Decreased density of beta-adrenergic and muscarinic cholinergic receptor sites in the vasa nervorum of aged rats

The pharmacological profile and the anatomical localization of beta-adrenergic and muscarinic cholinergic receptors of the vasa nervorum were studied in sections of sciatic nerve using radioreceptor binding and light microscope autoradiography techniques. Sprague—Dawley rats of 4 and 24 months of age were used. [³H]Dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) were used to label beta-adrenergic and muscarinic cholinergic receptors, respectively. The ligands were bound to sections of rat sciatic nerve in a manner consistent with the labelling of beta-adrenergic or muscarinic cholinergic receptors in the 2 age groups investigated. The dissociation constant (K_d) values (about 1.37 nM for [³H]DHA and 0.75 nM for [³H]QNB) did not significantly change between 4- and 24-month-old rats. The maximum concentration of binding sites (B_max) for [³H]DHA was decreased by about 35% in 24 in comparison with 4-month-old rats. The B_max value autoradiogaphy revealed the development of specific silver grains in the medial layer of epineurial and perineurial arteries in sections of sciatic nerve exposed either to [³H]DHA or [³H]QNB. The number of silver grains developed in epineurial and perineurial arteries of rats of 24 months is significantly lower than in animals of 4 months. The above results suggest the occurrence of an age-dependent loss in the density of beta-adrenergic and muscarinic cholinergic receptors of vasa nervorum.     

Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.     

Modulation of the spontaneous histamine release by adrenergic and cholinergic drugs

Experiments have been reported on the possible modulation of the spontaneous histamine release by adrenergic and cholinergic drugs.Adrenergic drugs increase the spontaneous histamine release in vivo, and in neoplastic mast cells, in vitro. The mechanism of histamine release appears to be dependent upon the activation of alpha-adrenoceptors.Cholinergic drugs activate the release of histamine in many secretory processes in vivo; in vitro, acetylcholine is one of the most powerful histamine releasers in isolated purified rat mast cells.The release of histamine evoked by acetylcholine in rat mast cells is a calcium-requiring, temperature-dependent exocytosis. The physiological relationship of the sympathetic, parasympathetic and histamine-containing cells are discussed.Lecture held at the Sixth Annual Meeting of the European Branch of the Histamine Club, London, April 20–22, 1977.     

Expression of muscarinic cholinergic receptors on lymphocytes during rheumatoid arthritis

The level of muscarinic receptors on lymphocytes from rheumatoid arthritis (RA) patients in comparison with healthy individuals was determined by the binding studies of a specific muscarinic ligand [3H]-QNB in the presence of the competitive antagonist atropine. The response to phytohaemagglutinin and the influence of acetylcholine on this process in patients' lymphocytes was also studied. We have found direct correlation among PHA stimulation indices, the level of muscarinic receptors, and the influence of acetylcholine on PHA stimulation indices. Differences in these parameters occurring in systemic forms of RA as compared to other RA patients are also described and their possible mechanism discussed. The influence of cholinergic stimulation on the reactivity of lymphocytes after PHA in different forms of RA could depend on the number of muscarinic receptors present on the lymphocyte surface, as well as on the activity of soluble factors, which could modulate the functional state of the receptors.     

Muscarinic receptor binding is inhibited by quinacrine.

Quinacrine altered the equilibrium binding of the muscarinic antagonist quinuclidinyl benzilate (QNB) to the muscarinic receptor in membranes from N18TG2 neuroblastoma cells. At 1 microM quinacrine, the apparent Kd for [3H]QNB binding was shifted from 48 to 210 pM without a decrease in the maximum binding. The competition binding profile of quinacrine indicates that it displaced [3H]QNB binding with an IC50 of 460 nM in a manner consistent with competitive inhibition. Quinacrine blocked the muscarinic inhibition of cyclic AMP accumulation by carbachol in cultured N18TG2 neuroblastoma cells without affecting arachidonic acid release or Gi coupling to the adenylate cyclase. The disruption of muscarinic receptor function by quinacrine may provide an explanation for its blockade of the muscarinic response in cells.     

Expression of muscarinic cholinergic receptors during T cell maturation in the thymus

Thymocytes at various stages of their ontogeny have been studied in relation to their ability to bind [3H]quinuclidinyl benzilate [( 3H]QNB), a specific radioligand of the muscarinic cholinergic receptors. [3H]QNB-specific binding to thymocytes from 15-19-day fetal, newborn and adult thymuses of mice and rats was compared and correlated. Our experiments showed that the kinetics of [3H]QNB binding to thymocytes at 37 degrees C was similar to that of the lymph node lymphocytes (LNL) with maximum after 5 min of incubation and subsequent decrease to 10% of the maximum after 90 min of incubation. Maximal binding for the entire thymocyte population was twice lower than for the cortisone-resistant thymocytes (CRT) or for LNL. Binding of [3H]QNB carried out at 4 degrees C resulted in disappearance of the maximum, but did not alter the difference between CRT and entire thymocyte population. Depletion of CRT detectable [3H]QNB-specific binding to thymocytes until 18th day of gestation but the maximal binding increased up to 20% at the day 19 and reached 90% of adult level on the third day after birth. Moreover, carbamylcholine (a muscarinic agonist) treatment in vivo induced a significant decrease in [3H]QNB binding to the thymocytes. We thus suggest that a subpopulation of thymocytes bearing muscarinic receptors in the periphery acquired these receptors in the thymus as one of the last steps of their maturation. We cannot exclude the possibility that cholinergic stimulation might trigger these lymphocytes to leave the thymus.     

Effect of cold stress on cholinergic receptors in the rat adrenal gland

The effects of cold exposure on cholinergic binding sites in the rat adrenal gland were assessed by examining the binding of [125I]alpha-bungarotoxin (BTX), a nicotonic receptor antagonist and [3H]quinuclidinyl benzilate (QNB), a muscarinic receptor antagonist, to adrenal tissue homogenates. Cold exposure resulted in significant alterations in both nicotinic and muscarinic binding. Exposure to cold for 4 and 7 days resulted in a significant decrease in QNB binding. Scatchard analysis indicates that this alteration is due to a decrease in binding sites (Bmax) rather than a change in ligand affinity (Kd). In contrast, chronic cold exposure produced a significant increase in BTX binding sites. These results indicate that adrenal cholinergic receptors are altered in reciprocal fashion by chronic cold exposure, and that this change may represent a key event in the sympathoadrenal system's adaptive response to chronic cold stress.     

Heterogeneity of presynaptic muscarinic receptors involved in modulation of transmitter release

In order to extend the characterization of muscarinic receptors at presynaptic sites their inhibitory effect on the stimulation-evoked release of [3H]noradrenaline and [3H]acetylcholine from different axon terminals was studied and the dissociation constants and potencies of different antagonists were estimated, in guinea-pig and rat. While oxotremorine reduced the release of [3H]acetylcholine and [3H]-noradrenaline in a concentration-dependent manner from different release sites (Auerbach plexus, noradrenergic neurons in the right atrium, cerebral cortex), McN-A 343, an M1 receptor agonist, enhanced their release evoked by field stimulation. When the inhibitory effect of oxotremorine on transmitter release was studied, pancuronium, pirenzepine and atropine were competitive antagonists of presynaptic muscarinic receptors located on the noradrenergic axon terminals of the atrium. While atropine and pirenzepine inhibited the muscarinic receptors of cholinergic axon terminals in the Auerbach plexus, pancuronium and gallamine had a very low affinity. Significant differences were found in the affinity constants of antagonists for muscarinic receptors located in the cholinergic axon terminals of Auerbach plexus and cerebral cortex, and noradrenergic axon terminals of the atrium. While atropine and pirenzepine exerted similar effects on these presynaptic sites, pancuronium, gallamine and (11-(2-[diethylamino)-methyl)-1-piperidinyl)acetyl)-5, 11-dihydro-6(1-pyrido(2,3-b)(1,4)-benzodiazepin-6-on) were much more effective on muscarinic receptors controlling acetylcholine release from the cerebral cortex and noradrenaline release from the heart. There was more than 100-fold (2.0 pA2 units) difference in affinities of these antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonists of luteinizing hormone releasing hormone bind to rat mast cells and induce histamine release

It was reported previously that administration of certain synthetic antagonists of LHRH to rats produced allergy-like symptoms that were attributed to their histamine releasing action. In the present study the interaction of LHRH analogs with rat peritoneal mast cells was investigatedin vitro. Potent antagonists of LHRH showed strongin vitro histamine releasing activity from rat peritoneal mast cells. Membrane preparations of rat pituitary glands showed specific binding of radioiodinated LHRH antagonist as well as LHRH agonist. However, rat peritoneal mast cells and membrane preparations from those cells bound antagonist but not the agonist. Furthermore, the LHRH antagonist did not bind to membranes prepared from tissues such as prostate, liver, kidney, and brain. Competitive displacement curves of the [¹²⁵I]-antagonist with different LHRH analogs showed that the ability of the analogs to compete for binding sites on mast cells was related to their histamine releasing activity. We conclude that histamine release from rat mast cells induced by LHRH analogs is mediated by specific binding of the active peptides to cell membranes. Furthermore, using rat mast cells, the binding assay in conjunction with histamine releasing assay may be utilized to predict thein vivo histamine releasing potential of new LHRH peptides which are of clinical importance.     

Autoradiographic distribution of [3H]acetylcholine binding sites in the cervical spinal cord of man and some other species

The distribution of [3H]acetylcholine ([3H]ACh) and [3H]ACh co-incubated with 1-mM nicotine (muscarinic receptor), and [3H]ACh co-incubated with 1.5 microM atropine (nicotinic receptor) binding sites were studied in man and compared to monkey, cat and rat using quantitative in vitro autoradiography. The highest density of total [3H]ACh binding sites was found in laminae II-III, IX (motor neuron areas) and X close to the central canal. The distribution pattern of the muscarinic cholinergic binding sites was similar to that of the total cholinergic binding. In general the number of nicotinic binding sites in the spinal cord was relatively small. The largest number of such binding sites was found in laminae II-III of the dorsal horn and in laminae X around the central canal. It is evident that the spinal cord has a 2-3 times higher number of muscarinic than of nicotinic cholinergic receptors.