Regulation of putative muscarinic cholinergic receptor subtypes in rat brain

Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [³H](−)quinuclidinyl benzilate {[³H](−)QNB}. Pirenzepine competition for [³H](−)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M₁ receptors) and low affinity (putative M₂ receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus.Chronic scopolamine treatment produced a 48% increase in the B_max of [³H](−)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [³H](−)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5′-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics.Carbachol competition for [³H]pirenzepine binding to putative M₁ receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M₂ [³H](−)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M₁ and M₂ muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to supress oxotremorine-induced tremors in treated and in control animals. ED50's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified.     

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [³H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [³H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.     

Effect of organophosphates on dopamine and muscarinic receptor binding in rat brain

The effect of Soman, Sarin and Vx, known potent cholinesterase inhibitors, on the binding of several neurotransmitter receptors in various regions of brain was studied. Vx, exhibited considerable inhibition of binding of 3H-N-methylscopolamine (3H-NMS) to muscarinic receptors and of 3H-spiperone to dopamine D2 receptors in the striatum. 3H-NMS binding was 50% inhibited at 10(-6)M and 90% at 10(-3)M Vx. Inhibition of 3H-spiperone binding by Vx in striatum had an ID50 of 10(-5)M. KD of the treatment was affected more than Bmax. Binding inhibition of both 3H-NMS and 3H-spiperone in post-mortem brain of rats pre-treated with Vx confirmed the specificity of the organophosphates effect, since other organophosphates and ligands failed to show any activity.     

Allosteric effect of gallamine on muscarinic cholinergic receptor binding

Gallamine interacts with an allosteric site on muscarinic acetylcholine receptor complexes in rat brain membranes, thereby slowing the dissociation of a radiolabelled ligand ([³H]N-methylscopolamine) from the receptor complex. This effect involves the elimination of the fast component of the biphasic dissociation curve. The allosteric effect of gallamine is equally prominent in membranes containing predominantly Ml (cerebral cortex) and M2 (brainstem) subtypes of muscarinic receptor. Gallamine's action is not affected by a variety of treatments which influence the conformational state of the receptor as reflected by agonist binding affinity, including treatments with heat,N-ethymaleimide and trypsin. A guanine nucleotide (5-guanylylimidodiphosphate), however, moderates the effects of gallamine on muscarinic receptors in brainstem, but not in cortical, membranes.     

Influence of phospholipase C on muscarinic acetylcholine receptor binding in rat brain

Treatment of neural membranes from rat cerebral cortex with phospholipase C (phosphatidylcholine cholinephosphohydrolase) inhibited the binding of radiolabelled antagonists to muscarinic acetylcholine receptors. This inhibition was incomplete, was not competitive, and did not appear to be related to the production of inhibitory products. The affinity of carbamylcholine for cortex muscarinic receptors was increased by phospholipase C action. The distribution of receptors between states of high and low affinity was not affected by phospholipase C; rather, the affinity for carbamylcholine of the lowest affinity receptors was selectively increased. This suggests that membrane lipids influence the interaction of the receptor binding subunit with other structures in the synaptic membrane.     

Increased number of muscarinic binding sites in brain following chronic barbiturate treatment to rat

Rats received as their only drinking fluid a solution of sodium barbital (3.33 mg/ml) for more than 40 weeks. In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice. Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C). Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate (³H-QNB) (0.2 nM) for 60 min. A significantly increased number of ³H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C). Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity.     

Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester

Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43±2% in the right and 26±2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleoylacetylglycerol decreased the amount of cell-surface mAChR by 37±2% and 25±2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25±1.5% and 33±2% in right and left cortical cells, respectively). These changes occurred without modification of the Kd_app for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.Abbreviations used A23187 ionophore A23187 - ATRO atropine - CARB carbamoylcholine - DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer - PZ pirenzepine - LCC left cerebral cortex - mAChR muscarinic acetylcholine receptor - OAG oleoylacetylglycerol - PDB phorbol dibutyrate - RCC right cerebral cortex     

Hormone effects on muscarinic cholinergic binding in bovine and rat sympathetic superior cervical ganglia

Muscarinic receptors were assessed by [3H]-quinuclidinyl benzilate (QNB) binding in 900 xg supernatants of bovine superior cervical ganglia (SCG). At 30 degrees C half maximal binding was reached within 3 min and equilibrium within 30 min. Scatchard analysis revealed a single population of binding sites with dissociation constant (Kd) = 0.15 +/- 0.01 nM and site concentration (Bmax) = 101 +/- 4 fmoles/mg prot. Binding was specific for muscarinic drugs. Incubation of bovine SCG with different hormones (10(-7)M) indicated that LH, TRH and testosterone depressed significantly Bmax, and that prolactin decreased both Kd and Bmax of [3H] -QNB binding. Several other hormones tested (TSH, GH, FSH, LHRH, angiotensin II, bradykinin, melatonin, estradiol, thyroxine and triiodothyronine) did not affect QNB binding. Hormone effects were not due to a direct interference with radioligand binding to membrane. The injection of LH to orchidectomized rats depressed Bmax of SCG QNB binding without changing the Kd. These results suggest that muscarinic cholinergic neurotransmission in SCG may be affected by hormones.     

Parasympathetic denervation decreases muscarinic receptor binding in rat parotid.

After unilateral postganglionic parasympathetic denervation of rat parotid glands for 3, 6 or 16 days, the binding of [³H] quinuclidinylbenzilate, a potent and specific muscarinic antagonist, was decreased by 28–45%. The largest part of the decrease was already present at 3 days, and thus appeared to coincide with the loss of parasympathetic terminals (as shown by a 94% reduction in choline acetyltransferase activity). The decrease in binding was expressed as a reduced number of membrane sites, with no significant change in affinity, and could be accounted for only in part by a reduction in the size of denervated glands. The rapid loss of binding suggests the existence of muscarinic receptors on parasympathetic terminals. The clear absence of any receptor increases at longer periods of denervation, when parotid glands are reported to give supersensitive secretory responses, suggests that mechanisms other than receptor increases are important in denervation supersensitivity in exocrine glands.     

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Autoradiographic quantification of muscarinic cholinergic synaptic markers in bat,shrew, and rat brain

We employed radioligand binding autoradiography to determine the distributions of pre- and postsynaptic cholinergic radioligand binding sites in the brains of two species of bat, one species of shrew, and the rat. High affinity choline uptake sites were measured with [³H]hemicholinium, and presynaptic cholinergic vesicles were identified with [³H]vesamicol. Muscarinic cholinergic receptors were determined with [³H]scopolamine. The distribution patterns of the three cholinergic markers were similar in all species examined, and identified known major cholinergic pathways on the basis of enrichments in both pre- and postsynaptic markers. In addition, there was excellent agreement, both within and across species, in the regional distributions of the two presynaptic cholinergic markers. Our results indicate that pharmacological identifiers of cholinergic pathways and synapses, including the cholinergic vesicle transport site, and the organizations of central nervous system cholinergic pathways are phylogenetically conserved among eutherian mammals.Special issue dedicated to Dr. Bernard W. Agranoff.     

Alterations in the rat forebrain apoptosis following exposure to ionizing radiation

Ionizing radiation commonly used in the radiotherapy of brain tumours can cause adverse side effects to surrounding normal brain tissue. The most significant response of adult brain to radiation damage is induction of apoptosis. The adult mammalian subventricular zone (SVZ) of the brain lateral ventricles (LV) and their subsequent lateral ventricular extension, the rostral migratory stream (RMS), is one of the few areas, which retains the ability to generate new neurons and glial cells throughout life. Taking into account the fact, that ionizing radiation is one of the strongest exogenous factors affecting cell proliferation, the aim of the present study was to investigate the occurrence of radiation-induced apoptosis in this neurogenic region. Adult male Wistar rats were investigated 1, 5 or 10 days after single whole-body gamma irradiation with the dose of 3 Gy. Apoptotic cell death was determined by in situ labelling of DNA nick ends (TUNEL) and fluorescence microscopy evaluation of TUNEL-positive cells. Considerable increase of apoptotic TUNEL-positive cells was observed 24 hrs after irradiation in caudal parts of RMS; i.e. in the vertical arm and elbow of RMS. Initial increase was followed by strong reduction of apoptosis in the RMS and by secondary over-accumulation of apoptotic cells in the animals that survived ten days after exposure. Results showed, that the proliferating population of cells, arisen in SVZ are highly sensitive to radiation-induced apoptosis. This observation should have implications for clinical radiotherapy to avoid complications in therapeutic brain irradiation.     

Muscarinic cholinergic receptor binding in rat hindlimb somatosensory cortex following partial deafferentation by sciatic nerve transection.

Peripheral nerve injury or amputation leads to extensive changes within the central representations of the mammalian body surface. The mechanisms responsible for post-traumatic reorganization of these maps in adults may also, at least partly, underlie a more general feature of the somatosensory system--the capacity for stimulus-dependent plasticity. Acetylcholine has been implicated in both of these processes. We studied the binding of the ligands [3H]QNB and [3H]pirenzepine in rat hindlimb somatosensory cortex from 1 to 14 days following sciatic nerve transection. Although the [3H]QNB binding was not different from normal levels in tissue homogenates of the affected somatosensory cortex, differences were demonstrated when binding was measured on a layer-by-layer basis. [3H]QNB binding was changed only in certain layers, at certain times. The predominant effects appeared to be a decrease in binding in the middle layers from 4 to 14 days after the transection. Combining the [3H]QNB data with data obtained from the more M1-selective ligand [3H]pirenzepine suggested that complex changes occur among several muscarinic receptors, including receptors with non-M1 subtype characteristics. Moreover, unilateral nerve transection affects the hindlimb somatosensory regions in both hemispheres.     

Interaction of amitriptyline with muscarinic receptor subtypes in the rat brain

The affinity of amitriptyline for muscarinic receptors in rat brain areas was studied using autoradiographic techniques including image analysis. As shown by competitive inhibition of [³H]-l-quinuclidinyl benzilate binding, amitriptyline was found to be a potent inhibitor of muscarinic receptors throughout the rat brain. Muscarinic receptors in the external layers of the cortex displayed a high affinity for amitriptyline (IC₅₀ = 65.8 ± 2.1 nM), while the hippocampal regions had somewhat lower affinities (e.g. IC₅₀ = 96.3 ± 3.4 nM). Amitriptyline bound with lower affinity in the thalamus and various midbrain regions, such as the paraventricular nucleus of the thalamus and the superior colliculus, which had IC₅₀ values of 112 ± 6.8 and 117 ± 32.6 nM, respectively. Other midbrain regions displayed higher affinities, for example, the substantia nigra had an IC₅₀ value of 62.8 ± 0.9 nM. The data show that amitriptyline binds with high affinity to muscarinic receptors with a modest subtype selectivity that is unlike that of either pirenzepine or AF-DX 116. In addition, amitriptyline at concentrations of 10 nM-1 μM antagonized the oxotremorine-induced inhibition of acetylcholine release in cortical nerve endings, demonstrating activity at M₂ autoreceptors.     

Development of muscarinic cholinergic receptor binding in the visual system of monocularly deprived and dark reared rats

In 25 day old rats monocularly deprived by unilateral eyelid suture on postnatal day 10 (MD), [³H]quinuclidinyl benzylate (³H-QNB) binding was significantly reduced in the visual cortex (VC) of both sides, but elevated in both superior colliculi (SC). Muscarinic receptor binding in the frontal cortex (FC), a non-visual brain area, in the lateral geniculate nucleus (LGN), and in the retina was not affected. In 25 day old rats raised in complete darkness from birth (DR) similar changes in³H-QNB binding were found in VC and SC. However, binding levels were also decreased in the FC and significantly increased in the retina. In adult (6 month old) MD and DR rats the differences in³H-QNB binding as compared to age-matched controls had disappeared completely in all visual brain areas studied. Detailed Scatchard analyses indicate that the alterations in the³H-QNB binding were due to changes in receptor number only.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

3H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart

The in vitro binding properties of the novel muscarinic antagonist [3H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [3H]AF-DX 116 binding were rapid at 25 degrees C (2.74 and 2.70 X 10(7) min-1 M-1 for K+1; 0.87 and 0.93 min-1 for k-1) but 20-40 times slower at 0-4 degrees C (0.13 and 0.096 X 10(7) min-1 M-1 for k+1; 0.031 and 0.022 min-1 for k-1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (Kds) were estimated to be 31.8 nM and 30.9 nM at 25 degrees C; 23.1 nM and 0-4 degrees C for the cerebral cortex and heart, respectively. In saturation studies, [3H]AF-DX 116 labeled 29 percent of the total [3H](-)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with Kd values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [3H]AF-DX 116 binding in a rank order of potency of atropine greater than dexetimide greater than AF-DX 116 greater than PZ greater than levetimide in both tissues. Except for PZ/[3H]AF-DX 116 and AF-DX 116/[3H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [3H]AF-DX 116 binding in both tissues. Regional distribution studies with [3H](-)QNB, [3H]PZ and [3H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striatum are of the M1 subtype while those in the brainstem, cerebellum and other lower brain regions are of the M2 subtype. These results indicate that [3H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors.     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.