Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes

The equilibrium dissociation constant and the kinetic rate constants were determined for the binding of (R)-[3H]3-quinuclidinyl benzilate ([3H]QNB) and [125I]3-quinuclidinyl-4-iodobenzilate ((R,R)- and (R,S)-[125I]IQNB) to transfected cell membranes expressing one single muscarinic acetylcholine receptor (mAChR) subtype. The association and dissociation kinetics for the m2 subtype were more rapid than for the m1 and m3 subtypes. The differential kinetic properties may be useful for the single photon emission computed tomographic (SPECT) evaluation of regional mAChR subtype alterations in disease states.     

A Point Mutation in the Coding Sequence of the β-Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2-Gangliosidosis Variant

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.     

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.     

Molecular mechanism of iodide transport by thyroid plasmalemmal vesicles: cooperative sodium activation and asymmetrical affinities for the ions on the outside and inside of the vesicles

The 125I- uptake by plasmalemmal vesicles from porcine thyroid was measured by a Millipore filtration method using 2 mM ClO4- as a reaction stopper. Effective uptake occurred in the presence of high concentrations of extravesicular Na+ (Na+o). In the presence of Na-ionophores such as monensin and nigericin, no uptake was observed and the accumulated I- was released. The initial rate of I- uptake increased with the concentration of extravesicular I- (I-o) according to simple saturation kinetics and [I-o] giving a half-maximum rate of about 5 microM. The dependence of the rate on [Na+o] showed cooperativity with a Hill coefficient of 1.8, and a KNa value of 0.0064 M2, suggesting that the binding of at least 2 Na+ ions to a carrier molecule was required to transport an I- ion. Further kinetic data were consistent with a mechanism in which bindings of the ions were rapid and the Na+ binding occurred prior to the I- binding. Intravesicular Na+ inhibited the I- uptake and the inhibition constant (KiNa) was about 4 mM, independently of [I-o] and [Na+o]. Intravesicular I- inhibited the I- uptake with an apparent KiI value of about 100 microM. The results suggest that the differences in the Na+- and I- -binding modes between outside and inside of the vesicles are important factors causing the I- uptake against its concentration gradient.     

Pharmacokinetic simulations of SPECT quantitation of the M2 muscarinic neuroreceptor subtype in disease states using radioiodinated (R,R)-4IQNB.

Alzheimer's disease (AD) involves selective loss of muscarinic M2, but not M1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of M2 receptors in AD is limited by the fact that there is currently no available M2-selective radioligand which can penetrate the blood-brain barrier. However, by taking advantage of the different pharmacokinetic properties of (R,R)-[123I]IQNB for the M1 and M2 subtypes, it may be possible to estimate losses in M2. It has previously been hypothesized that the difference between an early study and a late study should provide information on the M2 receptor population. In order to test this hypothesis, we present here the results of pharmacokinetic simulations of the in vivo localization of (R,R)-[123I]IQNB in brain regions containing various proportions of M1 and M2 subtypes. These results permit us to conclude that SPECT imaging of (R,R)-[123I]IQNB localization can potentially be used to quantitate changes in the M2 subtype in a disease state within a brain region for which the ratio M2/M1 is sufficiently high in normal individuals.     

Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.     

Phospholipase D activity and phosphatidylethanol formation in stimulated HeLa cells expressing the human m1 muscarinic acetylcholine receptor gene

The human m1 and m2 muscarinic acetylcholine receptor (AChR) genes were subcloned, permanently expressed in HeLa cells and analyzed for their pharmacological and biochemical profiles. Both subtypes displayed saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) which was displaced by muscarinic agonists and antagonists. Stimulation of intact HeLa cells expressing the human m1 AChR gene by the muscarinic agonist oxotremorine-M, in the presence of ethanol, resulted in the activation of phospholipase D (PLD) and the formation of phosphatidylethanol (PEt). In contrast, oxotremorine-M did not activate PLD in the HeLa cells expressing the human m2 AChR subtype. These data suggest that the human m1 AChR is linked to the signal transduction mechanism of PLD activation, whereas the human m2 AChR interacts with a different guanine nucleotide regulatory binding protein (G-protein) which does not cause the activation of PLD or the formation of PEt.     

A snake venom inhibitor to muscarinic acetylcholine receptor (mAChR): isolation and interaction with cloned human mAChR

An inhibitor to the muscarinic acetylcholine receptor (mAChR) was purified from the venom of Crotalus atrox (western diamondback rattlesnake). The inhibitor was found to be a 30-kDa homodimer protein with phospholipase A2 activity. In order to determine the subtype selectivity of the purified inhibitor, the inhibitory effect on the binding of two orthosteric antagonists, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine methyl chloride ([3H]NMS), to five subtypes of cloned human mAChR was tested. The purified inhibitor reduced the binding of [3H]QNB and/or [3H]NMS to all subtypes of the mAChR while showing the highest inhibitory effect on the M5 subtype. The Kd values of the receptors for the antagonists were increased in the presence of the inhibitor; however, the Bmax values were not changed. The effects of the purified inhibitor on the dissociation of [3H]NMS from the receptors were also investigated. Dissociation of the antagonist was remarkably slowed down by addition of the inhibitor. These findings may suggest an allosteric action of the purified inhibitor. In addition, the present study indicates that the presence of mAChR inhibitors is quite common in snake venoms.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.     

A Novel Subtype of Prostacyclin Receptor in the Central Nervous System

Recently, in the course of our search for the prostacyclin receptor in the brain, we found a novel subtype, designated as IP2, which was finely discriminated by use of the specific ligand (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15R-TIC) and specifically localized in the rostral part of the brain. In the present study, the tritiated compound 15R-[15-(3)H]TIC was synthesized and utilized for more specific research on IP2. The specificity of binding to rat brain regions was confirmed by use of several prostacyclin derivatives including 15S-TIC. Mapping of 15R- and 15S-[3H]TIC binding in adjacent pairs of frozen sections of rat brain demonstrated a quite similar pattern of distribution in almost all rostral brain regions, indicating that the regions may contain only the IP2 subtype. On the other hand, 15R-[3H]TIC binding was very faint as compared with 15S-[3H]TIC binding in the caudal medullary region. High densities of 15R-[3H]TIC binding sites were shown in the dorsal part of the lateral septal nucleus, thalamic nuclei, limbic structures, and some of the cortical regions. Scatchard plot analysis showed two components of high-affinity 15R-[3H]TIC binding in the rostral regions, one with a K(D) value at approximately 1 nM and the other with approximately 30 nM. These results strengthen our previous finding that a different subtype of prostacyclin receptor is expressed in the CNS, and the map with 15R-[3H]TIC obtained here could guide further studies on the molecular and functional properties of the IP2.     

125I] 3-quinuclidinyl 4-iodobenzilate: a high affinity, high specific activity radioligand for the M1 and M2-acetylcholine receptors

We have prepared a radioiodinated ligand which binds with high affinity to the muscarinic acetylcholine receptor (m-AChR). A derivative of 3-quinuclidinyl benzilate, [125I] labeled (R) 1-aza-bicyclo(2.2.2)oct-3-yl (R,S)-alpha-hydroxy-alpha-(4-[125I]iodophenyl)phenyl acetate (4- IQNB ) exhibits an affinity for the m-AChR from corpus striatum higher than that of (R) [3H] QNB. Additionally, [125I] 4- IQNB exhibits receptor selectivity for the M1 receptor since the affinity for the receptor from dog and rat heart is lower than that using dog or rat corpus striatum.     

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

Asymmetric synthesis and preliminary evaluation of (R)- and (S)-[11C]bisoprolol, a putative beta1-selective adrenoceptor radioligand

(+/-)-1-[4-(2-Isopropoxyethoxymethyl)-phenoxy]-3-isopropylamino-2-propanol (bisoprolol) is a potent, clinically used beta(1)-adrenergic agent. (R)-(+) and (S)-(-) enantiomers of bisoprolol were labelled with carbon-11 (t(1/2)=20.4 min) as putative tracers for the non-invasive assessment of the beta(1)-adrenoceptor subtype in the human heart and brain with positron emission tomography (PET). The radiosynthesis consisted of reductive alkylation of des-iso-propyl precursor with [2-11C]acetone in the presence of sodium cyanoborohydride and acetic acid. The stereo-conservative synthesis of (R)-(+) and (S)-(-)-1-[4-(2-isopropoxyethoxymethyl)-phenoxy]-3-amino-2-propanol to be used as the precursors for the radiosynthesis of [11C]bisoprolol enantiomers was readily accomplished by the use of the corresponding chiral epoxide in three steps starting from the commercially available hydroxybenzyl alcohol. The final labelled product (either (+) or (-)-1-[4-(-isopropoxyethoxymethyl)-phenoxy]-3- [11C]isopropylamino-2-propanol) was obtained in 99% radiochemical purity in 30 min with 15+/-5% (EOS, non-decay corrected) radiochemical yield and 3.5+/-1 Ci/micromol specific radioactivity. Preliminary biological evaluation of the tracer in rats showed that about 30% of heart uptake of [11C](S)-bisoprolol is due to specific binding. The high non-specific uptake in lung might mask the heart uptake, thus precluding the use of [11C](S)-bisoprolol for heart and lung studies by PET. The remarkably high uptake of the tracer in rat brain areas rich of beta-adrenergic receptors such as pituitary (1.8+/-0.3% I.D. at 30 min) was blocked by pre-treatment with the beta-adrenergic antagonists propranolol (45%) and bisoprolol (51%, p<0.05). [11C](S)-bisoprolol deserves further evaluation in other animal models as a putative beta(1) selective radioligand for in vivo investigation of central adrenoceptors.     

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.