New photoaffinity labels for rat brain muscarinic acetylcholine receptors

Localization of the ligand binding site on muscarinic acetylcholine receptors is one of the new fields of interest opened by the recent determination of their primary structures. Owing to their interesting photochemical properties, aryldiazonium salts may be considered as appropriate tools for "tagging" the agonist/antagonist binding domain and to get precise identification and positioning of covalently labelled residues along the primary sequence of these receptors. A series of aryldiazonium derivatives and some of their azido-analogs were synthesized and their reversible muscarinic binding component was assessed through competition experiments involving either the whole population of receptor sites [( 3H]QNB assay) or the super high affinity of their agonist binding sites [( 3H]OXO-M assay). Three compounds fulfilled the criteria for efficient photolabels, allowing substantial and irreversible occupation of the receptor sites to be obtained. Interestingly, the two diazonium derivatives which were selected have been previously described as potent photoprobes of the peripheral nicotinic receptor and of acetylcholinesterase, though displaying lower binding affinities for these acetylcholine binding proteins than for the muscarinic receptors. These findings, together with the all-to-none photolabelling efficiency observed for a quinuclidine derivative, substituted either by an azido or a diazonium group, are discussed. Finally, the apparent lack of binding selectivity of these new photo-affinity probes towards muscarinic receptor affinity states or subtypes should allow comparative studies of the acetylcholine binding site on different muscarinic receptor proteins, obtained either through purification procedures or expression of separate gene products.     

Competitive interaction of pirenzepine with rat brain muscarinic acetylcholine receptors

In the present work, we studied the details of the interaction of the nonclassical muscarinic receptor antagonist pirenzepine with [3H]quinuclidinyl benzilate binding sites in rat brain homogenates. Pirenzepine showed biphasic competition curves with a Hill coefficient lower than unity, and these curves were better described according to a two-site receptor model. The affinities and the relative preponderance of these sites were constant at different ligand concentrations, in accordance with a competitive type of interaction. Similarly, pirenzepine did not influence the rate of dissociation of the [3H]quinuclidinyl benzilate-receptor complex, even at relatively high concentrations. However, although low concentrations of pirenzepine decreased the affinity of [3H]quinuclidinyl benzilate for the receptor without affecting the density of the binding sites, higher concentrations of the antagonist decreased the receptor number in a reversible fashion. Schild plots of these data indicated an apparent deviation from simple competition in this experimental design, an observation which can be attributed to the selectivity of pirenzepine for different receptor subtypes. Furthermore, pirenzepine, at concentrations high enough to saturate both its high- and low-affinity sites protected [3H]quinuclidinyl benzilate binding sites in the brain against irreversible alkylation by propylbenzilylcholine mustard. Therefore, our data support a competitive nature of interaction of pirenzepine with rat brain muscarinic receptors.     

Age-related changes in rat brain muscarinic receptors and beta-adrenoreceptors

1. In experiments on 2-, 10- and 22-month old rats, it was found that the Bmax values for muscarinic receptors and beta-adrenoreceptors increased in the cerebral cortex, striatum and hippocampus of 10-month old rats as compared to those in 2-month old rats. 2. The Bmax values for both receptor types significantly decreased in the same brain structures of 22-month old rats as compared to those in 10-month old rats. In the striatum and hippocampus of 22-month old rats the binding capacity decreased as compared also to those in 2-month old rats. 3. In the hypothalamus there was also a tendency towards increasing the binding capacity of 10-month old rats and towards decreasing the binding capacity of 22-month old animals only for muscarinic receptors. The beta max of beta-adrenoreceptors remained unchanged in all age groups studied. 4. The receptor affinity of both receptor types was in most cases unaltered with advancing age. The Kd values were slightly increased only in the striatum and hippocampus of 22-month old rats as compared to 10-month old rats. 5. The role of age for the changes in the activity of brain muscarinic and beta-adrenoreceptor systems is discussed.     

Quantitative autoradiographic localization of the M1 and M2 subtypes of muscarinic acetylcholine receptors in the monkey brain

The distribution of muscarinic acetylcholine receptors (mAChR) was investigated in the monkey brain by means of quantitative in vitro autoradiography. 3H-QNB, 3H-pirenzepine (PZ) and 3H-AF-DX 116 were used for labelling total mAChR, M1 and M2 receptors, respectively. 3H-PZ and 3H-AF-DX 116 showed specificity to each receptor subtype in the monkey brain. On sections containing the putamen and globus pallidus, the sum of Bmax values of 3H-PZ and 3H-AF-DX 116 binding sites was almost close to that of 3H-QNB binding sites. Autoradiographic distributions of muscarinic subtype receptors in the monkey brain were similar to those reported in the rat brain; that is, M1 receptors were dominant in most areas of the telencephalon, while M2 receptors were richly distributed in the brainstem and cerebellum. However, some nuclei of the brainstem such as the central gray matter, superior colliculus, substantia nigra, nucleus of the oculomotor nerve, pontine nucleus and inferior olivary nucleus, had relatively high ratios of M1 receptors in the monkey brain. In addition, the cortical lamminar distribution of M2 receptors noticed in the rat was not observed in the monkey brain. Knowledge about the localizations of M1 and M2 receptors in various brain regions in the monkey brain will increase our understanding of the functions of the brain cholinergic system in the primate.     

Tetrahydroaminoacridine induces opposite changes in muscarinic and nicotinic receptors in rat brain

Rats were treated with 10 mg/kg tetrahydroaminoacridine (THA) twice daily for 14 days. THA (10 mg/kg) induced a significant decrease in the number of muscarinic receptors (both M1 and M2) in the cortex and striatum, whereas the number of nicotinic receptors in the cortex and hippocampus increased. Rats treated with physostigmine (0.9 mg/kg) showed a reduced number of muscarinic receptors, but no change in nicotinic receptors. The results indicate that treatment with cholinesterase inhibitors can induce opposite changes in brain muscarinic and nicotinic receptors in vivo.     

Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree.

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]Quinuclidinyl benzylate [( 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (Kd) of 0.41 +/- 0.3 nM in WKY rats and of 0.37 +/- 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.     

Transition and heavy metal inhibition of ligand binding to muscarinic acetylcholine receptors from rat brain.

Several divalent heavy metals interact with muscarinic acetylcholine receptors in neural membranes from rat forebrains in vitro to inhibit the the binding of [³H]3-quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. Mercury is the most potent inhibitor with an ID50 value of 10^?7m for the inhibition of the binding of 10^?10m QNB. Several other metals are less effective with ID50 values between 10^?5 and 2 × 10^?4m increasing in the following order: iron < silver < copper < lead < cadmium < terbium < zinc. Cobalt, manganese, lanthanum, nickel, and tin are less potent inhibitors (ID50 > 10^?3m). Binding inhibition by copper, mercury, and cadmium is competitive, and the inhibition by all the metals can be reversed by decreasing the free metal concentration through dilution or chelation by ethylenediaminetetraacetate, dimercaprol, or penicillamine. The effects of metals on muscarinic binding are largely independent of temperature. Chaotropic anions (100 mm iodide, thiocyanate, trichloroacetate, or nitrate) are similarly without effect on the metal-receptor interaction. The limited effects of sulfhydryl reducing (dithiothreitol, 2-mercaptoethanol, and glutathione) and alkylating (N-ethyl maleimide) reagents do not indicate a prominent role for sulfhydryl groups in mediating metal inhibition of muscarinic receptors.     

Agonist-induced desensitization of muscarinic acetylcholine receptor in rat brain

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This preparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity (B _max ) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37 °C but was not apparent in incubation at 15 °C. In addition, it was abolished when the receptors were blocked by atropine. The decline in (³H)N-methylscopolamine((³H)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that ‘tru’ receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist (³)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggests that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.     

The binding of some antidepressant drugs to brain muscarinic acetylcholine receptors.

1 The binding of some antidepressant drugs, including some new drugs of atypical structure (flupenthixol, iprindole, maprotiline, mianserin, nomifensine, tofenacine and viloxazine) to muscarinic acetylcholine receptors in the brain has been studied by displacement of [3H]-atropine. 2 Many of the drugs are potent muscarinic antagonists. 3 Some correlation can be made between the affinity for binding to the muscarinic acetylcholine receptor and the incidence of anticholinergic side effects in clinical usage.     

cGMP formation and phosphoinositide turnover in rat brain slices are mediated by pharmacologically distinct muscarinic acetylcholine receptors.

The cGMP response and the accumulation of inositol monophosphate (IP) induced by carbachol were compared in slices of different rat brain structures. Basal cGMP and the responses of cGMP to carbachol appeared dependent on the concentration of added Ca2+, suggesting that distinct Ca(2+)-mediated and Ca(2+)-sensitive muscarinic receptor-mediated mechanisms stimulate guanylate cyclase. Regional responses of cGMP to carbachol or to direct stimulation of guanylate cyclase with sodium nitroprusside were markedly distinct, indicating that a major proportion of guanylate cyclase in the cortex, an intermediate proportion in other forebrain regions, and only a minor proportion in the brainstem is sensitive to muscarinic receptor stimulation. The regional patterns of IP and cGMP responses to carbachol were different in the forebrain. Maximal IP accumulation was found in the cortex, whereas cGMP responses were highest in the hippocampus. Moreover, IP and cGMP formation in the hippocampus were differently antagonized by atropine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), the M2-receptor subtype-preferring antagonist AF-DX 116 and the M1-selective antagonist pirenzepine. These data support the notion that the IP formation induced by carbachol in the forebrain predominantly is mediated by muscarinic receptors of the M1 subtype, and indicate the involvement of muscarinic receptors of the M3 subtype in the carbachol-induced cGMP formation.     

Affinity changes of rat striatal dopamine receptors in vivo after acute bupropion treatment

The effect of bupropion (Wellbutrin) was studied on in vivo [3H]spiperone binding in rat striatum and cerebellum. The compound increased binding dose dependently in striatum whereas no effect was observed in cerebellum. Saturation analyses of in vivo binding in the striatum revealed an increased affinity of the receptors rather than changes in the number of binding sites. These results are the first demonstration of an increased sensitivity of central dopamine receptors after acute bupropion treatment.     

Altered muscarinic acetylcholine receptor subtype binding in neonatal rat brain following exposure to chlorpyrifos or methyl parathion.

The neurodevelopmental effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPS) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in neonatal rat brain. Animals were orally gavaged using an incremental dosing regimen from postnatal day 1 (PND1) until PND8 with a low, medium, and high dosage for both CPS and MPS. On PND4 and PND8, ChE activity was measured in whole brain while the total and subtype densities of mAChRs were measured in three brain sections: area anterior to optic chiasma (anterior forebrain), area from the optic chiasma to the medulla/pons (posterior forebrain); and the medulla/pons excluding the cerebellum. The ligands 3H-pirenzepine, 3H-AF-DX 384, 3H-4-DAMP, and 3H-QNB were used to measure the maximal binding of the M1, M2/M4, and M3 subtypes and total mAChR receptors, respectively. In the anterior and the posterior forebrain, the levels of all mAChRs nearly doubled from PND4 to PND8, while in the medulla/pons, M1- and M3-subtype mAChR densities were low and did not increase and M2/M4 subtype and total mAChR slightly increased from PND4 to PND8. Reduction of ChE activity and mAChR binding by CPS or MPS was more evident in rats at PND8 than at PND4. With respect to mAChR binding, the greatest effects were observed in the medulla/pons and the least effects were observed in the posterior region of the forebrain. These results demonstrate that OPs exert adverse effects on rat central nervous system development through the cholinergic system in an age- and region-dependent manner.     

Halothane effects on muscarinic acetylcholine receptor complexes in rat brain

Muscarinic acetylcholine receptors in membranes from rat cerebral cortex or brainstem were equilibrated with halothane (0.5 to 5%). Halothane did not affect the number of [3H]methylscopolamine [( 3H]MS) binding sites. [3H]MS binding affinity, however, was increased in the presence of halothane (KD, air = 0.41 nM; KD, 2% halothane = 0.26 nM). This increase reflected a decrease in the dissociation rate constant (from 13 X 10(-3) min-1 to 6.5 X 10(-3) min-1) rather than a change in the bimolecular rate constant of association (1.8 and 1.9 X 10(7) M-1 min-1 in the absence and presence of 2% halothane respectively). Carbamylcholine affinity for brainstem or cortical muscarinic receptors was not affected by halothane. The ability of a guanine nucleotide to lower carbamylcholine affinity for brainstem receptors, however, was eliminated after equilibration with 2% halothane.     

The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding.