Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells.

G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M(2) muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[(3)H]methylscopolamine and [(3)H]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M(2) antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M(2) muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.

M1 muscarinic receptor signaling in mouse hippocampus and cortex.

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.

Changes of the responses of single sympathetic ganglionic neurones to substance P following desensitization.

1. The neuropeptide substance P (SP) exerts an excitatory effect on sympathetic neurones by inhibiting a time- and voltage-dependent potassium current. During prolonged application of SP, the response desensitizes. The changes in kinetics of the SP response in single neurones after desensitization have been studied in an attempt to gain some insight as to the molecular mechanism of desensitization in live, functioning neurones. 2. Desensitization to SP resulted in subsequent SP responses being smaller, but the time course was unchanged in desensitized cells compared with non-desensitized cells. 3. Experimental manipulations were performed to decrease receptor and G protein function for comparison to desensitization. Intracellular application of GDPbetaS, to decrease G protein function, led to successive responses to agonist becoming smaller and slower. When functional muscarinic receptors were decreased by extracellular application of propylbenzilylcholine mustard (PrBCM), the response to muscarine became smaller, but the time course was unchanged compared with the change in time course produced by PrBCM vehicle alone. 4. The results have also been compared with simulations from a mathematical model of drug-receptor-G protein interactions. Under a constrained set of conditions, the model predicts that decreasing the size of the G protein pool will decrease both the magnitude and the time course of the response to agonist. Decreasing receptor levels results in a more efficient decrease in the magnitude of the response but no change in the time course of the response. 5. These data provide evidence that desensitization of the response to SP in single neurones results from a decrease in functional receptors.

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors.

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.

Endocytosis and recycling of muscarinic receptors.

Agonist stimulation causes the endocytosis of many G protein-coupled receptors, including muscarinic acetylcholine receptors. In this study we have investigated the agonist-triggered trafficking of the M3 muscarinic receptor expressed in SH-SY5Y human neuroblastoma cells. We have compared the ability of a series of agonists to generate the second messenger Ins(1,4,5)P3 with their ability to stimulate receptor endocytosis. We show that there is a good correlation between the intrinsic activity of the agonists and their ability to increase the rate constant for receptor endocytosis. Furthermore, on the basis of our results, we predict that even very weak partial agonists should under some circumstances be able to cause substantial receptor internalization. Receptor endocytosis occurs too slowly to account for the rapid desensitization of the Ca2+ response to carbachol. Instead, receptor endocytosis and recycling appear to play an important role in resensitization. After an initial agonist challenge, the response to carbachol is fully recovered when only about half of the receptors have been recycled to the cell surface, suggesting that there is a receptor reserve of about 50%. Removal of this reserve by receptor alkylation significantly reduces the extent of resensitization. Resensitization is also reduced by inhibitors of either endocytosis alone (concanavalin A) or of endocytosis and recycling (nigericin). Finally, the protein phosphatase inhibitor calyculin A also reduces resensitization, possibly by blocking the dephosphorylation of the receptors in an endosomal compartment.

Allosteric effects of four stereoisomers of a fused indole ring system with 3H-N-methylscopolamine and acetylcholine at M1-M4 muscarinic receptors.

We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.

Protein kinase C mediates increase of Ca2+ sensitivity for contraction by cholinoceptor partial agonist in ileal longitudinal muscle of guinea pig.

1. Experiments were designed to study the roles of protein kinase C in carbachol- and pilocarpine-induced contraction and the increase in cytosolic Ca2+ concentration ([Ca2+]i) in guinea pig ileal longitudinal muscle. 2. The protein kinase C inhibitors, GF 109203X (10 microM), calphostin C (10 microM) and H-7 (10 microM), reduced the maximum of the concentration response curve produced by pilocarpine more effectively than that produced by carbachol. 3. The slopes of the regression lines between [Ca2+]i and tension development for pilocarpine and carbachol in tissues treated with GF 109203X were significantly gentler than those for untreated tissues. 4. The protein kinase C alpha- and beta 1 selective inhibitor Goe 6976 (1 microM) decreased both [Ca2+]i and contraction, but did not affect the slopes of the regression lines for pilocarpine and carbachol. 5. These results suggest that protein kinase C (both n- and/or a-type) plays an important role in the increase of Ca2+ sensitivity of the contractile element, and that pilocarpine mainly activates the protein kinase C-dependent pathways for contractile mechanisms in guinea pig ileal longitudinal muscle.

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci.

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 >> AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

Cardiac muscarinic receptors. Relationship between the G protein and multiple states of affinity.

An expanded version of the mobile receptor model has been assessed in studies on the binding of N-[3H]methylscopolamine and [35S]GTPgammaS to cardiac muscarinic receptors and their attendant G proteins in ventricular membranes from hamster. The model comprises two pools of receptor, one of which lacks G proteins, and a heterogeneous population of G proteins that compete for the receptor within the G protein-containing pool. To guide the formulation of the model itself and to define the various parameters, data were combined from assays performed under various conditions with native membranes and following irreversible blockade of about 80% of the receptors with propylbenzilylcholine mustard. Multiple G proteins are indicated primarily by multiple states of affinity evident in the dose-dependent effect of guanyl nucleotides on the binding of carbachol; G protein-free receptors are indicated by sites of low affinity for carbachol that survive treatment with the mustard. The expanded model generally succeeds where more frugal schemes have been inadequate, but it nevertheless fails to yield a mechanistically consistent description of the data. Guanyl nucleotides and partial alkylation do not affect the inhibitory potency of carbachol in a manner consistent with their supposed effect on the equilibrium between uncoupled and G protein-coupled receptors. As inferred from the model, G proteins are lost upon alkylation of the receptor, and their numbers are regulated by guanyl nucleotides. Parameters estimated via N-[3H]methylscopolamine are wholly inconsistent with the same parameters estimated via [35S]GTPgammaS. The failure of the model suggests that multiple states of affinity may not arise from a ligand-regulated equilibrium between free receptors and G proteins on the one hand and one or more RG complexes on the other.

Drug receptor mechanisms in smooth muscle: beta-chloroethylamine-sensitive and -resistant receptor mechanisms.

Both alpha1-adrenoceptors and M3-cholinoceptors can be divided into two subtypes discriminated by the beta-chloroethylamines, chloroethylclonidine and propylbenzilylcholine mustard (PrBCM), only in the presence of GTP. The full agonists interact with both subtypes to induce responses. The partial agonists activate one of them to induce responses but behave as competitive antagonists when they interact with the other. The responses mediated through the receptors that are activated by the partial agonists are resistant to myosin light chain kinase inhibitors, while the response through the activation of the other receptors are suppressed by the inhibitors. The receptor stimulations through alpha1A-adrenoceptor and PrBCM-sensitive M3-cholinoceptor subtypes mainly activate the myosin light chain-phosphorylation-independent pathway mediated through protein kinase C and low molecular weight GTP-binding protein, whereas the stimulations through alpha1B-adrenoceptors and the PrBCM-phosphorylation-dependent pathway are directly related to Ca2+/calmodulin.

Muscarinic receptor reserve for inhibition of cAMP accumulation in bovine trachealis cells.

In airway smooth muscle, muscarinic agonists inhibit synthesis of adenosine 3',5'-cyclic monophosphate (cAMP). The goal was to characterize the relationship between agonist occupancy of muscarinic receptors and regulation of cAMP for bovine trachealis cells. For intact cells dispersed by enzyme, carbachol maximally inhibited 58 +/- 4% (mean +/- SE, n = 5) of isoproterenol-stimulated cAMP accumulation at low concentrations [log half-maximal effective concentration (EC50) = -8.34 +/- 0.16]. In radioligand binding experiments, carbachol competed for [3H]quinuclidinyl benzilate (n = 7) and [N-methyl-3H]scopolamine (n = 3) binding sites on intact cells with both low (log KL = -4.26 +/- 0.06 and -4.50 +/- 0.20, respectively) and high affinities (log KH = -5.91 +/- 0.24 and -6.39 +/- 0.19, respectively). In separate experiments, a fraction of the muscarinic receptors on intact cells were inactivated with either phenoxybenzamine (POB) or propylbenzylcholine mustard (PBCM). We compared equally effective concentrations of carbachol before and after partial inactivation of receptors, and the calculated equilibrium dissociation constants for agonist (log KA = -4.36 +/- 0.42 to -3.20 +/- 0.40 for POB; log KA = -4.27 +/- 0.45 for PBCM) were much greater than the half-maximally effective concentration of carbachol in control cells (log EC50 = -8.34 +/- 0.16). Based on these equilibrium dissociation constants, we calculated that maximum responses (EC95) to carbachol were obtained by occupancy of 0.8% of the receptors coupled to cAMP regulation. We concluded that muscarinic inhibition of cAMP accumulation is characterized by a muscarinic receptor reserve.

Muscarinic M2 receptor synthesis: study of receptor turnover with propylbenzilylcholine mustard.

We have investigated the rate and the functional responsiveness of the newly synthesised M2 muscarinic receptors in HEL 299 cells following propylbenzilylcholine mustard treatment at 37 degrees C. Propylbenzilylcholine mustard induced a dose-dependent loss of the hydrophilic ligand [3H]N-methylscopolamine binding sites with 80% inactivation at 0.1 microM. The rate of muscarinic receptor synthesis in these cells, estimated from wash-out experiments following propylbenzilylcholine mustard treatment, was very slow and returned to control values after 36 h of propylbenzilylcholine mustard removal. The recovery of muscarinic receptors was blocked by the cycloheximide pre-treatment, indicating the synthetic pathway for the new receptors. In control cells as well as in cells treated with propylbenzilylcholine mustard and allowed to recover for 12 h, carbachol still inhibited forskolin-induced cAMP accumulation. These results show that (i) the rate of M2 muscarinic receptor synthesis is slow (ii) the recovery of receptors is mainly through increased synthesis and (iii) the newly synthesised receptors retain their full functional activity.

Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells.

PURPOSE: The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. METHODS: Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. RESULTS: A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinculidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either m1 or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. CONCLUSIONS: The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists.

1. A comparative study of receptor activation by ten full and partial muscarinic agonists was undertaken on the five subtypes of human muscarinic receptors expressed at similar receptor densities in Chinese hamster ovary (CHO-K1) cells. In addition, m1, m2 and m3 receptors were expressed in mouse fibroblast A9L cells in order to compare the influences of cell type on agonist activation of these receptors. 2. Receptor-effector coupling efficiencies were greater in CHO than A9L cells and agonists displayed greater potencies and similar or greater intrinsic activities at CHOm1 and CHOm3 than A9Lm1 and A9Lm3 receptors. Although m2 receptor density was 6 fold higher in A9L than CHO cells, carbachol elicited significantly greater inhibition of adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in CHOm2 cells. These data suggest that not only receptor density but receptor-effector coupling and/or coupling efficiencies play significant roles in agonist-induced responses. 3. In CHO cells, receptor-effector coupling efficiencies were m3 = m1 > m5. Although CHOm5 receptors were the least efficiently coupled, some partial agonists displayed higher intrinsic efficacies at m5 than m3 receptors suggesting that, in CHO cells, m5 and m3 receptors may activate different G proteins and/or effectors to stimulate inositol monophosphate (IP1) formation. 4. McN-A-343 was a functionally selective m4 agonist. It had little or no agonist activity at m3 receptors expressed in either A9L or CHO cells. The slopes of McN-A-343 concentration-response curves inCHOm2 cells were significantly lower than the slopes obtained with this compound in CHOm4 cells suggesting that the mode of activation by McN-A-343 differed between the two muscarinic receptors negatively coupled to adenylyl cyclase.5. Cloned receptors provide valuable tools for the study of agonist-receptor interaction and agonist receptor activation but caution should be applied in assuming that the results are valid for all cell types or for tissue-expressed receptors.

Development of muscarinic acetylcholine receptors in the ferret retina.

The development of muscarinic acetylcholine receptor protein in the ferret retina was studied using biochemical, autoradiographic, and light and electron microscopic immunohistochemical techniques. The development of retinal muscarinic cholinergic receptor proteins involves transient shifts in their number and distribution, as well as changes in the relative abundance of two molecular weight variants. Receptor binding assays demonstrate changes in the number and affinity of retinal binding sites for the muscarinic cholinergic ligand [3H]quinuclidinylbenzilate ([3H]QNB). Light microscopic immunohistochemical studies reveal the presence of muscarinic acetylcholine receptor-like (mAChR-like) immunoreactivity in the adult inner plexiform layer. During development, the mAChR-like immunoreactivity appears in a number of other retinal layers. Electron microscopic immunohistochemical studies indicate that muscarinic acetylcholine receptor-like immunoreactivity is found at amacrine-amacrine cell contacts. Both autoradiographic and gel slice electrophoretic studies were carried out after labeling of developing and adult retinal muscarinic receptors with [3H]propylbenzilylcholine mustard ([3H]propylbenzilylcholine mustard ([3H]PrBCM), which irreversibly labels the muscarinic acetylcholine receptor. Polyacrylamide gel electrophoresis under reducing, denaturing conditions resolved two peaks of radioactivity corresponding to [3H]PrBCM-labeled protein; both were eliminated by pre- and co-incubation of labeled adult retinas with excess atropine. Combined with the results of earlier studies, these observations suggest that the subtypes, number and distribution of muscarinic receptor proteins changes during retinal synaptogenesis.

Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: requirement of endogenous acetylcholine for outgrowth initiation.

To better understand the mechanisms of skin re-epithelization, we developed a simple technique that assays the outgrowth of human keratinocytes. Second-passage foreskin keratinocytes were inoculated at high cell density into 3-mm wells cut from agarose gels in standard 6-well tissue culture dishes. The cells settled on the dish bottom and formed a confluent colony. The cells at the periphery of the colony flattened, spread their cytoplasm, and moved away over the dish surface under the agarose gel. The morphology of migrating keratinocytes was observed microscopically through the transparent agarose, and the migration distance was measured after the gels were removed and after cells were fixed and stained. To determine which cell activities were involved in the outgrowth, the effects of cholinergic compounds on keratinocyte outgrowth were compared with their effects on keratinocyte proliferation, cell-plastic attachment, and spreading measured in separate sets of experiments. Outgrowth was inhibited by the specific inhibitor of acetylcholine synthesis bromoacetylcholine (0.05 mM) and restored by 5 mM exogenous acetylcholine. The irreversible muscarinic antagonist propylbenzilylcholine mustard (0.05 mM) abolished the restorative effects of exogenous acetylcholine, and also inhibited outgrowth of intact keratinocytes. In keratinocyte cell cultures, bromoacetylcholine stopped cell division. Propylbenzilylcholine mustard increased cell number, but interfered with cell-plastic attachment and spreading. This suggests that cell-matrix attachment, spreading, and locomotion of human keratinocytes, but not mitosis, mediate the earliest stages of skin re-epithelization, and that endogenous acetylcholine regulates these keratinocyte functions. Specifically, keratinocyte acetylcholine is required to initiate outgrowth.

Absence of receptor reserve at hippocampal muscarinic autoreceptors which inhibit stimulus-dependent acetylcholine release.

The extent of reserve among inhibitory muscarinic autoreceptors on hippocampal cholinergic nerve terminals was examined in superfused calcium-naive synaptosomes. The tissues were treated with the irreversible muscarinic cholinergic receptor antagonist propylbenzilycholine mustard (PrBCM) and then used to assess the functional status of autoreceptors through acetylcholine (ACh)-induced inhibition of calcium-evoked [3H]ACh release. PrBCM treatment caused a marked reduction in the density of high-affinity [3H]quinuclidinyl benzilate binding sites (46%, 72% and 90% reductions after 3, 6 or 10 nM PrBCM, respectively) but had no apparent influence on the binding affinities or relative proportions of high- and low-affinity binding sites for the M1-selective antagonist pirenzepine or the agonist ACh. In vehicle-treated tissues, ACh was a potent (EC50 = 240 nM) and efficacious (maximal inhibition of stimulated [3H]ACh release = 65%) agonist at muscarinic autoreceptors. However, after PrBCM treatment, the maximal inhibition for ACh was greatly attenuated (35% and 17% for 3 and 6 nM PrBCM, respectively) with no concurrent changes in the EC50 or slope factor. Comparisons of equieffective agonist concentrations before and after receptor occlusion revealed a direct linear relationship between autoreceptor occupancy and inhibition of [3H]ACh release with close agreement between the calculated agonist dissociation constant (KA = 220 nM) and the EC50 for ACh. Pretreatment with 100 nM atropine methylbromide completely prevented PrBCM-induced reductions in muscarinic cholinergic receptor binding and autoreceptor function. These results support the conclusion that muscarinic autoreceptors on hippocampal nerve endings exhibit little or no reserve for inhibition of ACh release by the endogenous neurotransmitter.

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells.

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit.

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.