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.

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.

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.

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.

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.

Characterization of subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle.

The subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle was examined using four selective muscarinic antagonists, pirenzepine, AF-DX 116, himbacine and 4-DAMP. The pA2 values of the four antagonists against pilocarpine were not different from their values against carbachol after the treatment with PrBCM and was identified with the values for the m3 subtype. These results suggest that the subtype of PrBCM-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle is the m3 subtype only and not other subtypes.

[Transition of drug receptor mechanisms].

It is generally accepted that the agonists, full agonist and partial agonist, interact with the same receptors according to the classical receptor mechanisms. We tried to modify the drug receptor mechanisms in muscarinic cholinoceptors, alpha 1-adrenoceptors and beta-adrenoceptors. In the muscarinic cholinoceptor, there are two subtypes of M3-cholinoceptors, propylbenzilylcholine mustard (PrBCM)-sensitive receptors and PrBCM-resistant ones. The full agonists contract the longitudinal muscle through the interaction of two cholinoceptors, PrBCM-sensitive and-resistant ones, while the partial agonists produce the contraction through only the activation of PrBCM-sensitive ones. Upon activation PrBCM-sensitive receptors may use cytosolic Ca2+ more effectively than PrBCM-resistant receptors. In the alpha 1-adrenoceptor, the full agonist induces contraction through both alpha 1A and alpha 1B subtypes and the partial agonist through only alpha 1A subtype. The adrenoceptors activated by full agonist may be partly different from that by partial agonist in the arteries. In both the common iliac artery and thoracic aorta treated with the irreversible antagonist, phenoxybenzamine the slopes of schild plots of the results obtained from an antagonism between full agonist (phenylephrine) and alpha 1A-selective competitive antagonist (WB4101) equal to 1, suggesting that phenoxybenzamine preferably interacts with alpha 1B subtype. In the beta-adrenoceptor, the pD2-values of the partial agonists obtained from the concentration-response curves are significantly different from their pA2-values against full agonist (isoprenaline). The Scatchard plot of the specific [3H]befunolol (the partial agonist) binding showed two affinity sites of the receptors in the absence of Gpp(NH)p but the low affinity site was reduced while the high affinity site was not affected in the presence of Gpp(NH) p. The beta-adrenergic partial agonists are able to discriminate these two different binding sites of the beta-adrenoceptors. Our results suggest that the receptors activated by full agonists are partly different from those by partial agonists in muscarinic cholinoceptors, alpha 1- and beta-adrenoceptors, and that the irreversible antagonist can discriminate between the sites interact with full agonists and those with partial agonists in muscarinic cholinoceptors and alpha 1-adrenoceptors.

Identification of muscarinic receptor subtypes present in cerebellar granule cells: prevention of [3H]propylbenzilyl choline mustard binding with specific antagonists.

Subtypes of muscarinic receptors (possible m1 to m5) can be identified by their molecular size, specific effector systems and antagonist specificity. In membranes prepared from primary cultures of cerebellar granule cells, [3H]propylbenzilylcholine mustard [( 3H]PBCM) irreversibly binds to muscarinic receptive proteins, having two major molecular sizes, 92 and 66 kDa. With relatively short periods of incubation (approx. 30 min, 30 degrees C) of [3H]PBCM with atropine, a nonspecific competitive receptor antagonist, the irreversible labeling of these muscarinic proteins by [3H]PBCM could be prevented. Methoctramine, a specific competitive antagonist at muscarinic receptors coupled to inhibition of adenylate cyclase, protected most of the muscarinic receptors having a molecular size of 66 kDa from binding of [3H]PBCM. These 66 kDa receptive proteins are suggested to be muscarinic m2 and m4 subtypes. (-)Quinuclidinyl xanthene-9-carboxylate [(-)QNX], a somewhat specific competitive antagonist at muscarinic receptors coupled to hydrolysis of phosphatidylinositol, prevented the binding of [3H]PBCM to 92 kDa muscarinic receptive proteins and some 66 kDa muscarinic receptive proteins. The 92 kDa receptive proteins are suggested to be the muscarinic m3 subtype and the 66 kDa proteins could be either the m2 or m4 receptor subtype. Lastly, pirenzepine, a nonspecific antagonist at muscarinic receptors mediating inhibition of adenylate cyclase and hydrolysis of PI in these cultures, resembled (-)QNX in preventing binding of [3H]PBCM to the 92 kDa receptive proteins and some 66 kDa receptive proteins. The suggested subtypes of muscarinic receptors, specifically bound by pirenzepine should be the m3 (92 kDa) and the m4 (66 kDa) subtypes, since pirenzepine reportedly exhibits a low affinity for the muscarinic m2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

Location in muscarinic acetylcholine receptors of sites for [3H]propylbenzilylcholine mustard binding and for phosphorylation with protein kinase C.

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.

Autoradiographic visualization of muscarinic receptors on rat paratracheal neurons in dissociated cell culture.

An autoradiographic method was used to determine the distribution of muscarinic receptors on cells cultured from the trachealis muscle of 12-13-day-old rats. Cells identified in these culture preparations included neurones, fibroblasts, smooth muscle, and glial and epithelial cells. The cultured cells were incubated with the specific, irreversible ligand [3H]propylbenzylylcholine mustard, and the autoradiographs generated showed that most, if not all, of the paratracheal neurones observed in these cultures were specifically labelled. Both the neuronal cell body and associated neurites were evenly labelled over their entire surface. Neither the pattern nor the density of neuronal labelling appeared to be influenced by close association with other cultured cell types. Autoradiographic grains for muscarinic receptors also appeared to be uniformly distributed over smooth muscle cells and epithelial cell groups in culture. In contrast, no specific labelling was associated with cultured fibroblasts, glial cells and other non-neuronal supporting cells. The precise localization of muscarinic receptors on different cell types in culture may prove to be useful knowledge in the design of an effective and specific antimuscarinic bronchodilator.

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells.

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.

Muscarinic cholinergic receptors on the endothelium of human cerebral arteries.

To characterize the muscarinic cholinergic receptors on the endothelium of human cerebral arteries, isometric tension measurement and receptor autoradiographic studies were performed. Acetylcholine (ACh) induced dose-dependent relaxation of human cerebral arteries precontracted by 10(-5) M serotonin, with an EC50 of 1.9 +/- 0.6 X 10(-6) M (n = 7). The relaxation was abolished by 10(-5) M hemoglobin. Autoradiography, using the muscarinic antagonist [3H]propylbenzilycholine mustard, demonstrated the high density of muscarinic cholinergic receptors on the endothelial cells of human cerebral arteries, especially on the luminal surface of the endothelium. These findings suggest that ACh-induced relaxation mediated by muscarinic cholinergic receptors on the endothelium has a physiological function in human cerebral arteries.

Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function.

Muscarinic acetylcholine receptors contain a region encompassing the second and third transmembrane domains that is rich in conserved aspartic acid residues. To investigate the role of four conserved aspartic acids at positions 71, 99, 105, and 122 in muscarinic receptor function, point mutations in the rat m1 muscarinic receptor gene were made that converted each Asp to Asn, and wild type or mutant genes were stably expressed in Chinese hamster ovary cells that normally lack muscarinic receptors. Substitution of Asp71 or Asp122 with Asn produced mutant receptors that displayed high affinity for carbachol but decreased efficacy and potency, respectively, in agonist-induced activation of phosphoinositide hydrolysis, suggesting that these residues may mediate receptor-GTP binding protein interactions. Substitution of Asp99 or Asp105 with Asn produced marked decreases in ligand binding affinities and/or covalent incorporation of [3H] propylbenzilylcholine mustard, suggesting that these residues may be involved in receptor-ligand interactions.

Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor.

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors.

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.

Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation.

Significant progress has been made in determining regional expression of neurotransmitter receptors within the CNS, but little information is available at the neuronal level. In the current study, to begin characterizing cellular and subcellular aspects of receptor differentiation, we have localized ACh receptors on neurons isolated from the chicken CNS. Localization was determined autoradiographically using 2 cholinergic receptor probes: 3H-propylbenzilylcholine mustard (PrBCM) a muscarinic antagonist, and 125I-alpha-bungarotoxin (BTX), a putative nicotinic ligand in the avian CNS. To isolate neurons, we incubated embryonic chicken retinas (E13-E19) in Ca2+/Mg2+-free buffer containing 8 units/ml papain for 20 min and then gently agitated the tissue by trituration. Large numbers of dendrite-rich neurons, belonging to recognizable morphological subpopulations (e.g., multipolar neurons of various sizes, small bipolar and unipolar neurons), were collected on slides for autoradiography. Cell isolation had no effect on ligand binding levels, and a high ratio of specific to nonspecific binding allowed us to associate silver grains with receptor position. Muscarinic-receptor-positive cells comprised a recognizable subpopulation that had small rounded cell bodies (6-7 micron) and a single emergent arbor. The cells had an axial or elongated appearance. Muscarinic receptors were abundant over dendrites but absent from cell bodies. Segregation to dendrites was complete by E13, the age when synapses reportedly first begin to appear. Cells labeled with 125I-BTX were more heterogeneous in morphology. The most striking BTX-positive cells comprised neurons with large cell bodies (approximately 15 micron) and multiple processes. Dendrites were profusely labeled, but only sporadic labeling was seen on cell bodies, and often this was at sites crossed by labeled dendrites. Maximum labeling occurred in the distal, smallest-caliber ends of the dendritic arbors. All limbs of BTX-positive multipolar neurons expressed abundant receptors. Occasionally, a thin uniform-caliber process was seen branching from a primary dendrite, and such processes, which may have been axons, were never labeled. Very small neurons with bipolar morphology also showed minimal or no labeling on one process, despite dense labeling on the other. Photoreceptors and Mueller cells were never labeled with BTX or PrBCM. The data show that, within a discrete CNS region, specific subpopulations of neurons independently regulate expression of ACh receptors and that, even early in development, control mechanisms segregate receptors to physiologically appropriate regions of the cell surface.

Muscarinic acetylcholine receptors from avian retina and heart undergo different patterns of molecular maturation.

Muscarinic acetylcholine receptors (mAChRs) from the avian CNS exist in two molecular weight forms whose concentrations change during development. Here, we have compared the development of mAChRs from embryonic hearts with those of the CNS. Analysis of [3H]-propylbenzilylcholine mustard (PrBCM)-labeled retina and heart mAChRs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two atropine-sensitive peaks for each tissue. Apparent molecular masses of retina mAChRs, 86 +/- 0.7 kilodaltons (kDa) and 72 +/- 0.7 kDa, were different from those of heart mAChRs, 77 +/- 1.0 kDa and 52 +/- 0.9 kDa. During retina development, the major receptor type changed from 86 kDa to 72 kDa. No such change occurred during heart development. Furthermore, the 52-kDa species appeared to be generated by endogenous proteolysis, as prolonged incubation of heart membranes at 37 degrees C increased the amount of 52-kDa peptide with a decrease of 77-kDa peptide. Protease inhibitors blocked this conversion. Incubation of retina membranes at 37 degrees C did not result in a conversion of the 86-kDa peptide into the 72-kDa peptide, but it did cause the appearance of a minor amount of 52-kDa peptide. The proteolysis of retina mAChRs was not enhanced by cohomogenizing them with heart tissue, arguing against the presence of releasable proteases in heart. Membrane-bound retina and heart mAChRs displayed similar sensitivity to exogenous (Staphylococcus aureus V8) protease, indicating that heart receptors were not unusually susceptible to proteolytic attack; analysis of the labeled polypeptides with the V8 protease showed different patterns of digestion for the retina and heart receptors.(ABSTRACT TRUNCATED AT 250 WORDS)