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The lysosphingolipids sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) reportedly increase free cytosolic Ca2+ concentration ([Ca2+]i) in a variety of cell types, apparently by activating G protein-coupled plasma membrane receptors. We investigated whether and how sphingolipids modulate Ca2+ homeostasis in the insulinoma cell line RINm5F. The addition of SPPC and glucopsychosine (GPS) did not affect basal [Ca2+]i but inhibited the KCl (30 mM)-induced increase in [Ca2+]i in a pertussis toxin-insensitive and concentration-dependent manner (EC50 approximately 5 micro M). Similar inhibitory effects were observed with dihydro-SPPC and psychosine, whereas SPP and various N-acylated sphingolipids (at 10 micro M each) had little or no effect on the KCl-induced [Ca2+]i increase. Because in RINm5F cells the primary pathway for depolarization-induced [Ca2+]i increase are L-type Ca2+ channels, we studied whether sphingolipids reduce L-type Ca2+ current (ICa.L). When added to the bath, GPS and SPPC, but not SPP (10 micro M each), rapidly reduced maximal ICa.L by approximately 35%, similar to the alpha2-adrenoceptor agonist clonidine (30 micro M). However, when applied internally, GPS had no effect on ICa. L. When the electrode solution contained the stable GDP analog guanosine-5'-O-(2-thio)diphosphate (1 and 10 mM), the inhibitory effect of GPS was abolished. In conclusion, a novel cellular action of lysosphingolipids is observed in RINm5F cells (i.e., a guanine nucleotide-sensitive inhibition of L-type Ca2+ currents). The pharmacological profile of this inhibition is unique and unlike any known lysosphingolipid receptor-mediated action.

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein-coupled receptors with high affinity for sphingosine 1-phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration-response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

Besides its role as a putative second messenger releasing Ca2+ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+]i), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+]i elevations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+]i by 83%, in line with the previously reported involvement of G proteins of the Gi/o family in SPP signalling in other cell types. In contrast to other [Ca2+]i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca2+. Furthermore, SPP also did not cause activation of either phospholipase D or A2. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+]i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca2+]i differed by more than two orders of magnitude, with the EC50 values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a Gi/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.

We analyzed the role of receptor internalization and recycling in muscarinic acetylcholine receptor (mAChR) desensitization and resensitization. Incubation of Chinese hamster ovary cells stably expressing the m4 mAChR with 1 mM carbachol for 1 hr reduced cell surface receptor number by 50-60% with no change in total receptor number. Pretreatment of the cells with 450 mM sucrose, which did not affect the ability of m4 receptors to inhibit forskolin-stimulated cAMP accumulation, completely blocked receptor internalization. On the other hand, the carbachol treatment reduced the ability of m4 receptors to inhibit cAMP accumulation in both sucrose-treated and untreated cells, with a similar onset and to a similar extent. The EC50 value for carbachol was increased approximately 10-fold, and maximal inhibition determined at 100 microM carbachol was reduced approximately 50%. In contrast, thrombin-induced inhibition of cAMP accumulation was not affected. Recycled receptors in cells not treated with sucrose remained refractory to carbachol stimulation for > or = 2 hr after agonist removal, even though cell surface receptor number had recovered completely within 1 hr. In contrast, resensitization of receptor function was very rapid in cells treated with sucrose. Ten minutes on removal of agonist, mAChRs in the plasma membrane of sucrose-treated cells were fully resensitized. Also, an internalization-defective m4 mAChR mutant, T399A, that was found to desensitize similar to the wild-type receptor, resensitized more rapidly than the wild-type receptor. We conclude that desensitization and resensitization of m4 mAChRs in Chinese hamster ovary cells can occur at the plasma membrane and that receptor internalization strongly delays the process of resensitization of desensitized receptors.

We investigated the validity of streptolysin O (SLO)-permeabilized Madin-Darbin canine kidney (MDCK) cells which express muscarinic acetylcholine receptors (mAChRs) coupled to pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) for the study of the molecular machinery that regulated mAChR internalization and recycling. Exposure of SLO-permeabilized cells to carbachol-reduced cell surface receptor number by up to 40% without changing total receptor number. The kinetics and maximal extent of receptor internalization as well as the potency of carbachol to induce receptor internalization were almost identical in SLO-permeabilized and non-permeabilized cells. Using this semi-intact cell system, we studied the effect of various agents affecting components potentially involved in receptor trafficking. Internalization was prevented by treatment of the SLO-permeabilized MDCK cells with (i) the stable ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and adenylylimidodiphosphate, to block ATP-dependent processes, and (ii) heparin to block G protein-coupled receptor kinases. Inclusion of the stable GTP analogue, guanosine 5'-O-(3-thiotriphosphate), increased the rate but not the extent of receptor internalization. None of the membrane-impermeant agents affected receptor internalization in intact MDCK cells. This model system also allowed recycling of internalized receptors back to the plasma membrane. After removal of the agonist, cell surface receptor number in SLO-permeabilized cells returned to control values within 90 min with the same kinetics as seen in intact cells. Inclusion of guanosine 5'O-(3-thiotriphosphate) shortened the recovery time. These data suggest that both ATP-dependent kinases including G protein-coupled receptor kinases and G proteins participate in receptor internalization and recycling. In summary, the SLO-permeabilized MDCK cell is a feasible model system for the study of mAChR internalization and recycling and allows manipulation of the intracellular milieu with membrane-impermeable macromolecules.

The sphingolipids, sphingosylphosphorylcholine (SPPC) and sphingosine-1-phosphate (SPP), induce a rapid and transient rise in intracellular free calcium concentration ([Ca2+]i) in a variety of cell lines via activation of pertussis toxin-sensitive G protein-coupled receptors. We investigated whether these sphingolipids act on different receptors by testing the effect of varying concentrations of SPPC on [Ca2+]i in human leukemia HL-60 cells, which have been found to be nonresponsive to SPP. SPPC potently (EC50 = 1.5 microM) and rapidly increased [Ca2+]i in HL-60 cells in a pertussis toxin-sensitive manner. Differentiation of HL-60 cells through treatment with dibutyryl cAMP into granulocyte-like cells did not change the magnitude or the pertussis toxin sensitivity of the SPPC-induced [Ca2+]i rise, indicating that the receptor for SPPC is constitutively expressed in HL-60 cells. SPPC did not activate phospholipase C or D in HL-60 cells. However, SPPC, but not SPP, stimulated the generation of superoxide anions in dibutyryl cAMP-differentiated HL-60 cells as well as in human neutrophils, suggesting that the SPPC receptor may play a role in the inflammatory defense against invading microorganisms. On the basis of these results, we conclude that there apparently is a heterogeneity of G protein-coupled receptors for sphingolipids in mammalian cells.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine-1-phosphate (Sph-1-P) was studied in isolated cultured guinea-pig atrial myocytes using whole-cell voltage clamp. 2. Sph-1-P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph-1-P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph-1-P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph-1-P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre-incubation of the cells with carbachol did not affect the response to Sph-1-P; likewise, pre-incubation of the cells with Sph-1-P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre-incubation with either Sph-1-P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph-1-P in atrial myocytes is induced by binding to a novel G protein-coupled phospholipid receptor.

Many guanine-nucleotide-binding-protein-coupled receptors contain consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site-directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR-mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist-induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhibitor H-8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor-mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-protein-coupled receptors.

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

Activation of muscarinic acetylcholine receptors (mAChR) in human embryonic kidney (HEK) cells stably expressing the human m3 subtype leads to stimulation of both phospholipase C (PLC) and D (PLD). mAChR-stimulated PLD was turned off after 2 min of receptor activation with either the full (carbachol) or partial agonist (pilocarpine) and remained completely suppressed for at least 4 h. Partial recovery was observed 24 h after agonist removal. This rapid arrest of PLD response was not due to a loss of cell surface receptors and was also not caused by negative feedback due to concomitant activation of protein kinase C, tyrosine phosphorylation, increase in cytosolic calcium, or activation of Gi proteins. Furthermore, PLD stimulation by directly activated protein kinase C and GTP-binding proteins was unaltered in carbachol-pretreated cells. Finally, neither prevention of PLD stimulation during carbachol pretreatment by genistein nor inhibition of protein synthesis by cycloheximide, added before or after carbachol challenge, resulted in recovery of mAChR-stimulated PLD. The short term carbachol pretreatment nearly completely abolished agonist-induced binding of guanosine 5'-O-(3-thiotriphosphate) to membranes or permeabilized adherent cells. Full recovery of this response was achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by endogenously expressed purinergic receptors was also fully blunted after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to either receptor agonist partially, but not completely, reduced PLD stimulation by the other agonist. In contrast to desensitization of PLD stimulation, 2 min of carbachol treatment led to a sensitization, by up to 2-fold, of mAChR-stimulated inositol phosphate formation. This supersensitivity was also observed with pilocarpine, which acted as a full agonist on PLC. On the basis of these results, we conclude that the m3 mAChR stimulates PLD and PLC in HEK cells with distinct efficiencies and with very distinct durations of each response. The rapid and long lasting desensitization of the PLD response is apparently not due to a loss of cell surface receptors or PLD activation by GTP-binding proteins, but it may involve, at least initially, an uncoupling of receptors from GTP-binding proteins and most likely a loss of an as yet undefined essential transducing component.

Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by 125I-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild-type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin-stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264-394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264-394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des-(264-394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine-nucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild-type receptor and des-(264-394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264-394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi-protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization.

Molecular cloning studies have demonstrated the existence in mammals of five genes encoding distinct muscarinic acetylcholine receptors which are heterogeneously expressed in the central nervous system and the target organs of the autonomic nervous system. In order to determine the factors responsible for regulation of receptor expression, we isolated a genomic clone encoding the mouse m4 muscarinic acetylcholine receptor. The gene contains no introns in the coding sequence, and encodes a polypeptide of 479 amino acids that is able to bind various muscarinic ligands with affinities typical for mAChRs and to couple efficiently to inhibition of adenylyl cyclase.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.

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.

Muscarinic receptor and beta-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [3H]-L-quinuclidinyl benzilate and [125I]-(-) iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,1-propranolol on [3H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of 1-quinuclidinyl benzilate on [125I]-(-)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.

Beta adrenoceptor function in central airway smooth muscle of patients with chronic airflow obstruction was investigated by radioligand binding studies and isoprenaline relaxation experiments. Receptor characteristics were determined in tracheal smooth muscle preparations obtained at necropsy from 12 patients and in bronchial tissue obtained at thoracototomy from 21 patients with chronic airflow obstruction. Receptor characteristics were compared with those obtained in airway tissue preparations from 65 control subjects without chronic airflow obstruction. The number of beta adrenoceptors, their binding affinity for the radioligand [125I]-(-)-cyanopindolol, and the tissue binding characteristics of isoprenaline were similar in tissue from patients with chronic airflow obstruction and from control subjects. Isoprenaline induced relaxation of tracheal smooth muscle without precontraction by methacholine showed slightly (though not significantly) less sensitivity to isoprenaline in patients with chronic airflow obstruction than in control subjects (mean (SEM) pD2--the negative logarithm of the concentration producing 50% relaxation--6.32 (0.16) v 6.62 (0.15)). The same pattern of pD2 values was found in segmental bronchial strips without precontraction by methacholine (chronic airflow obstruction 6.55 (0.27), control 7.14 (0.12)). Isoprenaline relaxation in segmental bronchial strips when contracted maximally was significantly less in the patients with airflow obstruction than in the control subjects (pD2 value 5.99 (0.18) v 6.45 (0.07)). These results suggest that beta adrenoceptors in airway smooth muscle of patients with chronic airflow obstruction are not abnormal in number or in binding affinity but that there is less effective coupling between components of the relaxant system distal to the beta adrenoceptor. The possibility that the reduced isoprenaline sensitivity is a consequence of previous bronchodilator treatment cannot be excluded.

Specific binding of [125I]-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (Kd = 5.3 +/- 0.9 pmol/l and RT = 78 +/- 7 fmol/g tissue). The beta 1-selective antagonists atenolol and LK 203-030 inhibited specific [125I]-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous beta 2-adrenoceptor population. In one subject using LK 203-030 a small beta 1-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pKH- and pKL-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD2-value of 6.63 +/- 0.19.

Muscarinic receptors in human tracheal smooth muscle were characterized by radioligand binding and functional studies. Specific [3H]-(-)-quinuclidinylbenzilate ([3H]-(-)-QNB) binding to tracheal smooth muscle membranes was reversible, stereoselective and of high affinity (Kd = 47 +/- 4 pmol/l; RT = 920 +/- 120 fmol/g tissue). Inhibition of specific [3H]-(-)-QNB binding by the M-1 selective antagonist pirenzepine was found to occur at relative high concentrations classifying the muscarinic receptor population as belonging to the M-2 subclass. Inhibition of specific [3H]-(-)-QNB binding by muscarinic agonists revealed the presence of high and low affinity sites in nearly equal proportions. 5'-Guanylylimidodiphosphate converted high affinity sites into low affinity sites although its effect was minimal. Log dose-contraction curves of methacholine had Hill coefficients of 1.10 +/- 0.04 with pD2-values of 6.75 +/- 0.02. Inhibition of specific [3H]-(-)-QNB binding by methacholine, however, was best described by a two binding site model with pKi-values considerably lower. The difference between these affinity values points to the presence of substantial receptor reserve.

To visualize muscarinic receptors in human bronchi, the stripping film method was used which permits direct autoradiographic localization of tissue labeling. Cryostate sections of human bronchi were fixed in 0.5% glutaraldehyde in Krebs-Ringer buffer, pH 7.0 for 30 min at 0 degrees C, washed in Krebs-Ringer buffer for 20 min at 0 degrees C and incubated with (-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] for 90 min at 37 degrees C. Specific (-)-[3H]QNB binding to tissue sections was saturable (receptor density of 0.14 +/- 0.03 fmol/tissue section) and of high affinity (Kd of 40 +/- 9 pM). For autoradiography, labeled tissue sections were covered with stripping film and exposed for 5 months. Muscarinic receptors in human bronchi were located predominantly in submucosal glands and parasympathetic ganglia. There was less labeling in smooth muscle cells and nerve bundles. Epithelium and blood vessels located within the bronchial wall were devoid of specific labeling.

Sucrose gradient centrifugation was used to isolate membranes enriched in muscarinic receptors from bovine brain stem. Unlike the receptors in crude synaptosomal preparations of this tissue, the enriched preparations displayed only low-affinity pirenzepine binding. Similar results were obtained when purified preparations were preincubated for 1 hr in pH 7.0 buffer at 37 degrees C; however, preincubation in a pH 5.0 buffer partially restored the high-affinity pirenzepine binding. These results suggest that an endogenous factor, which is present in the crude synaptosomes of the brain stem and is removed by sucrose gradient centrifugation, is involved in maintenance of the high-affinity pirenzepine binding of the muscarinic receptors.

The hypothesis of an increased muscarinic receptor sensitivity in airway musculature of patients with asthma, chronic obstructive bronchitis and emphysema was investigated through methacholine-induced contraction of isolated airway smooth muscle strips. Contractile responses were recorded isotonically in tracheal smooth muscle preparations of 5 patients with chronic obstructive bronchitis, 2 patients with emphysema and 1 patient with allergic asthma, as well as in bronchial tissue preparations of 7 patients with chronic obstructive bronchitis. The responses were compared to those obtained in airway tissue preparations of 25 control subjects. The sensitivity to methacholine was normal in all groups of patients. This suggests that muscarinic receptor behaviour is normal in airway smooth muscle of patients with obstructive airway disease.

Chemical modification of muscarinic M1 receptors in a synaptoneurosomal preparation of rat cerebral cortex by a hydrophilic histidyl-group-specific reagent, diethylpyrocarbonate (DEP), reduces the number of [3H]-4NMPB binding sites in a dose-dependent way. The effect can be reversed by hydroxylamine treatment. No such effect is observed when carbethoxylation with 2.5 mM DEP is carried out in the presence of atropine, 4NMPB, pirenzepine or carbachol. These findings indicate that DEP specifically modifies histidyl residue(s) positioned at the binding site in members of the M1 receptor family. However, treatment with 2.5 mM DEP in the presence of various muscarinic ligands significantly disturbs the binding state of agonists. The results suggest that M1 receptors may have more than one histidyl residue of importance in ligand binding.

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (-)-[3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.

The hypothesis of an increased muscarinic receptor sensitivity in airway musculature of patients with chronic airflow obstruction (CAO) has been investigated by in vitro radioligand binding studies. The receptor binding profiles were determined in membrane homogenates of tracheal and main bronchial smooth muscle, as well as in segmental bronchial tissue preparations. The number of receptors was measured by binding of the radioactive muscarinic antagonist [3H]-(-)-quinuclidinyl benzilate ([3H]-(-)-QNB). The affinity for agonists was investigated by studying the inhibition of [3H]-(-)-QNB binding by the full muscarinic agonist methylfurtrethonium. Binding characteristics were determined (1) in tracheal smooth muscle from 7 patients with chronic airflow obstruction (2) in main bronchial smooth muscle of 5 patients with CAO and (3) in segmental bronchial tissue of 10 patients with CAO. The receptor binding properties were compared with values obtained on airway tissue from 31 subjects without CAO. Muscarinic receptors in smooth muscle preparations of trachea and main bronchus were present in normal density, and showed normal [3H]-(-)-QNB binding affinity and methylfurtrethonium binding properties. In segmental bronchial tissue preparations which contain smooth muscle and glandular tissue, also normal receptor numbers and [3H]-(-)-QNB affinity values were found. Morphometric examination of these preparations revealed normal amounts of smooth muscle and submucosal glands. This suggests that the excessive mucus production of CAO is not accompanied by an increased muscarinic receptor density in submucosal glands. These observations suggest unaltered muscarinic receptor characteristics in central airway smooth muscle of patients with chronic airflow obstruction.