Advanced glycation end products (AGEs) activate mast cells.

BACKGROUND AND PURPOSE: Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. EXPERIMENTAL APPROACHES: Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. KEY RESULTS: Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca(2+) increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. CONCLUSIONS AND IMPLICATIONS: Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.

Activation of CD47 receptors causes histamine secretion from mast cells.

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.

Heptahelical and other G-protein-coupled receptors (GPCRs) signaling.

Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G-proteins) which transduce most signals through their alpha and betagamma subunits to effectors, enzymes and ion channels. Of the 367 heptahelical receptors for endogenous ligands, about 330 are potential targets for drug discovery with agonist, antagonist or inverse agonist properties. The term G-protein-coupled receptors (GPCRs) is a broader functional definition rather than a structural one referring to heptahelical receptors specifically. Non-heptahelical putative GPCRs include some transmembrane receptors with tyrosine-kinase activity on their cytosolic endings (EGF, insulin and IGF-1 receptors), other transmembrane receptors (mannose-6-phosphate/IGF-2 receptor and integrin-associated protein IAP or CD47), and some receptors belonging to the class of glycosylphosphatidylinositol (GPI)-anchored proteins and located on the outer face of the plasma membrane. Also, activators of G-protein signaling (AGS) proteins that regulate vesicular trafficking activate heterotrimeric G-proteins in the Golgi independently of receptor activation. Main effectors activated through their direct interactions with alpha subunits or betagamma dimers of heterotrimeric G-proteins include adenylylcyclases, cGMP-phosphodiesterase, phospholipases Cbeta, phosphoinositide 3-kinase gamma, Ca(V2) calcium channels, GIRK/Kir3 potassium channels, and guanine nucleotide exchange factors RasGEF and RhoGEF leading to small G-proteins and MAP-kinases activation. Current signaling cascades leading to final cell responses are depicted.

Heterotrimeric G proteins control diverse pathways of transmembrane signaling, a base for drug discovery.

Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G proteins) which transduce most signals through their alpha and betagamma subunits to effectors including adenylylcyclases, ion channels, phospholipases Cbeta, and phosphoinositide 3-kinases. The diversity of G proteins, their effectors and regulators (RGS proteins), supports the interest of these protein families as potential drug targets.

Activation of betagamma subunits of G(i2) and G(i3) proteins by basic secretagogues induces exocytosis through phospholipase Cbeta and arachidonate release through phospholipase Cgamma in mast cells.

Mast cells are activated by Ag-induced clustering of IgE bound to FcepsilonRI receptors or by basic secretagogues that stimulate pertussis toxin-sensitive heterotrimeric G proteins. The cell response includes the secretion of stored molecules, such as histamine, through exocytosis and of de novo synthesized mediators, such as arachidonate metabolites. The respective roles of G proteins alpha and betagamma subunits as well as various types of phospholipase C (PLC) in the signaling pathways elicited by basic secretagogues remain unknown. We show that a specific Ab produced against the C-terminus of Galpha(i3) and an anti-recombinant Galpha(i2) Ab inhibited, with additive effects, both exocytosis and arachidonate release from permeabilized rat peritoneal mast cells elicited by the basic secretagogues mastoparan and spermine. A specific Ab directed against Gbetagamma dimers prevented both secretions. Anti-PLCbeta Abs selectively prevented exocytosis. The selective phosphatidylinositol 3-kinase inhibitor LY 294002 prevented arachidonate release without modifying exocytosis. Gbetagamma coimmunoprecipitated with PLCbeta and phosphatidylinositol 3-kinase. The anti-PLCgamma1 and anti-phospholipase A(2) Abs selectively blocked arachidonate release. Protein tyrosine phosphorylation was inhibited by anti-Gbetagamma Abs, LY294002, and anti PLCgamma1 Abs. These data show that the early step of basic secretagogue transduction is common to both signaling pathways, involving betagamma subunits of G(i2) and G(i3) proteins. Activated Gbetagamma interacts, on one hand, with PLCbeta to elicit exocytosis and, on the other hand, with phosphatidylinositol 3-kinase to initiate the sequential activation of PLCgamma1, tyrosine kinases, and phospholipase A(2), leading to arachidonate release.

[Control of bronchial contractility, regulation of receptor activity and expression]. / Contrôle de la contractilité bronchique, régulation de l'activité et de l'expression des récepteurs.

Heptahelical receptors mediate contractile and relaxant responses of bronchial smooth muscle cells to mediators and drugs. The activity of these receptors is constitutively regulated through the equilibrium between their inactive conformation R and their active conformation R*, coupled to G proteins. This equilibrium is shifted to R* by agonists, with a relaxant response to beta-adrenergic agonists and a contractile response to muscarinic agonists. In contrast, inverse agonists shift this equilibrium to R with, for instance, a putative relaxant response to muscarinic inverse agonists. The expression of heptahelical receptors at the plasma membrane correspond to the equilibrium between the endocytosis of membrane receptors and the synthesis of receptor mRNA and protein. Endocytosis is activated by agonists and decreased by inverse agonists. The transcriptional regulation of heptahelical receptors in bronchial smooth muscle cells is tightly controlled by pro-inflammatory cytokines. The variety of the involved control pathways might explain the spasmogen-selective hyperresponsiveness, allowing a reappraisal of the dogma of non selective bronchial hyperresponsiveness in asthma.

Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential.

Inverse agonists (negative antagonists) are ligands that stabilize the inactive conformation (R) of receptors according to the two-state receptor model. The active conformation (R*) of heptahelical receptors, i.e. G protein-coupled receptors, has high affinity for G proteins. According to ternary complex models of receptor activation, the R*G complex is in equilibrium with R + G, with spontaneous activity in the absence of agonist. Inverse agonists, having a higher affinity for R, shift R*G towards R + G, decreasing the spontaneous activity of receptors. Agonists have the opposite effect, with a higher affinity for R*. Neutral antagonists have the same affinity for R and R* and compete for both agonists and inverse agonists. Inverse agonists have been recently proposed for a variety of heptahelical receptors. Methods to detect inverse agonists among antagonists are based on the determination of ligand affinity at R and R* with binding experiments, and on the modulation of G protein activity (GTP binding and hydrolysis) or of effector activity. Receptor inverse agonists, but also G protein antagonists and GTPase inhibitors, decrease spontaneous G protein activity corresponding to R*G. Receptor agonists, G protein agonists and GTPase inhibitors increase effector basal activity, but receptor inverse agonists decrease it. The therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors and may be extended to diseases related to wild-type receptor over-expression leading to the increase of R*. Some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Inverse agonists lead to receptor upregulation, offering new approaches to tolerance and dependence to drugs.

PLA2 phosphorylation and cyclooxygenase-2 induction, through p38 MAP kinase pathway, is involved in the IL-1beta-induced bradykinin B2 receptor gene transcription.

We hypothesized that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity to bradykinin observed in asthmatic patients. We reported previously that IL-1beta induced a prostanoid-dependent increase in the density of bradykinin B2 receptors in cultured human bronchial smooth muscle cells. Our experiments demonstrate that the rapid prostaglandin E2 (PGE2) synthesis induced by IL-1beta is abolished by cycloheximide, suggesting the involvement of protein synthesis. The formation of PGE2 is preceded by the phosphorylation of cPLA2 and the expression of cyclooxygenase-2 (Cox-2). The inhibition of p38 MAP kinase inhibited PGE2 synthesis, cPLA2 phosphorylation and abolished Cox-2 expression. The inhibition of Cox-2 expression correlated with a decrease of bradykinin B2 receptor expression. These data demonstrate that the activation of p38 MAP kinase elicited by IL-1beta leads to the phosphorylation of cPLA2 and Cox-2 overexpression, allowing rapid synthesis of PGE2 as a prerequisite for bradykinin B2 gene expression in human bronchial smooth muscle cells which could explain the hyperresponsiveness of asthmatic patients to bradykinin.

Effect of NMDA receptor ligands on mast cell histamine release, a reappraisal.

Natural polyamines have been proposed to induce histamine release from mast cells through a direct interaction with G proteins. Alternatively, the polyamine binding site of ionotropic N-methyl-D-aspartate (NMDA) receptors has been suggested as a target for spermine on mast cells. We reexamined both hypotheses. Incubation of rat peritoneal mast cells with spermine resulted in a concentration-dependent histamine release (EC50 270 microM). Incubation with NMDA receptor agonists, glutamate or NMDA, associated to the co-agonist glycine, did not induce secretion. Western blot experiments did not reveal NMDA R1, R2a, R2b or R2c subunit expression in rat peritoneal mast cell membranes. The NMDA receptor antagonist at the glycine site, L-689,560, did not modify, at relevant concentrations, the spermine-induced secretion. The NMDA receptor antagonists, ifenprodil and LY 235959, and the NMDA channel blocker, MK801, slightly inhibited, at high concentrations, the secretory effect of spermine. The polyamine arcaine, an antagonist of the NMDA receptor polyamine binding site, induced histamine secretion (EC50 350 microM). Both spermine- and arcaine-induced effects were independent upon extracellular calcium and were largely inhibited by treatment of mast cells with pertussis toxin or benzalkonium chloride. The response to spermine and arcaine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase, and was restored by permeabilization of the plasma membrane with streptolysine-O, indicating that polyamines act intracellularly. These results confirm the involvement of pertussis toxin-sensitive G proteins in the secretory effect of polyamines and demonstrate the absence of NMDA receptors on rat peritoneal mast cells. Nonselective effects of some NMDA receptor ligands on mast cells cannot be excluded.

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine.

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.

Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors.

1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*<R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.

Glucocorticoids increase bradykinin B2 receptor gene transcription in cultured guinea-pig tracheal smooth muscle cells.

We investigated the effect of the glucocorticoid methylprednisolone on the modulation of the expression of the bradykinin B2 receptors in cultured, guinea-pig, tracheal, smooth muscle cells. These receptors are implicated in the pathogenesis of human asthma. Untreated cells expressed a single population of binding sites for [3H]bradykinin with a dissociation constant, Kd, of 87.7+/-12.0 pM and a maximum binding site density, Bmax, of 245.4+/-71 fmol/mg protein. Treatment of the cultured guinea-pig tracheal smooth muscle cells with methylprednisolone 10(-5) M for 6 h increased the number of bradykinin receptors; this response reached a maximum of 78% and returned to the basal value after 12 h. Bradykinin (10(-12) M) elicited a six-fold higher calcium level in treated cells than in control cells. To investigate bradykinin B2 receptor mRNA expression in guinea-pig cells, we used the reverse transcription polymerase chain reaction (RT-PCR) technique to synthesize a specific bradykinin B2 cDNA probe of 296 bp corresponding to nucleotides 456-751 of the human sequence. This guinea-pig cDNA had 88%, 86% and 83% homology with the corresponding human, mouse and rat sequences, respectively, but no homology with any other known sequences. Following methylprednisolone treatment, Northern blot hybridization indicated that mRNA increased fourfold after 3 h compared with control cells, and returned to basal level within 7 h. The rate of gene transcription, assessed by nuclear run-on assays, increased fourfold after 3 h treatment with 10(-5) M methylprednisolone. These results indicate that glucocorticoids induce early up-regulation of bradykinin B2 receptors in cultured guinea-pig tracheal smooth muscle cells by increasing the rate of transcription of the bradykinin B2 receptor gene.

Glucocorticoids inhibit the bradykinin B2 receptor increase induced by interleukin-1beta in human bronchial smooth muscle cells.

We studied the effect of the glucocorticoids, dexamethasone and budesonide, on the interleukin-1beta-induced increase of bradykinin B2 receptors in cultured human bronchial smooth muscle cells, a cellular model of bronchial hyperreactivity. Both compounds prevented the increase of the bradykinin B2 mRNA and the bradykinin-induced inositol phosphate accumulation. These results demonstrate a direct effect of glucocorticoids on airway smooth muscle hyperresponsiveness mediated through inhibition of the over-expression of receptors for contractile mediators induced by inflammatory mediators.

Interleukin-1beta induces bradykinin B2 receptor gene expression through a prostanoid cyclic AMP-dependent pathway in human bronchial smooth muscle cells.

We investigated the hypothesis that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity of asthmatic patients to bradykinin. In cultured human bronchial smooth muscle cells, IL-1beta elicited a rapid and transient increase in the density of bradykinin B2 receptors without affecting their affinity for ligands. The increase in B2 receptors was correlated to an enhancement of inositol phosphate formation elicited by bradykinin, indicating its relevance to the contractile response of smooth muscle cells to bradykinin. The increase in receptor density was related to an increase in B2 receptor mRNA level corresponding to a 5-fold enhancement of the transcriptional rate and to a lengthened half-life of mRNA. These effects of IL-1beta were largely inhibited by indomethacin, suggesting the involvement of a prostanoid pathway in IL-1beta transduction process. An increase in prostaglandin E2 levels preceded the mRNA increase, confirming this involvement. Moreover, IL-1beta and prostaglandin E2 led to cAMP formation. We propose this predominant transduction pathway of IL-1beta to stimulate the transcription of the bradykinin B2 gene in human bronchial smooth muscle cells as a major mechanism involved in the hyperresponsiveness of asthmatic patients to bradykinin.

The M2 muscarinic receptor antagonist methoctramine activates mast cells via pertussis toxin-sensitive G proteins.

Methoctramine, a selective M2 muscarinic cholinergic receptor antagonist, has been reported to activate phosphoinositide breakdown at high concentrations. Its polyamine structure suggests a putative activation of guanine nucleotide-binding proteins (G proteins). Incubation of methoctramine with rat peritoneal mast cells resulted in a dose-dependent noncytotoxic histamine release, with an EC50 of 20 microM and a maximum effect at 1 mM. Atropine, pirenzepine and HHSiD neither inhibited methoctramine-induced histamine release nor stimulated histamine release. Histamine release and inositol phosphates generation induced by methoctramine were both inhibited by pertussis toxin pretreatment. Benzalkonium chloride, a selective inhibitor of histamine secretion induced by basic secretagogues, inhibited the secretory response to methoctramine. [p-Glu5, D-Trp7,9,l0]-SPs5-11 (GPAnt-2), a well-characterized antagonist of G proteins, blocked the methoctramine-induced histamine release when the antagonist was allowed to reach its intracellular target by streptolysin O-permeabilization. The response to methoctramine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase. The response of mast cells was restored by permeabilization of the plasma membrane. These results demonstrate that methoctramine, following its entry into the cell and the involvement of pertussis toxin-sensitive G proteins, activates phosphoinositide hydrolysis leading to mast cell exocytosis.

Substance P-related inhibitors of mast cell exocytosis act on G-proteins or on the cell surface.

[p-Glu5,D-Trp(7,9,10)]substance P-(5-11) inhibited mastoparan-stimulated GTPase activity in homogenized rat peritoneal mast cells and decreased histamine secretion induced by mastoparan from streptolysin O-permeabilized mast cells (IC50 of about 30 microM), but not from intact cells. In contrast, [D-Pro4,D-Trp(7,9,10)]substance P-(4-11) inhibited the secretion from intact cells (IC50 of about 10 microM) but had no effect on histamine secretion from permeabilized cells, suggesting that this peptide exerts its inhibitory effect on the plasma membrane, whereas [p-Glu5,D-Trp(7,9,10)]substance P-(5-11) interacts with G proteins. Pretreatment of mast cells with neuraminidase led to an inhibition of the secretory response to mastoparan and related triggers. This response was restored following cell permeabilization, demonstrating the role of the cell surface on the entry of mastoparan and related triggers and on their ability to reach G proteins sensitive to pertussis toxin and [p-Glu5,D-Trp(7,9,10)]substance P-(5-11).

Drugs interacting with G protein alpha subunits: selectivity and perspectives.

Extracellular signal molecules as diverse as hormones, neurotransmitters and photons use a signal transduction pathway involving a receptor, a G protein and effectors. Compounds that interact directly with G proteins can mimic the receptor-G protein interaction or can block the activation of G proteins by receptors. Several binding sites exist on the G alpha protein that may be exploited for the design of synthetic stimulatory or inhibitory ligands. The effector binding site is regulated by endogenous proteins and appears to be a target for selective exogenous ligands. The GTP binding site presents a large homology within the G protein families and therefore the nucleotide analogs might not be considered as a tool to discriminate between the G protein subclasses. In contrast, different experimental strategies have substantiated the specificity in the interaction between a receptor and a G protein, the receptor binding site of G proteins should be considered as potential drug targets. Drugs interfering with this site such as mastoparan and related peptides, GPAnt-2 and suramin, are lead compounds in the design of selective G protein antagonists. Benzalkonium chloride and methoctramine have agonist or antagonist properties, depending on G protein subtypes. Such compounds would be very useful to delineate the functions of G proteins and G protein-coupled receptors, to understand some side effects of drugs used in therapy and to develop new therapeutic agents.

Effect of WIN 64338, a B2 bradykinin receptor antagonist on guinea-pig tracheal smooth muscle cells in culture.

This study investigated the effect of the non-peptidic B2 bradykinin (BK) receptor antagonist WIN 64338 on BK binding and BK-induced inositol phosphate formation on guinea-pig tracheal smooth muscle cells in culture. The presence of specific and saturable binding sites for BK was demonstrated using [3H]BK. Scatchard analysis indicates a single population of binding sites for [3H]BK with a maximal density (Bmax) of 245.4 +/- 71 fmol/mg of protein and an equilibrium dissociation constant (Kd) of 87.7 +/- 0.12 pM. The order of potency of] B2 BK receptor ligands was Hoe 140 = NPC 17731 > BK > WIN 64338 > D- Arg0[Hyp3, D-Phe7]-BK > > des-Arg9-BK, while B1 BK receptor antagonist, des-Arg9-[Leu8]-BK, was without effect on [3H]BK binding. These results demonstrate the presence of B2 Bk receptors on cultured tracheal smooth muscle cells. The cells were stimulated by BK, and inositol phosphate formation was determined by anion exchange chromatography. The stimulating effect of BK on inositol phosphate formation was concentration dependent (1 nM to 10 microM). The B1 BK agonist des-Arg9-BK did not induce inositol phosphate formation. The order of potency of B2 antagonists to inhibit BK-induced inositol phosphate formation was Hoe 140 = NPC 17731 > WIN 64338 > D-Arg0[Hyp3, D-Phe7]-BK. This study demonstrates that WIN 64338 is able to displace [3H]BK binding and to inhibit B2-BK-induced inositol phosphate formation on cultured guinea-pig tracheal smooth muscle cells.

Glucocorticoid modulation of muscarinic and beta-adrenergic receptors in guinea pig lung.

We investigated the effect of the in vivo treatment of guinea pigs with methylprednisolone, 10 mg/kg daily, on lung muscarinic and beta-adrenergic receptors. Receptor densities were assessed by saturation experiments of tritiated N-methylscopolamine and dihydroalprenolol binding to lung membranes. After 3 h of treatment, methylprednisolone induced a decrease of 19.2% (P < 0.05) of muscarinic receptors but was without effect on beta-adrenergic receptor density. After 24 h, an increase of 39.7% (P < 0.01) and 16.9% (P < 0.05) was observed for muscarinic and beta-adrenergic receptors, respectively. For muscarinic receptors, this increase reached 53.4% (P < 0.01) within 48 h and stayed at this level until 96 h. The increase of beta-adrenergic receptors was maximal (24.9%) after 72 h and returned to the control value after 96 h. The dissociation constant (Kd) values of both ligands were not affected by the glucocorticoid treatment. Functional studies showed that the 96 h treatment did not affect the contractile response of guinea pig lung parenchymal strips to carbachol since the 50% concentration value (EC50) and the maximal contraction value (Emax) were not significatively different from control values. These data show that glucocorticoids control the expression of both muscarinic and beta-adrenergic receptors in guinea pig lung but with different time courses and to a larger extent for muscarinic receptors. The glucocorticoid treatment did not modify the contractile response of lung strips to carbachol, confirming the absence of effect on the affinity of muscarinic receptors and suggesting that the receptor reserve exceed the increase of their density by the steroid.

M2-muscarinic receptors: how does ligand binding affinity relate to intrinsic activity?

In this study we looked for evidence regarding a correlation between M2-muscarinic receptor binding affinity and ligand intrinsic activity. Guanine nucleotide-binding protein-coupled receptors have been shown to exist in both a high affinity and a low affinity, agonist state. The agonist [3H]Oxotremorine-M, was used to determine the affinity of compounds for the high affinity state and the antagonist, [3H]N-methylscopolamine, plus GppNHp, was used to determine the affinity for the low agonist state. The magnitude of the difference in the affinity a compound has for the high versus the low agonist state of the receptor has been related to the intrinsic activity of the compound. NMS/Oxo-M ratios were established for muscarinic agonists, partial agonists and antagonists. NMS/Oxo-M ratios varied from 1695 for the agonist carbachol to 1.9 for the antagonist AFDX-166 with intermediate values for the partial agonists oxotremorine-M, pilocarpine and RS86 (233, 36 and 17 respectively). Intrinsic activity was assessed by receptor-mediated Gi-protein GTPase activity. Indeed, a close correlation (r=0.92) was found between the NMS/Oxo-M ratios of the ligands on the one hand, and their ability to activate the M2-receptor coupled Gi-protein on the other.