Pharmacological characterization of the muscarinic receptor subtype mediating contraction of human peripheral airways

The postjunctional muscarinic receptors mediating contraction of human bronchial smooth muscle have been characterized using four nonselective muscarinic receptor agonists and eight subtype selective and nonselective muscarinic antagonists. Carbachol, methacholine, oxotremorine M and (+)-cis-dioxolane all caused concentration-related contractions of human bronchial smooth muscle with a rank order of potency (pD2) of (+)-cis-dioxolane (7.3 +/- 0.2) > oxotremorine M (6.7 +/- 0.2) > carbachol (6.4 +/- 0.1) > methacholine (5.8 +/- 0.2, n = 5 for all). Maximum contractions were not significantly different between agonists, whether expressed as absolute my tension changes or as a percentage of the maximum response to 0.3 mM histamine. Antagonist apparent affinities (pKB) were determined against carbachol-induced contractions and the following rank order was obtained; 4-DAMP (9.4 +/- 0.3) > or = atropine (9.1 +/- 0.1) > zamifenacin (7.6 +/- 0.1) > hexahydrosiladifenidol (HHSiD; 7.1 +/- 0.1) > or = himbacine (7.0 +/- 0.3) > or = pirenzepine (6.8 +/- 0.2) > para-fluoro-hexahydrosiladifenidol (p-F-HHSiD; 6.7 +/- 0.1) > methoctramine (5.3 +/- 0.2). This rank order of antagonist affinities is consistent with activation of M3 receptors. The affinities of HHSiD, p-F-HHSiD and zamifenacin were, however, lower than those reported in guinea pig trachea.     

Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization

The most popular pretreatment method of plasma samples for the measurement of ascorbate (AsA) and dehydroascorbate (DHA) has been an acidic deproteinization via metaphosphoric acid or trichloroacetic acid. In general, DHA is absent in plasma samples prepared from human blood in a conventional manner. However, when these plasma samples were subjected to acidic deproteinization, DHA was detected in the acidified sample solutions. In the present study, we demonstrate that the oxidation of AsA to DHA in the solutions was promoted by at least two mechanisms, one involving catalysis by ferric ion released from transferrin, and the other involving catalysis by plasma hemoglobin. In the acidified transferrin solution by trichloroacetic acid, an oxidation of AsA to DHA proceeded with standing time, whereas the oxidation was not observed in that by metaphosphoric acid. This oxidation appeared to be catalyzed by ferric ion released from transferrin. In contrast, plasma hemoglobin functioned as a catalyst for AsA oxidation in both metaphosphoric acid and trichloroacetic acid solutions. Therefore, DHA content in the trichloroacetic acid-treated plasma sample was markedly higher than that in the metaphosphoric acid-treated one. These results suggest that DHA detected in acidified plasma samples is an artifact resulting from AsA oxidation.     

Immunoelectronmicroscopic analysis of the ligand-induced internalization of the somatostatin receptor subtype 2 in cultured human glioma cells

We analyzed the internalization of the receptor subtype 2 (sst2) for the neuropeptide somatostatin in glioma cells at the ultrastructural level using an antibody against an extracellular amino acid sequence. Intact cells derived from solid human gliomas or those of the human glioma cell line U343 were receptor-labeled (a) by classical gold immunocytochemistry using a 15-nm gold-labeled second antibody, (b) directly with the sst2 antibody adsorbed to 5-nm colloidal gold, and (c) with the physiological ligand somatostatin conjugated to 5-nm colloidal gold. The receptor was predominantly internalized via uncoated vesicles budding from the cell membrane but only rarely via coated pits, which has been mostly reported for G-protein-coupled, seven transmembrane-domain receptors. In the presence of ligand and sst2 antibody vesicles, tubule-like structures, and multivesicular bodies were labeled in superficial and in perinuclear portions of the cells within the first 30 min. Lysosomal labeling was observed after 30 min and especially after an hour of internalization time. This internalization route is also used to study the directly labeled sst2 antibody or the labeled ligand. However, the late endosomal compartment appears to be reached more rapidly in these latter experiments.     

Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes

We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell lines, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytohemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in autocrine and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.     

Endothelin subtype A receptor antagonist induces osteopenia in growing rats

Previous studies suggested that endothelin (ET) peptides are involved in bone metabolism. We examined the effects of long-term blockade of the ET(A) receptor, a receptor subtype primarily involved in the anabolic actions of ET, on bone mineral status in growing rats. Eight-week-old rats injected intraperitoneally with FR139317 50 mg/kg body weight, a specific ET(A) receptor antagonist, for 2 or 4 weeks were compared with control rats injected with vehicle only. Treatment with FR139317 caused a significant decrease in bone mass in the lumbar spine as determined by dual-energy x-ray absorptiometry (DXA). FR139317-induced osteopenia was associated with a significant decrease in the serum osteocalcin concentration but no change in the urinary excretion of pyridinium cross-links of collagen. Our findings indicate that long-term blockade of the ET(A) receptor reduces bone formation and induces osteopenia in growing rats. Our results suggest that ET produced by vascular endothelial cells plays an important role in bone growth and metabolism in vivo.     

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.     

A novel epitope tag for the detection of rabGTPases

Rab GTPases are localized on the cytoplasmic surface of most intracellular organelles where they play a role in the regulation of vesicular transport. As it has been difficult to detect endogenous rab proteins by morphological methods, their localizations were often inferred from transfection experiments using epitope-tagged constructs. Because most of the available epitope tags are only recognitzed by mouse monoclonal antibodies they are often not suitable for double or triple label immunocytochemistry. To overcome this problem, we generated antibodies against a novel 10 amino acid X31 influenza hemagglutin epitope (NH). We here characterized these antibodies and document their utility for detecting early endosomal rab proteins N-terminally tagged with the NH decapeptide in morphological and biochemical assays.     

Molecular modeling of the interaction of diagnostic radiopharmaceuticals with receptor proteins: m2 antagonist binding to the muscarinic m2 subtype receptor

Models of the m2 muscarinic receptor have been built and acetylcholine and an antagonist of the quinuclidinyl benzilate family docked to the putative active site. We have incorporated aspects of homology, site-directed mutagenesis studies and structure-activity studies of specific lead compounds in the construction of our receptor models with a primary focus on the structure of the binding sites. We have observed a deep pocket binding of 5-BrQNT, suggesting a plausible explanation for the observation that agonists and antagonists do not bind competitively. The results of these computational studies are interpreted within the context of the observed in vitro results. Our goal is to assist in the development of subtype receptor selective radiopharmaceuticals for use in PET and SPECT.     

The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

Agonist and antagonist-dependent internalization of the human vasopressin V2 receptor

In this report we demonstrate that in HEK293 cells stably expressing the human V2 vasopressin receptor, ligand-induced internalization of the hormone receptor occurs via the clathrin-dependent pathway. Studies of receptor trafficking either by direct visualization of the V2 receptor by confocal microscopy or binding experiments show a rapid internalization (half-time 6-7 min). Blocking of the clathrin-dependent pathway by hypertonic sucrose increased vasopressin-induced cellular cAMP production and decreased the desensitization of the V2 receptor-adenylyl cyclase system. Thus, internalization appears to be a major regulatory mechanism terminating vasopressin action in HEK293 cells. Two antagonists of the vasopressin V2 receptor exerted different effects on receptor internalization, as determined by confocal fluorescence microscopy. The nonpeptidic antagonist OPC31260 did not induce any visible receptor internalization, whereas the peptidic antagonist d(CH2)5[D-Tyr(Et)2,Val4,Lys8,Tyr-NH29]VP induced a slow but substantial receptor internalization. These results suggest that long-term treatment with peptidic V2 receptor antagonists might lead to desensitization.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.     

Corticotropin-releasing hormone receptor subtype 1 is significantly up-regulated at the time of labor in the human myometrium

Circulating concentrations of CRH rise late in human pregnancy, reaching a peak at labor. The presence of functional CRH receptors, CRH-R1 and CRH-R2, in the human myometrium suggests that CRH may modulate uterine activity. We hypothesized that the number of CRH receptors would be higher in myometrium than fetal membranes (FM) and would change during labor. Myometrial samples were collected from the lower segment (LS) in nonpregnant, preterm (32 +/- 2 weeks), and term (39 +/- 1.6 weeks) pregnant patients before and at labor. Fundus and LS samples were also collected from nonpregnant, pregnant, laboring, and postpartum women. FM were collected at term and at labor. We identified CRH receptors in myometrium and FM by semiquantitative RT-PCR and immunohistochemistry. CRH-R1 messenger ribonucleic acid (mRNA) in the LS was decreased in pregnancy and increased significantly in both preterm and term labor (P < 0.05), but remained unchanged in the fundus. CRH-R2 mRNA was present in 28% of LS myometrium with no change at labor. CRH-R1 and CRH-R2 protein was localized to myometrial smooth muscle in nonpregnant and laboring patients, with lower levels at term. CRH-R1 mRNA was present in chorion and decidua, but CRH-R2 was undetectable in these tissues. We conclude that CRH-R1 is expressed preferentially in myometrium and FM. Changes in CRH receptors during labor are consistent with CRH mediating effects on myometrial activity.     

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons

Agonist-induced endocytosis and recycling of G protein-coupled receptors contributes to desensitization and resensitization of the receptors. In this study, we have used fluorescence immunohistochemistry, confocal microscopy and digital image analysis to quantify the proportion of receptor in the cytoplasm and on the surfaces of nerve cells in the guinea-pig ileum. With these methods we examined the dynamics of internalization of the neurokinin 1 receptor in response to agonist, return of receptor to the cell membrane and its capacity to be re-internalized in response to further exposure to agonist. The basal level of neurokinin 1 receptor immunoreactivity in the cytoplasm was 12-15% of total cellular immunoreactivity. Concentration-response relations were generated for neurokinin 1 receptor internalization after incubation of isolated ileum with 10(-11) to 10(-6) M substance P at 4 degrees C and warming to 37 degrees C for 20 min. The threshold concentration for cytoplasmic receptor to exceed baseline was 10(-11) M and the proportion of receptor in the cytoplasm increased with increasing substance P concentration. The effect of two exposures to agonist was studied using 10(-8) M and 10(-6) M substance P. After equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C with no substance P, neurokinin 1 receptor immunoreactivity in the cytoplasm increased significantly from 12% to 36+/-3% for incubation with 10(-8) M and to 64+/-3% for 10(-6) M. When return of receptor to the surface was blocked with monensin (10(-5) M), 90% of the receptor was in the cytoplasm after 1 h at 37 degrees C following exposure to 10(-6) M substance P. After 60 min without substance P and no monensin, receptor in the cytoplasm decreased to 19+/-2% (10(-8) M) and 38+/-4% (10(-6) M). A second period of equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C, without substance P, resulted in a second wave of endocytosis; the fractions of receptor in the cytoplasm were 47+/-2% (10(-8) M) and 70 2% (10(-6) M). These results indicate that most of the receptors on the cell surface are available for internalization and that the receptors that return to the cell surface after endocytosis rapidly regain their ability to bind ligand and undergo endocytosis.     

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.     

Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis

We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post-transplant and declined after 6 weeks, while human anti-human platelet antibodies were detected 2-8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross-reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP.     

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin-receptor interaction. The muscarinic antagonist N-methyl-[3H]scopolamine was used as a 'reporter' ligand. Evidence for a mechanism of toxin-receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin-receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.     

Structure of the m1 muscarinic acetylcholine receptor gene and its promoter

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.     

Alpha 1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists

Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha1-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha 1A receptors were created by systematically substituting individual transmembrane domains from alpha 1D adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha 1A/D(TM2), and moderate reductions in affinity versus constructs alpha 1A/D(TM5), alpha 1A/B(TM5), and alpha 1A/D(TM6). Antagonist affinities for these chimeras exceeded those found for wild type alpha 1D and alpha 1B. Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha 1A receptor have a major impact on receptor subtype selectivity.