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.     

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.     

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.     

Phospholipase A2 from Naja naja sputatrix venom is a muscarinic acetylcholine receptor inhibitor

DNA topoisomerase I (topo I) is the molecular target for the camptothecin group of anticancer drugs. These drugs are showing activity against a wide array of human tumors. Many data have indicated that the sensitivity of a tumor cell to the camptothecins is dependent on tumor topo I levels. Drug-sensitive cells have high levels of topo I. Unfortunately, there is still a relative lack of information on topo I levels in human malignancies. Because of this, we investigated topo I activity and immunoprotein levels in a variety of normal murine and human tissues, as well as tissues obtained from several carcinomas, lymphomas, and sarcomas. Flow cytometric analysis was also performed on the neoplastic specimens to determine the percentage of cycling cells. Topo I catalytic activity was detected in all normal tissues at a fairly constant level. The average topo I catalytic activity in normal mammalian tissues was 2.7 +/- 1.3 x 10(4) units/mg protein (range 1.1 to 5.0 x 10(4)). Topo I catalytic activity was much more variable in human malignancies and ranged from a low of 1.4 x 10(4) units/mg protein in a rhabdomyosarcoma to a high of 160 x 10(4) units/mg protein in a poorly differentiated ovarian carcinoma. Western blot analysis with either a mouse monoclonal antibody or scleroderma antibodies directed against topo I revealed that the elevated topo I catalytic activity levels in the malignant tissues are due to elevated amounts of topo I immunoprotein. It is possible that the high topo I levels that characterize several different types of human malignancies might indicate that these tumors would be sensitive to many of the new drugs that target topo I.     

Antagonist discrimination between ganglionic and ileal muscarinic receptors

The effects of four antagonists on the depolarization of isolated superior cervical ganglia and the contraction of isolated ileal segments of the rat were compared. pA2 values estimated from Schild plots indicated significantly higher affinities of stercuronium (x100) and pirenzepine (x23) and a significantly lower affinity of 4-diphenylacetoxy-N-methylpiperidine methiodide (x0.39) for the ganglion than for the ileum. The affinities of N-methylscopolamine for the two tissues were not significantly different. It is concluded that the two types of muscarinic receptor are not identical.     

Combined treatment with a 5HT1A receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior

In the present study, the effects of PACAP27, PACAP38 and VIP in a concentration range from 10(-13) to 10(-6) M were studied in vitro on the spontaneous and directed mobility of lymphocytes from rat spleen and thymus. The results show that VIP and both PACAPs inhibit significantly and in a similar way the mobility of lymphocytes from thymus and spleen, and the maximal effects were observed at 10(-9) M and 10(-8) M. The three neuropeptides significantly increased cAMP concentrations. Moreover, incubation with increasing PMA concentrations showed a progressive enhancement of chemotaxis of lymphocytes, which was partially prevented by VIP, and both PACAPs. Incubation with forskolin caused decrease in the chemotaxis of thymocytes and splenocytes, and the presence of VIP or PACAP peptides was not synergistic in the inhibitory effect on lymphocyte chemotaxis, suggesting that the three neuropeptides and forskolin mediate their actions by the same intracellular pathway. This study showed the ability of the VIP receptor antagonist (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 to partially reverse the inhibitory effect of both PACAPs and VIP on chemotaxis, suggesting that PACAP receptors are identical or very similar to VIP receptors in both thymocytes and splenocytes. These data suggest that PACAP27 and PACAP38 can be included as two novel immunoregulatory peptides that can modulate cell mobility on central and peripheral lymphoid organs.     

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.     

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Allele-specific population structure of Drosophila melanogaster alcohol dehydrogenase at the molecular level

The history of the Drosophila melanogaster alcohol dehydrogenase (ADH) Fast/Slow polymorphism was studied by recording molecular variation and inversion polymorphism in 233 chromosomes from European and African populations. Silent molecular variation in the Slow allele was very different between standard chromosomes and chromosomes bearing the In(2L)t inversion. Within populations, inverted Slow haplotypes were more variable than standard Slow haplotypes. Between populations, geographical structure was almost nonexistent for inverted Slow haplotypes and highly significant for standard Slow. All Fast haplotypes occurred on standard chromosomes. They showed little variation within and between populations. They were highly significantly closer to standard Slow haplotypes from Europe. These results suggest that the current range of Fast and In(2L)t Slow haplotypes is recent and that an older genetic differentiation between populations was followed by allele-specific gene flow.     

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.     

On the unique binding and activating properties of xanomeline at the M1 muscarinic acetylcholine receptor

We investigated the molecular nature of the interaction between the functionally selective M1 muscarinic acetylcholine receptor (mAChR) agonist xanomeline and the human M1 mAChR expressed in Chinese hamster ovary (CHO) cells. In contrast to the non-subtype-selective agonist carbachol, xanomeline demonstrated M1 mAChR binding that was resistant to extensive washout, resulting in a significant reduction in apparent N-[3H]methylscopolamine saturation binding affinity in intact cells. Functional assays, using both M1 mAChR-mediated phosphoinositide hydrolysis and activation of neuronal nitric oxide synthase, confirmed that this persistent binding resulted in elevated basal levels of system activity. Furthermore, this phenomenon could be reversed by the addition of the antagonist atropine. However, pharmacological analysis of the inhibition by atropine of xanomeline-mediated functional responses indicated a possible element of noncompetitive behavior that was not evident in several kinetic and equilibrium binding experimental paradigms. Taken together, our findings indicate for the first time a novel mode of interaction between an mAChR agonist and the M1 mAChR, which may involve unusually avid binding of xanomeline to the receptor. This yields a fraction of added agonist that is retained at the level of the receptor compartment to persistently bind to and activate the receptor subsequent to washout. The results of the current study suggest that elucidation of the mechanism or mechanisms of interaction of xanomeline with the M1 mAChR is particularly important in relation to the potential therapeutic use of this agent in the treatment of Alzheimer's disease.     

Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

New mutants of Drosophila melanogaster

Characteristics are given of 57 Drosophila melanogaster mutants catched in the South and Soeth-West Iran.     

Volatile anaesthetics have differential effects on recombinant m1 and m3 muscarinic acetylcholine receptor function

Muscarinic acetylcholine signalling plays major roles in regulation of consciousness, cognitive functioning, pain perception and circulatory homeostasis. Halothane has been shown to inhibit m1 muscarinic signalling. However, no comparative data are available for desflurane, sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also prominent in the brain) been studied. Therefore, we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes expressing recombinant m1 or m3 muscarinic or (for comparison) AT1A angiotensin II receptors were voltage clamped, and Ca(2+)-activated Cl- currents (ICl(Ca)) induced by acetyl-beta-methylcholine (Mch) or angiotensin II were measured in the presence of clinically relevant concentrations of halothane, sevoflurane, desflurane or isoflurane. To determine the site of action of the volatile anaesthetics we compared anaesthetic effects on m1, m3 and AT1A receptor function and studied the effects of volatile anaesthetics on signalling induced by intracellular injection of the second messenger IP3. Desflurane had a biphasic effect on m1 signalling, enhancing at a concentration of 0.46 mmol litre-1 but depressing at 0.92 mmol litre-1. A similar, although not significant, trend was observed with m3 signalling. Isoflurane had no effect on m1 signalling, but profoundly inhibited m3 signalling. Sevoflurane depressed the function of m1 and m3 signalling in a dose-dependent manner. Halothane, similar to its known effect on m1 signalling, dose-dependently depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four anaesthetics. Similarly, none of the anaesthetics tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on muscarinic signalling most likely result from interactions with the m1 or m3 receptor molecule. Multiple interaction sites with different affinities may explain the biphasic response to desflurane. Anaesthetic-specific effects on closely related receptor subtypes suggest defined sites of action for volatile anaesthetics on the receptor protein.     

An hypothesis concerning the molecular structure of the nicotinic acetylcholine receptor

An hypothesis is presented concerning the molecular structure of the nicotinic acetylcholine receptor at the neuromuscular junction based on the actual amino acid sequence of the N-terminal segment of the alpha-subunit and the Chou and Fasman prediction of secondary structure from the primary sequence. This is mainly in the form of two alpha-helices cross-linked by four ionically bound complementary amino acids (arg/lys to glu). This structure (R) is complementary to a wide range of ACh agonists and to the antagonist beta-erythroidine. If the ionic cross-links are disrupted the two segments can separate by 2-3 A. This new conformation (R1) is now complementary to antagonists of the type of histrionicotoxin. A further separation (approximately 8 A) gives a conformation complementary to antagonist of the type of decamethonium. Experiments to test the hypothesis are suggested.     

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.     

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.     

Calcium and the muscarinic receptor

Pharmacological receptors may be viewed as composed of two linked functions, a recognition site through which the specificity and selectivity of ligand action is expressed and an amplification or catalytic site which translates the ligand-recognition site interaction into response. The successful elucidation of receptor mechanisms requires analysis of both sites. The muscarinic cholinergic receptors of guinea-pig ileal longitudinal smooth muscle will be discussed from this two function stand point and the source and utilization of Ca2+ in excitation-contraction coupling discussed.     

The soluble alpha-mannosidases of Drosophila melanogaster

The alpha-mannosidases are implicated in both the catabolism of carbohydrates and the N-linked glycosylation pathway in insects, but little is known of the biochemistry of these glycosidases. In order to study the soluble alpha-mannosidases of Drosophila melanogaster we have used artificial fluorogenic substrates for detection of activity in situ following non-denaturing gel electrophoresis. This approach also permitted examination of the mannosidases present in different tissues and the sensitivity of the enzymes to known mannosidase inhibitors. Fluorogenic substrates were also used to determine the pH optima of partly purified mannosidases. We report that D. melanogaster contains several soluble alpha-mannosidase activities. Acidic mannosidases were detected in the gut, fat body and haemolymph of third-instar larvae. The major activity detected in larval guts was a neutral mannosidase presumed to be involved in digestion.