Jakob Schmidt

Autoradiographic localisation of α-bungarotoxin-binding sites in the central nervous system

SNAKE α-toxins have high specificity and affinity for nicotinic receptors, and have been used widely for the characterisation of peripheral acetylcholine receptors of electric organs and skeletal muscle. In some cases autoradiography has shown the extent and local distribution of bound neurotoxins1–5. Although α-bungarotoxin (α-BTX) does not pass the blood-brain barrier and therefore does not enter the central nervous system (CNS) to a measurable extent6, central nicotinic acetylcholine receptors may bind α-toxins in vitro, and several investigators have used this toxin to characterise brain receptors7–9. Its specificity for central nicotinic receptors has been inferred not only from its peripheral action, but also from cytological and pharmacological observations: binding activity is highest in synaptosomal preparations, and is inhibited by various nicotinic drugs. Studies on the localisation of toxin-binding sites in brains, not so far reported, would be valuable, not only to corroborate neurotoxin specificity, but also to provide information on the regional distribution of nicotinic receptor sites in the CNS.

Radioautographic localization of125I α-bungarotoxin binding sites in the retinas of goldfish and turtle

There is substantial evidence for the action of acetylcholine (ACh) as a neurotransmitter in the vertebrate retina. Chotineqic mimics and antagonists alter the response properties of optic nerve fibers in rabbit (Ames and Pollen, 19691, cat (Straschill, 1968) and the ERG of frog (Val’tsev. 1966). The hydrolytic enzyme acetylcholinesterase (AChE) is found in the retinas of a wide variety of vertebrates (Francis. 1953; Nichols and Koelie, 1968; Dickson, Fiumerfelt. Hollenberg and Gwyn, 1971). In mammalian and avian retinas AChE is confined largely to the inner plexiform layer (IPL) and amacrine cells (Nichols and KoeIle. 1968). However, in the newt. an amphibian, AChE was found in the outer plexiform layer (OPL) where it was associated with horizontal and bipolar ceII processes (Dickson et al.. 1971). Since the mere presence of AChE is not conclusive proof that a neuron utilizes ACh as a transmitter, additional histological markers are needed if cholinergic transmission is to be demonstrated more convincingly and localized more accurately in the retina. Such an approach has become possible with the discovery that r-bungarotoxin (r-BTX), the principal component of the venom from the banded krait, Btmynrus mdticinctus, binds to nicotinic receptors with high affinity (Chang and Lee, 1963) With radioactively-labeled &TX and the related r-toxin from fVaja nigricollis, the ACh receptor sites at neuroeffector junctions of electric organ (Bourgeois, Ryter, Menez, Fromageot, Boquet and Changeux, 1972) and muscle (Porter, Chiu, Wieckowski and Barnard, 1973; Fertuck and Salpeter, 1974) were localized and quantified. Also, toxin binding sites were described in the chick optic tectum, rat hippocampus, and other structures of the central nervous system (Polz-Tejera, Schmidt and Karten, 1975). It seems reasonable to expect that if ACh is a retinal transmitter, the location ofnicotinic receptors sites could be found by radioautography utilizing labeied Z-BTX.

Two types of receptors for α-bungarotoxin in the synaptic layers of the pigeon retina

Summary Pigeon retinae were analyzed for binding [ 125 I]α-bungarotoxin (αBgt) by radioautographic and biochemical methods. Toxin binding, localized to the outer and inner plexiform layers (OPL and IPL), was inhibited by micromolar concentrations of native αBgt and d -tubocurarine and by 1 m M acetyl- and butyrylcholine in both synaptic layers. Nicotine, at comparable concentrations affected only the IPL. In vitro drug competition experiments showed that the pigeon retina contains two types of receptors for αBgt which differ in sensitivity to inhibition by nicotine by 4 orders of magnitude. The results suggest that: (1) the receptor for αBgt in the IPL is a nicotinic receptor, (2) the receptor for αBgt in the OPL may be involved in an unusual cholinergic system, and (3) ability to bind αBgt is not a sufficient criterion for identifying nicotinic-cholinergic receptors.

Biochemistry of nicotinic acetylcholine receptors in the vertebrate brain.

Publisher Summary This chapter focuses on nicotinic receptors of the vertebrate central nervous system. Receptor are considered to be a ligand-binding protein, regardless of physiological function and signal transduction mechanism, acetylcholine receptor (AChR) to be an acetylcholine (ACh)-binding protein with a synaptic function, and nicotinic receptor denotes a binding protein with a preference for nicotinic compounds. The biochemical characterization of ion channels, such as the one contained within the nicotinic AChR of muscle and suspected as an integral constituent of the neuronal receptor, faces a unique dilemma, namely, the mutually exclusive requirements for solubilization (as a prerequisite for purification) and for maintenance of membrane-bounded compartments (for testing physiological function). This problem is solved by redefining the receptor as a ligand-binding protein and by demonstrating channel properties after purification using suitable membrane reconstitution protocols. The biophysical reconstitution technique, involving insertion of the purified receptor into lipid bilayers or vesicles and analysis of ligand-induced ion fluxes, has over the past several years given way to a molecular biological one, whereby appropriate messages are expressed in Xenopus laevis oocytes to produce functional channels measureable by patch clamp techniques.

Nicotinic receptors in sensory ganglia

Alpha-bungarotoxin (aBuTX) has been used as a probe not only for nicotinic acetylcholine receptors in skeletal muscle, but also to label specific binding sites on neuronal membranes. Although the functional significance of the neuronal aBuTX receptor has not been established with certainty, it appears to be a constituent c f nicotinic cholinergic synapses 15. In a previous communication we reported the presence of aBuTX binding sites in dorsal root ganglia and the trigeminal ganglia of the rat la. The existence of a putative synaptic constituent in tissue presumed to be devoid of synaptic contacts prompted us to carry out a more detailed analysis. Here we describe biochemical and autoradiographic findings. Adult Sprague-Dawley rats were anesthesized with ether, decapitated and the trigeminal or dorsal root ganglia dissected. For biochemical analysis, freshly dissected trigeminal ganglia were homogenized in 10 vol. of 10 mM sodium phosphate pH 7.4, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and 0.02 ~ sodium azide. The binding of mono-[125I]aBuTX (approximately 106 Ci/mol, depending on age of preparation; prepared as previously reported 9) and [3H]quinuclidinylbenzilate (QNB) (Amersham, 44,000 Ci/mol) to particulate fractions was analyzed by means of a centrifuge assay as described previously I°. For autoradiography, freshly dissected primary sensory ganglia, and in one instance a superior cervical ganglion, were immediately frozen in dry ice onto a cryostat chuck and cut into 16 Fm sections. The cryostat-sectioned tissue was incubated with 6.3 × 10 -8 M mono-[125I]aBuTX in 50 mM sodium phosphate pH 7.4 for 30 min at room temperature, washed, fixed and prepared for autoradiography as previously described 7. In vitro aBuTX binding analysis reveals low levels of specific, saturable binding sites in sensory ganglia (Fig. 1). The dissociation constant is about 2 × 10 -11 M, and receptor concentration is approximately 1.0 fmol/mg wet tissue. Nicotine and Dtubocurarine block toxin binding at low concentrations, with inhibition constants of 1.2 × 10 -6 M and 4 × 10 -6 M, respectively, thereby indicating the nicotinic nature of

Interaction of MyoD Family Proteins with Enhancers of Acetylcholine Receptor Subunit Genes in Vivo

The myogenic determination factors (MDFs) are transcriptional activators that target E boxes in many muscle-specific promoters, including those of the genes coding for the subunits of the acetylcholine receptor. It is not known, however, if in vivo a given E box in a transcriptionally active gene is occupied, either uniquely by one MDF or randomly by all MDFs. We have analyzed expression of MDF and acetylcholine receptor subunits in cultured mouse muscle cells and, using chromatin immunoprecipitation, have determined which individual MDFs reside at promoters of several receptor subunit genes. We find that before fusion, C2C12 cells express myf-5, MyoD, and myogenin, all of which take up residence at promoters of all subunits except ε. At this stage, herculin is present in limited amounts and is detected mainly at the γ and δ subunit genes. On myotube formation, herculin reaches high levels; concomitantly, the ε subunit gene becomes a common MDF target and begins to be expressed. In general, any MDF protein that is expressed also is present on transcriptionally active receptor genes; transcriptional activity of target genes correlates with occupancy by MDF, in particular, herculin.

Kinetics of expression of ACh receptor alpha-subunit mRNA in denervated and stimulated muscle.

Levels of the acetylcholine receptor (AChR) alpha-subunit mRNA were quantified in chick leg muscle, both after section of the sciatic nerve and following electrical stimulation of the denervated leg musculature. Whereas a lag period of approximately 17 h intervenes between the nerve section and the increase in message level, electrical stimulation leads to an immediate decline, which proceeds with a half-life of 3-4 h, similar to the decay induced by treatment with actinomycin D. The asymmetry in the kinetics of activation and repression can be accommodated by several regulatory schemes of which the simplest contains an autocatalytic loop as recently proposed by Changeux (The New Biologist 3: 413-429, 1991).

Trifluoperazine Stimulates Acetylcholine Receptor Synthesis in Cultured Chick Myotubes

Abstract: Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I‐α‐bungarotoxin. At drug concentrations of 5 to 10 × 10−6M, receptor appearance rate was significantly enhanced while receptor half‐life, cellular protein, net protein synthesis rate, and acetyl‐cholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 × 10−5M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KC1. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D‐600, phenothiazines may stimulate receptor synthesis by blocking a voltage‐gated calcium channel.

Rapid modulation of acetylcholine receptor synthesis.

Electrical stimu.lation of adult denervated muscle leads to inhibition of acetylcholine receptor synthesis [ 1,2]. How information about the activity of the sarcolemma is conveyed to the protein-synthesizing apparatus is largely unknown. Some progress has been made with the use of myogenic cells in culture, which resemble denervated muscle fibers and respond to specific drug treatments with characteristic changes in receptor synthesis rate. For instance, pharmacological blockage [3], or activation [4], of sodium channels, result in acceleration or slow-down, respectively, of acetylcholine receptor synthesis. Dantrolene, which blocks release of Ca 2÷ from the sarcoplasmic reticulum (SR), stimulates receptor synthesis [5], while ryanodine, when used at the low concentration at which it is thought to deplete calcium from the SR, has the opposite effect (L. P., J. S., unpublished). The notion that calcium may play a messenger role in acetylcholine receptor regulation is supported by several other experiments [6,7]. Subsequent steps in the signaling pathway are not known. We have studied the effect of various drugs on receptor synthesis in chick myotubes at high temporal resolution, to determine if receptor synthesis is shut off at the transcriptional or post-transcriptional level. The findings suggest that pharmacological agents capable of turning off receptor synthesis act on the protein synthesizing apparatus rather than on the genome.