The role of extracellular ATPin vivoand the various cellular responses mediated by P2 purinoceptors have not yet been fully elucidated, in part depending on the lack of subtype-specific high affinity antagonists. Here we describe the synthesis of a new class of compounds, peptidyl derivatives of adenosine 5′-carboxylic acid, among which some have inhibitory effects in certain P2 purinoceptor-carrying biological systems, e.g., glioma and smooth muscle cell lines and isolated smooth muscle tissue preparations from guinea pig vas deferens and urinary bladder.

Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [I25]α-bungarotoxin revealed a KD of 62 ± 2 nM, Bmax = 7.2 ± 1.8 × 107 binding sites/OHC, and a slowly reversible dissociation rate constant, k−1 = 2.2 ± 0.01 × 10−4 min−1. L-[3H]Nicotine bound reversibly (estimated KrmD ≈ 230 nM and Bmax ≈ 5 × 107) with kinetic rate constants of association k1 = 6.2 ± 0.06 x 104 min−1 nM−1 and dissociation k−1 = 0.23 ± 0.003 min−1. [3H]Strychnine bound to OHC with a KD of 35 ± 6 nM and Bmax = 2.6 ± 0.5 × 107, and binding increased 3–4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > nM concentrations, of [3H]3-quinuclidinyl benzilate (KD = 11.5 ± 5 nM; Bmax = 2.5 ± 0.6 × 106) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 ± 0.5 and 6.5 ± 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.

Outer (OHC) and inner (IHC) hair cells in the organ of Corti of the mammalian cochlea process sound. OHC and their efferent synapse are part of a feedback system assumed to control and modulate information carried by afferent neurons passing from IHC to the brain. Underlying mechanisms are not well understood. This paper discusses recent progress. In vivo and in vitro information is presented on structure, pharmacology, function and localization of the pre- and postsynaptic acetylcholine receptors (AChRs) at the efferent synapse. Recent data are given on a presynaptic M3 AChR subtype, probably an autoreceptor involved in transmitter release. Data from our lab on specific binding of [3H]3-quinuclidinyl benzilate ([3H]3-QNB) to non-enzymatically isolated guinea pig OHC reveal a KD several 100 x higher than that for any known muscarinic receptor subtype, including the above-mentioned presynaptic muscarinic AChR of the OHC efferent synapse. The extremely high concentrations of [3H]3-QNB needed for any binding at all to OHC thus rule out presynaptic membrane impurities as the cause of such binding, and also the presence of a typical mAChR subtype on OHC. The number of [3H]3-QNB binding sites (∼ 106/OHC) we found on OHC was 110th of that we found for binding of nicotinic ligands to OHC, further making it questionable that an ACh-binding site on OHC binds [3H]3-QNB. Observations may instead point to the possibility of another binding site, e.g. an (allosteric) site involved with the as yet not understood ‘weak’ muscarinic properties of the OHC AChR. Further new data on the OHC AChR confirm reversible α-bungarotoxin, nicotine and d-tubocurarine binding. [3H]α-Bungarotoxin and [3H]-nicotine binding sites are estimated at ∼6 · 107 sites/OHC. Strychnine, a glycine receptor blocker suggested to interfere with cholinergic sites of the efferent OHC synapse, was found to bind to OHC (cold strychnine for unspecific binding). This binding, not seen in the presence of high [glycine], increased in the presence of depolarizing [K+], while ACh (100 μM) had no significant effect. Results suggest strychnine binding to the outside of OHC, but also to sites accessible only after cell depolarization, possibly to the hyperpolarizing Ca2+-dependent K+ channel. Recent molecular cloning of the OHC AChR indicates a novel α-subunit. An often observed ACh-activated Ca2+-influx close to zero into OHC leaves an unanswered question. OHC also carry P2-purinergic receptors (P2Rs), a more rapid ionotropic P2zR-like subtype and a quantitatively dominating slow metabotropic P2yR subtype coupled to a G protein-phospholipase C cascade and not desensitized. Both contribute to increased cytoplasmic [Ca2+], from respectively external and internal sources. Whether or not such receptors are part of efferent synaptic activity is unknown; their localization on the OHC plasma membrane is so far only indirect and synaptic vesicles of the efferent nerve endings have not yet been analyzed for their ATP content. Localization, function and interaction of [Ca2+] increases triggered by, respectively, ATP and ACh are currently studied in this laboratory.

Outer hair cells (OHC) of the mammalian cochlea are thought to preprocess the sound signal by active movements, which can be induced by electrical or chemical stimulation, e.g. depolarization evoked by high [K +]or increased cytoplasmic [Ca24]. Extracellular ATP has been found to induce cytoplasmic [Ca2 +]increases in OHC but involved mechanisms have not been elucidated. Cytoplasmic [Ca2+]was measured in non-enzymatically isolated single OHC using Fura-2 microspectrometry. Results, using ATP/derivatives and other P2-purinergic receptor (P2R) ligands, as well as Ca2+-channel blockers and pertussis toxin, revealed several signal transduction pathways that increase cytoplasmic [Ca2 +]in OHC: a P2-purinergic receptor (P2 R) -G-protein - effector (phospholipase C or an ion channel) system and a voltage-dependent Ca2+ channel. Agonist potency studies denote a pattern analogous to that found in skeletal muscle, i.e. ATP-α-S > ATP = 2-methyl-S-ATP > ADP >α,β-methylene- but no activation by ADPβF or UTP, leaving a choice of P2y or P2zR subtypes. The latter possibility gained strength from calculations showing that up to 8% of ATP may have formed the P2zR agonist ATP4− in the experimental medium. Experiments in Ca2+-free medium and with pertussis toxin revealed that the main Ca2 + source was intracellular. Pertussis toxin did not affect [Ca2 +]increase induced by carbachol. Acetylcholine, administered a few seconds before ATP. did not affect total cytoplasmic [Ca2+]increases. Induced cytoplasmic [Ca2 +]increases were high enough ( > 500 nM at 50 μM ATP/ derivatives) to hyperpolarize the OHC membrane by opening K+-channels and decreased little with time. Artifacts may have been caused by the sustained Ca2+ levels, e.g. activation of proteases by the high cytoplasmic [Ca2+]. Similar events in vivo may have pathological consequences.

2-Alkylthio analogues of adenosine 5'-triphosphate were synthesized and evaluated as P2y purinoceptor agonists. ATP and analogues transiently increased intracellular Ca2+ levels in C6 glioma cells and in skeletal muscle derived myotubes in culture. Most derivatives were resistant to stepwise dephosphorylation by ecto-ATPases.

This chapter discusses the cholinesterases (ChEs), enzymes known to hydrolyse cholinesters. Cholinergic neurotransmission, implicated with neurotransmission in motor, autonomic, and central synapses, requires very rapid inactivation (fraction of milliseconds) of its transmitter acetylcholine (ACh). This is achieved mainly by acetylcholinesterase, the enzyme that prefers ACh to other cholinesters and that is present in the relevant synaptic clefts, including the neuromuscular junction (NMJ), where it is anchored to the basal lamina that runs between nerve terminal and the muscle membrane. Close control of the transmitter concentration during the various steps of the synaptic events must be of high importance for the proper function of cholinergic transmission and for the maintenance of the cells adjacent to the cleft. Therefore, it can be expected that its control includes all aspects of ACh synthesis, release, and hydrolysis, as well as those of receptor synthesis, membrane insertion, removal, and function, and further, those of enzyme concentration and activity. Their regulations are most probably not independent from each other. In summary, in spite of great progress, much remains to be done, especially as the cholinesterases seem to be models for hitherto almost unknown events and actions. The aim is a complete functional mapping of the enzymes and an understanding of the existence of the many diverse molecular species of AChE and ChE and of their relation to physiology and pathology.

The activation of two receptors of skeletal muscle and myotube in culture, the nicotinic acetylcholine receptor (nAChR) and the ATP-activated P2-purinergic receptor (P2R) resulted, in both cases, in increased intracellular levels of diacylglycerol (DAG). In the case of the receptor-ion channel macromolecule the intracellular DAG increases were seen after activation of nAChR by a cholinergic ligand and blocked by the nAChR inhibitors α-bungarotoxin or d-tubocurarine; they were dependent on the presence of external Ca2+, which points to the action of a phospholipase A2, present in the membrane and activated directly, probably via a G-protein, by nAChR. In the second case the P2R activates a G-protein-phospholipase C system which results in phosphoinositide turnover and a simultaneous increase in inositol phosphates and DAG, followed by intracellular Ca2+ movement and influx of Ca2+. It is discussed if DAG increases, when occurring close to the sarcolemma, might result in lipoxygenase products moving into the synapse and acting as “retrograde” signals. A preliminary experiment with arachidonic acid and a mouse phrenic nerve-diaphragm preparation was performed and showed no changes in MEPPs, while higher AA concentrations may have decreased the EPPs’ amplitudes.

In earlier experiments, using a fluorimetric method (fura-2), we found what seemed to be a decreased cytoplasmic [Ca2+] in neuroblastoma cells when 1,2,3,4-tetrahydro-9-aminoacridine (THA) was added, but discovered by repeating our work under cell-free conditions, that THA affected the Ca(2+)-fura-2 signal. The present study aimed at investigating the previously observed interference of THA with fura-2. 1 mM THA completely inhibits fluorescence of fura-2 and another Ca(2+)-indicator, indo-1, 1 microM each (apparent IC50 = 40 microM), presumably by absorbing excitation light.

A considerable rise in inositol phosphates was observed at the beginning of myoblast fusion. Extracellular ATP, through P2-purinergic receptors, induced inositol phosphate accumulation before and after fusion; however, no effect of ATP on phosphoinositol levels could be detected during the period of fusion. The possibility of ATP being a fusion signal is discussed.

Acetylcholine (ACh) appears to be the major neurotransmitter liberated from olivocochlear efferents terminating on outer hair cells (OHC). Recently, cholinergic receptor epitopes were visualized at the basal pole of the OHCs. To evaluate the ACh receptor type at OHC we performed binding studies with [125I]-labelled α-bungarotoxin (α-bgtx), a close to irreversibly acting blocker of the nicotinic acetylcholine receptor (nAChR) of skeletal muscle and of electrocytes of Torpedo and Electrophorus. An irreversible and saturable binding (80 nM) of the radiolabelled compound to OHCs was observed. The number of α-bgtx sensitive binding sites present on each OHC was calculated to be about 2×10−17 mol/OHC, which would amount to about 107 binding sites/cell. Preincubation with the reversibly acting cholinergic ligands, carbamylcholine (1 mM), nicotine (0.1 mM) and d-tubocurarine (1–100 μM) was found to inhibit α-bgtx binding to a varying degree. Atropine (0.05 mM), a muscarinic antagonist, had no influence on the binding of α-bgtx to OHCs. [3H]-QNB, a specific marker and antagonist for muscarinic AChR, and [125I]-κ-toxin, known to react with neuronal and ganglionic nAChR, showed no specific binding to OHCs. The data indicate that a peripheral type nAChR is present on OHCs mediating ACh-induced modulation of the biomechanics of the cochlea by influencing OHC motility.

Annals of the New York Academy of SciencesVolume 603, Issue 1 p. 456-457 Extracellular ATP Modifies Intracellular Free Ca2+ Levels in Skeletal Muscle via Activation of a Purinergic Receptor-G Protein-Phospholipase Cascade E. HEILBRONN, E. HEILBRONN Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this authorH. ERIKSSON, H. ERIKSSON Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this authorJ. HÄGGBLAD, J. HÄGGBLAD Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this author E. HEILBRONN, E. HEILBRONN Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this authorH. ERIKSSON, H. ERIKSSON Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this authorJ. HÄGGBLAD, J. HÄGGBLAD Unit of Neurochemistry and Neurotoxicology University of Stockholm S-10691 Stockholm, SwedenSearch for more papers by this author First published: December 1990 https://doi.org/10.1111/j.1749-6632.1990.tb37703.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume603, Issue1Biological Actions of Extracellular ATPDecember 1990Pages 456-457 RelatedInformation

Myotubes prepared from mice with muscular dysgenesis (mdg) were used to further elucidate the putative role of inositol triphosphate (InsP 3 ) in excitation‐contraction (E‐C) coupling of skeletal muscle. The mdg mutation is characterized by an uncoupling of the E‐C coupling. InsP 3 production in normal and mdg / mdg myotube cultures and its relation to the levels of cytosolic free calcium were analyzed. Basal and ATP‐stimulated levels of InsP 3 were equal in normal and mdg / mdg myotube cultures. In contrast, the transient increases of cytosolic free calcium in mdg / mdg myotubes in culture were generally much lower than those in normal ones. This suggests that the defect in dysgenic myotubes does not rest on the InsP 3 formation but on the InsP 3 ‐triggered transduction of excitation and/or the induction of calcium release from internal stores.

Publisher Summary This chapter reviews the future prospects of cholinergic research on neuromuscular transmission. Isolation of the nicotinic cholinergic receptor, in combination with molecular biology and new electrophysiological techniques, such as the patch-clamp, provides a unique insight into the molecular structure and function of the receptor. Neuropeptides, adenosine triphosphate (ATP), and other neuronal messengers that regulate synaptic formation and efficacy open new areas for research. Pre- and postsynaptic phosphorylation processes and the control of gene expressions in the muscle are areas for future research. The muscle secretes substances with the nerve growth promoting and modulating activities. The neuromuscular junction is a very specific synapse whose high speed of transmission can be understood in terms of the structure and location in the muscle cell membrane of a glycoprotein–ion channel neuroreceptor macromolecule. An increasing contribution from the molecular geneticist in explaining the basis of hereditary defects that affect the structure or function of the neuromuscular junction (NMJ) is anticipated. A better understanding of the molecular architecture and physiological properties of the voltage-sensitive calcium channel of the motor nerve terminal is presented in the chapter.

Extracellularly applied ATP mediates a biphasic calcium signal in cultured chick myotubes. A rapid and transient increase in cytosolic calcium was independent of extracellular calcium while a second signal, slower in onset and decay, was absent without extracellular calcium. In depolarized myotubes, the cytosolic [Ca2+] was increased more than ten times above baseline level. Addition of ATP to the incubation medium immediately increased the rate of return of cytosolic Ca2+ levels to baseline. The ATP effect was half-maximal at about 10 μM ATP and was mimicked by ATP S. This ATP-sensitive calcium influx was also rapidly stopped by addition of dihydropyridines such as PN 200-110, suggesting that it is the voltage operated Ca2+-channel that was inactivated by ATP.

Oat cells (of the small cell carcinoma of the lung) have been reported to generate calcium action potentials. The calcium channels have further been suggested to play a crucial role in the relation between oat cell carcinoma and the often associated myasthenic syndrome. We have examined cultured human oat cells (U-1690) under voltage-clamp conditions, using the patch-clamp technique. We found, contrary to previous reports, that the action potential was caused by sodium and potassium currents. No calcium current was detected under these conditions, which indicates that calcium channels, if present, are very rare. The findings restrict, but do not rule out, the hypothesis that calcium plays a key role in the carcinoma/myasthenic syndrome relation.

ATP, a trigger of P2‐purinoceptor‐mediated polyphosphoinositide (PI) turnover in cultured myotubes, increased cytosolic calcium levels in a time‐ and dose‐dependent manner (quin2 fluorescence). The calcium was released from intracellular stores, as acute addition of 5 mM EGTA was without significant effect. Adenosine 5′‐(3‐thiotriphosphate) and 5′‐adenylyl imidodiphosphate also increased intracellular levels of inositol phosphates (InsP) and cytosolic calcium levels. Treatment with cholera or pertussis toxin of myotube cultures did not affect the P2‐purinoceptor‐mediated InsP increase although PI turnover in permeabilized myotubes was stimulated by guanosine 5′‐(3‐thiotriphosphate). The results suggest that myotube P2‐purinoceptors trigger PI turnover and increase intracellular free calcium levels, via a mechanism insensitive to ADP‐ribosylation, by cholera or pertussis toxin of guanyl nucleotide‐binding (G) proteins. However, the presence of a phospholipase C‐coupled G‐protein was otherwise demonstrated.

M. Jiang, J. Häggblad and E. Heilbronn. Isolation and pharmacological characterization of a new α-neurotoxin (α-AgTx) from venom of the viper Agkistrodon halys (Pallas). Toxicon25, 1019 – 1022, 1987. — A hitherto unknown α-neurotoxin, α-agkistrodotoxin, was isolated from the venom of the pit viper Agkistrodon halys (Pallas). It's molecular weight was approx. 8000±80 (SDS-polyacrylamide electrophoresis). The toxin crossreacted with antiserum directed against α-bungarotoxin and inhibited binding of 125I-α-bungarotoxin to the nicotinic acetylcholine receptor of cultured myotubes (IC50 = 2 × 10−9M). The association and dissociation rates were 4.85 × 105 per mole per min and 3.55 × 10−4 per min, respectively, giving a Kd of 7.3 × 10−10 M. The toxin also inhibited carbachol-induced influx of cations through the nAChR (ic50 = 6 × 10−8M).