Waser Peter GaudenzInstitute of Pharmacology, University of Zurich, Zurich 8006 SwitzerlandPhone : Fax :
Peter Gaudenz Waser was born on 21 July 1918 in Zurich and died on 11 April 2010 in Schiers. Professor of pharmacology, dean, and rector at Zurich University. In the 50's he studied the effects of radioactive labelled curare on neuromuscular junctions. He organized in 1974 the second International Symposium on Cholinergic Mechanisms in Boldern Switzerland
The interaction of obidoxime (Toxogonin) with sarin was shown by different analytical methods. The UV spectrum of obidoxime at pH 7.4 yields two absorption maxima, lambda 1 = 284 nm and lambda 2 = 353 nm. The peak at lambda 2 = 353 nm is representative for the amount of zwitter-ionic obidoxime, i.e. the active form of obidoxime. By addition of sarin, lambda 1 shifts immediately to 278 nm and the intensity at lambda 2 decreases, thus indicating an interaction. TLC and 31P-NMR evidence shows that both mono-phosphonylated and diphosphonylated obidoximes are present. Decomposition of phosphonylated obidoxime in MOPS (3-[N-morpholino] propanesulfonic acid) buffered D2O at pH 7.4 occurs with t1/2 = 13.3 min at 24 degrees C. Decomposition of di-phosphonylated obidoxime is faster. It is suggested that decomposition of di-phosphonylated obidoxime occurs through the mono-phosphonylated form. Formation and decomposition of mono- and di-phosphonylated obidoxime is pH dependent. We conclude that obidoxime exerts a detoxifying effect by capturing free sarin molecules and thus increasing its polarity. Thereby the transition of sarin through the blood-brain barrier is restricted and its renal elimination facilitated.
The pharmacokinetics and pharmacodynamics of the oxime obidoxime (Toxogonin, 50 mg/kg iv) were investigated in anesthetized normal rats and in sarin-poisoned (50 micrograms/kg iv) rats. The kinetics were described by a two-compartment open model. The elimination half-life ranged from 35 min in normal rats to 86 min in sarin-poisoned rats. Obidoxime excretion occurred predominantly by the renal route, amounting to 4.6% of the administered dose in normal rats and to 0.9% in sarin-poisoned rats within the first hour of administration. The significantly diminished glomerular filtration rate confirmed the retardation of obidoxime excretion in sarin poisoning. The mean arterial blood pressure (MAP) response to obidoxime, measured in normal rats, was a transient hypotension, but to sarin an immediate hypertension. In sarin-poisoned rats the therapeutic sequence of administration of obidoxime and atropine (5 mg/kg iv) seemed to be important: the administration of atropine 10 min after and of obidoxime 20 min after sarin poisoning exerted a stabilizing effect on MAP. No serum albumin binding was found for obidoxime. Competition experiments at the isolated nicotinic receptor demonstrated the anticholinergic activity of obidoxime. The affinity of obidoxime was 1000 times smaller than that of acetylcholine. It is concluded that obidoxime, due to its prolonged residence time in the organism in sarin poisoning, exerts a "curare-like" inhibition and protection of the nicotinic acetylcholine receptor and, combined with atropine, a synergistic effect on blood pressure normalization.
Title: Distribution of the 4-aminopyridine derivative 3-methoxy-4-aminopyridine in mice Berger SG, Waser PG, Chang Sin-Ren A Ref: Neuropharmacology, 28:191, 1989 : PubMed
The tissue distribution of [14C]3-Methoxy-4-aminopyridine was studied after intravenous administration in mice using whole body and microautoradiography. Dense accumulation was found in cholinergically innervated, secretory organs. High radioactivity was detected in the adrenal medulla suggesting that the observed excitement and hyperglycemia are due to the stimulation of catecholamine secretion. [14C]3-Methoxy-4-aminopyridine quickly passes the blood brain barrier and predominantly accumulates in the hippocampus, the thalamic nuclei and the cortex.
Title: Effects of new 4-aminopyridine derivatives on neuromuscular transmission and on smooth muscle contractility Berger SG, Waser PG, Hofmann A Ref: Arzneimittelforschung, 39:762, 1989 : PubMed
The effects of new 4-aminopyridine (4-AP) derivatives were investigated in the isolated mouse phrenic nerve-hemidiaphragm preparation under single impulse stimulation and tetanic conditions. The basic 4-AP structure was modified in position 3 on the pyridine nucleus by introducing different substituents. Results were compared to those obtained with 4-AP and 3,4-diaminopyridine. The compounds were tested for their antagonistic effect against calcium antagonists and botulinum toxin A. The effect on smooth muscle was investigated on the isolated guinea-pig ileum. Physico-chemical parameters of the test compounds were determined by the partition coefficient and ionization constant. Finally structure-activity relationship analysis revealed that the activity was highly related to lipophilicity and the steric volume. So far 4-AP itself provided the most advantageous molecular structure.
Title: Purification and isolation of choline acetyltransferase from the electric organ of Torpedo marmorata by affinity chromatography Raeber AJ, Riggio G, Waser PG Ref: European Journal of Biochemistry, 186:487, 1989 : PubMed
Choline acetyltransferase (EC 2.3.1.6) catalyzes the synthesis of the neurotransmitter acetylcholine from acetylcoenzyme A and choline. It has been purified from the electric organ of Torpedo marmorata by a new double-affinity chromatography. Our rapid and specific purification procedure includes affinity chromatography on CoA-Sepharose and then a second affinity chromatography on the enzyme's inhibitor [2-[3-(2-ammonioethoxy)-benzoyl]ethyl]trimethylammonium bromide coupled to Sepharose via a six-carbon spacer arm. The final enzyme preparation has been purified 7300-fold to a specific activity of 73 mumol acetylcholine formed min-1 mg protein-1. The isolated enzyme gave a single band on disc polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The relative molecular mass was determined to be 68,300 +/- 2100.
Title: Muscarinic receptors on cultured cells of rat hippocampus: cholinergic regulation and presence of subtypes Rimvall K, Keller F, Waser PG Ref: European Journal of Pharmacology, 160:1, 1989 : PubMed
Muscarinic acetylcholine receptors in intact, cultured explants of rat hippocampus were investigated in binding experiments with tritiated quinuclidinyl benzilate ([3H]QNB) as ligand. Dissociation constants (Kd) were determined to 320-575 pM and maximal binding capacity (Bmax) to 67-87 fmol/explant. The KdS obtained in kinetic experiments were very similar. Hippocampal explants cultured alone contained more muscarinic receptors than hippocampal explants reinnervated by cholinergic fibers from co-cultured septal explants. Pretreatment of hippocampal explants with carbachol resulted in a down-regulation of receptor number which was counteracted by the simultaneous addition of atropine. Atropine added alone had no effect on receptor number in hippocampal explants cultured alone whereas it occasionally caused an up-regulation in co-cultured hippocampus. Displacement experiments with scopolamine and oxotremorine as competitors, showed that hippocampal explants cultured alone contain multiple types of muscarinic receptors. With atropine, pirenzepine and AF-DX 116, only one class of receptors could be detected.
Title: Interaction of quaternary ammonium compounds with acetylcholinesterase: characterization of the active site Thanei-Wyss P, Waser PG Ref: European Journal of Pharmacology, 172:165, 1989 : PubMed
The relation of the structure of 31 quaternary ammonium compounds (28 inhibitors; 3 substrate analogues) with their effects on the activity of acetylcholinesterase (EC 3.1.1.7; AChE) was studied. The compounds were structurally related to the natural substrate acetylcholine (ACh). All bear a trimethylammonium moiety as cationic head. The inhibitors include a variety of functional groups instead of an electrophilic ester group, making these substances suitable to probe the esteratic subsite. The inhibition constants and Km values were determined in kinetic experiments under steady state conditions (pH-stat method). Most of the substances acted as reversible, competitive inhibitors with KI in the range of 10(-6)-10(-3) M. The substrate analogues had Km values between (1.2-2.2) X 10(-4) M. The data allow the following main conclusions: (1) The quaternary trimethylammonium group of ACh is of high importance for substrate binding to AChE. It mediates association at the anionic site. (2) A poorer contribution to binding (two orders of magnitude lower) is attributable to the apolar methylene chain in ACh. It can be related to a hydrophobic interaction of the hydrocarbon chain at a region neighbouring the anionic site. (3) The ester group (both C = O and O) does not contribute to substrate binding. It is only responsible for reactivity.
Title: Isolation and purification of acetylcholine receptor proteins by affinity chromatography Waser PG, Bodmer DM, Hopff WH Ref: European Journal of Pharmacology, 172:231, 1989 : PubMed
In order to further molecular investigations on the binding capacity of acetylcholine receptors, a method was developed for the affinity chromatography of the nicotinic acetylcholine receptor. Reversibly binding cholinergic ligand groups were used as affinity ligands, instead of the well known snake venom alpha-toxins. These ligands are small in size, chemically well defined and fixed to long spacer chains (at least 40 nm). One ligand, with a pharmacologically stabilizing effect on the receptor, was a derivative of gallamine. Another, with a depolarizing effect, resembled carbamoylcholine and a third was a derivative of decamethonium. The receptor proteins were isolated from Torpedo marmorata electric organs. Preparation included solubilization with a non-ionic detergent, alkaline treatment to extract peripheral membrane proteins and affinity purification. The receptor proteins eluted from the three affinity resins were identical in their assembly of subunits (alpha, beta, gamma and delta) but of different purity. Receptor proteins were obtained on a large scale within a short time and under mild conditions for elution with the affinity ligands of the decamethonium or the gallamine type. This was a considerable advantage compared to the use of alpha-bungarotoxin.
Title: Recovery from opioid receptor alkylation: social conflict analgesia and brain [3H]etorphine binding in beta-chlornaltrexamine-treated mice Lazega D, Frischknecht HR, Waser PG, Siegfried B Ref: European Journal of Pharmacology, 155:333, 1988 : PubMed
The effects of beta-chlornaltrexamine (CNA, 5 mg/kg s.c.) on social conflict analgesia and brain opioid binding were investigated in mice at different times after the administration of the alkylating antagonist. The specific binding of [3H]etorphine to high-affinity binding sites and the stress-induced analgesia of attacked mice (50 bites) were prevented for 6 h after CNA administration. Stress-mediated inhibition of pain fully recovered within 3 days after CNA treatment. Brain opioid binding was still reduced to 45% at this time and reached control values 9 days after treatment.
Title: [14C]chloroacetylcholine as an advantageous affinity label of the acetylcholine receptor Bodmer DM, Sin-Ren AC, Waser PG Ref: J Recept Res, 7:799, 1987 : PubMed
The alkylating agent [14C]chloroacetylcholine perchlorate [( 14C] ClACh) was synthesized and used for affinity labelling of the nicotinic acetylcholine receptor from Torpedo marmorata. Solubilized and affinity-purified receptor proteins were reduced and alkylated according to the bromoacetylcholine-method. Covalent binding of [14C] ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the alpha-subunit. Halogen substitution of acetylcholine by chlorine and insertion of a 14C-isotope instead of the widely used 3H resulted in favourable properties of the affinity label.
Title: Choline and acetylcholine metabolism in slice cultures of the newborn rat septum Keller F, Rimvall K, Waser PG Ref: Brain Research, 405:305, 1987 : PubMed
The incorporation of [3H]choline into acetylcholine and other choline-containing compounds was investigated in slice cultures of the septal area of newborn rats. At choline concentrations in the range of the high affinity transport mechanism (0.1-1 microM) most of the labeled choline was incorporated into phosphorylcholine, followed by lipids, acetylcholine and the free choline pool. Hemicholinium-3 (1-10 microM) lead to a marked decrease of acetylcholine synthesis, whereas choline accumulation or phosphorylcholine synthesis were not decreased. Both basal and K+-induced release of acetylcholine were Ca2+ dependent. The efflux of choline was not stimulated by high K+. When choline was absent from the incubation medium, the slices were able to liberate significant amounts of the [3H]choline previously incorporated into phospholipids, and were also able to synthesize some acetylcholine. In choline-free medium, acetylcholine synthesis was greatly enhanced by depolarization. During the period in culture, there was a decrease of the incorporation rate of [3H]choline into phosphorylcholine and an increase of the incorporation rate into acetylcholine. The tissue structure was well preserved after several weeks in culture. After staining for acetylcholinesterase, the cholinergic neurons in the cultures showed a similar morphology to that seen in situ. The main conclusions of the present study are: cholinergic neurons in slice cultures develop and behave in a manner which is very similar to their in situ counterparts; the main divergence from previous studies of choline metabolism in tissue culture is the substantial incorporation rate of choline into acetylcholine at choline concentrations in the range of the high affinity uptake mechanism.
Title: Presynaptic effects of the pardaxins, polypeptides isolated from the gland secretion of the flatfish Pardachirus marmoratus Renner P, Caratsch CG, Waser PG, Lazarovici P, Primor N Ref: Neuroscience, 23:319, 1987 : PubMed
The effects of the two toxic proteins Pardaxin I and II isolated from the gland secretion of the flatfish Pardachirus marmoratus on frog neuromuscular transmission have been investigated and compared to those of the gland secretion. Pardaxin I and II showed pre- but not postsynaptic neurotoxic effects. They increased the frequency of the spontaneous release of transmitter quanta in a dose-dependent and temperature-influenced way up to more than 100 times control values. At the same time the quantal content of the evoked end-plate potentials was greatly elevated. Pardaxin I was about 5 times more effective than Pardaxin II, and both were roughly in the same range of efficacy as the original gland secretion (w/v). The glycosteroids isolated from the same gland secretion were relatively ineffective in promoting neurotransmitter release; however, at high doses they had postsynaptic effects, as shown by a diminution of the amplitude of the evoked end-plate potentials. They did not reinforce the effect of the Pardaxins. At higher doses both the Pardaxins and the gland secretion induced depolarization of postsynaptic membranes, muscle cell contractions which could not be blocked by (+)-tubocurarine or by tetrodotoxin, and eventually also physical disruption of muscle cells. No effects on nerve conductance were observed. Pore-forming activity of the Pardaxins has already been demonstrated. It is suggested that their presynaptic effects are a result of a possible affinity to the nerve terminals, of their hydrophobicity and mainly of this pore-forming activity. These toxins might be valuable tools in neuroscience research.
Title: Subtypes of muscarinic receptors in cultured explants of the hippocampus of the rat Waser PG, Rimvall K Ref: Neuropharmacology, 26:1027, 1987 : PubMed
Using the tritiated, muscarinic antagonist quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic receptors have been identified and characterized in intact, cultured explants of the hippocampus of the rat. Competition studies with scopolamine and oxotremorine indicated a certain heterogeneity in the population of muscarinic receptors, whereas atropine and pirenzepine competed with [3H]QNB in a manner consistent with only one binding site for these substances. Thus, the observed heterogenity does not fit in with the M1/M2 receptor concept. Extended studies, with the aim of determining to what extent these putative subtypes of receptors are functional, would be of interest.
The distribution and kinetics of 14C-vecuronium were studied in rats and mice. 14C-Vecuronium accumulated rapidly in the liver. Both unchanged and metabolized vecuronium were excreted with the bile into the intestines and stomach. Reabsorption in the gut was probably responsible for an enterohepatic increase in radioactivity in the liver after one hour. Excretion through the kidneys increased continuously from low values after the initial peak. Binding in compartments with acid mucopolysaccharides such as cartilage, connective tissue etc., was less important. Blood-brain barrier and placenta were permeable only to a small degree.
Title: Muscarinic receptors in slice cultures of rat brain Rimvall K, Keller F, Waser PG Ref: Neuropharmacology, 25:221, 1986 : PubMed
Muscarinic acetylcholine receptors in organotypic slice cultures of hippocampus of the rat, have been examined using the tritiated muscarinic antagonist quinuclidinylbenzilate [( 3H]QNB) as a as a marker. Maximum specific binding of [3H]QNB in mature explants of hippocampus amounted to 316 fmol/mg protein and a dissociation constant (KD) of 185 pM was determined. Scatchard analysis suggested binding to one single binding site. In younger cultures smaller KDs were registered. This decrease in ligand affinity in maturer cultures possibly reflects a decrease in the turnover of acetylcholine. Muscarinic antagonists inhibited the total binding of [3H]QNB significantly, whereas muscarinic agonist, nicotinic antagonists and cholinesterase inhibitors had no influence whatsoever on the total binding of [3H]QNB. The content of muscarinic acetylcholine receptors varied between cultures with explants from different brain areas: hippocampus greater than striatum greater than septum greater than spinal cord greater than cerebellum. These in vitro results are generally in good agreement with results obtained in situ by other investigators and suggest that the binding of [3H]QNB observed in these cultures is indeed correlated to specific muscarinic receptor sites.
Title: Development of cholinergic projections in organotypic cultures of rat septum, hippocampus and cerebellum Rimvall K, Keller F, Waser PG Ref: Brain Research, 351:267, 1985 : PubMed
ChAT and AChE activity in the hippocampus originate primarily in axons from cholinergic neurons located in the medial septum. The development of cholinergic projections in organotypic explant cultures of rat septum, hippocampus, cerebellum and habenula was studied using AChE histochemistry and biochemical ChAT and AChE determinations. Hippocampal and cerebellar explants cultured without a septum contain negligible amounts of ChAT after 6 days of culture. When the hippocampus was cultured for several days in the presence of a septal explant, a massive increase in ChAT was observed in the hippocampal explant. When co-cultures were stained for AChE, AChE-positive projections were seen to grow out from the septum to invade the hippocampal explant. To a certain extent this ingrowth of septal cholinergic fibers into the hippocampus is target-specific, since cerebellar explants cultured with septum showed neither an ingrowth of AChE-containing septal fibers, nor an increase in ChAT activity. Also, habenular AChE-positive fibers fail to grow into a co-cultivated hippocampal explant. Further, in septal explants co-cultivated with hippocampal explants an increase in ChAT activity was seen as compared to septal explants cultivated alone. The possible factors responsible for the observed specificity and the increase in ChAT activity under co-culture conditions are discussed.
Title: Morphology and molecular function of the cholinergic synapse Waser PG Ref: European Journal of Anaesthesiology, 2:105, 1985 : PubMed
The effect of the oxime reactivator obidoxime chloride (obidoxime) on single frog neuromuscular junctions has been studied in order to clarify its action on the acetylcholine receptor (AChR) and on the acetylcholine esterase (AChE), both before and after blocking its enzymatic activity with the organophosphorus compound sarin. Experiments iontophoretic application of obidoxime to end-plates demonstrated that it has a weak direct depolarizing effect. Furtheron, the drug is shown to possess a potentiating effect on the ACh-induced depolarization. After the AChE activity had been inhibited with sarin, obidoxime on the contrary decreases the depolarization induced by ACh. Both effects are fully reversible. It is concluded that obidoxime acts as an inhibitor of the AChE and as a partial antagonist of the AChR. The antagonistic effect on the receptor is usually masked by the predominating anticholinesterase effect. The effect of obidoxime on miniature end-plate potentials in long-time experiments on sarin-poisoned muscles, showed only weak signs of recovery from the action of the AChE inhibitor. Only focally higher concentration of the drug produced a more marked but short term recovery of the mepps, which is, however, supposed to be dependent on the AChR antagonism. It is still unclear how much of the varying therapeutic usefulness of obidoxime in clinical cases is due to its AChE reactivation and how much to the antagonistic effect on the AChR.
In spite of worldwide research efforts in the search for the treatment of organophosphate poisoning, the substances with practical antidotal capabilities remain to be discovered. This problem has generally been approached by attempting to reactivate the inhibited acetylcholinesterase. Our approach consisted of reducing the amount of the lethal agent acetylcholine by blocking its synthesizing enzyme cholineacetylase with methyl methane thiol sulfonate (MMTS). We have taken into consideration that we are dealing with acute toxicological problems. This applies for poisoning as well as for treatment, and therefore in the present stage we can only present minimal results. The time from sarin (2 mg/kg) injection to death in rats (controls) was 2:59 min. With a MMTS dosage of 133.5 mg/kg prior to sarin, it was prolonged to 20:55 min (p less than 0.01). With the same dosage of MMTS under identical conditions, the time from soman (2 mg/kg) injection to death was prolonged from 6:08 to 14:48 min (p less than 0.01). Although MMTS cannot be used as a therapeutic agent, our attempt has demonstrated a utility in treating organophosphate poisoning in mice and rats and points in a direction where further work might be fruitful.
Title: Sarin poisoning in guinea pigs compared to reactivation of acetylcholinesterase in vitro as a basis for therapy Hopff WH, Riggio G, Waser PG Ref: Acta Pharmacologica et Toxicologica (Copenh), 55:1, 1984 : PubMed
Contrary to the large number of publications dealing with treatment of organophosphate poisoning in a variety of animal species, there is no logic reason in the preference of one species, for this purpose. Guinea pigs were reported to respond better to treatment by oximes, than mice and rats. However, in the analysis of data on the effect of obidoxim and atropine or benactyzine on sarin poisoning it is demonstrated, that guinea pigs do not respond differently from mice and rats. Subcutaneous LD50's of sarin in mice ranged from 0.06 to 0.207 mg/kg, and those of guinea pigs from 0.04 to 0.112 mg/kg. The difference in the LD50's may be related to the different susceptibility of various animal species. The importance of "in vivo" dosage, mode of application, kinetics of antagonists, in correlation to the ability to reactivate "in vitro" is discussed.
Cholinergic binding proteins were purified from torpedo electric organ. The preparation comprises: solubilization by non-ionic detergents followed by unspecific prepurification. For prepurification the double reversed technique proved to be very useful. Finally we applied affinity chromatography. For the affinity purification we used resins with chemically well defined small ligand groups from the depolarizing type (carbachol- and decamethonium-analogue), and from the stabilizing type (gallamine amide amine). The purified receptor proteins from all three resins showed different subunit compositions and different properties of alpha-bungarotoxin binding.
Title: Slice cultures confirm the presence of cholinergic neurons in the rat habenula Keller F, Rimvall K, Waser PG Ref: Neuroscience Letters, 52:299, 1984 : PubMed
The opinion that the medial habenular nuclei contain cholinergic perikarya has recently been questioned, mainly on the basis of the difficulty to detect choline acetyltransferase (ChAT) immunoreactivity in cell bodies of these nuclei. We decided therefore to determine ChAT activity in long-term cultures of the embryonal rat habenula. In these cultures, all extrinsic fiber systems are expected to degenerate a few days after explanation. ChAT activity increased markedly during the first 3 weeks. Control cultures of adjacent thalamic tissue, which is devoid of intrinsic cholinergic neurons, displayed a 150-fold lower ChAT activity. Immunocytochemical staining with a monoclonal antibody revealed the presence of tightly packed, ChAT-containing cell bodies in the habenular slices. These two findings, together with the observation that habenular cultures show an extensive outgrowth of acetylcholinesterase-containing fibers, lead us to the conclusion that at least some cholinergic perikarya must be present in the habenular nuclei.
Title: Choline acetyltransferase in organotypic cultures of rat septum and hippocampus Keller F, Rimvall K, Waser PG Ref: Neuroscience Letters, 42:273, 1983 : PubMed
Choline acetyltransferase (CAT) activity in the hippocampus originates almost exclusively in axons from neurons located in the medial septum. In the rat, the development of CAT in the hippocampus takes place during the first 3 weeks after birth. The development of CAT was studied in organotypic cultures of fetal rat septum and early postnatal rat hippocampus. In some septal explants, enzyme activity increased up to 10-fold during the first 3-4 weeks in vitro. Acetylcholinesterase (AChE) histochemistry showed the presence of AChE-positive cells and fibers in many explants. Thus it appears that septal cholinergic neurons develop CAT and AChE activity even without making contact with their target cells. However, the development of CAT was accelerated by the presence of hippocampal tissue. No CAT activity was found in the hippocampal cultures, confirming that there are few, if any, intrinsic cholinergic cell bodies in the hippocampus.
Title: Incorporation of small unilamellar liposomes loaded with horseradish peroxidase into isolated nerve endings from electric organ of Torpedo marmorata Muller U, Munz K, Waser PG Ref: Journal of Neurocytology, 12:507, 1983 : PubMed
A population of small unilamellar liposomes loaded with horseradish peroxidase, an electron dense marker, was prepared by passing the lipid-protein-detergent micelles through Sephadex G-50. By electron microscopy it was shown that these artificial lipid vesicles were incorporated into the cytoplasm of isolated pure cholinergic nerve endings from the electric organ of Torpedo marmorata. This liposome carrier system may be useful in manipulating the internal parameters involved in presynaptic processes in the electric organ.
Title: Electron microscopic localization of choline acetyl transferase activity in the electric organ of Torpedo marmorata Munz K, Muller U, Waser PG Ref: Histochemistry, 78:339, 1983 : PubMed
The light microscopic method for demonstration of choline acetyltransferase (CAT) activity based on the formation of a lead mercaptide of free SH-acetyl Coenzyme A was adapted for electron microscopy. In samples of electric organ of Torpedo marmorata CAT activity was found to be restricted to synaptic vesicles and cysternae. The precipitate formed was mostly fine grained and distributed more or less evenly throughout the vesicles. Generally, the reaction product seemed not to adhere to the inner side of the vesicle membrane. CAT activity was found only in the presynaptic region of the synapse, neither the synaptic cleft nor the postsynaptic region reacted positively. CAT activity was found also within synaptic vesicles in nerve endings prepared from electric organ. Samples of Torpedo brain reacted positively too. Complete suppression of CAT activity with inhibitors, judged on the basis of lead mercaptide deposited, was rather difficult to achieve. From a group of 10 presumed enzyme inhibitors, only 2 compounds reacted satisfactorily, namely trans-1,2-dihydro-2-imino-4-(1-naphthylvinyl)-1-pyridine-ethanol hydrobromide and 5,5-dithio-bis-(2-nitrobenzoic acid) (3,3'-6). On the whole, the results obtained show the viability of the method used and furthermore it offers also some new insight into the turnover of acetylcholine, since it may be deduced from the results that under certain circumstances acetylcholine may be synthesized in synaptic vesicles.
Title: Recent advances in identification and isolation of acetylcholinesterase and a cholinergic receptor protein Waser PG, Hopff WH, Schaub MC, Hofmann A Ref: Adv Biochem Psychopharmacol, 36:7, 1983 : PubMed
Title: Quantitative correlation between complete block of nicotinic acetylcholine receptors and saturation of the motor endplate with 14C-toxiferine Caratsch CG, Waser PG, Spiess C, Schonenberger E Ref: Naunyn Schmiedebergs Arch Pharmacol, 306:17, 1979 : PubMed
Title: Morphometric analyses of the electric organ of Torpedo: the influence of different fixative modes on the vesicle diameter Naef W, Munz K, Waser PG Ref: Histochemistry, 58:193, 1978 : PubMed
The influences of various fixatives on the vesicle size of the electric organ of Torpedo marmorate were investigated. Thin section and freeze etched preparations were examined under the electron microscope. In thin section increased vesicle diameters were observed compared with the freeze etched preparations. The same experiments in different torpedo fish led to significantly different vesicle sizes observed. Variations of the molarity, the pH and osmolarities result in particularly high differences in vesicle diameters. Using Karnovsky's method (1965) and a fixative consisting of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, results in vesicle sizes comparable to those reported by other authors. Results obtained from freeze etched preparations are not comparable in general with results from thin section experiments with the same fixative.
Title: The influence of taipoxin on cholinergic synapses in the electric organ of Torpedo marmorata Naef W, Waser PG Ref: J Neural Transm, 42:263, 1978 : PubMed
The influences of the snake venom taipoxin on the cholinergic synapses of the electric organ of Torpedo mamorata were investigated. 1. In ultrathin sections presynaptic membrane indentations are noted under the influence of taipoxin/NaCl. 2. The presynaptic membranes often show small interruptions. 3. In taipoxin/horseradish peroxidase incubated organ pieces typical omega-shaped indentations are found, but on the postsynaptic side. 4. The vesicle density decreased under the influence of the taipoxin in contrast untreated cholinergic synapses. 5. In freeze etched preparations no membrane indentations and interruptions are observed, but only a small vesicle density was seen.
Title: The Endocytotic Role of Vesicles at the Cholinergic Synapse Waser PG, Naef W Ref: Advances in Behavioral Biology, 24:347, 1978 : PubMed
Electrophysiological experiments were done to investigate the effect o p-nitrophenyl diazonium fluoroborate (p-NPD) on motor endplates of the frog's m. cutaneus pectoris. The compound has no direct depolarizing effect on the postsynaptic membrane and stabilizes it irreversibly when added to the bath. Longtime iontophoretical applications of p-NPD produce a biphasic effect: initially a potentiation of the depolarizations due to acetylcholine (ACh) (both iontophoretically applied and presynaptically liberated), and subsequently an inhibition of the response to ACh. When the acetylcholinesterase (AChE) is inactivated previously, only the inhibiting effect of the compound is demonstrable. The association constant of p-NPD to purified AChE and to membrane fragments of electroplax was determined by biochemical methods. The compound's affinity to the AChE was found to be about 20 times greater than to the acetylcholine receptor (AChR). Iontophoretical application of p-NPD to cholinergic neurons in the hippocampal cortex of the cat also produced the characteristic biphasic effect on ACh-induced activity of these investigated neurons. The results suggest that the biphasic effect depends on the capacity of p-NPD to combine with both the AChE and the AChR. The AChE is first inhibited with low concentrations thereby potentiating the ACh response. At higher concentrations the AChR's are progressively inhibited too, thereby diminishing the excitability of the postsynaptic membrane up to a complete block.
Title: Characterization of the active site of acetylcholinesterases by application of sterically modified acetylcholine homo1ogues Hopff WH, Riggio G, Hofmann A, Waser PG Ref: Croatica Chemica Acta, 47:309, 1975 : PubMed
Title: Affinity-labelling of the muscarinic receptor of the guinea-pig illeum Lebbin C, Hofmann A, Waser PG Ref: Naunyn Schmiedebergs Arch Pharmacol, 289:237, 1975 : PubMed
1. The activities of some cholinergic compounds were investigated on the terminal ileum of the guinea-pig and their muscarinic potencies were found to be in the following order: acetylcholine (ACh) greater than diazoacetylcholine (DACh) greater than iodoacetylcholine (IACh) greater than azidoacetylcholine (AACh). 2. Protection experiments with atropine showed a decreased affinity of the four cholinergic compounds for the muscarinic receptor, demonstrating a direct interaction of the drugs and the receptor. 3. The maximal contractions (intrinsic activity) caused by DACh were greater than those casued by ACh, IACh and AACh. 4. Incubation of the ileum with ACh was followed by a reversible loss of its ability to contract (desensitization). The time course of recovery was similiar to that after incubation with AACh. 5. IACh caused a partial (50%), irreversible paralysis of the muscle. 6. The furaziridines seem to react partially irreversibly (30%), whereas paranitrophenyldiazonium fluoborate (p-NDP) caused a complete, irreversible blockade of the muscarinic receptor.
Title: Studies about the specificity of the histochemical localization of choline acetyltransferase Lebbin C, Waser PG Ref: Histochemistry, 45:309, 1975 : PubMed
1. Tissues examined for the histochemical localization of choline acetyltransferase are best fixed by acetone. 2. Topographical identification of choline acetyltransferase by its reaction products is not only substrate-dependent because a slight staining also occurred in the absence of the substrates choline and acetyl-coenzyme in the incubation medium. 3. Histochemical localization of choline acetyltransferase was inhibited by sarin but not by DFP or eserine. 4. According to Burt and Silver's method cells and cell organelles, of which the enzymatic content is doubtful, where stained. 5. Chloroacetylcholine-perchlorate did not inhibit the histochemical localization of choline acetyltransferase. 6. The staining of acetylcholinesterase showed a different topographical distribution although both methods were inhibited by sarin.
Title: Binding studies on membrane fragments of electroplax from Torpedo marmorata Walser JT, Schaub MC, Waser PG Ref: Cholinergic.Mechanisms, Raven Press, :365, 1975 : PubMed
Title: Tricyclic antidepressants: action on synaptosomal acetylcholinesterase and ATPase in the brain of guinea pigs and subcellular distribution Caratsch CG, Waser PG Ref: Neuropharmacology, 12:563, 1973 : PubMed
Title: Electron microscopic studies of degeneration and regeneration of rat neuromuscular junctions Gonzenbach HR, Waser PG Ref: Brain Research, 63:167, 1973 : PubMed
Title: [Electron microscopic studies on degeneration and regeneration of the nervus phrenicus of the white rat] Gonzenbach HR, Nickel E, Waser PG Ref: Microsc Acta, 71:159, 1972 : PubMed
Title: The action of some neuro- and psychopharmacological agents on the membrane ATP-ase of cortical synaptosomes Waser PG, Schaub E Ref: Advances in Cytopharmacology, 1:397, 1971 : PubMed
Title: [Why may reactivators be harmful? Transacted on the example of reactivation of the blockaded acetylcholinesterase] Hopff WH, Waser PG Ref: Pharm Acta Helv, 45:414, 1970 : PubMed
Title: [Influence of denervation of muscle end plates on the localization of acetylcholinesterase and fixation of decamethonium] Truog P, Waser PG Ref: Naunyn Schmiedebergs Arch Pharmacol, 266:101, 1970 : PubMed
Title: On receptors in the postsynaptic membrane of the motor endplate. In: Molecular properties of drug receptors Waser PG Ref: Ciba Found Symp, :59, 1970 : PubMed
Title: [Affinity labelling of the cholinergic receptor of the motor end plate with diazoacetylcholine] Waser PG, Hofmann A, Hopff WH Ref: Experientia, 26:1342, 1970 : PubMed
Title: Action of some neuro- and psychopharmaco on membrane ATP-ase and acetylcholinesterase of cortical synaptosomes Waser PG, Schaub E Ref: Drugs and Cholinergic Mechanisms in the CNS, :359, 1970 : PubMed
Title: Electronmicroscopic and autoradiographic studies of normal and denervated endplates Waser PG, Nickel E Ref: Prog Brain Res, 31:157, 1969 : PubMed
Title: [Distribution and metabolism of C14-decamethonium in cats] Luthi U, Waser PG Ref: Archives Internationales de Pharmacodynamie et de Therapie, 156:319, 1965 : PubMed
Title: [Designation of the number of active centers of acetylcholinesterase in motor endplates] Waser PG, Reller J Ref: Experientia, 21:402, 1965 : PubMed
Title: [Pharmacology and clinical use of the short-term muscle relaxant diallyl-nor toxiferine] Waser PG, Harbeck P Ref: Anaesthesist, 11:33, 1962 : PubMed
Title: [Effect of muscarine and acetylcholine on isolated iris muscles] Kuenzle CC, Waser PG Ref: Archives Internationales de Pharmacodynamie et de Therapie, 109:8, 1957 : PubMed
Title: Autoradiography of end-plates with carbon-14-calabash-curarine I and carbon-14-decamethonium Luthi U, Waser PG Ref: Nature, 178:981, 1956 : PubMed
Title: [Autoradiographic detection of C14-calabash curarine and C14-decamethonium in endplate] Waser PG, Luthi U, Huber P Ref: Helv Physiol Pharmacol Acta, 14:C55, 1956 : PubMed
