Interaction of obidoxime with sarin in aqueous solution.

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.

Pharmacokinetics and pharmacodynamics of obidoxime in sarin-poisoned rats.

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.

Purification and isolation of choline acetyltransferase from the electric organ of Torpedo marmorata by affinity chromatography.

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.

Isolation and purification of acetylcholine receptor proteins by affinity chromatography.

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.

Effects of new 4-aminopyridine derivatives on neuromuscular transmission and on smooth muscle contractility.

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.

Interaction of quaternary ammonium compounds with acetylcholinesterase: characterization of the active site.

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.

Distribution of the 4-aminopyridine derivative 3-methoxy-4-aminopyridine in mice.

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.

Muscarinic receptors on cultured cells of rat hippocampus: cholinergic regulation and presence of subtypes.

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.

Recovery from opioid receptor alkylation: social conflict analgesia and brain [3H]etorphine binding in beta-chlornaltrexamine-treated mice.

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.

Opioids and behavior: genetic aspects.

Three animal models, based on genetic differences in endogenous opioid peptides and opioid receptors, are described. Obese mice and rats, whose pituitary opioid content is elevated, may be used to investigate eating disorders. Recombinant inbred strains of mice, which differ in brain opioid receptors and analgesic responsiveness, can be used for study of opioid- and nonopioid-mediated mechanisms of pain inhibition. Individual reactivity to opioids can be examined in C57BL/6 and DBA/2 inbred strains of mice. A model that combines a variety of opioid effects is offered and suggests the existence of a genetically determined dissociation of opioid effects on locomotor activity and pain inhibition. In addition, stimulatory locomotor responses in the C57BL/6 reaction type are linked to a high risk of drug addiction and facilitatory effects on adaptive processes, while high analgesic potency in the DBA/2 reaction type is accompanied by a low proneness to drug abuse and amnesic properties of opioids.

Social conflict-induced changes in nociception and beta-endorphin-like immunoreactivity in pituitary and discrete brain areas of C57BL/6 and DBA/2 mice.

The present study characterizes the time course of social conflict analgesia and its reversibility by opioid antagonist drugs in the C57BL/6 and DBA/2 inbred strains of mice and examines the relationship between alterations in brain and pituitary levels of beta-endorphin-like immunoreactivity (beta-ELIR) and the antinociception elicited by social stress. Data revealed statistically significant strain differences in regard to beta-ELIR in control animals. The pituitary content of beta-ELIR was higher in DBA/2, while the values in the periaqueductal grey (PAG) and in the amygdala were higher in C57BL/6 mice. No interstrain differences were found in the hypothalamus. Exposure to 50 attack bites resulted in a 6-fold higher analgesia in DBA/2 mice and in a strain-independent fall of beta-ELIR in pituitary (approximately 27%) and PAG (23%). PAG but not pituitary beta-ELIR levels in C57BL/6 mice correlated positively with the increase in tail-flick latency after attack. Mere confrontation with a non-aggressive opponent failed to induce analgesia and was associated in C57BL/6 mice with a significant reduction in the beta-ELIR content of both the pituitary and the PAG. The data are discussed in terms of genotype-dependent sensitivity of the beta-endorphin system to stress and its relation to analgesia.

Long-term analgesic reaction in attacked mice.

Four experiments were designed to characterize long-term analgesic (LTA) reaction in attacked mice. In Experiment 1 we showed that analgesic reaction in DBA mice, induced by the stress of being attacked (30 or 50 bites), is reinstated upon reexposure to seven bites 24 hr later. The magnitude of the LTA response depended on the level of analgesia on Day 1 and was smaller than the original response. In Experiment 2 we showed that LTA was prevented by naloxone or beta-chlornaltrexamine given before exposure (50 bites) on Day 1. Results of Experiment 3 revealed that naloxone or beta-chlornaltrexamine injected before reexposure to seven bites on Day 2 antagonized LTA measured 10 min, but not 1 min, after reexposure. In Experiment 4 we showed that morphine substituted for being attacked on Day 1 failed to produce LTA. We concluded that pain inhibitory mechanisms remain in a state of increased readiness for at least 24 hr after attack stress and that activation of opioid systems is necessary but not sufficient to produce LTA, a response that is only partly sensitive to opioid antagonists.

Presynaptic effects of the pardaxins, polypeptides isolated from the gland secretion of the flatfish Pardachirus marmoratus.

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.

Distribution and kinetics of 14C-vecuronium in rats and mice.

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.

Subtypes of muscarinic receptors in cultured explants of the hippocampus of the rat.

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.

Preexposure to a nonaggressive opponent prevents low-intensity, social-conflict analgesia in mice.

In a first experiment, exposure of DBA/2 mice to a small number of attack bites by a C57BL/6 mouse resulted in low-intensity analgesia as assessed by the tail-flick test. The analgesia dissipated within 10 min and was insensitive to naloxone (10 mg/kg, sc) but was antagonized by the irreversible opioid antagonist beta-chlornaltrexamine (5 mg/kg, sc). In a second experiment, preexposure to a nonaggressive C57BL/6 opponent prevented low-intensity analgesia induced by a small number of attack bites 24 hr later. The preexposure effect was abolished by naloxone (10 mg/kg, sc) given before the nonaggressive confrontation. This suggests that the release of endogenous opioids during preexposure interferes with the subsequent activation of endogenous opioid-mediated pain control mechanisms.

Choline and acetylcholine metabolism in slice cultures of the newborn rat septum.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of repeated as compared to single aggressive confrontation on nociception and defense behavior in C57BL/6 and DBA/2 mice.

Behavioral reactions (submissive postures, escape, immobility, activity, locomotion) in C57BL/6 and DBA/2 test mice were recorded during single (50 bites) or three repeated (3 X 50 bites, separated by 24 hr) aggressive confrontations, as well as during a nonaggressive confrontation 24 hr after the last aggressive confrontation with opponents of the opposite strain. Nociception (hot plate response latency) was measured 1 min after aggressive or nonaggressive confrontations. During repeated aggressive confrontation, DBA mice reacted with a stable pattern of escape and analgesia, whereas C57 mice failed to develop an analgesic response and changed their behavioral defense strategy during repeated aggressive confrontations (decrease of escape, increase of defensive upright). The conditioned display of submission and of escape behavior during nonaggressive confrontation did not change as a function of earlier repeated aggressive confrontations in DBA mice, while C57 mice showed a significant increase of defensive upright postures and immobility. Conditioned analgesia was not observed after nonaggressive confrontations. The results point toward a dissociation between attack-elicited behavior and antinociception and suggest that encounter-induced analgesia may influence the processing of aversive experience.

Selective kainic acid lesions in cultured explants of rat hippocampus.

The influence of the excitotoxin kainic acid (KA) on cultivated explants of rat hippocampus was investigated. Addition of 3 microM KA to the culture medium over 24-48 h induced a destruction of the pyramidal cells in the CA3 region, whereas the CA1 pyramidal cells and the granule cells were left undamaged. Higher concentrations (10-100 microM) of KA destroyed also the latter cell groups. The selectivity of the KA lesion at 3 microM was further indicated by the fact that the acetylcholinesterase-positive neurons in the hippocampus were not destroyed through KA administration and that the stereoisomer dihydrokainic acid was ineffective in inducing lesions. Application of tetrodotoxin did not protect the CA3 pyramidal cells from KA lesion, whereas gamma-glutamylaminomethylsulphonic acid (GAMS) only offered a very small, statistically not significant, protection. Baclofen protected the cultures slightly from KA lesions but not when added together with GAMS. Possible mechanisms responsible for the KA lesions in these cultures are discussed.