[Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus].

Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.

Mechanisms of cardiac muscle insensitivity to a novel acetylcholinesterase inhibitor C-547.

We compared the effects of the novel acetylcholinesterase (AChE) inhibitor C-547 on action potential configuration and sinus rhythm in the isolated right atrium preparation of rat with those of armin and neostigmine. Both armin (10(-7), 10(-6), and 10(-5) M) and neostigmine (10(-7), 10(-6), and 5 x 10(-6) M) produced a marked decrease in action potential duration and slowing of sinus rate. These effects were abolished by atropine and are attributable to the accumulation of acetylcholine in the myocardium. The novel selective AChE inhibitor C-547 (10(-9) to 10(-7) M), an alkylammonium derivative of 6-methyluracil, had no such effects. The inhibition constant of C-547 on cardiac AChE is 40-fold higher than that on extensor digitorum longus muscle AChE. These results suggest that C-547 might be employed to treat diseases such as myasthenia gravis or Alzheimer disease, without having unwanted effects on the heart.

[Modulation of the intensity of the non-quantal transmitter release by nitric oxide (NO) at the neuromuscular junction]. / Moduliatsiia intensivnosti nekvantovoi sekretsii mediatora v nervno-myshechnom soedinenii krysy oksidom azota (NO).

In the rat diaphragm muscle, nitric oxide (NO)--sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), as well as substrate for the NO synthesis L-arginine, decrease the level of hyperpolarization of the muscle fibre membrane after acetylcholine receptor blockade by the d-TC and irreversible acetylcholinesterase inhibition by armin (H-effect). Contrary to that, disruption of the NO synthesis in the muscle fibres by the NO-synthase inhibitor NG-nitrol-L-arginine methyl ester (L-NAME) results in enhancement of the H-effect both in vitro and in vivo. Inactivated SNP and inactive forms of arginine and NAME did not affect the H-effect magnitude. Haemoglobin, effectively binding the NO molecules, abolishes the suppressing effects of the SNP, SNAP and L-arginine upon the H-effect. The findings suggest that the NO could be acting as a modulator of nonquantal transmitter release at the mammalian neuromuscular junction.

[The effect of armin on neuromuscular transmission in the frog against a background of hexadecamethonium action]. / Vliianie armina na nervno-myshechnuiu peredachu liagushki na fone deistviia geksadekametoniia.

Hexadecamethonium as well as its alkylating derivatives significantly decrease the effect of organophosphorous acetylcholinesterase inhibitor armine on the amplitude and duration of the EPPs. It seems possible that these substances prevent inhibition of acetylcholinesterase. This property of the substances seems to be due to a polymethelene chain with two nitrogens as the basic part of the molecule structure.

[Non-quantal secretion of a mediator as factor determining consequences of acetylcholinesterase inhibition]. / Nekvantovaia sekretsiia mediatora kak faktor, opredeliaiushchii posledstviia ingibirovaniia atsetilkholinésterazy.

The mechanism of shortening MEPC decay phase after initial prolongation due to acetylcholinesterase inhibition by armine and neostigmine was studied by use of two-electrode voltage-clamp at the mice diaphragm Factors which switch off non-quantal secretion of acetylcholine from the nerve (acute denervation in vitro, ouabain, high concentration of magnesium ions) only slightly reduced the prolongation of MEPC caused by AChE inhibition. So, postsynaptic potentiation of MEPC by nonquantal ACh is not significant immediately after AChE inhibition. At the same time these factors abolished the process of shortening MEPC decay phase. It is concluded, that desensitization of the postsynaptic membrane induced by nonquantal ACh is the main mechanism of the MEPC shortening and that this mechanism can compensate insufficient AChE activity.

[Effect of the quantal composition, membrane potential and cholinoreceptor density on the temporal flow of the end plate current in the rat under conditions of acetylcholinesterase inhibition]. / Vliianie kvantovogo sostava, membrannogo potentsiala i plotnosti kholinoretseptorov na vremennoe techenie tokov kontsevoi plastinki krysy v usloviiakh ingibirovaniia atsetilkholinésterazy.

The factors determining the decay of multiquantal end plate currents (EPC) were studied in the diaphragm muscle of rat by the comparison of EPC and miniature EPC (MEPC) amplitude--temporal characteristics. The decay of EPC (quantal content 25-100) was 1.2 times slower than the decay of MEPC when AChE was active. The AChE inhibition by armine or neostigmine made this difference 10-100 times higher. In most synapses the decay of multiquantal EPC can be approximated by a sum of two or three exponents. It depended on the quantal content and 3-exponential EPC could be transformed in 2-exponential and later to monoexponential ones if increasing concentration of magnesium ions. A slow component of EPCs (but not of MEPC) decay was highly sensitive to concentration of magnesium ions and had 3 times higher dependence of the membrane potential value than that one of MEPC. The irreversible blocking of receptors by alpha-bungarotoxin (alpha-BuTX) accelerated the decay of MEPC but the decay of multiquantal EPC changed in two phases: it was prolonged at the beginning of alpha-BuTX action followed by its acceleration, but never the time of the decay of EPC had achieved the apparent open time of ACh-activated ionic channels. It is suggested that during the multiquantal EPC generation not only the synchronization of opening but the kinetic of ACh-activated channels is changed, probably by blocking of this channels by high concentrations of endogenous ACh.

Non-quantal acetylcholine release after cholinesterase inhibition in vivo.

After anticholinesterase treatment in vivo, depolarization of the postsynaptic muscle fibre membrane by about 4 mV develops due to non-quantally released acetylcholine from the motor nerve terminal. This conclusion was supported by experiments with the curarization of diaphragm slices from anticholinesterase treated mice during intracellular microelectrode recordings.

[The early postdenervation changes in the properties of the postsynaptic membrane of amphibian muscle fiber]. / Rannie postdenervatsionnye izmeneniia svoistv postsinapticheskoi membrany myshechnogo volokna amfibii.

The amplitude-temporal characteristics of miniature end-plate currents (MEPC) were studied in denervated frog cutaneous-pectoral muscles. After the nerve cutting, the initial shortening of MEPC decay time was followed by its prolongation. In 6-8 days after the denervation, the MEPC sensitivity to membrane potential decreased, while the average open time of ACh-activated channels increased 2-fold. The autocorrelation function of ACh-induced current noise revealed two components at some of the synapses. The denervation of the frog cutaneous-pectoral muscle seems to make the postsynaptic membrane to contain two types of ACh receptors (AChR): normal synaptic AChR which gradually disappear, and new synthesized AChRs which open the channels with much longer mean time and a lesser sensitivity to membrane potential changes.

[Nonquantal mediator secretion in the synapses of the mouse diaphragmatic preparation to the administration of sublethal doses of armin]. / Nekvantovaia sekretsiia mediatora v sinapsakh diafragmal'nogo preparata myshi pri vvedenii subletal'nykh doz armina.

During in vivo experiments on the mouse diaphragm preparations the effect of a single sublethal dose of armin (1/2 of LD50) on the dynamics of changes in acetylcholinesterase activity, frequency and time of half-decay of miniature end-plate potentials (MEPP) was studied as compared with the H-effect value. It was found that the increase of the H-effect correlated (r = 0.902) with the decrease of acetylcholinesterase activity, the increase of MEPP frequency occurring later than the changes in the enzyme activity and the H-effect value. The data obtained reveal the possible of nonquantal secretion of the mediator from the motor nerve endings of the skeletal muscles in the pathogenesis of acute poisonings with armin.

[The effect of cholinergic substances on the development of delayed hypersensitivity]. / Vliianie kholinergicheskikh veshchestv na formirovanie giperchuvstvitel'nosti zamedlennogo tipa.

The formation of delayed-type hypersensitivity (DTH) to ram erythrocytes was studied in BALB/c mice on different models during cholinergic stimulation induced by armin (0.3 mg/kg) and a M-cholinolytic atropine (50 mg/kg). The relationship between the formation of this reaction and the contents of T-lymphocytes and T-suppressors in the spleen and peripheral blood was also investigated. When administered one hour before immunization by ram erythrocytes, armin was shown to reduce the formation of DTH. Under the same conditions atropine failed to influence DTH reaction. In the local adoptive DTH armin increases and atropine decreases the reaction formation. The character of DTH on different models is related mainly to the specific features of migration of T-suppressors from the spleen and their content in peripheral blood.

[The effect of "nonquantal" acetylcholine on miniature end-plate currents in the mouse]. / Vliianie "nekvantovogo" atsetilkholina na miniatiurnye toki kontsevoi plastinki u myshi.

The development of postsynaptic potentiation (PSP) and desensitization (DS) under the nonquantal secretion (NS) of acetylcholine (ACh) was studied using characteristics of miniature end-plate currents (MEPCs) in mice diaphragm muscle. The H-effect (the measure of the NS level) fell to zero during 3 hours after acetylcholinesterase (AChE) inhibition by armine at 20 degrees C. The MEPCs decay time constant (tau) decreased in parallel with the H-effect, though there was no reduction of the MEPCs amplitude in this case; tau did not change when NS was absent (early denervation). The maximal prolongation of tau after AChE inhibition was the same both in innervated (NS was normal) and denervated (NS was absent) muscle. The reduction of tau accelerated with the temperature rise and occurred after AChE inhibition by neostigmine. There was no changes in tau when AChE was active. It is suggested that nonquantal ACh decreases tau due to the DS development. NS induces no significant PSP after the AChE inhibition, but can do it later according to DS development, so that signs of DS may partially be masked by PSP.

[The characteristics of the action of calcium ions on miniature end-plate currents after the disruption of mediator hydrolysis]. / Osobennosti deistviia ionov kal'tsiia na miniatiurnye toki kontsevoi plastinki posle narusheniia gidroliza mediatora.

Effect of calcium on the miniature end-plate currents (MEPC) at the frog neuromuscular junction was studied by the voltage-clamp technique. Rise of the calcium concentration in the Ringer solution up to 9 mmol/l caused a decrease of the MEPC amplitude which was related to the reduction of the end-plate channel conductance. Calcium had no effect on the time course of MEPCs at the active acetylcholinesterase (AChE) but accelerated MEPC decay by 26% after AChE inhibition by neostigmin or armin. It is supposed that the shortening effect of calcium on the MEPC decay phase is based on the ability of calcium to modulate the block of ionic channels by acetylcholine or to accelerate the process of desensitization of the postsynaptic membrane.

[Cerebral blood flow function during altered cholinergic mediation]. / Sostoianie mozgovogo krovotoka pri izmenenii kholinergicheskoi mediatsii.

The study of cerebral blood flow was performed with the aid of hydrogen clearance technique and the cinema-television method in alteration of cholinergic transmission by means of phosphorus-organic inhibitors of cholinesterase and a cholinolytic agent in unanesthetized rats. The enzyme inhibition by 60-70% sharply increased the velocity of blood flow in the microvessels of cerebral cortex and in the sagittal sinus. A considerable vasodilation of pial vessels occurred. Atropine administration reduced the velocity of both the cortical and the total blood flow, increasing the lumen of pial vessels in intact animals. Whereas atropine administration against the background of the inhibitors action normalized the blood low velocity with no elimination of the vasodilation of pial arteries. The findings suggest that atropine widely used in poisoning with various inhibitors of cholinesterase, does not normalize the tonus of cerebral vessels.

[Restoration of neuromuscular transmission in the rat diaphragm after the action of an organophosphorus inhibitor of acetylcholinesterase by using curare]. / Vosstanovlenie nervno-myschechnoi peredachi v diafragme krysy posle deistviia fosfororganicheskogo ingibitora atsetilkholinésterazy s pomoshch'iu kurare.

The acetylcholinesterase phospho-organic inhibitor armine (4.10(-7) M) reduces the muscle response to a single nerve stimulus (up to 50%) and the nerve-induced tetanic tension in the rat phrenic-diaphragm muscle preparation. Tubocurarine exerts a similar effect. Substitution of armine by 0.5.10(-7) or 1.10(-7) M tubocurarine for 60-90 min leads to almost complete restoration of neuromuscular transmission. In the presence of armine, tubocurarine exerts a considerably lesser effect. Possible pre- and post-synaptic mechanisms of the tubocurarine action, are discussed.

[Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition]. / Sravnitel'noe issledovanie sinapticheskoi peredachi v simpaticheskikh gangliiakh krolika i liagushki v usloviiakh ingibirovaniia atsetilkholinésterazy.

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.

[Effect of hyaluronidase on the end-plate miniature currents and potentials in the frog]. / Vliianie gialuronidazy na miniatiurnye potentsialy i toki kontsevoi plastinki liagushki.

The effect of 0.1% hyaluronidase on miniature end-plate potentials and currents (MEPP and MEPC) was studied in the frog cutaneous-pectoris muscle. The action of hyaluronidase on armin-pretreated muscles caused a decrease in the amplitude, duration of half-decay time and rising phase of MEPPs and MEPCs. The positive correlation between the amplitude and half-decay time of MEPPs and MEPCs was diminished. Hyaluronidase treatment of preparations with active acetylcholinesterase caused the half-life time of MEPCs to increase without any changes in the amplitude and rising phase of MEPCs. It is suggested that enzymatic destruction of a part of the glycocalix of cells forming the neuromuscular junction and of a part of the extracellular matrix results in a weakening of the nonspecific acetylcholine binding, thus facilitating the acetylcholine diffusion into the synaptic cleft.