Adenosine depresses spontaneous transmitter release from frog motor nerve terminals by acting at an A1-like receptor

Adenosine (1 microM to 1 mM) depressed spontaneous transmitter release from frog motor nerve terminals without producing any observable postsynaptic effects. Since this action of adenosine was blocked by 20 microM theophylline and 1 microM 8-phenyltheophylline, adenosine probably acts at a specific receptor on motor nerve terminals to reduce spontaneous transmitter output. The effects of the adenosine analogs, L-N6-phenylisopropyladenosine (L-PIA, 100 pM to 1 microM), D-PIA (100 nM to 100 microM), and 5'-N-ethylcarboxamidoadenosine (NECA, 10nM to 100 microM), were tested on spontaneous transmitter release at the frog neuromuscular junction. L-PIA depressed mepp frequency at a threshold concentration of about 1 nM, was thirteen times more potent than NECA, and was 294 times more effective than D-PIA. The rank-order potency of these analogs indicates that adenosine acts at an A1-like receptor to depress spontaneous transmitter release. Inhibitory actions of maximally effective concentrations of adenosine and L-PIA were also blocked by the A1-specific antagonist, 1-3-dipropyl-8-cyclopentylxanthine (DPCPX) at a concentration of 100 nM. Micromolar concentrations of NECA, an agonist with approximately equal affinity for the A1 and A2 receptors, produced biphasic effects on mepp frequency. Thus, a second adenosine receptor, perhaps of the A2 subtype, may be present on motor nerve terminals and may mediate an increase in spontaneous transmitter release.     

Mediator secretion in the nerve-muscle synapse in the frog following long-term effect of calcium-free solutions

The changes of spontaneous and evoked transmitter release in condition of long time (1-4 hours) incubation in Ca-free solution with EGTA adding, were investigated with extracellular recordings in experiments on the nerve-muscular junction of the frog cutaneous-pectoris muscle. Using the method of three extracellular microelectrodes recordings of the monoquantal postsynaptic signals, it was shown that during action of Ca-free solutions the topography of transmitter release changed, the specific spatial organization of points of transmitter release was disrupted. These changes remained after returning to the initial solution. The obtained data suggest that the Ca2+ free solution leads to disruption of active zones of nerve ending. In condition of low initial extracellular Ca2+ concentrations (0.15-0.4 mmol/l), the active zones disorganization led to decreasing of average amplitude of the end-plate currents (EPC) by decreasing their quantal content, increasing their time-course and decreasing the frequency of the miniature end-plate currents (MEPC). The sharp displacement of dependence of quantal contents of EPC in extracellular Ca2+ concentration to a higher Ca2+ concentration without significant changes of slope was revealed. In condition of high (1.8 mmol/l) concentration of Ca2+, the long action of Ca-free solutions leads to decreasing of amplitude of EPC too, but it was less obvious than in condition of initial low Ca2+ concentration. It is supposed that intra- and extracellular Ca concentration provides the support of the typical morpho-functional organization of the mechanisms of transmitter release at the nerve ending of the frog. The disorganization of active zones leads to separation of the elements, which take part at the transmitter release process and reduces the efficiency of secretion.     

Effects of cholinergic compounds on spontaneous quantal transmitter release at the frog neuromuscular junction

The effects of nicotinic and muscarinic mimetics and lytics on spontaneous quantal transmitter secretion from the motor nerve endings were investigated during experiments on theRana temporaria sartorius muscle. Acetylcholine and carbachol reduced the frequency of miniature endplate potentials both in a normal ionic medium and in one with potassium ion concentration raised to 10 mM. Similar effects were produced by nicotinic agonists, namely nicotine, tetramethylammonium, and suberyldicholine, whereas muscarinic mimetics — methylfurmetide, oxotremorine, and F-2268 (L- and D-stereoisomers) — did not affect transmitter release. Neither d-tubocurarine, benzohexonium, nor atropine abolished the presynaptic effects of carbachol and acetylcholine. It is concluded that nicotinic cholinoreceptors are present at the frog motor nerve endings which modify spontaneous transmitter release and differ in their pharmacological properties from recognized N-cholinoreceptors of the motor and autonomic systems of the higher vertebrates.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 586–593, September–October, 1986.     

Termination of the presynaptic effect of carbacholine by tubocurarine]

The presynaptic action of carbacholine (Cch) was studied in experiments on the frog sartorius muscle neuromuscular preparation. Cch proved to decrease the quantum content of the end plate potential (EPP); this effect was induced by a direct Cch action on the motor nerve endings. d-tubocurarine decreased the sensitivity of the nerve endings to Cch significantly. Both d-tubocurarine and Cch had concurrent antagonistic interrelations in respect to their action on the quantum content of EPP. Atropine in low concentrations had no influence on the presynaptic effect of Cch. It was concluded that Cch decreased the quantum content of EPP through the N-cholinergic structures of the motor nerve endings.     

Mechanism of carbachol and adenosine action on spontaneous quantal mediator release at frog neuromuscular junction

Experiments on the frog sartorius muscle showed that nonhydrolisable acetylcholine analog carbachol (CCh) depresses spontaneous quantal mediator release via muscarinic M2 receptors of nerve ending. Adenosine (Ade) acting via inhibitory A1 receptors is another strong spontaneous quantal release modulator. Inhibition of pertussis toxin (PTx)-sensitive G-proteins only partly eliminated CCh and Ade depressive action. It means metabotropic A1 and M2 receptors of the frog nerve ending regulate spontaneous quantal release via activating of both PTx-sensitive and PTx-insensitive inhibitory mechanisms.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Action of D-alpha aminoadipic acid on the sensory organs of the inner ear in the frog

Following the suggestions in the literature that glutamate or aspartate may be the transmitter at the primary afferent synapses of acoustico-lateralis organs, we have employed the "selective" excitatory amino acid antagonist. D-alpha amino adipate (DAA) as a tool with which to shed further light on this problem in the labyrinthine organs of the frog. DAA produces a dose-responsive, reversible depression of spontaneous activity in the afferent nerves of the posterior semicircular canal, saccule and basilar papilla. These structures are examples of ampullar, otolithic and auditory organs, respectively. The drug effect seems qualitatively the same throughout the labyrinth. The most interesting finding was that of a presynaptic (hair cell) effect of DAA on the semicircular canal. The means of recording did not permit detection of a presynaptic effect in the other organs examined. All the observed effects of DAA could be explained by a presynaptic action to inhibit transmitter release. Therefore, the ability of DAA to reduce transmission at primary afferent synapses of the frog labyrinth must not necessarily be interpreted to imply that the transmitter is an excitatory amino acid. A presynaptic action to reduce the release of a transmitter (of unknown structure) could explain all our results.     

Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Electrophysiological study of mediator secretion in the frog neuromuscular synapse under a colchicine block of axoplasmic transport

Two weeks after colchicine nerve treatment the evoked transmitter release was blocked in part of the frog sartorius synapses, with spontaneous activity being absent from some of them. In the synapses with evoked and spontaneous transmitter release preserved within this period of time, the magnitudes of the absolute refractory phase of nerve terminals were significantly higher than the control ones, while in part of synapses, the frequency of miniature end plate potentials (MEPP) was considerably increased. Nerve stimulation (5 imp.s-1) led to a rise of the amplitude of evoked potentials and of MEPP frequency followed by irreversible blockade of synaptic activity. It is concluded that substances transported by rapid axonal flow control the level of membrane potential of nerve terminals and are fairly important for presynaptic membrane integrity.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

Desensitisation of postsynaptic membrane in the neuro-muscular junction due to an increase in spontaneous quantal release of a mediator

Dependence of the amplitude of miniature end-plate currents on frequency of spontaneous quantal release modulated by the elevation of K+ concentration was studied in the frog voltage clamped neuromuscular junctions. A sharp increase of mEPC frequency (not less than approximately 50 per sec) was followed by an obvious fall in both their amplitude and acceleration of decay only in the presence of 3 microM prostigmine (acetylcholinesterase inhibitor) and 5 microM proadiphene, these agents promoting a desensitization of cholinergic postsynaptic membrane. Probable depletion of transmitter store is not involved in the phenomenon observed which is mainly due to the repetitive activation of the postsynaptic zones and the increase of the desensitized cholinoreceptor number.     

The action of the venom of the spider Eresus niger on the neuromuscular synapses of the frog

The effect of Eresus niger spider venom on frog neuromuscular preparations has been studied using the intracellular microelectrodes. The spider venom has been found to block both spontaneous and elicited transmitter release and possesses the phospholipase activity. It has been suggested that the venom blocking synaptic transmission results from its phospholipase activity by analogy with the action of snake presynaptic neurotoxins.     

Differences between Spontaneous and Evoked Monoquantum Signals in the Frog Neuromuscular Junction

In experiments on the frog cutaneous-pectoris muscle, the amplitude-temporal parameters of monoquantum end-plate currents (EPC) and miniature EPC (mEPC) were investigated using extracellular recording. A significant dependence of the risetime of the signals on their amplitude was found after analyzing mEPC; at the same time, such dependence was absent for EPC. Approaches leading to disorganization of the active zones (AZ) of the nerve ending (NE), prolonged action of a Ca-free solution, and denervation resulted in an increased dependence of the risetime of the monoquantum signals on their amplitude; moreover, these dependences were similar for both mEPC and monoquantum EPC. Mathematical simulation showed that the obtained data could be explained by the spatial heterogeneity of the sites of spontaneous and evoked transmitter release within the regions of the AZ. A new hypothesis interpreting spontaneous and evoked transmitter release is proposed.     

Pre- and post-synaptic activity of glutamate on preparations of the frog semicircular canal

Glutamate (Glu) has at least two sites of action in the frog semicircular canal: the hair cell (presynaptic) and the primary afferent nerve fibres (postsynaptic). Glu's action on the hair cell results in an increased release of the natural transmitter which is responsible for a substantial increase in the frequency of firing in primary afferents. Glu produces a long-lasting depolarization in the afferent nerve fibres which does not by itself elicit any afferent discharge of impulses when the release of the natural transmitter is prevented. The difficulty of reconciling some of the observations made of the effects of Glu in semicircular canals with its presumed role as an afferent transmitter in this organ is discussed.     

Effects of carbamazepine on nerve activity and transmitter release in neuroblastoma-glioma hybrid cells and the frog neuromuscular junction

Effects of the antiepileptic drug carbamazepine on nerve action potential and transmitter release in mouse neuroblastoma-glioma hybrid cells (NG108-15) and the frog neuromuscular junction were studied. Carbamazepine within a concentration range of 0.1–0.5 mmol/L reduced the peak height of the action potential of the NG108-15 cells, whereas the membrane potential and membrane resistance were unaffected. Voltage clamp revealed that the decrease in the action potential was due to the blockage of the Na⁺, delayed K⁺ and transient Ca²⁺ currents. Carbamazepine did not affect Ca²⁺-activated and A type K⁺ currents and long-lasting Ca²⁺ current. In the frog neuromuscular junction, carbamazepine decreased the mean quantal content by a parallel shift in the frequency augmentation–potentiation (FAP) relation. It is concluded that carbamazepine blocks the voltage-dependent Na⁺, delayed K⁺, and transient Ca²⁺ currents and quantal transmitter release through a decrease of nerve excitation.     

The effects of pentachlorophenol (PCP) at the toad neuromuscular junction

1. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul. 2. Within the concentration 0.003-0.1 mM, PCP caused a dose-time-dependent block of evoked transmitter release acompanied by an increase in the rate of spontaneous quantal release. 3. PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2(+)-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores. 4. The present data, together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4-diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca2+ influx occurs during depolarization of motor nerve terminals.     

Effect of substances blocking calcium channels (verapamil,D-600, manganese ions) on transmitter release from motor nerve endings in frog muscle

Experiments on isolated frog nerve-muscle preparations showed that manganese ions (0.4–5.0 mM) inhibit evoked transmitter release by reducing the quantum composition of the end-plate potentials, and they intensify spontaneous transmitter release to a certain extent by increasing the frequency of miniature potentials. Verapamil (1 · 10^–6–5·10^–5 g/ml) and D-600 (2.5·10^–5 g/ml), by contrast with manganese ions, do not inhibit evoked release, but also intensify spontaneous release of the transmitter. All the agents tested prevent the potentiating effect of imidazole (3 mM). During repetitive stimulation, verapamil disturbs action potential generation in the motor nerve. Manganese ions had no such action. It is concluded that between the calcium channels of motor nerve endings and the calcium channels of heart muscle or the neuron soma there are molecular differences, expressed as sensitivity to the blocking action of verapamil and D-600.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 415–422, July–August, 1977.     

Effects of a neurotoxin isolated from the sea anemone, Anemonia sulcata, at frog neuromuscular junction (author's transl)]

ATX II is a toxin extracted from tentacles of Anemonia sulcata. It was known that this protein displays neurotoxic effects on frog isolated neuromuscular preparation (Fig. 1, 2) and that muscular contractures observed with ATX II are blocked by d-tubocurarine (Fig. 3) or on a 40-days-denervated gastrocnemius (Fig. 4). Part of these experiments has already appeared. 1. These effects of ATX II depend on calcium concentration in the bathing medium, as is the case for transmitter release. The same results were observed when we substituted strontium to calcium. 2. On an intact sciatic sartorius preparation, ATX II does not act on the amplitude of the miniature endplate potentials (mepps, Fig. 6). The muscular action potential is not modified by this toxin. 3. ATX II increases the frequency of the mepps (Fig. 5). The evoked transmitter release (quantal content) after ATX II is also largely increased (Fig. 7). 4. In conclusion, it is suggested that ATX II acts indirectly on the muscle through an increase in acetylcholine release from the motor nerve terminals.     

Effect of the venom of Glycera convoluta on the spontaneous quantal release of transmitter

A neurotoxin able to increase the spontaneous release of transmitter was found in the venom glands of the polychaete annelid Glycera convoluta. We studied the effect of this venom on the frog cutaneous pectoris muscle, where its application produced a prolonged (20-h), high-frequency discharge of miniature potentials. After 5 h of action, the initial store was renewed several times but no detectable ultrastructural changes were observed. After 19 h of sustained activity, nerve terminals with their normal vesicular contents were infrequent; others were fragmented and contained swollen mitochondria, abnormal inclusions, and vesicles of various sizes. In the noncholinergic crayfish neuromuscular preparation, the venom triggered an important increase in spontaneous quantal release that subsided in 1 h. An activity higher than that in resting conditions then persisted for many hours. This high electrical activity was not accompanied by any detectable structural modifications after 3 h. In the torpedo electric organ preparation, the venom elicited a burst of activity that returned to control levels in 1 h. The release of ACh (evaluated by the efflux of radioactive acetate) paralleled the high electrical activity. No morphological changes or significant depletion of tissue stores were detected. The venom of Glycera convoluta appears to enhance considerably the release of transmitter without impairing its turnover. The venom effect is Ca++ dependent and reversible by washing, at least during the first hour of action. Because the high rate of transmitter release appears dissociated from the later-occurring structural modifications, it is possible that the venom mimics one component of the double mode of action proposed for black widow spider venom.