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E M Silinsky

Publications and source records attributed to E M Silinsky.

At least 37 records · Page 2Linked to original sources
PubMed1992

ATP mediates excitatory synaptic transmission in mammalian neurones.

Adenosine 5'-triphosphate (ATP, 0.1-100 microM), produced inward currents in patch-clamped coeliac neurones from guinea-pig when studied in either the whole cell configuration or in excised (outside-out) patches. The P2-purinoceptor antagonists suramin (80-230 microM) or reactive blue 2 (2-20 microM) depressed the ATP-induced currents but not those produced by acetylcholine. Excitatory post-synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current-voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.

Acetylcholine↗
PubMed1992

Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog.

1. The effects of adenosine (50 microM) and 2-chloroadenosine (1-25 microM) were studied on Ca2+ currents in frog motor nerve endings. 2. Ca2+ currents associated with the synchronous, neurally evoked release of acetylcholine (ACh) were measured using either perineural or patch recording methods. Tetraethylammonium and/or 3,4-diaminopyridine were employed to block K+ currents. 3. Ca2+ currents were depressed by omega-conotoxin (1.5-2.5 microM), Cd2+ (100 microM-2 mM), Co2+ (500 microM-5 mM) or by a reduction of the extracellular calcium concentration. Such currents were also observed when Sr2+ was substituted for Ca2+. Both ACh release and Ca2+ currents at motor nerve endings have been reported to be insensitive to 1,4-dihydropyridine antagonists in this species. 4. Adenosine receptor agonists did not affect Ca2+ currents at concentrations that produced maximal inhibition of ACh release. 5. The effects of adenosine receptor agonists were examined on asynchronous K(+)-dependent ACh release under conditions in which the Ca2+ concentration gradient is likely to be reversed (Ca(2+)-free Ringer solution containing 1 mM EGTA). ACh release was measured by monitoring the frequency of occurrence of miniature endplate potentials (MEPPs). In Ca(2+)-free solutions containing 1 mM EGTA, high K+ depolarization caused a decrease in MEPP frequency, presumably because it elicits the efflux of Ca2+ from the nerve ending via membrane Ca2+ channels in a reverse Ca2+ gradient. 6. The Ca2+ channel blocker Co2+, which blocks the exit of Ca2+ from the nerve ending, increased the frequency of MEPPs in a concentration-dependent manner in a reverse Ca2+ gradient. 7. Adenosine or 2-chloroadenosine inhibited ACh release in a reverse Ca2+ gradient. 8. The results suggest that blockade of Ca2+ entry is not responsible for the inhibitory effects of adenosine at frog motor nerve endings.

2-Chloroadenosine↗
PubMed1990

The effects of TMB-8 on acetylcholine release from frog motor nerve: interactions with adenosine.

The putative intracellular calcium (Ca) antagonist TMB-8 was shown to reduce postjunctional sensitivity and quantal acetylcholine (ACh) release at low micromolar concentrations. At 10-fold higher concentrations, TMB-8 also blocked caffeine-induced Ca release (as monitored electrophysiologically by changes in ACh release) but did not impair the ability of adenosine to inhibit quantal ACh release. This last result implies that TMB-8 and adenosine exert their inhibitory actions at different steps in the depolarization-secretion coupling sequence.

Acetylcholine↗
PubMed1990

The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings.

1. The importance of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2. Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3. The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4. All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5. The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30-100 microM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6. H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250 microM). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7. H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8. The results suggest: (i) that the adenylyl cyclase-cyclic AMP-PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP-PK A system modulates rather than mediates the inhibitory effects of adenosine.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Pertussis toxin prevents the inhibitory effect of adenosine and unmasks adenosine-induced excitation of mammalian motor nerve endings.

Pertussis toxin (PTX), which blocks certain classes of guanine nucleotide binding proteins (G proteins), consistently blocked the inhibitory effects of adenosine (100 microM-250 microM) on quantal acetylcholine (ACh) secretion in rat phrenic nerve hemidiaphragm preparations. PTX pretreatment also highlighted long-lasting increases in evoked ACh release elicited by adenosine. The results suggest that specific G proteins are involved in mediating the inhibitory effects of adenosine at motor nerve endings.

Acetylcholine↗
PubMed1988

The influence of 2-chloroadenosine on potassium-evoked and neurally-evoked acetylcholine secretion from normal or from latent active zones in the frog.

1. It has been suggested that adenosine receptor agonists do not impair K-dependent acetylcholine (ACh) secretion at motor nerve endings. If true, this result would be discordant with the conventional theories of adenosine action at the neuromuscular junction. It was thus decided to examine the effect of 2-chloroadenosine on quantal ACh release evoked by different K concentrations at frog motor nerve endings. 2. Quantal ACh release evoked by mild increases in the extracellular K concentration (from 2 mM to 6-11 mM) was inhibited by 2-chloroadenosine (10 microM) in a manner similar to the inhibition of neurally-evoked ACh release. 3. ACh secretion evoked by prolonged exposure to 20 mM K Ringer was also inhibited by adenosine derivatives. Under these conditions, alterations in the structure of the secreting active zones have been reported whereby the original release sites now release only a small proportion of the total quantal ACh output. 4. Preparations were bathed for several hours with Ca-free Ringer containing Mg to examine further the importance of intact active zones on inhibition produced by adenosine receptor agonists. This procedure has been reported to produce latent sites of ACh secretion and persistent derangement of the active zones. Shortly after this treatment, neurally-evoked ACh release in normal Ringer solution was found to be inhibited by 2-chloroadenosine (1-5 microM) or adenosine (50 microM). 5. The results suggest that (a) K-evoked ACh release is inhibited by adenosine derivatives even when quantal secretion occurs outside the original active zone and that (b) the cytoskeletal or membrane structures which maintain the structural integrity and lateral regularity of the active zones are not the target sites for inhibition by adenosine derivatives.

2-Chloroadenosine↗
PubMed1988

The effect of reduced temperature on the inhibitory action of adenosine and magnesium ion at frog motor nerve terminals.

1. A study was made to exclude the notion that adenosine receptor agonists exert a direct physical blockade of the depolarization-secretion process. Reduced temperature was employed as a tool for distinguishing between physico-chemical processes (such as those which mediate evoked transmitter release) and biochemical mechanisms (such as those which involve second messenger substances) in the action of adenosine. Adenosine and 2-chloroadenosine were used as agonists in this electrophysiological study of the release of acetylcholine (ACh) from frog motor nerve terminals. 2. The ability of these two adenosine receptor activators to reduce neurally-evoked ACh release was prevented or greatly attenuated by maintaining the preparation at temperatures between 5 and 10 degrees C. Such low temperatures inhibit the activation of receptors coupled to second messengers via guanine nucleotide binding proteins (e.g. adenylate cyclase). Low temperature alone did not substantially alter evoked ACh secretion under the conditions of these experiments. 3. Inhibition of evoked ACh release by the extracellular Ca antagonist Mg, which acts directly to block Ca channels, was not affected by low temperature. 4. The results are consistent with the hypothesis that a temperature-sensitive second messenger system controls the intracellular events linked to extracellular adenosine receptor activation.

Acetylcholine↗
PubMed1987

Independent control of channel closure and block of open channels by methylxanthines at acetylcholine receptors in frog.

1. A series of related methylxanthines were studied for their effects on the kinetics of decay of end-plate currents (e.p.c.s) and miniature end-plate currents (m.e.p.c.s) at motor end-plates of the frog. 2. Isobutyl methylxanthine (IBMX, 50 microM-3 mM) produced a concentration-dependent depression of the peak e.p.c. and m.e.p.c. amplitude and a change in the kinetics of e.p.c. and m.e.p.c. decay from the normal single-exponential to a double-exponential function. Drug effects of this nature are generally attributed to open-channel blockade. 3. After wash-out of IBMX, the decay of the e.p.c. or m.e.p.c. was restored to a single-exponential function but with a significantly prolonged time constant. 4. Caffeine or theophylline derivatives (0.1-4 mM), during exposure to drug, produced effects similar to those observed after the application of IBMX; namely a prolongation of the time course of e.p.c.s and m.e.p.c.s without changing the single-exponential nature of the function. 5. Computer simulations were made of the m.e.p.c.s in IBMX. The effects of IBMX could be fitted to the sequential model of channel block only if the prolonged time constant observed upon wash-out was used for the rate constant of channel closure. Independent calculations of the rate constant of channel closure during IBMX application were in agreement with those measured during wash-out. 6. The theophylline derivative 8-phenyltheophylline, a selective adenosine receptor blocker with minimal effects on phosphodiesterase (PDE), increased the time constant of e.p.c. decay in a manner similar to theophylline and caffeine. Non-xanthine PDE inhibitors, either had no effect on m.e.p.c. decay (papaverine) or decreased the time constant of decay (RO 20-1724). It is thus unlikely that PDE inhibition is responsible for the post-junctional effects of IBMX. 7. IBMX (50 microM-2 mM) increased quantal ACh release in the virtual absence of extracellular calcium and also increased the efficacy of adenosine derivatives in inhibiting ACh release. Adenosine (10-100 microM) or 2-chloroadenosine (1-10 microM) had no effect on the time constant of e.p.c. decay nor did these adenosine receptor agonists alter the post-junctional actions of IBMX. The effects of IBMX on end-plate channel kinetics are thus not due to the blockade of adenosine receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

The effects of an adenylate cyclase inhibitor on the electrophysiological correlates of neuromuscular transmission in the frog.

The presynaptic and postsynaptic effects of MDL 12,330A, an adenylate cyclase inhibitor in several biological tissues, were studied at motor endplates in frog cutaneous pectoris nerve-muscle preparations. This agent increased both spontaneous quantal acetylcholine (ACh) release and neurally-evoked ACh release approximately twofold during the first 20-40 min of application. The increased ACh release was accompanied by a profound irreversible depression in the amplitudes of the miniature endplate potentials (m.e.p.ps) and endplate potentials (e.p.ps). The response to iontophoretically-applied ACh was reduced in parallel with the amplitude of the spontaneous m.e.p.ps, indicating that the depression of synaptic transmission was postsynaptic in origin. Endplates were voltage-clamped to study the postsynaptic depression in more detail. It was observed that the peak endplate current (e.p.c.) was depressed without concomitant changes in: the kinetics of e.p.c. decay, the relationship between peak e.p.c. and membrane potential, the ACh equilibrium potential or the voltage sensitivity of the e.p.c. decay. This suggests that MDL 12,330A reduces the postsynaptic sensitivity to ACh by a voltage-dependent block of the cholinoceptor. The presynaptic enhancement and the postsynaptic depression of junctional transmission produced by MDL 12,330A are discussed in conjunction with current theories of the role of adenylate cyclase and cyclic nucleotides at nicotinic cholinergic synapses.

Acetylcholine↗
PubMed1984

On the mechanism by which adenosine receptor activation inhibits the release of acetylcholine from motor nerve endings.

The process by which adenosine receptor agonists inhibit the evoked release of acetylcholine (ACh) was studied at motor nerve endings to frog skeletal muscle. Adenosine and 2-chloroadenosine were employed as agonists. Each agonist reduced the mean number of ACh packets released synchronously in response to a nerve impulse (m). Adenosine was from one to two orders of magnitude less potent than 2-chloroadenosine as an inhibitor of this release. Focally recorded nerve terminal action potentials were unaffected by either adenosine receptor agonist. In normal Ca (1.8 mM), addition of sufficient Mg to reduce m to less than half the control value did not alter the degree of inhibition produced by adenosine receptor agonists. ACh release evoked by methods that do not require Ca entry through Ca channels (Ca-containing liposomes, La) was inhibited by either 2-chloroadenosine or adenosine. In Ca-free solutions containing Ba, the magnitude of neurally evoked asynchronous ACh release (miniature end-plate potential frequency = m.e.p.p.f) was depressed by either adenosine receptor agonist without change in the rate constant of decay of m.e.p.p.f; the m.e.p.p.f decay is thought to reflect the rate of clearance of Ba from regions of ACh release. Agents which displace Ca from storage sites and also inhibit phosphodiesterases increased m.e.p.p.f in the virtual absence of extracellular Ca and increased the level of inhibition produced by adenosine receptor agonists. RMI 12,330A (7 X 10(-6) to 7 X 10(-5) M), an adenylate cyclase inhibitor, occluded the effects of adenosine receptor agonists on ACh release. The results are consistent with the hypothesis that activation of extracellular adenosine receptors on adenylate cyclase inhibits evoked ACh release by reducing the affinity for Ca of an intracellular component of the secretory apparatus.

2-Chloroadenosine↗
PubMed1982

Effects of calcium and strontium in the process of acetylcholine release from motor nerve endings.

1. The effects of Ca and Sr ions on synchronous acetyleholine (ACh) secretion (the impulsive, physiologically functional form of secretion which produces an end-plate potential in response to a single nerve impulse) and on asynchronous ACh secretion (the delayed, residual increase in miniature end-plate potential frequency evoked by repetitive nerve impulses or by accumulation of intracellular divalent cations) were studied at frog neuromuscular junctions.2. In a comparison of their extracellular effects, Ca was far more effective than Sr in supporting synchronous ACh secretion but less effective than Sr in mediating asynchronous release evoked by repetitive nerve impulses.3. In studies of their intracellular effects, Sr and Ca were delivered to the nerve terminal cytoplasm using liposomes as a vehicle. Ca-containing liposomes, although producing effects on asynchronous ACh secretion that were indistinguishable from those of equimolar Sr-containing liposomes, were more effective than Sr-containing liposomes in increasing synchronous release.4. Extracellular Ca behaved as a potent competitve inhibitor of asynchronous, neurally evoked release mediated by Sr. In contrast, intracellular Ca (i.e. liposomal Ca), whilst increasing synchronous ACh release, failed to antagonize evoked asynchronous release.5. The results demonstrate that synchronous and asynchronous secretion have different sensitivities to alterations in intracellular divalent cation concentrations. It is suggested that selectivity for Ca over Sr may occur at intraterminal sites responsible for synchronous ACh secretion but not at sites responsible for asynchronous ACh release. Furthermore, Ca appears to bind with high affinity as an antagonist at the external surface of the nerve ending. These results are discussed in conjunction with current theories of depolarization-secretion coupling.

Acetylcholine↗
PubMed1982

Properties of calcium receptors that initiate depolarization-secretion coupling.

The relationship between the binding of divalent metal (Me) activators Ca and Sr and the secretion of acetylcholine (ACh) was studied quantitatively at frog motor nerve terminals using conventional electrophysiological methods. Experiments were designed to evaluate the assumption that maximal secretion requires occupancy of all receptors by testing for the presence of spare Ca receptors on nerve endings. Such a receptor reserve for Ca would invalidate the simple mass action approach to ACh secretion. Experimental log [Me]-ACh secretion curves constructed to saturation for Ca Sr were consistent with the presence of spare Ca receptors. La3+ (greater than or equal to 0.5 microM) and 2-chloroadenosine (25 microM) were employed as irreversible antagonists of depolarization-secretion coupling. Despite the irreversible occlusion of a proportion of Me receptors increases in the extracellular [Ca] overcame this antagonism while increases in [Sr] did not. These results suggest that Ca can produce maximal ACh release while leaving a proportion of receptors unoccupied or spare. Further support for this contention is provided by the excellent agreement between the values of the equilibrium affinity constant for Sr calculated by methods that do or do not require the assumption of spare receptors. The equilibrium affinity constant for Ca and the efficacies (efficacy reflects the ability of the Me species once bound to evoke ACh secretion) for both Ca and Sr were determined experimentally by using the mathematical framework of receptor theory. These constants were then employed to generate theoretical curves of log [Me]-ACh secretion. The theoretical relationships were similar to the experimental results, which suggests that the motor nerve endings behaves as a pharmacological receptor for Me agonists and antagonists. It is speculated that spare Ca receptors are equivalent to spare Ca channels and the efficacy may reflect the affinity of Me for an intraterminal site associated with ACh release.

2-Chloroadenosine↗
PubMed1981

On the calcium receptor that mediates depolarization-secretion coupling at cholinergic motor nerve terminals.

1 The behaviour of the divalent cations Ca and Sr as agonists for receptors that mediate the synchronous evoked secretion of acetylcholine (ACh) was studied in the hope of determining whether the relationship between Ca binding and ACh secretion is determined only by the law of mass action or by the mathematical framework of receptor theory. Experiments were designed to evaluate the assumption that maximum effect requires occupation of all receptors by testing for the presence of spare Ca receptors on presynaptic terminals. Frog cutaneous nerve-muscle preparations were employed in conjunction with conventional electrophysiological methods.2 Curves of log [Ca] or log [Sr] against the mean number of ACh quanta released (m) were constructed to saturation. The log [Sr]-m relationship was shifted to the right and had a smaller maximum than the log [Ca]-m curve. This suggests that Ca has a higher efficacy than Sr and raises the possibility that spare binding sites are present for Ca.3 As a qualitative test for spare Ca receptors, La(3+) (>/=0.5 mum) or 2-chloroadenosine (25 mum) was employed as an irreversible antagonist of the effects of extracellular Ca on evoked ACh release. Despite the irreversible blockade of a proportion of receptors, increases in the [Ca] overcame this antagonism and produced a parallel shift in the log [Ca]-m relation to the right. This suggests an apparent receptor reserve for Ca. Antagonism of Sr-mediated ACh release by either La(3+) or 2-chloroadenosine could not be overcome by increasing the [Sr].4 As a quantitative test for spare Ca binding sites, the equilibrium affinity constant for Sr(K(Sr)) as a competitive inhibitor of Ca was determined and compared with values for K(Sr) calculated by two other methods which invoke the spare receptor assumption. All three methods produced comparable results. (K(Sr) = 0.24-0.27 mm(-1)).5 The equilibrium affinity constant for Ca (K(Ca)) was calculated by comparing reciprocal plots of the concentrations of Ca that produce equal levels of ACh release in the presence and absence of La(3+) (0.5 mum-3 mum). K(Ca) was estimated to be between 0.02 and 0.06 mm(-1).6 Efficacy (e), which is thought to reflect the ability of Ca or Sr once bound to receptors to support ACh release, was determined by the modified occupation theory of Stephenson (1956). The e(Ca) was estimated to be 9-20 and e(Sr) was 0.2-0.5.7 The experimentally determined values for K(Ca), K(Sr), e(Ca), e(Sr) along with the assumptions that spare Ca binding sites exist and that the non-linearities in the log [Ca] or log [Sr]-m curves are introduced beyond the sites of binding and efficacy were used to generate theoretical log [Me]-m curves. The theoretical relationships were similar to the experimental results.8 The results suggest that spare Ca receptors are present at motor nerve endings and that receptor theory provides an accurate quantitative description of the lumped events between Ca binding and ACh secretion. The possible physical correlates of affinity and efficacy are discussed.

Animals↗
PubMed1981

Intracellular magnesium does not antagonize calcium-dependent acetylcholine secretion.

1. The effects of intracellular application of Ca and Mg ions on evoked acetylcholine secretion at frog motor nerve terminals were studied. Ca and Mg were applied to the nerve-ending cytoplasm using liposomes as a vehicle. 2. Under conditions in which intracellular application of Ca produced many-fold increased in evoked acetylcholine release. 3. When Mg was applied to the nerve-ending cytoplasm concurrently with Ca, acetylcholine release was further increased above the level produced by introducing Ca alone. 4. The results suggest that intracellular Mg does not antagonize depolarization-secretion coupling and that antagonism of transmitter release by extracellular Mg occurs only at the external surface of the nerve ending.

Acetylcholine↗