Biomedical subjects

F Vyskocil

Publications and source records attributed to F Vyskocil.

At least 19 recordsLinked to original sources

PubMed2006

Different sensitivity of miniature endplate currents of the rat extensor digitorum longus, soleus and diaphragm muscles to a novel acetylcholinesterase inhibitor C-547.

A novel derivative of 6-methyluracil, C-547, increased the amplitude and prolonged the duration of miniature endplate currents (MEPCs) which is typical for acetylcholinesterase inhibition. In the soleus and extensor digitorum longus significant potentiation was detected at nanomolar concentrations. In contrast, in the diaphragm muscle, the increase in the amplitudes of the MEPCs and the decay time constant appeared only when the concentration of C-547 was elevated to 1 x 10(-7) M. Possible consequences for the exploitation of this drug, which can selectively inhibit AChE in particular synapses, are discussed.

Animals↗

PubMed2005

Calcium dependence of uni-quantal release latencies and quantal content at mouse neuromuscular junction.

Uni-quantal endplate currents (EPC) were recorded at mouse diaphragm neuromuscular synapse by extracellular microelectrode during motor nerve stimulation. The probability of release expressed as quantal content m(o), and variability of synaptic latencies expressed as P90 were estimated in the presence of extracellular calcium ([Ca2+]o) varying between 0.2 and 0.6 mM in the bathing solution. At 0.2 mM ([Ca2+]o), m(o) was low (0.10) and many of long-latency EPCs were present during the late phase of the release (P90 = 2.44 ms). No change in m(o) was found when ([Ca2+]o) was 0.3 mM, but P90 decreased by 39 %. For latency shortening, saturating concentration of ([Ca2+]o) was 0.4 mM, when P90 was 1.49 ms and latencies did not further change at 0.5 and 0.6 mM ([Ca2+]o). In the latter concentrations, however, an increase of m(o) was still observed. It can be concluded that the early phase of the secretion did not significantly change when ([Ca2+]o) was raised and that only the late phase of the release depends on extracellular calcium up to 0.4 mM.

Action Potentials↗

PubMed2005

Spontaneous quantal and non-quantal release of acetylcholine at mouse endplate during onset of hypoxia.

At 20 (0)C, both quantal and non-quantal spontaneous acetylcholine release (expressed as miniature endplate potential frequency [f-MEPPs] and the H-effect, respectively) increased during the first 30 min of hypoxia in solution with normal extracellular calcium ([Ca(2+)](o) = 2.0 mM). The hypoxia-induced tenfold increase of the f-MEPPs was virtually absent in low calcium solution([Ca(2+)](o) = 0.4 mM) whereas there was still a significant increment of non-quantal release. This indicates that each of these two processes of acetylcholine release is influenced by mechanisms with different oxygen sensitivity. The rise of f-MEPPs during the onset of hypoxia apparently requires Ca(2+) entry into the nerve terminal, whereas the non-quantal release can be increased by another factors such as a lower level of ATP.

Acetylcholine↗

PubMed2004

Activation and modulation of ligand-gated ion channels.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Allosteric Regulation↗

PubMed2003

Different degree of cooperativity in adult, embryonic and mutated mouse muscle nicotinic receptors.

Adult and embryonic nicotinic receptors expressed in COS cells have similar affinities for acetylcholine but differ in their Hill coefficient. Parameters of wild-type receptors were compared with those of receptors with mutated delta and gamma subunits in selected negatively charged amino acids, which were expected to participate in agonist binding. A tentative scheme of affinities, allosteric interactions and channel gating efficacy was used for assessing the role of mutated amino acids in the channel function. In three models, the parameters of wild-type embryonic and adult receptors were compared with those of receptors with mutated delta and gamma subunits. The analysis of different models of channel activation indicates that negatively charged amino acids which were mutated in the delta subunit in embryonic receptors participate in channel gating and in allosteric interactions between subunits rather than directly in agonist binding. Changes in the gamma subunit in the embryonic receptors and delta subunit in the adult receptors could equally affect agonist binding, allosteric coupling between subunits or channel gating.

Acetylcholine↗

PubMed2003

Temperature effect on proximal to distal gradient of quantal release of acetylcholine at frog endplate.

The conduction velocity of the nerve terminal, mean quantal content, and release latencies of uniquantal endplate currents (EPCs) were recorded in proximal, central, and distal parts of the terminal by extracellular pipettes located 5, 50, and 100 microm from the end of myelinated nerve trunk. The spike conduction velocity, minimal latency, modal value of the latency histograms, and time interval during which 90% of EPCs released (P90) at distal, central, and proximal part of the frog nerve terminal have different temperature dependency between 10 degrees and 28 degrees C. As shown by the size and time-course of reconstructed multiquantal EPCs, the secretion synchronization, which is greatest in distal parts, compensates at least partly for the progressive slowing of spike conduction velocity in the proximodistal direction, in particular at lower temperatures.

Acetylcholine↗

PubMed2003

Long release latencies are increased by acetylcholine at frog endplate.

Uni-quantal endplate currents (EPCs) were recorded extracellularly at the frog neuromuscular synapse and their latency dispersions expressed as P(90) were estimated in the presence of acetylcholine. Stimulation-evoked EPCs with long release latencies increased in number when acetylcholine was applied. P90, which is designated as the interval between the minimal synaptic delay and the time at which 90 per cent of all measured uni-quantal EPCs had occurred, was significantly and reversibly increased by 66 per cent from 0.51 ms to 0.85 ms in the presence of 5x10(-4) M acetylcholine. This indicates that the evoked release pattern is less synchronous and the increased asynchrony leads to a substantial drop (by 28 per cent) in the amplitude of reconstructed multi-quantal currents.

Acetylcholine↗

PubMed2003

Chloride cotransport in the membrane of earthworm body wall muscles.

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).

Animals↗

PubMed2002

Citalopram inhibits L-type calcium channel current in rat cardiomyocytes in culture.

Selective serotonine reuptake inhibitors (SSRI) are believed to be less dangerous in the treatment of depressive disorder in comparison with tricyclic antidepressants (TCA) due to their relative lack of cardiotoxicity. Thus, we investigated the effect of citalopram (SSRI) on membrane electrophysiology in rat cardiomyocytes in tissue culture. The results were compared with those from amitriptyline (TCA). The whole-cell configuration patch-clamp technique was used. Both citalopram and amitriptyline exhibited the concentration-dependent inhibition of the L-type calcium channel current (ICa). Citalopram in concentrations of 3 microM and 10 microM inhibited peak calcium current by 2.7% and 8%, respectively. We demonstrated the same potency of citalopram and amitriptyline to inhibit ICa. These observations led us to conclude that citalopram and amitriptyline are drugs, which exhibit a similar potency for causing concentration-dependent inhibition of ICa.

Animals↗

PubMed2002

[Early post-denervation depolarization: effect of NO, acetylcholine and glutamate on chloride transporter].

Resting non-quantal acetylcholine (ACh) and probably glutamate (Glu) release from nerve endings activates M1- and NMDA-receptor mediated Ca2+ entry into the sarcoplasm with following activation of NOS and production of NO. This is a trophic message from motoneurones which keeps the Cl- transport inactive in the innervated sarcolemma. After denervation, the secretion of ACh and Glu at the neuromuscular junction is eliminated within 3-4 h and the production of NO in the sarcoplasm is lowered. As a result, the Cl- influx is probably activated by dephosphorylation of the Cl- transporter with subsequent elevation of intracellular Cl- concentration. The equilibrium Cl- potential becomes more positive and the muscle membrane becomes depolarized.

Acetylcholine↗

PubMed2001

The role of desensitisation in decay time of miniature endplate currents in frogs Rana ridibunda and Rana temporaria.

A new comparative characteristic of endplate microphysiology has been introduced. It is the feasibility of receptors to become desensitised as demonstrated on two frog species, Rana temporaria and Rana ridibunda: the decay times (tau(MEPC)) of single quantum miniature endplate currents (MEPCs) in the sartorius muscles of both species were about 1 ms and were not affected by the desensitisation-promoting agent proadifen when AChE was active. However, when the desensitisation was induced by anticholinesterase neostigmine and promoted by proadifen, the prolongation of tau(MEPC) from 1 ms was almost twice as great in Rana temporaria (tau(MEPC) = 4.4 ms) than in Rana ridibunda (tau(MEPC) = 3.1). This indicates that desensitisation reduces the number of available receptors and lowers the number of available ACh molecules for repetitive binding by trapping them by desensitised, high-affinity receptors significantly more in Rana ridibunda than in Rana temporaria. The application of proadifen, a promoter of desensitisation, decreased the prolongation of MEPCs in both species, but this shortening was more rapid in Rana ridibunda than in Rana temporaria. It is concluded that the desensitisation-induced reduction in the density, and the number of postsynaptic receptors is significantly higher at Rana ridibunda than in Rana temporaria endplates.

Animals↗

PubMed2001

Temperature effect on carbachol-induced depression of spontaneous quantal transmitter release in frog neuromuscular junction.

The effects of carbachol (CCh) on the frequency (f) of the miniature endplate potentials were tested at temperatures between 5 and 30 degrees C. Higher CCh concentrations, 1 x 10(-5) and 5 x 10(-6) M, reduced the f to 60% and the temperature dependence was negligible. However, an inverse temperature dependence was found when low concentrations 3 x 10(-7) and 6 x 10(-7) M were applied. The depression of f was 40-50% in 5-10 degrees C but only 10-20% of the control in the 25 and 30 degrees C. During application of CCh, the new steady of f was reached at temperatures between 5 and 30 degrees C within 17-20 min (Q10 = 1.07). Much greater temperature dependence of recovery was observed during washing out CCh (Q10 = 1.6). The temperature-independence of the steady state effects of CCh, good agreement with Langmuir adsorption-desorption theory and non-steady kinetics indicate that physical rather than receptor-mediated events are responsible for the depression of f.

Animals↗

PubMed2001

ATP but not adenosine inhibits nonquantal acetylcholine release at the mouse neuromuscular junction.

The postsynaptic membrane of the neuromuscular synapse treated with antiacetylcholinesterase is depolarized due to nonquantal release of acetylcholine (ACh) from the motor nerve ending. This can be demonstrated by the hyperpolarization produced by the application of curare (H-effect). ATP (1 x 10-5 M) decreased the magnitude of the H-effect from 5 to 1.5 mV. The membrane input resistance and the ACh sensitivity were unchanged, and so changes in these cannot explain the ATP effect. Adenosine alone was without effect on the nonquantal release. On the other hand, both ATP and adenosine depressed the frequency of spontaneous miniature endplate potentials, to 56% and 43% respectively. The protein kinase A inhibitor Rp-cAMP or the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one did not affect the inhibitory influence of ATP on the H-effect, whereas staurosporine, an inhibitor of protein kinase C, completely abolished the action of ATP. Suramin, an ATP antagonist, enhanced the H-effect to 8.6 mV and, like staurosporine, prevented the inhibitory effect of ATP. ATP thus suppresses the nonquantal release via a direct action on presynaptic metabotropic P2 receptors coupled to protein kinase C, whilst adenosine exerts its action mainly by affecting the mechanisms underlying quantal release. These data, together with earlier evidence, show that nonquantal release of ACh can be modulated by several distinct regulatory pathways, in particular by endogenous substances which may or may not be present in the synaptic cleft at rest or during activity.

Acetylcholine↗

PubMed2001

[Effect of glutamate on spontaneous secretion of acetylcholine in the nerve-muscle synapse in rats].

In rats, glutamate was shown to exert no effect on the mean frequency, character of interstimuli distribution, amplitude and temporal parameters of the miniature EPPs. Glutamate suppressed nonquantal release. The glutamate effect depended on its concentration and was abolished by blockade of NMDA receptors, NO-synthase inhibitoin, and NO molecules binding by haemoglobin in extracellular medium. Glutamate seems to modulate the nonquantal acetylcholine secretion by initiation of the NO synthesis in muscle fibres via activation of the NMDA receptors.

Acetylcholine↗

PubMed2000

Carbachol and acetylcholine delay the early postdenervation depolarization of muscle fibres through M1-cholinergic receptors.

The resting membrane potential (RMP) of denervated muscle fibres of rat diaphragm muscle is depolarized by approximately 8-10 mV during the first 3 h after nerve section and this early postdenervation depolarization is reduced substantially by the presence of 5x10(-8) M acetylcholine (ACh) or carbachol (CB). The muscarinic antagonist atropine (Atr; 5x10(-9) to 5x10(-6) M) reduced the effect of CB in a dose-dependent manner (K(i)=7x10(-8) M) and increased the rate of the early postdenervation depolarization. In lower doses (5x10(-7) M), Atr acted only in the presence of an allosteric stabilizator hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium] (W-84). Also pirenzepine, a specific inhibitor of the M1 subtype of muscarinic receptor, blocked the action of CB in a dose-dependent manner with an apparent inhibition constant K(i)=1x10(-7) microM. DAMP, a specific M3 antagonist, was without effect on the muscle hyperpolarization induced by CB. CB also hyperpolarized the membrane potentials of muscles which were denervated for 1-3 days. It is concluded that ACh and CB protect the muscle fibres from early depolarization through M1-cholinergic receptors on the muscle membrane. These particular receptors can apparently mediate the 'trophic', non-impulse regulation of RMP in skeletal muscles when they are activated by acetylcholine released non-quantally.

Acetylcholine↗

PubMed2000

Effect of nitric oxide and NO synthase inhibition on nonquantal acetylcholine release in the rat diaphragm.

After anticholinesterase treatment, the postsynaptic muscle membrane is depolarized by about 5 mV due to nonquantal release of acetylcholine (ACh) from the motor nerve terminal. This can be demonstrated by the hyperpolarization produced by the addition of curare (H-effect). The magnitude of the H-effect was decreased significantly to 3 mV when the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) were applied to the muscle, or when NO production was elevated by adding L-arginine, but not D-arginine, as a substrate. The H-effect was increased to 8-9 mV by inhibition of NO synthase by L-nitroarginine methylester (L-NAME), or by guanylyl cyclase inhibition by methylene blue and 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ). ODQ increased the H-effect to 7.3 +/- 0.2 mV and diminished the SNP-induced decrease of the H-effect when applied together with SNP. The effects of NO donors and L-arginine were eliminated by adding reduced haemoglobin, an extracellular NO scavenger. The present results, together with earlier evidence for the presence of NO synthase in muscle fibres, indicate that nonquantal release of ACh is modulated by NO production in the postsynaptic cell.

Acetylcholine↗

PubMed2000

The effect of carbachol and alpha-bungarotoxin on the frequency of miniature endplate potentials at the frog neuromuscular junction.

The effects of an acetylcholine analogue, carbachol (CCh), and a purified irreversible nicotinic antagonist, -bungarotoxin (BTX), on the frequency of the miniature endplate potentials (mEPPs) at the neuromuscular junction of the frog were tested at 20 and 10 C. CCh (5 10-6 m) reduced the frequency of mEPPs to about 60 %; this reduction was not affected by 1 10-7 g ml-1 BTX. BTX also reversibly decreased the mEPP frequency by 40 %, but not in the presence of CCh or in Ringer solution with 0 or 8 mM Ca2+. The present data show that BTX, which inhibits a class of nicotinic ACh receptors, does not block the decrease of mEPP frequency evoked by CCh and can itself suppress the frequency of spontaneous quantal release.

Animals↗

PubMed2000

[Modulation of the intensity of the non-quantal transmitter release by nitric oxide (NO) at the neuromuscular junction].

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.

Acetylcholine↗