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Biomedical subjects

F Vyskocil

Publications and source records attributed to F Vyskocil.

At least 73 records · Page 4Linked to original sources
PubMed1990

The effects of nerve terminal activity on non-quantal release of acetylcholine at the mouse neuromuscular junction.

1. Local endplate depolarization induced by anticholinesterase application to mouse nerve-diaphragm preparations was taken as a measure of non-quantal release of acetylcholine. 2. Non-quantal acetylcholine release occurred within 20-60 s after anticholinesterase application, either spontaneously or evoked by nerve stimulation. Non-quantal release declined with time and disappeared after 3-5 min. 3. The amplitude of stimulation-evoked non-quantal release increased with the frequency of stimulation and was maximal at frequencies above 50 Hz. Two stimuli were sufficient to evoke the maximal effect. 4. Micromolar concentrations of atropine, pirenzepine and vesamicol reduced the amplitude and shortened the duration of non-quantal release. Oxotremorine (10(-8) M) enhanced the amplitude and ouabain (10(-4) M) prolonged the duration of non-quantal release. 5. Our results support the idea that the non-quantal release is due to the vesicular acetylcholine transport system which becomes transiently a part of the nerve terminal during exocytotic release of quantal acetylcholine.

Acetylcholine↗
PubMed1989

Single K+ currents during differentiation of embryonic muscle cells in vitro.

After 3-7 days in culture, chicken myotubes possess five types of K+ channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K+ channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K+ channels predominate.

Adenosine Triphosphate↗
PubMed1989

Effect of Mg2+ on non-quantal acetylcholine release at the mouse neuromuscular junction.

The effects of extracellular concentrations of Mg2+ on the non-quantal release of acetylcholine (ACh) from nerve terminals was studied by extra- and intracellular electrophysiological methods. Anticholinesterase-treated mouse diaphragms were used in vitro. In the presence of Ca2+, the non-quantal release was maximal in the absence of Mg2+ and was inhibited by 3 mmol/l Mg2+. The inhibitory effect of Mg2+ was antagonized by ouabain and was absent in Ca2+-free (EGTA) solutions. The non-quantal release of ACh was found to be more sensitive to inhibition by Mg2+ than the quantal one which was measured as the amplitude of miniature endplate currents.

Acetylcholine↗
PubMed1989

Development of desensitization during repetitive end-plate activity and single end-plate currents in frog muscle.

1. The amplitudes of end-plate currents (EPCs) in short trains of fifteen to seventeen EPCs at 10 Hz were depressed in the presence of 10 microM-proadifen when acetylcholinesterase (AChE) was inhibited. 2. The proadifen-induced EPC depression was voltage-dependent and the effect was more pronounced at negative membrane potentials. 3. In the presence of proadifen, the mean amplitude of miniature end-plate currents (MEPCs) was reduced by 36% 5 s after the EPC train as compared with MEPCs before the train. 4. Without proadifen, but with inhibited AChE, an increase of temperature from 20 to 26 degrees C and elevation of external Ca2+ from 1.8 to 2.5 mM led to EPC amplitude depression in the train, which was also potential-dependent. 5. After AChE inhibition, proadifen (10 microM) progressively shortened MEPC decay without significant reduction of amplitude up to 40 min of exposition. MEPCs were not affected by proadifen when AChE was active. 6. It is concluded that these postsynaptic effects of proadifen can be explained neither by its action on the resting acetylcholine receptors (AChR) nor on open ion channels but are due to its desensitization-promoting action.

Animals↗
PubMed1988

Inhibition of the electrogenic Na,K pump and Na,K-ATPase activity by tetraethylammonium, tetrabutylammonium, and apamin.

The K+-induced hyperpolarization of Na-loaded mouse diaphragm muscle, enzymatic activity of Na,K-ATPase and 3H-ouabain binding to rat brain microsomes was measured in the presence of K+ channel blockers tetraethylammonium (TEA), tetrabutylammonium (TBA) and apamin. TBA, and to a lesser extent TEA in millimolar concentrations, inhibited the electrogenic effect of the Na,K pump, Na,K-ATPase activity, and 3H-ouabain binding. The inhibition of 3H-ouabain binding by TEA or TBA was more evident in the presence of ATP and Na+ ions. Apamin in nanomolar concentrations inhibited the electrogenic effect of Na,K pump and Na,K-ATPase but not the 3H-ouabain binding. The hyperpolarizing effects of insulin and NADH, but not that of noradrenaline, were also prevented by apamin. The inhibition of Na,K pump by TEA and TBA is apparently due to both competition with K+ for a binding site on the Na,K-ATPase and a reduction in the number of transporting sites. The site of action of apamin on Na,K-ATPase is different from that of tetra-alkylammonium compounds; it apparently decreases the turnover rate of the enzyme.

Animals↗
PubMed1988

A possible role for the acetylcholine transport system in non-quantal release of acetylcholine at the rodent myoneural junction.

The effects on the spontaneous, non-quantal release of acetylcholine (ACh) from motor nerve terminals of substances known to inhibit the ACh transport system present in cholinergic synaptic vesicles have been investigated. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine (dTC) in muscle endplates that had been treated by an anticholinesterase was partly or completely abolished by 2-(4-phenylpiperidino) cyclohexanol (AH5183, 10(-7)-10(-6)M), quinacrine (10(-7)M) and tetraphenylborate (10(-6) M). Since the sensitivity of the endplate to ACh was not changed, the block of the dTC induced hyperpolarization indicated an inhibition of the spontaneous, non-quantal release of ACh. This was confirmed by direct measurement of the ACh released by rat diaphragm. The release of ACh from the innervated diaphragm was decreased by about 50% by AH5183 (10(-8)-10(-6) M) and by 42% by quinacrine (10(-7)-10(-6) M). The ACh released was presumably neural, since the release of ACh from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of ACh from the motor nerve terminals is probably mediated by a carrier which may be the vesicular transport system responsible for moving ACh into the vesicle. The transport system is likely incorporated into the membrane of the nerve terminal during exocytosis.

Acetylcholine↗
PubMed1988

The changes in conformation of (Na+ K+)-ATPase from rat brain membranes are accompanied by changes of protein segment movements in the nanosecond range.

Differential polarized phase fluorometry of fluorescein-5-isothiocyanate (FITC) showed that the activation of (Na,K)-ATPase in crude plasma membranes from rat brain by 10 mmol.l-1 K+ and 100 mmol.l-1 Na+ significantly increased the rotational relaxational rate (R) of enzyme-bound FITC. This increase was blocked by both ouabain (0.1 mmol.l-1) and vanadate (0.1 mmol.l-1). In the absence of ATP, R was increased less after adding of 10 mmol.l-1 K+ to the membranes. The shifts in the nanosecond movements of the protein segments measured as R during the activation of (Na,K)-ATPase suggest that this type of movement might be of some functional importance.

Animals↗
PubMed1987

Arachidonate activates muscle electrogenic sodium pump and brain microsome Na+,K+-ATPase under suboptimal conditions.

Arachidonate 5 x 10(-5) mol.l-1 increased the rate of hyperpolarization induced in Na+-loaded mouse diaphragm fibers by 5 mmol.l-1 K+. When applied to Na+-loaded muscles without potassium, arachidonate 1 x 10(-6) and 5 x 10(-5) mol.l-1 induced a ouabain-sensitive hyperpolarization of the muscle fibers. The activity of rat brain microsomal Na+,K+-ATPase was stimulated by 1 x 10(-7)-5 x 10(-6) mol.l-1 arachidonate in reaction media with reduced amounts of ATP or K+ and after short-lasting sonication of the samples. It was concluded that, under particular conditions, arachidonate might serve as a Na+,K+-ATPase activator or inhibitor regulating its ion transport and electrogenicity.

Animals↗
PubMed1987

Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells.

Single channels in mouse neuroblastoma cells with a high conductance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels were selective for Cl- as compared to cations and exhibited a linear I-V relationship between +40 and -40 mV. These Cl- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of Cl- channels described was the dissipation of their conductance into several substates during the course of individual recordings.

Animals↗
PubMed1987

A study on early post-denervation changes of non-quantal and quantal acetylcholine release in the rat diaphragm.

The d-tubocurarine (dTC) induced hyperpolarization of antiesterase-treated muscles at the endplate zone, miniature endplate potentials (mepps), resting membrane potentials (RMPs) and the input resistances of single muscle fibres (Rin) were measured in rat diaphragm at various times after denervation. The dTC-induced hyperpolarization decreased in two phases: 2 h after denervation it decreased transiently to 25%, after 4 h it had partially recovered to 60% and from 6 h it progressively decreased up to 12 h after which time it changed to depolarization. The initial fall and recovery were also present in muscles from sham-operated animals. The frequency of mepps decreased by 25% and the amplitude diminished by 10% within the first 2-4 h. After 10 h the frequency had decreased by 35% and the amplitude by 65%. After 12 h no mepps were present. The RMP was not significantly changed during the first 16 h after denervation. From 16 to 24 h the membrane became depolarized at a rate of about 1 mV/h. The input resistance of a single muscle fibre was constant for 12 h after denervation and from 12 to 24 h it increased by 25%. It is concluded that the early decrease in the dTC-induced hyperpolarization is probably due to the desensitization of acetylcholine (ACh) receptors caused by stress-activated non-quantal ACh release. The later decrease of dTC-hyperpolarization reflects a fall in the non-quantal ACh release. The depolarization of the resting membrane after denervation is related to the decrease in passive membrane permeability which is a secondary consequence of transmission failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1987

The action of excitatory amino acids on chick spinal cord neurones in culture.

1. Membrane currents evoked by N-methyl-D-aspartate (NMDA), L-aspartate, L-glutamate, quisqualate and kainate were studied in cultured neurones from the embryonic chick spinal cord by the patch-clamp technique and by employing a quasi-step microperfusion technique. 2. Application of NMDA, aspartate, glutamate and quisqualate induced currents which exhibited an initial peak which declined to a plateau level with a time constant of 2 s and then remained constant or slowly decreased. The discontinuation of the application was followed by an after-current. The individual components of the responses were insensitive to TTX (2 X 10(-6) M) and were present in neurones which did not exhibit any sign of synaptic activity. The responses induced by kainate were monophasic and declined slowly during long-lasting application. 3. The responses induced by NMDA, aspartate and glutamate were voltage dependent, while those induced by kainate were linear between -80 and +80 mV. The equilibrium potential for all components of the responses to all excitatory amino acids was close to zero. 4. From dose-response curves the half-maximum effective dose (ED50) for glutamate and kainate was 3 X 10(-5) and 2 X 10(-4) M respectively. The Hill coefficients for the glutamate and the kainate were calculated to be 1.8 +/- 0.1 (n = 4) and 1.9 +/- 0.5 (n = 4) respectively. Thus two molecules may be interacting with each of the receptor-activated ion channels. 5. Interaction between kainate and quisqualate or kainate and NMDA was studied at both negative and positive holding potentials. No summation of the responses was found when kainate at concentrations close to those required for evoking the maximum response was applied simultaneously with quisqualate or NMDA. On the contrary, a diminution of the membrane currents was observed. A marked decrease in membrane currents was also observed when glutamate (10(-4) M) was applied simultaneously with aspartate (10(-4) M). 6. Glutamate-activated single-channel currents were recorded in the cell-attached configuration with electrodes filled with glutamate (20 microM) in five neurones and a conductance approximately 50 pS was found. 7. It is suggested that differences in the potency of the different excitatory amino acids as open-channel blockers may be one of the mechanisms which contribute to the diversity in the action of excitatory amino acids and that at least some of the effects of NMDA, aspartate, glutamate, quisqualate and kainate may be mediated by a common receptor-channel complex.

Action Potentials↗
PubMed1987

Arachidonate has a positive effect on the electrogenic sodium-potassium pump in the mouse diaphragm.

Sodium arachidonate 5 X 10(-5) mol X l-1 shortened the time course of hyperpolarization caused by the electrogenic Na+-K+ pump in intact muscle fibres in the mouse diaphragm preincubated in a K+-free physiological solution. Contrary to experiments on membrane fragments, no inhibition of the ouabain-sensitive Na+-K+ ATPase was observed. It is unlikely that the arachidonate may be identical with the endogenous "ouabain-like" substance (Bidard et al. 1984).

Animals↗
PubMed1986

Vanadyl ions increase the order parameter of plasma membranes without changing the rotational relaxation time.

Differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene showed that vanadyl ions (VO2+) increased its limiting anisotropy (order parameter) in crude plasma membranes from brown adipose tissue of the golden hamster (Mesocricetus auratus). This was about 10(3) times larger than the effect of Ca2+ and was several times greater than the action of Co2+. Vanadate anions were without any effect. During the membrane treatment with VO2+, the rotational relaxational time of diphenylhexatriene did not change. This results suggest a possible positive influence of tetravalent vanadium on the stability of cell membranes.

Adipose Tissue, Brown↗
PubMed1986

Spider venom of Araneus opens and desensitizes glutamate channels in chick spinal cord neurones.

Whole cell currents induced by the excretions of venom glands from the spider Araneus diadematus (ASV) were tested in neurones from the embryonic spinal cord of the chick in culture by the patch clamp technique. ASV in a dose of one bite in 1 ml induced a large membrane current of the same polarity as the excitatory amino acids, which decreased during long-lasting application. The effects were, to a large extent, rapidly reversible. It is suggested that ASV activates and desensitizes the receptor channels for excitatory amino acids.

Amino Acids↗
PubMed1986

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block.

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.

Amino Acids↗
PubMed1986

Effect of catecholamines and metal chelating agents on the brain and brown adipose tissue Na,K-ATPase.

Catecholamines stimulate Na,K-ATPase activity in the microsomal membranes of the brain and brown adipose tissue. This stimulation is apparent in the absence of soluble, cytosolic inhibitors and exhibits the same characteristics in both tissues: it occurs at high concentrations (10(-6)-10(-4) M) only; there is no difference in potency between isoprenaline, norepinephrine and epinephrine (EC50 = 1-2 X 10(-5) M); the D-stereoisomer of isoprenaline is equally as effective as the L-form; stimulation of Na,K-ATPase may also be achieved by the metal chelators EDTA, EGTA and desferal; the hydrophobic beta-blockers, propranolol and alprenolol, inhibit both the norepinephrine-stimulated and basal levels of enzyme activity at concentrations of 10(-5)-10(-3) M; phenoxybenzamine, an irreversible alpha-adrenergic blocker, inhibits basal Na,K-ATPase as well as norepinephrine-stimulated enzyme activity (EC50 = 2.5 X 10(-5) M). Because none of these observations can be related to the properties of the stereospecific adrenergic receptor (alpha or beta), it may be concluded that the catecholamine-Na,K-ATPase interaction is not mediated by the receptor. More probably, catecholamines may antagonize the Na,K-ATPase inhibition caused by some tightly membrane-bound metals (but not vanadium) via the ortho-catechol moiety of the catecholamine molecule. The stimulation of brown fat Na,K-ATPase by catecholamines does not have much relevance to the norepinephrine-stimulated thermogenesis in this tissue.

Adipose Tissue, Brown↗