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V I Skok

Publications and source records attributed to V I Skok.

At least 19 recordsLinked to original sources
PubMed2004

[Alfa-subunit composition of neuronal nicotinic acetylcholine receptors of the submucosal plexus in the guinea-pig small intestine].

The alpha-subunit composition of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using the whole-cell patch-clamp technique and affinity-purified antibodies (Abs) against alpha3-, alpha4, alpha5 and alpha7-subunits of nAChRs. The independent addition of anti-alpha3 and anti-alpha5 Abs to extracellular bath solution induced depression of the acetylcholine (ACh)-evoked currents by 60% +/- 1.56% and 65% +/- 1.62% correspondingly. Successive application of anti-alpha5 Abs in the presence of anti-alpha3 Abs did not have any additional blocking effect on ACh-evoked currents. Anti-alpha7 Abs evoked depression of Ach-induced currents by 24% +/- 1.51% in 80% of investigated neurons and by 67% +/- 1.5% in 20% of neurons. The addition of anti-alpha4 Abs to extracellular bath solution did not have effect on membrane currents of the investigated neurons. Our data provide evidence of participation of alpha3-, alpha5 and alpha7-containing receptors in the response to ACh. Alpha3- and alpha5-subunits are associated in the same functional nAChRs that provide greater part of response to ACh in the most of submucosal neurons. However, in some neurones alpha7-containing nAChRs provide greater part of response to agonist.

Acetylcholine↗
PubMed2004

Functional nicotinic acetylcholine receptors in the neurons of rat intracardiac ganglia.

The alpha subunit composition of nicotinic acetylcholine receptors (nAChRs) expressed in the neurons of intracardiac ganglia of the rat was investigated using monoclonal and polyclonal antibodies against alpha3, alpha4, alpha5 and alpha7 nAChR subunits. The acetylcholine-induced membrane potentials in the neurons of the subepicardial plexus isolated from the left atrium of the heart were studied by intracellular recording performed in the presence of subunit-specific nAChR-blocking antibodies applied either separately or in various combinations. It was found that intracardiac ganglia neurons express alpha3alpha5; alpha7; alpha7(alpha5) and alpha4-containing nAChRs. The neurons were heterogeneous as to the nAChR subtypes expressed that possibly indicated their functional differences.

Acetylcholine↗
PubMed2001

Nicotinic acetylcholine receptor subtypes in rat superior cervical ganglion neurons as studied by sequential application of two alpha-subunit-specific antibodies.

All cultured neurons of rat superior cervical ganglion (SCG) were stained with nicotinic acetylcholine receptor (nAChR) alpha5- or alpha7-subunit-specific oligoclonal antibodies (Abs) and could additionally bind alpha3-subunit-specific monoclonal antibody (mAb). About 60% of the neurons were stained with alpha4-specific Ab and could not bind alpha3-specific mAb. The acetylcholine-induced membrane currents recorded with the whole-cell patch clamp method and partially blocked with alpha3-specific mAbs, could be additionally blocked with alpha5- and alpha7- specific Abs, and vice versa. The results suggest that: (1) each neuron of rat SCG expresses several nAChR subtypes with different alpha-subunits; (2) the alpha3-, alpha5- and alpha7-subunit-containing nAChRs are probably located far enough from each other thus enabling joint binding to the cell of the corresponding alpha-subunit specific Abs, in contrast to the alpha4-subunit-containing nAChRs which are probably located too close to the alpha3-containing ones to allow their joint binding.

Acetylcholine↗
PubMed1999

Modulation of nicotinic acetylcholine receptor activity in submucous neurons by intracellular messengers.

The effects on acetylcholine-induced membrane currents (ACh currents), produced by agents known to modify the activity of intracellular messengers, were studied in the neurons of the guinea-pig ileum submucous plexus (SMP) using a whole-cell patch clamp recording method. The ACh currents were not affected by forskolin, the adenylate cyclase activator, regardless of whether or not ATP and GTP were present in the intracellular solution, and by phorbol 12-myristate 13-acetate, the protein kinase C activator. The ACh currents were strongly suppressed by thapsigargin, the microsomal calcium ATPase inhibitor, and genistein, the tyrosine protein kinase inhibitor. They were also suppressed by 3-isobutyl-1-methylxanthine, the cyclic-AMP phosphodiesterase inhibitor, regardless of the presence of forskolin in the extracellular solution and ATP and GTP in the intracellular solution. In addition, the currents were suppressed by activation of P2 purinoceptors with ATP, which could not be explained by a direct effect of ATP on nicotinic acetylcholine receptors (nAChRs). Reactive blue 2, the P2y purinoceptor antagonist, did not abolish inhibition of the ACh current by ATP. Alpha,beta-Imido-ATP and adenosine caused no membrane current responses and did not influence the ACh currents. These results suggest that the activity of the nAChRs in the SMP neurons is strongly suppressed by raised intracellular Ca2+ level, without involvement of protein kinases A and C, and may involve the participation of tyrosine kinase. The activity of nAChRs is also influenced by the activity of P2 purinoceptors; the mechanisms responsible for this influence are not yet clear. So, the activity of the SMP neuronal nAChRs is relatively independent on the intracellular signaling known to influence many other groups of transmitter-gated receptors of neuronal membrane.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies.

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different alpha subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the alpha(181-192) region of alpha3, alpha4, alpha5, and alpha7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among alpha3, alpha4, alpha5, and alpha7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the alpha3, alpha5, and alpha7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly alpha4-positive. In contrast, only about one-third of the intracardiac neurons were alpha3-positive, about 50% were alpha4-positive, one-seventh were alpha5-positive, and one-fifth were alpha7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that alpha3, alpha5, and alpha7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas alpha4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

Acetylcholine↗
PubMed1999

[The effect of SR 142801, a blocker of tachykinin NK-3 receptors, on synaptic transmission in the rat caudal mesenteric ganglion].

Effects of tachykinin NK-3 receptors antagonist SR 142801 on the synaptic transmission in the rat's caudal mesenteric ganglion were investigated using extracellular recording of compound action potentials (CAPs) from the ganglion nerves. It is shown that antagonist of NK-3 receptors SR 142801 (2.5.10(-7) mol/l) decreased the amplitude and square of CAPs obtained by stimulating of intermesenteric nerve (IMN) and leading off from the colonic nerve, after high-frequency stimulation of IMN. It is suggests that tachykinin NK-3 receptors take part in SP-ergic synaptic transmission through the rat's caudal mesenteric ganglion.

Action Potentials↗
PubMed1999

[The pharmacological blockade of the cardiac and gastric enteric ganglia by a new parasympatholytic].

The blocking effects of a newly synthetized compound N-decyltropine bromide (IEM-1556), on pentagastrin-, carbachol- and histamine-induced gastric secretion in chronic dogs, on stress-induced changes in gastric mucosa in rats, on vagus-induced effect in heart and on arterial blood pressure in rats were studied. The effects were compared with those produced by hexamethonium, a conventional ganglionic blocking agent. IEM-1556 inhibited gastric secretion and acid output for a much longer time than hexamethonium did. It also effectively protected gastric mucosa against stress-induced erosions and hemorrhages. IEM-1556 much more effectively blocked the vagus-induced reduction of the heart rate than hexamethonium did, and, in contrast to latter, did not reduce the arterial blood pressure. The results suggest that IEM-1556 is a highly selective blocking agent for parasympathetic and enteric ganglia versus sympathetic ganglia.

Animals↗
PubMed1998

Patch-clamp recordings of membrane currents evoked during natural synaptic activity in sympathetic neurons.

Membrane currents elicited by colonic distension and by electrical stimulation of the intermesenteric nerve containing colonic afferent nerve fibres were recorded from neurons of the mouse superior mesenteric ganglion at 20 degrees C with the whole-cell patch-clamp method. Electrically-evoked excitatory postsynaptic currents reversed at -3.5 mV. At membrane holding voltages of -70 mV and -110 mV, the excitatory postsynaptic currents were characterized by a single exponential decay with a mean (+/- S.E.M.) time-constant of 17.5 +/- 1.3 ms and 15.5 +/- 2.3 ms, respectively. Colonic distension evoked a series of the excitatory postsynaptic currents which ranged in amplitude from 10 to 700 pA (at a membrane holding voltage of -70 mV). Hexamethonium (100 microM) applied only to the ganglion abolished both electrically- and distension-evoked excitatory postsynaptic currents, suggesting activation of nicotinic acetylcholine receptors. The decay time-course of distension-evoked single excitatory postsynaptic currents was characterized by one, or, less commonly, by two exponentials. The decay time-constant histograms of distension-evoked single excitatory postsynaptic currents exhibited main kinetic components of 8.1 +/- 2.3 ms and 8.2 +/- 2.5 ms (peak +/- S.D.) at -70 and -110 mV membrane holding voltages, respectively. Longer time-constants ranging up to 51 ms were also observed. The number of the distension-evoked excitatory postsynaptic currents with a decay time-constant higher than 20 ms, as well as their mean amplitude, were significantly lower at -110 mV than at -70 mV membrane potential levels, in contrast to the currents with a decay time-constant lower than or equal to 20 ms. The results suggest that colonic afferent nerve fibres activate in the mouse superior mesenteric ganglion neurons a few populations of the postsynaptic nicotinic acetylcholine receptors with different channel kinetics, which are characterized by a lack of voltage sensitivity within -70 to -110 mV membrane potential range, except those with comparatively slow channel kinetics, which are possibly blocked by membrane hyperpolarization.

Animals↗
PubMed1998

[Intracellular regulation of neuronal nicotinic cholinergic receptors].

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.

1-Methyl-3-isobutylxanthine↗
PubMed1995

The ionic channel of neural nicotinic acetylcholine receptors is funnel-shaped.

Membrane currents evoked by iontophoretically applied acetylcholine were recorded from non-dissociated neurons of rat superior cervical ganglion using the whole-cell patch-clamp recording method. Blocking effects produced by a series of specially synthesized organic compounds, the blockers of the open channel of ganglionic nicotinic acetylcholine receptors, used as tools, were studied, and dimensions of the channel were deduced from correlation between the sizes of the blocking molecules and their blocking activities. Two channel cross-profiles were found, small and large, as approximated by the rectangles with the most probable dimensions 5.8 x 8.0 A and 7.0 x (8.4 - 9.0) A, at intra- and extracellular sides of the channel, correspondingly, in addition to the 6.1 x 8.3 A (medium) cross-profile found in our earlier work and localized between the two above. These findings indicate that the channel is funnel-shaped in the area where the open channel blockers are bound. The binding site is localized at the level of the medium cross-profile or between it and small cross-profile. The small cross-profile is probably a selectivity filter.

Acetylcholine↗
PubMed1994

Mechanism of long-lasting block of ganglion nicotinic receptors by mono-ammonium compounds with long aliphatic chain.

The effect of long-chain mono-ammonium compounds (long-chain MACs), t-butyldecylammonium (IEM-1078), 2,2,6,6-tetramethyldecylpiperidine (IEM-1559), and diisopropyldecylammonium (IEM-1194), on nicotinic acetylcholine receptors (nAChRs) was studied in sympathetic ganglion neurons using the patch clamp method. Long-chain MACs (1-10 microM) strongly inhibited acetylcholine (ACh)-induced current (ACh-current); the block persisted for hours after washing the drugs out. Short-chain MACs had a much weaker and completely reversible blocking effect. Suppression of ACh-current by MACs was dose- and voltage-dependent; it was absent at low ACh doses or at potentials > or = 60 mV and increased with higher ACh doses or hyperpolarization. The second of two ACh-currents induced by paired application of ACh was inhibited by long-chain MACs more strongly than the first. This use-dependent block also persisted for hours after washing the drugs out. Additional inhibition of the second ACh-current was reduced by lengthening the time interval between ACh applications in the pair. Time constants of the recovery of the second ACh-current in the presence and after washing out of long-chain MACs were similar, ranging from 45 to 140 s at -50 mV for different long-chain MACs, and decreased with de- or hyperpolarization. The use-dependent block produced by long-chain MACs could be prevented by another long-chain MAC with a small ammonium head (IEM-1195, 75-100 microM) or trimethaphan (30 microM), a competitive antagonist of ACh in ganglia. Neither the short-chain MAC (IEM-1405, 100 microM) nor ACh (100 microM) could exert this protective effect. Long-chain MACs did not exert any use-, dose- or voltage-dependent suppression of ACh-current when applied intracellularly. Single-channel conductance was not affected by IEM-1194 (3-10 microM). We suggest that inhibition of ACh-current by long-chain MACs is accounted for by (i) a long-lasting, apparently irreversible, binding of the drug near the channel of nAChR via its long aliphatic chain and (ii) a slow reversible block of the nAChR channel with the MAC's ammonium head.

Acetylcholine↗
PubMed1992

Synaptic transmission in rat cardiac neurones.

Intracellular recordings of spontaneous synaptic activity and synaptic responses to fibre tract stimulation were taken from neurones of ganglia isolated from the left atrium and interatrial septum of the rat. In six out of 57 neurones studied, spontaneous fast excitatory postsynaptic potentials (EPSPs) were recorded. Single stimulation of fibre tracts approaching the ganglion resulted in an all-or-none response consisting of an EPSP, from which an action potential abruptly appeared. This response disappeared in Ca(2+)-free/high-Mg2+ solution, indicating that it was orthodromic in origin. EPSPs were markedly exaggerated and prolonged by neostigmine (1-5 microM). EPSPs produced by high-frequency (0.1-20 Hz) fibre tract stimulation were markedly attenuated when compared with responses to single fibre tract stimulation, although they usually remained suprathreshold for spike initiation. High concentrations of hexamethonium (1 mM) and d-tubocurarine (300 microM) failed to inhibit responses to single fibre tract stimulation, although they completely abolished responses to high-frequency stimulation. Responses to single fibre tract stimulation were abolished by trimetaphan (greater than or equal to 100 microM). No slow synaptic responses were detected during single or high-frequency fibre tract stimulation. All cardiac neurones that responded orthodromically were highly excitable: they had a short post-spike after-hyperpolarization (AHP) and responded with multiple firing to prolonged membrane depolarization. It is concluded that cardiac neurones, in the region of the heart studied here, receive single 'strong' cholinergic inputs from some fibre tracts approaching the ganglion that elicit EPSPs accompanied by spikes. EPSPs are rather resistant to ganglion-blocking agents and subject to frequency modulation.

Action Potentials↗
PubMed1992

Neuronal mechanisms responsible for ongoing activity of rabbit superior cervical ganglion neurons.

The action potentials evoked by stimulation of single preganglionic nerve fibres or of single neurons of the ganglion were recorded from postganglionic nerves of rabbit superior cervical ganglion. The averaging technique was used to improve the nerve signal-to-noise ratio. It was found by dividing the area by the area that single preganglionic nerve fibre discharges, as an average, 15 neurons of the ganglion. Action potentials were also recorded from preganglionic nerves, either appearing synchronously with the intracellular spikes recorded during ongoing activity of the ganglion neuron, or being evoked by stimulation of a single preganglionic nerve fibre. It was found by dividing the area by the area that each ongoing spike in the ganglion neuron is preceded by firing, as an average, of three preganglionic nerve fibres. Thus, 45 ganglion neurons are expected to be discharged by a preganglionic volley during their ongoing activity. This number is much lower than that found in our previous experiments (about 100 neurons). The difference suggests that in the majority of the ganglion neurons the ongoing discharges need summation of excitatory effects produced by a few converging preganglionic nerve fibres (multiple input) rather than being evoked by a single preganglionic nerve fibre (single input). Methacine, a selective muscarinic antagonist (0.3-0.5 mg/kg, i.v.), decreased by about 10% the rate of the multiple input-induced ongoing spikes while not affecting the single input-induced ongoing spikes in the ganglion neurons. This effect is probably due to the increase in the duration of after-hyperpolarization observed after the application of methacine.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

Acetylcholine receptors in rat cardiac neurones.

Membrane potential changes produced by acetylcholine (ACh), and their underlying mechanisms, were studied in neurones of isolated cardiac ganglia of the rat by means of intracellular microelectrodes. Five components of membrane potential change could be detected in cardiac neurones following 1-5 s micro-application of ACh: (i) fast depolarization resulting from an activation of nonselective cationic conductance; (ii) slow depolarization associated with a decreased membrane conductance, presumably for potassium ions; slow hyperpolarization which consisted of (iii) early and (iv) late parts resulting from an activation of calcium-sensitive potassium current and from inhibition of steady-state inward current, respectively; and (v) delayed slow hyperpolarization associated with an increased conductance, most likely for potassium ions. Components (i), (iii) and (iv) persisted in the presence of atropine and were inhibited by nicotinic antagonists. Thus they were due to activation of nicotinic ACh receptors. However, the sensitivity of component (i) to ganglion-blocking agents appeared to be rather low: IC50s for inhibiting (i) were 226 +/- 34.2 microM, 31.2 +/- 4.31 microM and 15.3 +/- 3.27 microM for hexamethonium, d-tubocurarine, and trimetaphan, respectively. Components (ii) and (v) were abolished by atropine (1 microM) and mimicked by muscarine (component (ii) also persisted in d-tubocurarine), hence they resulted from activation of muscarinic ACh receptors. It is concluded that cardiac neurones are endowed with both nicotinic and muscarinic ACh receptors. Their activation leads to membrane depolarization and discharges followed by hyperpolarization and inhibition of discharges.

Animals↗
PubMed1992

Molecular mechanisms of open-channel blockade in nicotinic acetylcholine receptors of autonomic ganglia neurons.

The results of recent attempts to estimate the dimensions of ionic channels in nicotinic acetylcholine receptors of sympathetic and enteric ganglia neurons are reviewed. The channel dimensions, obtained from comparison of the sizes of the open-channel blocking molecules with their blocking activities, are 6.1 x 8.3 A (1 A = 0.1 nm) in both sympathetic and enteric ganglia. None of the competitive ganglionic blockers fit within this channel size. In addition, a chemical structure that binds the open-channel blockers in ganglionic nicotinic acetylcholine receptors is suggested to be formed by serine and threonine residues, as found by comparing the differences between the structures of the neuronal and muscle nicotinic acetylcholine receptors with the differences in their pharmacology.

Amino Acid Sequence↗
PubMed1991

Dimensions of the ion channel in neuronal nicotinic acetylcholine receptor as estimated from analysis of conformation-activity relationships of open-channel blocking drugs.

Relationship between the size of the molecule in the series of organic ions Et3+N--(CH2)5--+NR1R2R3 (Ri--alkyl or cycloalkyl substituents) and their abilities to block nicotinic acetylcholine receptors (AChRs) due to their open-channel blockade in the neurons of autonomic ganglia and in frog end-plate was analyzed. All low-energy equilibrium conformations of the drugs were calculated by the molecular mechanics method. A unique rectangular channel profile 6.1 x 8.3 A, for which the best correlation between blocking activity of the drugs and total population of their conformations being able to penetrate into the channel, was deduced from all those tested.

Animals↗