Facilitation and Depression of Neuromuscular Transmission under Conditions of Blockade of Ryanodine Receptors

In our experiments on mice, end-plate currents (EPC) evoked by stimulation of the phrenic nerve were intracellularly recorded in neuromuscular synaptic junctions of the phrenic muscle. We studied the effects of a specific blocker of ryanodine receptors, ryanodine (10 to 20 M), on the amplitude and time parameters of EPC under conditions of tetanic facilitation and depression of synaptic transmission at frequencies of stimulation of 4 to 200 sec^-1. Ryanodine inhibited facilitation at stimulation frequencies of 7 to 70 sec^-1 (with maximum effect at 20 sec^-1) and accelerated depression. In the presence of ryanodine, an initial rundown of the EPC amplitude in the course of depression of transmission increased at high frequencies of stimulation (50 to 100 sec^-1), whereas the EPC amplitude at the plateau level decreased already at low frequencies (4 to 7 sec^-1). We concluded that the changes in facilitation and depression resulted from blocking of the presynaptic ryanodine receptors by ryanodine. It seems probable that calcium release from the calcium stores in murine motor terminals is a factor involved in the control of processes of transmitter secretion during short-term rhythmic activation of the junction.     

Changes in the Asynchronicity of Transmitter Release at the Neuromuscular Junction during Prolonged High-Frequency Stimulation

In experiments on neuromuscular junctions in the frog m. cutaneous-pectoris, changes in the intensity and asynchronicity of transmitter release during high-frequency (10 and 50 sec^-1) rhythmic stimulation of the motor nerve were investigated using extracellular recording. At low extracellular Ca²⁺ concentrations, rhythmic stimulation resulted in a gradual enlargement of the quantum content of end-plate currents (EPC), the so-called facilitation. The latter phenomenon was accompanied by an increase in the average value and variance of synaptic delays of single-quantum EPC, a shift of the main mode of their distribution towards greater values, and an increase in the latency of the nerve ending responses. The above-described changes reduce the magnitude of facilitation in the neuromuscular synapse.     

Estimation of the Temporal Course of Evoked Secretion of Transmitter Quanta in the Frog Neuromuscular Junction

In experiments on the cutaneothoracic muscle of the frog, we recorded, using the technique of two-electrode voltage clamp at a normal Ca²⁺ concentration (1.8 mM), multiquantum end-plate currents (EPC) and miniature uniquantum EPC (mEPC). Multiquantum signals, when compared with uniquantum currents, were characterized by longer leading and trailing edges. The quantum composition of multiquantum signals estimated according to the ratios of EPC and mEPC amplitudes was, on average, 27% lower than that calculated according to the ratios of their integral values (areas). These data demonstrate that stimulus-evoked transmitter secretion from the motor nerve endings is noticeably asynchronous. Based on the parameters of the experimental EPC and mEPC, we estimated the temporal course of evoked secretion using various techniques: spectral analysis, a system of linear equations, and Van der Kloot's method. Using convolution with uniquantum signals, we found that spectral analysis is the best technique for such estimation. Calculated parameters of the temporal course of secretion were the following: risetime 0.20 msec and decay time constant 0.33 msec. The respective distribution significantly differed from that of the synaptic delays of extracellularly recorded uniquantum EPC by longer durations (150-200%) of the leading and trailing edges. We hypothesize that these differences are related to the geometry of the junction and the temporal sequence of switching on of the active zones in the nerve ending upon their activation by spreading action potentials. Factors influencing the temporal course of evoked secretion of the transmitter in the junction under study (its asynchronicity, in particular) are discussed.     

Changes in Electrogenesis in the Motor Nerve Terminal with Long-Lasting High-Frequency Activation as a Factor of the Control of Transmitter Release

Using the technique of extracellular recording from the region of the neuromuscular junction in the cutaneous-sternal muscle in the frog under conditions of a reduced concentration of Ca²⁺ in the surrounding milieu, we demonstrated that long-lasting (10 min) rhythmic stimulation of the motor nerve with a frequency of 10 sec^{− 1} leads to a gradual increase in the evoked transmitter release. These changes are accompanied by a decrease in the amplitude of electrical responses of the nerve terminal (NT) and by a retardation of its second phase, as well as by a diminution of the third phase. Under conditions of long-lasting (5 min) stimulation with a frequency of 50 sec⁻¹, we observed a two-phase change in the intensity of transmitter release: on the 2nd min, the initial rise was replaced by inhibition. Modifications of the response of the NT with different stimulation frequencies were qualitatively similar, but with a frequency of 10 sec⁻¹ they were clearly expressed. Mathematical simulation of ion currents in the NT demonstrated that voltage-dependent potassium and sodium channels are inactivated in the course of long-lasting high-frequency excitation; the shape of the action potential is modified with changes in the rate of such inactivation. This leads to either an increase or a decrease of the inward calcium current. We conclude that the change in electrogenesis in the NT with long-lasting high-frequency activation of neuromuscular junctions exerts a significant influence on the dynamics of transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 108–115, March–April, 2005.     

Role of Potassium Channels in Facilitation of Transmitter Release from Frog Motor Nerve Ending (Electrophysiology and Mathematical Simulation)

In experiments on neuromuscular junctions in the frog m. thoraco-cutaneous, we studied changes in the transmitter release and shape of the nerve ending (NE) response related to high-frequency (10 or 50 sec^-1) rhythmic stimulation of the motor nerve; an extracellular recording technique was used. At a low extracellular Ca²⁺ concentration, rhythmic stimulation resulted in a gradual increase in the quantum content of end-plate currents, i.e., in facilitation. Simultaneously, the third (positive) phase of the NE response became smaller, the amplitude of the second (negative) phase of this response also decreased, while the duration of this phase increased. Modifications of the NE response upon stimulation with a 10 sec^-1 frequency were more clearly expressed than those at 50 sec^-1 stimulation. In Ca²⁺-free solutions, rhythmic stimulation was accompanied by analogous modifications of the shape of NE responses, and the dynamics of these changes were the same at both the above-mentioned stimulation frequencies. When 0.5-1.0 mM tetraethylammonium was applied, 10 sec^-1 stimulation was accompanied by no facilitation of transmitter release; at 50 sec^-1 stimulation, this phenomenon was observed but was weaker than in the control, and the shape of NE responses underwent only mild changes. Simulation of electrogenesis in the studied structure showed that modifications of the NE response shape related to rhythmic 10 sec^-1 stimulation can develop in the case of a gradual decrease in the voltage-dependent potassium membrane conductivity, which results in prolongation of the de- and repolarization phases of action potentials and increases in the amplitude and duration of the inward calcium current. At higher stimulation frequencies (50 sec^-1), this mechanism is accompanied by a gradual increase in the Ca²⁺-dependent potassium conductivity, due to an increase in the intracellular Ca²⁺ concentration. These data allow us to conclude that the intensity of facilitation of transmitter release from the frog motor NE is related not only to accumulation of residual calcium, but also to changes of presynaptic calcium current due to modification of the kinetics of functioning of the potassium channels.     

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.     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

Are steady-state temporal correlations between evoked stochastic releases of synaptic vesicles as informative as suggested?

Using a stochastic model, we found that the steady-state temporal correlation between synaptic responses evoked by successive presynaptic spikes under conditions of high-frequency repetitive stimulation (50–100 sec⁻¹) is always negative. Therefore, the sign of this correlation cannot be used as a criterion that allows one to distinguish the univesicular vs multivesicular modes of neurotransmitter release in an active zone or the univesicular releases with low vs high probabilities of vesicle release, as suggested earlier [7]. For lower stimulation frequencies (15–20 sec⁻¹), positive correlation between release events evoked by consecutive stimuli is observed only in those cases where the number of ready-releasable vesicles and the time constant of recovery from depression are sufficiently large. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 412–415, September–December, 2006.     

IFNα Serum Levels Are Associated with Endothelial Progenitor Cells Imbalance and Disease Features in Rheumatoid Arthritis Patients

Introduction

IFNα has been largely implicated in the ethiopathogenesis of autoimmune diseases but only recently it has been linked to endothelial damage and accelerated atherosclerosis in autoimmunity. In addition, proinflammatory conditions are supposed to be implicated in the cardiovascular status of these patients. Since a role for IFNα in endothelial damage and impaired Endothelial Progenitor Cell (EPC) number and function has been reported in other diseases, we aimed to evaluate the potential associations of IFNα serum levels on EPC populations and cytokine profiles in Rheumatoid Arthritis (RA) patients.

Methods

pre-EPC, EPC and mature EPC (mEPC) populations were quantified by flow cytometry analyzing their differential CD34, CD133 and VEGFR2 expression in blood samples from 120 RA patients, 52 healthy controls (HC), and 83 systemic lupus erythematosus (SLE) patients as disease control. Cytokine serum levels were measured by immunoassays and clinical and immunological data, including cardiovascular (CV) events and CV risk factors, were retrospectively obtained by reviewing clinical records.

Results

Long-standing, but not recent onset RA patients displayed a significant depletion of all endothelial progenitor populations, unless high IFNα levels were present. In fact, the IFN^high RA patient group (n = 40, 33%), showed increased EPC levels, comparable to SLE patients. In addition, high IFNα serum levels were associated with higher disease activity (DAS28), presence of autoantibodies, higher levels of IL-1β, IL-6, IL-10 and MIP-1α, lower amounts of TGF-β, and increased mEPC/EPC ratio, thus suggesting higher rates of endothelial damage and an endothelial repair failure. Finally, the relationship between high IFNα levels and occurrence of CV events observed in RA patients seems to support this hypothesis.

Conclusions

IFNα serum marker could be used to identify a group of RA patients with increased disease activity, EPC imbalance, enhanced proinflammatory profile and higher cardiovascular risk, probably due, at least in part, to an impaired endothelial repair.     

Asymmetry of Characteristics of the Background Activity of Neurons of the Medial Vestibular Nucleus in Rats After Long-Term Vibration

Using computer analysis, we compared characteristics of the impulse background activity (BA) generated by neurons in the right and left medial vestibular nuclei (MVN) of the rats under control conditions and on the 5th, 10th, and 15th day with everyday 2-h-long sessions of vibrational stimulation. In the control group, the BA frequency generated by left-side MVN neurons was, on average, higher than that in the right MVN (23.6 ± 1.5 and 16.6 ± 1.7 sec^-1, respectively); other main characteristics of the BA demonstrated no significant internuclear differences. Vibrational influences of different durations induced complex significant laterally specific modifications of the level of regularity and dynamic indices of BA generated by neurons of the right and left MVN, of proportions of the cells with different types of distribution of interspike intervals (ISI), and of coefficients of variations of ISI. The mean frequency of background spiking in the right MVN increased about twofold (to 31.6 ± 2.2 sec^-1) after 10 days with vibration sessions, but dropped on the 15th day to 20.6 ± 1.7 sec^-1. In the left MVN, the mean BA frequency monotonically decreased, to 11.6 ± 1.0 sec^-1 after 15 days with vibration sessions. Therefore, chronic vibrational stimulation results in differential shifts of the characteristics of the BA generated by neurons of two MVN and in the formation of a new significantly asymmetrical pattern of such activity. Possible reasons for lateral asymmetry of the impulsation of MVN neurons and modifications of this asymmetry after long-term vibrational influences are discussed. We suppose that such an asymmetry can be one of the factors responsible for the development of motor and autonomic manifestations of vibration-induced motion sickness.     

Postsynaptic effects of substance P in frog neuromuscular junction

We have studied the effect of substance P on the end-plate currents (EPC) and the miniature EPC (MEPC) after acetylcholine esterase (ACE) inhibition in the cut neuromuscular preparation of the frog sartorius muscle using the voltage-clamp technique. At concentrations of 5·10^–7–1·10^–6 moles/liter substance P had no effect on the amplitude and the time characteristics of single EPC and MEPC but promoted prolongation of EPC decay on repetitive stimulation of the nerve with a frequency of 10/sec, indicating intensification of postsynaptic potentiation. Elevation of the concentration of the given peptide to 5·10^–6 moles/liter led to the shortening of the decay of single EPC and a more marked depression of the EPC amplitude in the trains as compared to the control, reflecting a decrease in the sensitivity of the postsynaptic membrane to the mediator, i.e., development of desensitization.S. V. Kurashov State Medical Institute, Kazan. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 436–441, July–August, 1991.     

Kinetics of the acid hydrolysis of heparin from its Cu (II) complex and its relation to biological activity

The acid-catalyzed hydrolysis of heparin from Cu(II) complex was studied as a function of time and temperature. Four independent calculations showed that the hydrolysis, during the 5-hr period examined, obeys the first-order kinetic law. Specific rate constants, calculated at 50°C, 57°C, 65°C, 71°C, and 80°C, were 3.3 × 10^?5 sec^?1, 6.5 × 10^?5 sec^?1, 10.4 × 10^?5 sec^?1, 15.1 × 10^?5 sec^?1, and 26.6 × 10^?5 sec^?1, respectively. Arrhenius plots of the data yielded 14.7 kcal as the energy of activation. An independent run of the self-hydrolysis of heparin at 57°C also obeyed first-order kinetics and its specific rate constant of 6.4 × 10^?5 sec^?1 is in excellent agreement with that of the hydrolysis of Cu(II)-heparin at 57°C. The anticoagulant activity of heparin and of the Cu(II)-heparin are not appreciably different. Further, the inactivation of heparin closely parallels Cu(II) release from the Cu(II) complex which in turn parallels desulfation.     

Kinetics of the low pH decolorization of azurin

Reaction rates were measured for the low pH-induced Cu ligand modification of azurins from Pseudomonas aeruginosa and Alcaligenes faecalis. Loss of the intense absorption band at 625 nm obeyed a rate law:

where Az is the concentration of azurin in its native oxidized form possessing the 625 nm band. For Pseudomonas aeruginosa at 25°C, n = 1 and k = 4.0 × 10^-2 sec^-1 M^-1 in citrate buffer but 1.2 × 10^-2 sec^-1 M^-1 in phosphate buffer. For Alcaligenes faecalis, n = 3 and k = 1.5 × 10⁴ sec^-1 M^-3 in citrate and 2.6 × 10³ sec^-1 M^-3 in phosphate. In equilibrium experiments on Alcaligenes faecalis azurin in citrate buffer, the pH-dependent change to the low pH form exhibited an apparent transition pK of 3.1. The form of the rate law implies a mechanistic scheme that contains fast equilibrium protonation steps prior to a rate limiting ligand rearrangement.     

The Rate Constants of Valinomycin-Mediated Ion Transport through Thin Lipid Membranes

Electrical relaxation experiments have been performed with phosphatidylinositol bilayer membranes in the presence of the ion carrier valinomycin. After a sudden change of the voltage a relaxation of the membrane current with a time constant of about 20 μsec is observed. Together with previous stationary conductance data, the relaxation amplitude and the relaxation time are used to evaluate the rate constants of valinomycin-mediated potassium transport across the lipid membrane. It is found that the rate constants of translocation of the free carrier S and the carrier-ion complex MS⁺ are nearly equal (2·10⁴ sec^-1) and are of the same order as the dissociation rate constant of MS⁺ in the membrane-solution interface (5·10⁴ sec^-1). The equilibrium constant of the heterogeneous association reaction M⁺ (solution) + S (membrane) → MS⁺ (membrane) is found to be ~ 1 M^-1, about 10⁶ times smaller than the association constant in ethanolic solution.     

Myonemal contraction of Spirostomum. I. Kinetics of contraction and relaxation

A microphotometric method is introduced that allows measurement of the contraction-relaxation kinetics of Spirostomum in response to electrical stimulation. The time course of contraction includes a rapidly contracting phase of some 4–5 mS during which cells shorten at a rate in excess of 100 cell lengths sec^?1. While a stimulus strength-duration curve determines the threshold of the response, the response to above threshold stimuli of different strengths and to trains of stimuli suggest that contraction of Spirostomum may not be an all-or-none event. The kinetics of relaxation following high stimulating voltages and repetitive after contractions also induced by high voltages are explained by excitation-contraction coupling through a stimulus-dependent intermediate effector, possibly the release of calcium ions. Changes in resting membrane potential detected by intracellular recording do not influence the initiation of contraction, while microinjection of calcium buffers above 10^?5 M Ca²⁺ invariably induces contraction.     

Quenching Effect of Tocopherols on the Methyl Linoleate Photooxidation and Their Oxidation Products

The quenching effect of α-, γ- and δ-tocopherols on the methylene blue sensitized photo- oxidation of methyl linoleate was investigated, and the ¹O₂. quenching ability of tocopherols was determined. The ¹O₂ quenching rate constants for α-, γ- and δ-tocopherols in ethanol were estimated to be 2.6 × 10⁸ m^?1 sec^?1, 1.8 × 10⁸ m^?1 sec^?1 and 1.0 × 10⁸ m^?1 sec^?1, respectively. And the rate constants for the chemical reaction between each tocopherol and ¹O₂ were 6.6 × 10⁶ m^?1 sec^?1, 2.6 × 10⁶ m^?1 sec^?1 and 0.7 × 10⁶ m^?1 sec^?1 for α-, γ- and δ-tocopherols, respectively. The results show that α-tocopherol is the most effective compound toward ¹O₂ among the three tocopherols. The photooxidation of each tocopherol produced two peroxides which, after chemical reduction, were identified to be tocopherol hydroquinone by gas chromatography-mass spectrometry analysis. The photooxidation mechanism of these tocopherols was assumed to be different from that of autoxidation.     

Inactivation of taste receptor cell function by two cationic protein modification reagents

Two cationic protein modification reagents, 1-cyclohexyl-3-(2-morpholinylethyl) carbodiimide (CMCD) and dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB-dmS), inhibit taste receptor cell stimulation by NaCl, sucrose, and HCl. Modified inactive derivatives of the reagents under the same conditions are ineffective. Inactivation by HNB-dmS is essentially irreversible. The effects of inactivation by CMCD are reversible after about 10–15 minutes of a water rinse, however, when applied in the presence of glycine methyl ester, the inhibited response is stabilized and only recovers after about 1.5–3 hours. Glycine methyl ester alone has no inhibitory properties. The kinetics of inactivation by both HNB-dmS and CMCD are consistent with a second-order reaction with rate constants of 0.041 ± 0.001 M^?1 sec^?1 and 0.121 ± 0.012 M^?1 sec^?1, respectively. The rate of inactivation by both compounds is independent of NaCl concentration as well as degree of receptor stimulation. This, together with the observation that the response to stimulation by all effectors examined is altered, suggests the inactivation occurs at an event which is common to the transduction of the response from all three stimuli. The ether:water partition coefficients, as well as previous results from inactivation by N–substituted maleimides, indicate that hydrophilic reagents do not cross the cell membrane in significant concentrations within the time period of application. This suggests the site of modification by the cationic protein modification reagents is at the surface of the cell membrane. Significant residual NaCl, sucrose, and HCl activity remains after total inactivation. To account for this, a two-state membrane receptor system is postulated.     

Dependence of the Curative Effects of Phosphene Electrostimulation on Its Frequency in Patients with Myopia

We studied the curative effects of transcutaneous stimulation of the visual analyzer (phosphene electrical stimulation, PhES) in patients with uncomplicated myopia. PhES frequencies of 30 and 15 sec^-1 were used in groups I (45 persons) and II (64 persons), respectively. After the PhES course, means of the visual acuity increased in these groups by 12 and 69%. The reserves of accommodation for long distances and mesopic visual acuity increased in these groups by 22 and 65% and by 10 and 21%, respectively. The light sensitivity of the cone phase did not significantly change and in both groups remained within a normal range. We discuss possible mechanisms of PhES-induced changes in the visual analyzer and conclude that a certain correction of the generally used PhES frequency (its shift toward lower values) can noticeably increase the efficiency of this therapeutic technique.     

Inter-and intraganglionar synaptic connections formed by neurons of the <Emphasis Type="Italic">Helix</Emphasis> visceral ganglion

In experiments on the subpharyngeal complex of the Helix ganglia, we found an excitatory monosynaptic input to the pacemaker PPa2 neuron from an unidentified cell of the visceral ganglion and a polysynaptic inhibitory influence of another unidentified neuron of this ganglion on the PPa1 cell. In addition, we revealed three pairs of neurons synaptically connected with each other (excitatory connections) in the visceral ganglion. In the case where we used high-frequency (11 sec⁻¹) stimulation of presynaptic elements, synaptic transmission to the PPa2 neuron demonstrated the greatest efficiency and stability. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 32–36, January–February, 2007.     

Kinetic Characterization of [125I] Iodo-Prolactin Binding to Primary Monolayer Cultures of Rabbit Mammary Epithelium

Abstract

Monocellular suspensions of epithelial cells from mammary glands of rabbits at 20–22 days of pregnancy were prepared by sequential dissociation with collagenase-hyaluronidase followed by Pronase. Maintenance in D-valine-substituted minimum essential medium (D-valine-MEM) supplemented with 10% dialyzed calf serum yielded monolayers enriched for rabbit mammary epithelial cells (RMEC). RMEC specifically and reversibly bound bovine PRL with K_a = 1.41–1.85 × 10⁹M^-1. Association of lactogen with RMEC receptor followed bimolecular reaction kinetics with rate of 5.17 (±0.75) x 10⁵M^-1 sec^-1 at 24 C, and 1.03 (±0.11) x 10⁶M^-1 sec^-1 at 37 C. Dissociation was first order (k-₁ = 5.97 (±0.70) x 10^-5 sec^-1) and was unaffected by the presence of lactogen. Specific binding determined with an excess of unlabelled bPRL was 66–77% of the total binding, and was optimal at pH 7.4. The binding reaction reached equilibrium in 2 h at 37 C, in 3 h at 24 C, and after 24 h at 4 C. Studies of binding capacity revealed the presence of 4.6–6.3 × 10³ sites per cell, competition for which was limited to hormones demonstrating lacto-genic activity. Recovered lactogen was not degraded by incubation with or dissociation from RMEC. Approximately 25% of the radioactivity remained associated with the cells even upon prolonged incubation. These studies demonstrated several advantages of RMEC for the investigation of hormone-receptor interaction and receptor regulation.