Oleg N. Osipenko

Neurochemical, electrophysiological and immunocytochemical evidence for a noradrenergic link between the sympathetic nervous system and thymocytes

The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.

Metal ion-induced permeability changes in cell membranes: A minireview

Summary1. The paper summarizes the effects of the metal ions Cu2+, Pb2+, Ag+, Hg2+, Zn2+, and Cd2+ applied externally or internally to the surface membrane of different excitable cells.2. Conductance changes induced by metal ions, and metal ion-activated current, are compared with respect to their ion and voltage dependence.3. It is suggested that metal ion-induced effects can be realized through special structures of the cell membrane, the metal ion “receptors,” although other mechanisms, as, for example, competition for Ca-binding sites in the channel forming proteins, cannot be excluded.

Effect of Ag+ on membrane permeability of perfused Helix pomatia neurons.

1. Isolated, non‐identified neurons were voltage clamped using the internal perfusion technique. 2. Ions of Ag+ (1‐100 microM) introduced into the bathing solution activated a steady‐state inward current (IAg) in the soma. The effect of Ag+ was reversible when the concentration of Ag+ was less than 75 microM or the time of application was shorter than 10 min. 3. IAg was observed both in the presence and absence of Na+ ions in the extracellular saline. It could also be activated when Cs+ ions were substituted for Na+ ions. 4. The current‐voltage characteristics were linear in the voltage range ‐100 to 0 mV. The reversal potential in control saline was an average of 1.19 +/‐ 5.1 mV. 5. The application of Ag+ ions induces an elevation of intracellular free Ca2+ concentration by 10‐20 times in both Ca(2+)‐containing and Ca(2+)‐free extracellular salines, as revealed by Fura‐2 measurements. 6. Agents that increase the intracellular free Ca2+ concentration ([Ca2+]i), like thymol, caffeine and dinitrophenol, increased the amplitude of IAg. The effect was additive. Ruthenium Red, which blocks the release of Ca2+ from intracellular stores, decreased the Ag+ effect. 7. It is concluded that extracellularly applied Ag+ ions increase the cytoplasmic free Ca2+ concentration, which in turn activates non‐specific cationic channels. 8. Ag+ ions in 1‐10 microM concentration were able to decrease the voltage‐activated Ca2+ current amplitude. This decrease, however, was due to the increase of [Ca2+]i which caused Ca(2+)‐dependent inactivation.

Effect of deltamethrin on acetylcholine-operated ionic channels in identified Helix pomatia L. neurons

Abstract The main action of pyrethroids is to slow the gating kinetics of the Na channel of the axon and some. However, it was also found that pyrethroids may affect the neurotransmission as well, acting postsynaptically. It can be proposed, therefore, that pyrethroids may have their target at the synaptic site in addition to the axonal or somal Na channels. The aim of the present study was to examine the effect of deltamethrin on acetylcholine (ACh) receptors of snail neurons using a two-microelectrode voltage clamp. Ionophoretic application of ACh on the soma of identified Helix neurons produced inward current of which amplitude was dependent on the holding potential. Deltamethrin (1–100 μ M ) depressed the ACh-induced current in a concentration-dependent manner. The I–V curves, both in control and in deltamethrin-containing saline, were linear in the potential range of −100 and −25 mV. The reversal potential was unchanged. The data suggest a monomolecular binding of deltamethrin to the ACh-activated channel with a K D of 50 μ M . Deltamethrin increased the time of recovery from ACh desensitization. It can be concluded, therefore, that deltamethrin may contribute to its toxic effect by affecting the function of the ACh-receptors, although this effect could be secondary.

Effects of Cu2+, Pb2+ and Zn2+ on voltage-activated currents in Helix pomatia L. Neurons

Effects of Cu2+, Pb2+ and Zn2+ were studied on voltage-activated Na-, Ca-, and K-currents in snail neurons. It was found that: 1. In normal physiological saline Cu2+, Pb2+ and Zn2+ ions exerted complex changes on the total ionic currents; 2. All three metal ion have depressed the inward Na-currents but with different KD, moreover Pb2+ increased Na-current at low concentrations (5 μM); 3. The inward Ca-current was also reduced. The sequence of the blocking effect of metals was different: Pb>Cu>Zn, however the steady-state inactivation was influenced only by Cu2+; 4. Outward currents were decreased in all neurons by Cu2+, but the effects of Pb2+ and Zn2+ were either depression or enhancement in different indentified neurons; 5. The possibility of binding heavy metals to wide variety of membrane proteins and the observed effects on different ionic channels suggest that the metal effect is complex and cannot be taken as a specific one to a single channel type or site of location.

Lead ions close steady-state sodium channels in Helix neurons

Extracellularly applied Pb2+ (1-150 microM) induced an outward current (IPb) in intracellularly perfused snail neurons. The current-voltage relationship of the Pb(2+)-induced current was linear over the potential range of -100 to -40 mV with negative slope conductance. The Pb-induced current was strongly dependent on the Na+ gradient. The IPb in intra- or extracellular K+- and Cl(-)-free or -rich solutions was almost the same as in control external and internal salines. The negative slope of the I-V curve and the decreased conductivity during Pb2+ application suggested that IPb is owing to the blocking of the resting Na conductance. Data obtained from single-channel measurements also supported this conclusion. Patch-clamp data showed that the steady-state Na channel has a conductance of 14 pS and both closed and open time-distributions displayed single exponential character.

Opposite effects of molluscan neuropeptides CARP and MIP on LVA Ca-currents in Helix pomatia L. neurons

Abstract 1. Voltage-clamp recordings from identified neurons of Helix pomatia L. revealed that two Ca-current components may contribute to the inward current in Na-free saline. 2. It was found that the transient component of the inward Ca-current was largely blocked by Ni 2+ but not by Cd 2+ . 3. CARP (10 −6 M) selectively suppressed LVA currents. This effect is also reflected by tail current measurements, where in control saline the deactivation could be fitted by two exponentials, while after CARP treatment, by one. 4. MIP (10 −6 M) specifically enhanced the LVA component, or shifted the I-V curve of the composite Ca-current in hyperpolarizing direction. 5. It is concluded that peptides (CARP and MIP) isolated from Mytilus ganglia selectively affect the LVA Ca-channels, and that this effect is supposed to be species-specific.

Inositol-1,4,5-trisphosphate and non-hydrolysable GTP analogue induced calcium release from intracellular stores of the Helix pomatia neurons

1. Cytoplasmic Ca2+ concentration ICaIin and membrane potential of single Helix pomatia neurons was studied by Fura-2 fluorescence measurement and conventional current clamp methods. 2. Intracellular injection of inositol-1,4,5-trisphosphate (IP3) and nonhydrolysable GTP analogue (Gpp/NH/p) led to a rise of ICaIin; in contrast, GTP injection did not cause significant ICaIin changes. 3. We suggest that both IP3 and Gpp/NH/p directly activated Ca release from intracellular stores.

Modulatory action of oxytocin on electrical responses of the neurons in the snail Helix pomatia

1. 1. We have studied the influence of 10–100 nM oxytocin (OXT) added to the extracellular solution on electrical responses in Helix pomatia snail neurons. 2. 2. Certain neuronal responses were found to increase in the OXT-containing solution (OXT-solution). The following responses were studied: (a) to the stimulation of excitatory interneuron; (b) to application of the endogenous peptide; (c) to the OXT application; (d) to the intracellular cAMP injection. 3. 3. We observed a decrease of neuronal response to acetylcholine application in the OXT-containing solution and during cAMP injection. 4. 4. The intracellular injection of OXT increased the amplitude of the current induced by intracellular cAMP injection (cAMP-current). 5. 5. The OXT-solution effect on the cAMP-current was abolished by tolbutamide, a cAMP-dependent protein kinase inhibitor, added to the external solution. 6. 6. According to our finding, we suggest the existence in the Helix pomatia neurons intracellular OXT receptors. These receptors, likely, modulate the cAMP-dependent protein kinase activity, that could be underlye for modulatory OXT effects.

Depolarization and hyperpolarization of the neuronal membrane in the snail Helix pomatia identified neurons induced by oxytocin application to the soma of these neurons

Abstract 1. The inward and outward currents (OXT-currents) responsible for depolarization and hyperpolarization of the neuronal membrane induced by oxytocin (OXT) application to the soma of identified Helix pomatia neurons was investigated with conventional voltage-clamp technique. 2. The inward current (OXT 1-current) was increased in amplitude in low Cl- ions solution. This current associated with neuronal membrane conductance increase. The reversal potential of this current was near −40 mV. Addition to the bath solution of furosemide reversibly blocked the OXTl-current. 3. Two types of neurons were found with a different outward OXT-currents. The first type associated with a neuronal conductance decrease. Its reversal potential was near −40 mV. This current was increased by hyperpolarization and was reversibly blocked by furosemide (OXTZ-current). The second one associated with a conductance increase. Its reversal potential was near −70 mV, and with a 2-fold increase of external K+ ions concentration it shifted towards depolarization by 15mV. Addition of TEA was blocked this current (OXT3-current). 4. Extracellular addition of theophylline increased, while imidazole and tolbutamide decreased the amplitude of OXTl-current. The OXT2-current was decreased, while OXT3-current was unchanged, in tolbutamide-containing solution. 5. It is suggested that Helix pomatia neurons have different types of OXT receptors. Activation of some receptors cause an increase or a decrease in the membrane permeability for Cl-ions, probably through the system of cyclic nucleotides, in turn, the activation of another receptor, which increase K+ ions permeability probably, without cyclase system activation.