Different GTP-binding proteins mediate regulation of calcium channels by acetylcholine and noradrenaline in rat sympathetic neurons

In dissociated neurons of rat superior cervical ganglion (SCG), noradrenaline (NA) and acetylcholine (ACh) suppressed Ca2+ currents elicited by depolarizations to 0 mV from -60 mV. With GTP-gamma-S in patch electrodes, ACh and NA caused persistent inhibition of Ca2+ currents. Pretreatment of SCG cells with pertussis toxin abolished the action of ACh but not of NA. The results suggest that ACh and NA reduce the Ca2+ currents in SCG cells through different G proteins.     

The "trap"--a modification of the block of neuronal nicotinic cholinoceptors

The effect of IEM-1742, a pentaethonium derivative, on the currents induced by iontophoretic applications of acetylcholine was studied in rat superior cervical ganglion neurons using patch-clamp method in the whole-cell modification. Blocking action increased with membrane hyperpolarization and was removed by strong membrane depolarization. Apparent dissociation constant for the receptor-blocker reaction was found to be (2.9 +/- 0.6) 10(-6) M (n = 6) at -50 mV and 20-23 degrees C. IEM-1742 blocks the nicotinic acetylcholine receptor in its activated form. The dissociation of IEM-1742 from the receptor was drastically accelerated during its activation by agonist (trap-block). Trapped receptor was not released from the blocker only by membrane depolarization to the level at which any blocking effect is absent. The data obtained show that IEM-1742 in rat sympathetic ganglion neurons acts in the potential-dependent manner and displays a trap-effect.     

Two modes of activity of nicotinic acetylcholine receptor channels in sympathetic neurons

Spectral analysis of acetylcholine (ACh) noise was performed in voltage-clamped neurons of the isolated rabbit superior cervical ganglion at 34–37°C and at membrane potential −80 mV. Two modes of activity were found in the ionic channels of nicotinic ACh receptors, with mean channel life-times of τ_f = 1.1 ± 0.1 ms for fast-operating channels and τ_s = 5.6 ± 0.6 ms for slow-operating channels. Excitatory postsynaptic current (EPSC) decays exponentially with a time constant which is very close to τ_s, indicating that the slow-operating channel activity determines the duration of EPSC. The mean value of conductance of single nicotinic ACh-receptor channel is 36 ± 3 pS.     

The potential dependence of acetylcholine-activated membrane conductance in sympathetic ganglion neurons

Membrane conductance activated by acetylcholine (ACh-conductance) was studied in rat isolated superior cervical ganglion neurons by means of the patch-clamp method in the whole-cell recording mode. It was found that ACh-conductance was increased or decreased with membrane hyper- or depolarization, respectively. The decrease in ACh-conductance was not associated with the reversal of ACh-current or with the presence of Ca2+ ions in external solution. The time constant of voltage-jump relaxation of ACh-current revealed e-fold increase with membrane hyperpolarization by 70 mV, which corresponded to the voltage dependence of ACh-conductance. Basing upon these results it was concluded that the voltage dependence of ACh-conductance is mostly determined by the voltage dependence of nicotinic receptor channel gating kinetics.     

State-dependent block of rat Nav1.4 sodium channels expressed in xenopus oocytes by pyrazoline-type insecticides

Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Na(v)1.4 sodium channels at a membrane potential of -120 mV, but depolarization to -60 mV or -30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.     

Effects of T-588, a newly synthesized cognitive enhancer, on hippocampal CA1 neurons in rat brain tissue slices

The effects of a newly synthesized cognitive enhancer, (-)-R-alpha-[[2-(diethylamino) ethoxy] methyl] benzo [b] thiophene-5-methanol hydrochloride (T-588), on the membrane properties of hippocampal CA1 neurons were investigated in a rat brain slice preparation. T-588 produced a slow and long-lasting depolarization of CA1 neurons with an increase in membrane resistance; this action showed close similarity to that of acetylcholine (ACh). However, the action of T-588 was not affected by atropine, tetrodotoxin or DL-2-amino-5-phosphonovalerate, while the action of ACh was blocked by atropine. The estimated reversal potential of this T-588 effect was near -90 mV which is the reversal potential of potassium ions in CA1 neurons. In the whole-cell voltage-clamp study, T-588 produced a reversible block of the outward potassium current in CA1 neurons. T-588 did not block the afterhyperpolarization evoked by an intracellular current injection, while ACh suppressed it. These results suggest that T-588 has a direct effect on CA1 neurons independent of its cholinergic activity, resulting from blockade of a conductance carried predominantly by potassium ions.     

Single acetylcholine channel currents in sympathetic neurons

Single acetylcholine (ACh) channel currents were studied by the gigaohm patch-clamp technique in cultured sympathetic neurons of the bullfrog, Rana catesbeiana. Recordings were made at 22C oncell-attached and excised membrane patches. When ACh (0.5-1 μM) was present in the pipette, a single class of inward currents was observed with a chrod conductance of 30 pS and reversal potential of -2 mV. The mean channel open time was 11.6 ms at -65 mV and showed little or no voltage-dependence over the range -85 to -45 mV. These channels appear to mediate the fast nicotinic excitatory postsynaptic current.     

Steroids inhibit nicotinic acetylcholine receptors.

Application of progesterone to Xenopus oocytes expressing a cloned neuronal nicotinic acetylcholine (nAChR) revealed two effects. The first effect was a fully reversible reduction of the current induced by acetylcholine (ACh), its onset being nearly instantaneous. The second effect, which developed in a few hours, was an irreversible suppression of ACh-evoked currents. The transient inhibition had an apparent Ki of 7 microM when tested with 50 nM ACh, but the percentage of inhibition was positively correlated to the ACh concentration. A reduction of ACh-induced currents which appeared immediately upon progesterone application was also observed with muscle nAChR expressed in oocytes and with nAChR on membrane patches isolated from ciliary ganglion neurons. Thus nAChRs are modulated by progesterone and steroids may play an important role in nicotinic cholinoception.     

Mechanism of action of alpha-neurotoxins from snake venom on nicotinic cholinoreceptors of sympathetic ganglion neurons in the rabbit

Effect of alpha-neurotoxins from snake venoms (alpha-bungarotoxin and alpha-cobratoxin, 10(-6)M) on the ACh-induced current and on the fast excitatory postsynaptic current (EPSC) were studied in voltage-clamped neurons of the isolated rabbit superior cervical ganglion treated with atropine (10(-6)M) alpha-Neurotoxins produced potentiating or inhibitory effects on ACh-induced current and only inhibitory effect on EPSC. alpha-Neurotoxins did not change single channel current and lifetime for short-living nicotinic channels and prolonged the lifetime for long-living channels as well as EPSC decay. It is suggested that the inhibitor effect of alpha-neurotoxins on ACh-induced current and on EPSP is due to partial blockade of nicotinic Ach receptors and that their potentiating effect on ACh-induced current results from the prolonged lifetime for long-living channels.     

l-Carnitine: therapeutic strategy for metabolic encephalopathy

The effects of 4-aminopyridine (4-AP) on membrane electrical properties and synaptic transmission in neurons of the isolated rabbit superior cervical ganglion were investigated. 4-AP (0.03-0.1 mM) increased the amplitude of the fast excitatory postsynaptic potential (f-EPSP) without affecting appreciably either the acetylcholine (ACh) depolarization induced by iontophoresis of ACh or the passive and active membrane properties of the neurons. At concentrations of 1-5 mM, 4-AP reversibly depressed the amplitude of the f-EPSP as well as the ACh depolarization; a slight to moderate prolongation of the action potential duration was observed. In addition to the effects on evoked synaptic potentials, 4-AP induced spontaneous discharges which were abolished reversibly by curare, low Ca solution or Co. The results indicate that 4-AP at low concentrations facilitated evoked as well as spontaneous release of ACh by a presynaptic mechanism, whereas at higher concentrations it exerted a curare-like effect on the postsynaptic membrane.     

The mechanism of action of pachycarpine on the nicotinic cholinoreceptors of sympathetic ganglion neurons in the rat

The mechanism of the pachycarpine action was studied in isolated superior cervical ganglion of rat using the patch-clamp method in the whole-cell recording mode. Action of the pachycarpine is not potential-dependent in the membrane potential range from +30 to -50 mV, the blockade increases during hyperpolarization of the membrane (-50-90 mV). Changes in the "dose-effect" dependence show that the blocking of nicotinic acetylcholine receptors is competitive both within the potential-dependent interval and within the potential-independent one.     

Action of tubocurarine on non-activated nicotinic cholinoreceptors of neurons of sympathetic ganglia in the rat

Mechanism of tubocurarine (Tc) action on nonactive nicotinic acetylcholine receptors (NAChRs) of rat sympathetic neurons was studied by means of the patch-clamp whole-cell method. It was found that the blocking action of Tc on nonactive NAChRs augmented with the membrane hyperpolarization. The rate constant of Tc binding to nonactive NAChR (k + B) and the rate constant of dissociation of Tc-inactive NAChR complex (k-b) were found to be (9.0 +/- 1.5).10(3) M-1 s-1 and 0.11 +/- 0.05 s-1, respectively. Constant k + B decreased with the membrane depolarization (e-fold change in k + B corresponded to the potential shift approximately by 50 mV). In contrast, k-B slightly increased with the membrane depolarization (e-fold change in k-B corresponded to the potential shift approximately by 100 mV). It was concluded that Tc acted on the sympathetic neurons of rat noncompetitively as both open-channel and nonactive-receptor (closed-channel) blocker.     

Long-term potentiation of nicotinic synaptic transmission in rat superior cervical ganglia produced by phorbol ester and tetanic stimulation

The long-term potentiation of nicotinic synaptic transmission induced by both active phorbol ester (4beta-phorbol-12,13-dibutyrate, PdBu) and tetanic trains of preganglionic stimulation was studied in single neurons of the superior cervical ganglion (SCG) of the rat using intracellular recording techniques. PdBu significantly increased the mean amplitude of both the unitary evoked fast excitatory postsynaptic potentials (EPSPs) and the fast excitatory postsynaptic currents (EPSCs) to 17.0+/-3.3 mV (control 8.4+/-1.9 mV, n=5) and 2.8+/-0.4 nA (control 0.8+/-0.1 nA, n=10), respectively. There was no significant change in either the resting membrane potential, input resistance, or the threshold for the initiation of an action potential. The response to exogenously applied acetylcholine (ACh) was also not changed following exposure to PdBu. In low-calcium, high-magnesium solutions, PdBu significantly increased the quantal content of EPSPs approximately threefold from a control of 0.9+/-0.2 (n=5) to 2.6+/-0.6 (n=5). The quantal content of EPSCs was also increased to 1.3+/-0.2 (control 0.5+/-0.1, n=10). PdBu increased the frequency of miniature EPSPs (mEPSPs) to 196+/-47% (n=6) of control, while the amplitude, rise time, rate of rise, and decay of mEPSPs were not significantly changed. Tetanic stimulation significantly increased the amplitude of the unitary synaptic EPSPs and EPSCs without significantly changing the resting membrane potential, input resistance, threshold for initiation of an action potential, or the response to exogenously applied ACh. Tetanic stimulation significantly increased quantal content of EPSPs and EPSCs threefold. The results obtained with tetanically induced LTP are similar to the results obtained with phorbol ester-induced LTP in these ganglion neurons. These results suggest that both tetanically induced and phorbol ester-induced LTP, in the rat, share similar mechanisms which involve, at least in part, activation of PKC-dependent mechanisms to increase quantal release from sympathetic preganglionic axon terminals.     

The action of alpha-bungarotoxin on the acetylcholine responses of the membrane of snail neurons

The effect of alpha-bungarotoxin (alpha-BT) on two types of acetylcholine (ACh)-induced currents distinguished by their desensitization properties was studied on the isolated dialyzed Helix neurons by a concentration clamp method. It was shown that alpha-BT depressed both types of ACh responses and this action was reversible in the majority of experiments. The depressive action of alpha-BT on ACh-induced currents occurred only in the presence of albumin, though albumin itself had no effect on the ACh responses. The concentration dependence of the blocking effect of alpha-BT on ACh-induced currents in both types of neurons was studied. The calculated values of alpha-BT concentration which caused 50% depression of the ACh-induced current were (13.85 +/- 1.25) X 10(-8) and (5.56 +/- 1.0) X 10(-8) g/ml for A and B types of neurons, respectively.     

Desensitization kinetics of a chloride acetylcholine response in Aplysia

The kinetics of desensitization of acetylcholine-evoked Cl conductance increased response of Aplysia RC neurons of the abdominal ganglion were studied under voltage-clamp conditions for comparison with results of similar studies on acetylcholine Na and K responses. The response evoked by acetylcholine on RC neurons was an outward current at resting potential (about -45 mV) that reversed at about -65 mV and was blocked by D-tubocurarine and strychnine but not hexamethonium and was not activated by arecoline. From the current-voltage relation this response can be ascribed to a pure conductance increase to Cl. The apparent KD was 40.6 microM. Upon prolonged exposure to acetylcholine the response peaked within 200-400 ms, and then decayed to a plateau current in the continued presence of the agonist. The peak and plateau currents reversed at the same potential, indicating that there had not been significant redistribution of Cl. The current decay in every cell was best fit by a double exponential function plus a constant, and the average time constants were tau fast = 1.8 +/- 0.2 s and tau slow = 16.2 +/- 1.0 s. Both components were slowed by cooling. While tau fast did not change with dose, tau slow increased with dose. Both components accelerated with hyperpolarization and upon application of trifluoperazine (2 microM). These results are consistent with the interpretation that desensitization of the acetylcholine Cl response is composed of two independent processes. This conclusion is the same as that derived from studies of the acetylcholine Na and K responses, and is in general consistent with desensitization being a property of a common acetylcholine receptor, and independent of the ionic selectivity of the associated channel. There are, however, significant differences in voltage, temperature and trifluoperazine dependence of the two components of the three ionic responses which may reflect influence of the different ion channels and/or transduction mechanisms.     

Oxytocin modulation of the functional properties of 3 types of cholinoreceptors in mollusk neurons

It was found that 10(-8) mol/l oxytocin (OT) application exerted effects on functional properties of three types of acetylcholine (ACh) receptors in neurons identified in the ganglia of Helix pomatia under voltage clamp conditions. OT depressed ACh-induced sodium-potassium-calcium current in neuron RB3 without a shift of the reversion potential. The data obtained show that there are two types (subtypes) of ACh receptors connected with chloride channels. OT decreased the ACh-induced chloride current in neuron F4 and enhanced the ACh-induced chloride current and desensitization of ACh receptors in neurons D5, F86. Effects of OT and serotonin applications were reversible but not additional. Effects of OT injection and OT application were independent. The present results suggest that cyclic AMP may be the second messenger mediating the OT-induced modulation of functional properties of three types of ACh receptors.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglia neurons

Slow EPSPs evoked in the neurons of the rabbit isolated superior cervical ganglion were studied using intracellular microelectrodes. Two types of EPSPs occurring in different neurons were found. The type I slow EPSPs showed an increase during hyperpolarization of the membrane and a decrease during its depolarization. Input resistance of the neurons during the response either decreased or remained unchanged. The type II slow EPSPs were increased by depolarization and decreased by hyperpolarization with the reversal potential -78.9 +/- 3.6 mV. Depolarization evoked by acetylcholine or carbocholine was followed by an increase in the input resistance in 53% of neurons with reversal potential -83.2 +/- 6.7 mV. It is concluded that in the first group of the neurons the nature of the slow EPSP is similar to that of ordinary EPSP. The main component underlying the ionic mechanism of slow EPSP in the other group of the neurons is a decrease in potassium conductance of the membrane.     

The coupling of acetylcholine-induced BK channel and calcium channel in guinea pig saccular type II vestibular hair cells

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the alpha9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs(+) completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd(2+) and Ni(2+), respectively. The inhibition potency of these four calcium channel antagonists was nimodipine>nifedipine>cadmium>nickel. The L-type Ca(2+) channels agonist, (-)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.     

Modulatory effect of nitric oxide on acetylcholine-induced activation of cat petrosal ganglion neurons in vitro

The inhibitory effect of nitric oxide (NO) on carotid chemosensory responses to hypoxia has been attributed in part to an antidromic inhibition of chemoreceptor cells activity. However, NO may also modulate the activity of the primary sensory neurons because NO is produced in the soma of these neurons located in the petrosal ganglion. Since a population of petrosal neurons is selectively activated by acetylcholine (ACh), we studied the effects of NO-donor, sodium nitroprusside (SNP), and the NO-synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), on the responses evoked in the carotid sinus nerve (CSN) by ACh applied to the petrosal ganglion in vitro. ACh (1 microgram-1 mg) increased the frequency of action potentials recorded from the CSN in a dose-dependent manner. SNP (10-50 microM) reduced the sensibility and amplitude of the CSN response to ACh, although the maximal response appears less affected. The withdrawal of SNP from the superfusion medium increased the sensibility of the responses to ACh. l-NAME (1-2 mM) slightly increased the sensibility of the ACh-induced responses, effect that persisted after l-NAME withdrawal. These results suggest that NO may play a role as modulator in this autonomic primary sensory ganglion.     

The potential dependence of calcium current deactivation via the somatic membrane of sensory neurons in the mouse

Potential dependence of calcium inward current deactivation kinetics was studied in the somatic membrane of mouse dorsal root ganglion neurons by intracellular dialysis technique. The decay of the high-threshold calcium current upon repolarization was reasonably described by single-exponential process with the time constant tau less than or equal to 130 microseconds (V = = -80 mV), when the intracellular solution contained tris-PO4, and by two-exponential process (tau congruent to 0.1 and tau = 0.8 divided by ms, V = -80 mV), when the intracellular solution contained Cs-aspartate and EGTA. Both time constants were strongly voltage dependent. The amplitude of the fast component of the tail current had sigmoidal voltage dependence, and the slow component had V-shaped voltage dependence. The low-threshold calcium current deactivation occurs more slowly with high voltage dependent kinetics (tau = 1.1 divided by 1.2 ms, V = -160 mV). A dependence of low-threshold current deactivation time constant on the type of penetrating cation was observed. A kinetic model of calcium current deactivation was proposed considering three types of calcium channels presented in the somatic membrane of the neurons studied.