Ca-dependent regulation by Na, K-pump of post-tetanic sensitization of extrasynaptic choline receptors in Helix lucorum neurons

Posttetanic potentiation (by orthodromic stimulation) of cholinosensitivity in LPa3 and RPa3 Helix lucorum neurons that are command in respect to withdrawal behavior was shown earlier (Pivovarov et al., 1999). Now we studied the regulatory role of the Na,K-pump and intracellular free Ga2+ in the posttetanic potentiation (PTP) of cholinosensitivity in command neurons. Semiintact Helix preparation "CNS-visceral bag" was used in experiments. Acetylcholine-induced inward currents were recorded using two-electrode voltage clamp technique. Acetylcholine was applied to somata of the identified LPa3 and RPa3 neurons with a 10-min interval before and after electrical tetanic stimulation of the n. intestinalis (10.5 mA; 0.1 s; 2/s; 2 min). Ouabain (extracellular application, 70 mcM) blocked the PTP. Intracellular injection of BAPTA (1 mM), chelator of Ca2+ ions, prevented the PTP. The PTP was absent after the ouabain application against the background of preliminary intracellular injection of BAPTA. A conclusion war drawn about Ca-dependent participation of Na,K-pump in posttetanic potentiation of cholinosensitivity in command Helix lucorum neurons of withdrawal behavior.     

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.     

Synaptic transmission at visualized sympathetic boutons: stochastic interaction between acetylcholine and its receptors.

Excitatory postsynaptic currents (EPSCs) were recorded with loose patch electrodes placed over visualized boutons on the surface of rat pelvic ganglion cells. At 34 degrees C the time to peak of the EPSC was about 0.7 ms, and a single exponential described the declining phase with a time constant of about 4.0 ms; these times were not correlated with changes in the amplitude of the EPSC. The amplitude-frequency histogram of the EPSC at individual boutons was well described by a single Gaussian-distribution that possessed a variance similar to that of the electrical noise. Nonstationary fluctuation analysis of the EPSCs at a bouton indicated that about 120 ACh receptor channels were available beneath boutons for interaction with a quantum of ACh. The characteristics of these EPSCs were compared with the results of Monte Carlo simulations of the quantal release of 9000 acetylcholine (ACh) molecules onto receptor patches of density 1400 microns-2 and 0.41 micron diameter, using a kinetic scheme of interaction between ACh and the receptors similar to that observed at the neuromuscular junction. The simulated EPSC generated in this way had temporal characteristics similar to those of the experimental EPSC when either the diffusion of the ACh is slowed or allowance is made for a finite period of transmitter release from the bouton. The amplitude of the simulated EPSC then exhibited stochastic fluctuations similar to those of the experimental EPSC.     

Regulation of posttetanic increase in choline sensitivity in Helix neurons by Na,K-pump: the role of Na/Ca-exchange and of mobilized calcium

We studied the role of Na/Ca-exchange and intracellular mobilized calcium in ouabain-mediated suppression of potentiation of cholinosensitivity of somatic membrane in Helix LPa3 and RPa3 command neurons of defensive behaviour after electrical orthodromic tetanisation of n. intestinalis. Cholinosensitivity of neurons was assessed by the amplitude of the inward current evoked by acetylcholine. Inhibitor of a Na/Ca-exchange benzamil and specific inhibitor of Ca-ATPase in endoplasmic reticulum thapsigargin prevented the development of the posttetanic potentiation (PTP). PTP did not arise and at joint action of ouabain with benzamil or thapsigargin. It was concluded that Na/Ca-exchange and mobilized calcium are involved in development of PTP of cholinosensitivity in somatic neuronal membrane and its regulation by Na,K-pump.     

Posttetanic potentiation and skeletal muscle fatigue: interactions with caffeine

The purpose of this study was to determine the interaction of three factors that modify twitch contraction amplitude in the rat gastrocnemius muscle in situ: posttetanic potentiation, fatigue, and caffeine. Posttetanic (200 Hz for 1 s) twitch responses were observed before and after 15 Hz stimulation for 6 min (group FS), injection of caffeine (75 mg/kg dissolved in saline, group NC), a combination of both repetitive stimulation and caffeine injection (group FC), or no treatment (group NS). Developed tension increased significantly with posttetanic potentiation and caffeine injection and these potentiating factors were additive (group NC). Repetitive stimulation attenuated the twitch response and the fatigued muscle was still responsive to the potentiating factors. Posttetanic potentiation was accomplished primarily by a significant increase in the peak rate of force development whereas caffeine potentiation and fatigue were effected with a proportional change in contraction time. It seems likely that the mechanism of posttetanic potentiation is not the same as the mechanism of caffeine-induced potentiation. Caffeine-induced potentiation is known to be related to increased release of calcium. Because changes in contraction time with fatigue were opposite to those associated with caffeine potentiation, it is proposed that the attenuated twitch response in fatigue results from reduced release of calcium.     

Participation of the adenylate cyclase system in the postsynaptic mechanism of heterosynaptic facilitation

In an analysis of the postsynaptic mechanism of heterosynaptic facilitation, changes in the amplitude of the excitatory postsynaptic current (EPSC) and the current evoked by application of acetylcholine (ACh current), acting on the adenylate cyclase system of the LC-1 and RC-1 neurons of the molluskPlanorbis corneus, were compared. Both responses are n-cholinergic and depend on the membrane conductivity for Na⁺ and K⁺. Application of serotonin led to a 100–300% increase in the amplitude of the EPSC and (in most cases) the ACh current. However, in 30% of the cases, the increase in the EPSC was accompanied by a decrease in the ACh current. This is probably due to the different contributions of Na⁺ and K⁺ to the mechanism of activation of the conductivity of th channel-receptor complex of the nonsynaptic cell membrane. The influence of serotonin on the EPSC and ACh current was simulated by the action of phosphodiesterase blockers and adenylate cyclase activators. Phosphodiesterase activators and protein kinase blockers reversibly inhibited the EPSC and ACh current. Thus, activation of the adenylate cyclase system, mediated by the action of serotonin, promotes the development of a postsynaptic mechanism of formation of heterosynaptic facilitation of the EPSC in the command neurons of the mollusk.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 676–683, November–December, 1991.     

The function of motor nerves innervating slow tonic skeletal muscle in hens with delayed neuropathy induced by tri-o-tolyl phosphate

The reduction of neurogenic posttetanic potentiation in the slow twitch, soleus muscle is an index of impaired motor nerve function in cats with organophosphate-induced neuropathy. We have applied the measurement of posttetanic potentiation to study the functional state of the slow, tonic, plantaris muscle and its motor innervation in adult White Leghorn hens with tri-o-tolyl phosphate (TOTP)-induced neuropathy. At suitable intervals following single oral doses of vehicle or TOTP (500 mg/kg), nerve conduction velocity and posttetanic potentiation were measured in anesthetized hens. Conduction in the sciatic nerve was not altered by TOTP. The plantaris muscle of birds treated with vehicle (peanut oil) either failed to contract or responded to nerve stimulation at 0.4 Hz with very small twitches. Following nerve stimulation at frequencies inducing tetanus (50-140 Hz), the muscles responded with large, slow twitches that gradually decayed in amplitude. The area under the curve formed by the amplitude of these twitches over time (posttetanic potentiation) was directly proportional to the frequency and duration of nerve stimulation. In hens at 1,2, and 4 weeks following treatment with TOTP, the average amount of posttetanic potentiation was reduced concomitantly with the development of ataxia, paralysis, and pathological changes in the peripheral nerves. This difference between vehicle- and TOTP-treated hens was not significant, owing to large interbird variations. Since TOTP-treated hens showed greater disturbances in gait following moderate exercise, the fatigue of posttetanic potentiation with periodic neuronal stimulation was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Cationic Selectivity of the Nicotinic Channel at the Rat Ganglionic Synapse: A Role for Chloride Ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.     

The effect of metabotropic glutamate receptors on longitude of posttetanic reaction in spinal motoneurons of frog

Effects of metabotropic glutamate receptors of the duration of posttetanic changes in monosynaptic excitatory postsynaptic potentials (mEPSP), evoked by afferent and reticulospinal input stimulation, were investigated in lumbar motoneurons of the frog isolated spinal cord. It was found that application of MAP4 (25 microM), a selective antagonist of group III of these receptors, prolonged posttetanic potentiation and depression of synaptic transmission, whereas activation of this group of metabotropic glutamate receptors by L-AP4 (1 mM), a selective agonist of these receptors, suppressed the amplitude of synaptic responses, but did not affect the dynamics of development of posttetanic changes. The NMDA receptor antagonist AP5 (50 microM), added to the perfusing solution, blocked completely the effects produced by MAP4. Neither selective antagonist MCCG (400 microM), nor agonist tACPD (50 microM) of group II metabotropic glutamate receptors affected the terms of mEPSP posttetanic potentiation and depression, although the latter, in contrast to the antagonist, in most cases increased the synaptic potential amplitude. The data obtained permit to suggest that group III metabotropic receptors may control the duration of posttetanic changes of synaptic transmission in the frog spinal motoneurons. The long-term changes in the investigated synapses seem to be mediated by activation of postsynaptic metabotropic glutamate receptors (most likely, of group I receptors), which is normally masked with activation of group III presynaptic autoreceptors. The mechanism of such an induction essentially depends on activation of NMDA type of inotropic glutamate receptors.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

Methiothepin-sensitive serotonin receptors are involved in postsynaptic mechanism of sensitization of Helix lucorum escape reaction

Tetanic electric stimulation of Helix foot evokes sensitization of escape reaction. This behavioral sensitization and posttetanic potentiation (PTP) of acetylcholine-induced inward current (ACh-current) in command Helix neurons of escape behavior were similar. Antagonist of serotonin receptors methiothepin prevents the PTP of the ACh-current and behavioral sensitization. Serotonin disrupts the PTP of the ACh-current. It is suggested that the increase in cholinosensitivity of the command neurons with the involvement of methiothepin-sensitive serotonin receptors may be the cellular postsynaptic mechanism of behavioral sensitization of Helix escape reaction.     

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.     

Posttetanic changes in the afferent and efferent activity of monosynaptic reflex in kittens

Both the afferent volleys from the dorsal root and the monosynaptic reflex discharges from the corresponding ventral root were recorded with hook electrodes during stimulation of the nerves innervating the triceps surae muscles. The effects of conditioning high frequency tetanus on the magnitudes of these afferents and reflex volleys were examined in kittens of postnatal age 1-90 days and in adult cats. In young kittens under barbiturate anaesthesia, large-amplitude monosynaptic reflex discharge can be evoked without prior conditioning. The amplitude of this reflex discharge decreased with increasing age of the animal. Application of conditioning tetanic stimuli to the muscle nerves resulted in posttetanic depression followed by posttetanic potentiation of the monosynaptic reflex. The magnitude of posttetanic depression was much higher than that of potentiation in the first postnatal week. As the age increased, the magnitude of depression decreased while the magnitude of potentiation increased. The afferent volley showed a considerable posttetanic potentiation in older kittens and cats. No significant potentiation or depression was observed in the younger animals. Possible mechanisms contributing to posttetanic depression and potentiation are discussed.     

Humoral factor dependence of neuronal post-theta choline-sensitive potentiation in Helix Lucorum

Comparison of posttetanic changes of the acetylcholine-induced inward current (ACh-current) in command Helix lucorum neurones at different conditions (using stop flow of saline through the chamber with a ganglia preparation and using flow of saline) was made. Flow of saline reduces latency and degree of posttetanic increase of the ACh-current in neurones. Earlier and weak posttetanic potentiation of the ACh-current in command Helix lucorum neurones during flow of saline through the chamber with a ganglia preparation testifies to participation of the humoral factor in the mechanism of posttetanic potentiation of cholinosensitivity of somatic membrane in postsynaptic neurone.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.     

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

The effect of leucine enkephalin on the development of a convulsive afterdischarge in the sensorimotor cortex of rats]

The application of leucin-enkephalin solution (LEU) (2 micrograms/2 microliters) on stimulated region of sensomotor cortex did not influence threshold of direct and transcallosal cortex responses (DR and TCR). On coupling of repeated electrostimulation train (RET) (duration of impulse--0.1 ms; duration of train--10 s; frequency--10/s) with application of LEU (after every odd train) the changes of DR and TCR in course of even trains and latency of afterdischarge appearance were such as in control ones. Simultaneously LEU effectively depressed short posttetanic potentiation of DR and TCR and potentiation of amplitude and duration AD, evoked by RET. It is suggested that LEU released from neurons in the course of RET does not participate in initiation of seizure in sensomotor cortex. A possible role of LEU in sensomotor cortex is limitation of intensity and duration of seizures and prevention of status epilepticus.     

Nonselective cationic currents activated by acetylcholine in swine tracheal smooth muscle cells

Membrane currents in isolated swine tracheal smooth muscle cells were investigated using a pipette solution containing BAPTA-Ca2+ buffer and Cs+ as the major cation. With a pipette solution containing 100 nM free Ca2+, acetylcholine (ACh; 1-100 microM), in a concentration-dependent manner, activated a current without inducing shortening of cells, although neither 1 mM histamine nor 1 microM leukotriene D4 activated the current (n = 7, n is the number of cells). The effect of 100 microM ACh was suppressed by pretreatment with 100 microM atropine (n = 6) or intracellular application of preactivated pertussis toxin at a concentration of 0.1 microg x mL(-1) (n = 8). Genistein (0.1-100 microM), in a concentration-dependent manner, suppressed the activation of the inward current by 100 microM ACh, whereas it did not significantly suppress that of the outward current (n = 6-8). With a pipette solution containing 50 nM free Ca2+, outward current, but not inward current, was activated by 100 microM ACh (n = 10). When the pipette solution had free Ca2+ concentrations greater than 50 nM, the inward current together with the outward current was activated. The ratio between the amplitude of the inward and outward currents was significantly increased as the free Ca2+ concentration in the pipette solution increased. The steady-state activation curve of the ACh-activated current with the 50 nM free Ca2+ pipette solution was fitted by a single Boltzmann distribution (Vh = +69.8 mV, k = -11.9 mV, n = 10). The activation time constant became smaller as the membrane potential was more depolarized (164.3+/-5.9 ms at +40 mV to 92.4+/-6.3 ms at +120 mV, n = 10). The reversal potential was not significantly changed by reducing extracellular Cl- concentration to one-tenth of the control (n = 8), suggesting that the current is a nonselective cationic current. These results suggest that ACh activates an outward nonselective cationic current via pertussis toxin-sensitive G-protein(s) coupled with muscarinic receptors. Involvement of genistein-sensitive tyrosine kinase in the activation process of the current is unlikely.     

ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用

利用全细胞膜片钳技术研究乙酰胆碱(acetylcholine,ACh)对大鼠皮层体感区神经元延迟整流钾电流(IK)的调制作用。结果表明:(1)ACh(0．1、1、10、100 μmol/L)对大鼠皮层体感区神经元IK有抑制作用,并具有剂量依赖性关系(P<0．01)。 (2)ACh可使IK激活曲线的斜率变大,并使激活曲线向超极化方向移动。IK激活曲线的半数激活电压(V1/12)和斜率因子(k)分别由给药前的(-41．8±9．7)mV和(30．7±7．2)mV变为给药后的(-122．4±38．6)mV和(42．4±7．0)mV。(3)100 μmol/L的N受体拮抗剂筒箭毒碱(tubocurarine)可减弱ACh对IK的抑制作用,在指令电压+60 mV时tubocurarine+ACh组的IK幅度下降了(16．9± 13．8)％(n=8),与10 μmol/L ACh组引起的(36．5±7．8)％的IK下降幅度相比,有极显著差异(P<0．01)。10 μmol/L的M1受体拮抗剂哌仑西平(pirenzepin)拮抗ACh对IK的抑制作用不明显(n=7,P>0．05);而10 μmol/L的M3受体拮抗剂4-DAMP可部分拮抗ACh对IK的抑制作用,并且4-DAMP+ACh组使IK的电流值下降了(26．8±4．7)％(n=6),与ACh组引起的IK电流下降相比,有显著差异(P<0．05)。(4)蛋白激酶C(protein kinase C,PKC)阻断剂chelerythrine拮抗ACh对IK的抑制作用,PKC激动剂PDBu可增强ACh对IK的抑制作用(P<0．05)。综上所述,ACh对人鼠皮层体感区神经元IK的抑制作用主要是通过烟碱受体(nAChRs)和M3受体介导,并经过PKC信号途径。