Different actions of calcium channel blocking agents on resting membrane conductance in developing skeletal muscle

The effects of Co2+, Mn2+, and La3+ (2 mM) and verapamil (5 x 10(-6) M) on membrane conductance (Gm) and resting potential (Em) were studied in chick skeletal muscle fibres developing in culture. Cobalt and manganese had no effect on Gm at any time during myogenesis but verapamil caused a decrease in Gm in immature myotubes. This effect diminished with time and was absent by 3 days after myoblast fusion. Lanthanum caused an increase in Gm at all stages of development. All the agents studied caused a significant depolarization of Em. It is concluded that there is no resting calcium conductance in developing skeletal muscle but that there may be a resting sodium conductance which declines with maturation. Lanthanum may increase Gm by displacing membrane-bound calcium and destabilizing membrane structure. All the agents studied were thought to induce depolarization by an inhibitory action on (Na+ + K+)-ATPase.     

Substitution of extracellular sodium ions blocks the voltage-dependent decrease of input conductance evoked by L-aspartate

The response of cultured spinal cord neurones to L-aspartic acid consists of membrane depolarization accompanied by an apparent voltage-dependent reduction in input conductance (Gm). Depolarizations evoked by this amino acid and the decrease in Gm were reduced or eliminated when cultured spinal cord neurones were superperfused with solutions where Na was substituted with choline or Tris. Responses to L-aspartate recovered rapidly upon return to the control (Na-containing solution). The voltage-dependent component of the response to L-aspartate is dependent upon the presence of external Na.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate and blockade of desensitization by Con A

Primary afferent C fibers in rat dorsal roots are depolarized by the excitatory amino acids kainate and domoate. Under whole-cell voltage clamp, kainate and domoate increase membrane conductance in a subpopulation of freshly dissociated DRG neurons. In contrast to kainate currents observed in CNS neurons, responses to kainate and domoate in DRG cells desensitize with prolonged agonist exposure. Half-maximal activation is achieved with much lower concentrations of kainate and domoate in sensory neurons than in CNS neurons from cerebral cortex. Rapid applications of glutamate, quisqualate, and AMPA evoke a transient current in DRG neurons and desensitize cells to subsequent applications of kainate or domoate. Brief incubation with the lectin concanavalin A eliminates desensitization to excitatory amino acids; after treatment with concanavalin A, all five agonists gate sustained currents of similar amplitude via the same receptor.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Concentration-jump analysis of voltage-dependent conductances activated by glutamate and kainate in neurons of the avian cochlear nucleus.

We have examined the mechanisms underlying the voltage sensitivity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in voltage-clamped outside-out patches and whole cells taken from the nucleus magnocellularis of the chick. Responses to either glutamate or kainate had outwardly rectifying current-voltage relations. The rate and extent of desensitization during prolonged exposure to agonist, and the rate of deactivation after brief exposure to agonist, decreased at positive potentials, suggesting that a kinetic transition was sensitive to membrane potential. Voltage dependence of the peak conductance and of the deactivation kinetics persisted when desensitization was reduced with aniracetam or blocked with cyclothiazide. Furthermore, the rate of recovery from desensitization to glutamate was not voltage dependent. Upon reduction of extracellular divalent cation concentration, kainate-evoked currents increased but preserved rectifying current-voltage relations. Rectification was strongest at lower kainate concentrations. Surprisingly, nonstationary variance analysis of desensitizing responses to glutamate or of the current deactivation after kainate removal revealed an increase in the mean single-channel conductance with more positive membrane potentials. These data indicate that the rectification of the peak response to a high agonist concentration reflects an increase in channel conductance, whereas rectification of steady-state current is dominated by voltage-sensitive channel kinetics.     

Passive Proton Conductance Is the Major Reason for Membrane Depolarization and Conductance Increase in Chara buckellii in High-Salt Conditions

Chara buckellii G.O.A., a salt-tolerant alga, has a less negative membrane potential (Em) when cultured in saline medium (artificial Waldsea water) than when cultured in freshwater. The cell hyperpolarizes and membrane conductance (Gm) decreases when the external medium is changed from Waldsea control solution (WCS), a high-salt medium, to low-salt medium containing sufficient sorbitol to generate the same osmotic potential as WCS. Banding pattern and proton flux experiments show that C. buckellii has higher passive proton influx in the alkaline band in high-salt medium than in low-salt medium. Decrease of the passive proton influx by darkness or low external pH dramatically hyperpolarizes the membrane and decreases the conductance. The pH dependence curves of Em and Gm also indicate the existence of high passive proton conductance (GH) in C. buckellii. Ion substitution experiments show that Em and Gm of saltwater cells are not dependent on K+, Na+, Cl-, or SO42+. Mg2+ also affects Em and Gm, but its effect is probably on GH. We conclude that GH is the most important cause of the membrane depolarization and conductance increase in the saltwater alga C. buckellii.     

Action of excitatory amino acids and their antagonists on hippocampal neurons

Intracellular recordings were obtained from guinea pig hippocampal neurons maintained in vitro. Current- and voltage-clamp techniques were used to study the effect of microiontophoresis of excitatory amino acid agonists. Modification of agonist responses by bath application of known concentrations of antagonist agents was also examined. All agonists used, glutamate, aspartate, N-methyl-D-aspartic acid (NMDA), and quisqualate, depolarized hippocampal neurons and caused repetitive firing. NMDA was also noted to induce burst-firing in some neurons. Quisqualate and NMDA were more potent than glutamate or aspartate. In slices perfused with a nominally calcium-free saline containing tetrodotoxin and manganese, quisqualate application produced a depolarization associated with a conductance increase. Under those conditions, NMDA-induced depolarizations caused apparent decreases as well as increases in conductance. The apparent decreases in conductance were observed in the voltage range of -40 to -70 mV, whereas increases in conductance were observed at membrane potentials more positive than -35 mV. Under voltage-clamp conditions, quisqualate produced an inward current whose amplitude increased with hyperpolarization and decreased upon depolarization, reversing near 0 mV. The conductance change induced by quisqualate was independent of voltage. NMDA application resulted in an inward current that was maximal around the resting potential and decreased with both hyperpolarization and depolarization. Response reversal was not observed with hyperpolarization to -100 mV but was apparent with depolarization beyond 0 mV. Conductance changes induced by NMDA were voltage dependent, and the application of this agent was associated with the appearance of a region of negative slope conductance in the current-voltage relationship. Apparent decreases in conductance in response to NMDA were reduced when the extracellular magnesium concentration was lowered. Response amplitudes were not affected. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) was a potent and selective blocker of NMDA responses, whereas the antagonist DL-2-amino-4-phosphonobutyric acid (DL-APB) was less potent and did not select between NMDA and quisqualate responses. Analysis of iontophoretic dose-response curves indicated that DL-APV was a competitive antagonist. The results of these experiments indicate that hippocampal CA1 pyramidal neurons possess separate receptors for quisqualate and NMDA, with different pharmacological and electrophysiological profiles.     

Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons

The actions of cholecystokinin octapeptide (CCK) on the membrane properties of mouse spinal neurons grown in monolayer culture were examined using intracellular recording techniques. In a subpopulation of cells, application of CCK (0.2-100 micron) by pressure ejection from micropipettes produced a small (approximately 2 mV) membrane depolarization that was accompanied by a decrease in membrane conductance (approximately 11 percent). These effects were associated with an enhanced tendency of the cells to generate action potentials when stimulated with intracellular depolarizing current. The unsulfated analog of CCK, which possesses weak biological activity in the gut, had little or no effect on cultured spinal neurons. A number of differences were noted between the responses to CCK and the excitatory amino acid glutamate. First, the effects of CCK were more delayed in onset (approximately 17 sec) and prolonged in duration (approximately 124 sec). Second, the depolarizations produced by glutamate were of larger magnitude and associated with variable effects on membrane conductance. Third, the response to CCK showed tachyphylaxis with repeated applications whereas glutamate remained effective as often as it was applied. It is concluded that CCK facilitates the excitability of spinal neurons in a manner distinct from that of the conventional excitant glutamate.     

Responses of pyriform cortex neurons to excitatory amino acids: Voltage dependence,conductance changes,and effects of divalent cations

The actions of ionophoretically applied N-methyl aspartate (NMA), quisqualate, and kainate, thought to activate three different types of excitatory amino acid receptors, were studied on pyramidal neurons of the rat pyriform cortex, maintained in an isolated, submerged, and perfused brain slice. Intracellular recordings were made with either K acetate or CsCl electrodes. In most neurons all three agonists elicited monophasic responses which could be evoked at 20-sec intervals. Some neurons showed biphasic responses, most commonly to kainate but, on occasion, also for quisqualate. The slower component appeared to be correlated with excitotoxicity and, consequently, was difficult to study. As a result the kainate responses studied were from neurons selected for having a single component. In neurons selected for having a linear current-voltage relationship or neurons loaded with Cs to suppress K conductance and linearize the current-voltage relationship, the average changes in resistance recorded during ionophoretic responses at resting potential were as follows: NMA, 131.2 +/- 6.7% of control; kainate, 104.7 +/- 5.8% of control; and quisqualate, 92.8 +/- 2.8% of control. The magnitude and direction of the conductance change were very reproducible in any one neuron, but especially for kainate some cells showed clear conductance increases, while others showed clear conductance decreases. Using CsCl electrodes it was possible to reduce K+ conductance and depolarize the neurons over a wider range. By passing depolarizing current it was possible to reverse the responses. The response to all three agonists reversed at the same depolarized potential. This observation indicates that while there are differences in the ionic channels associated with the three agonists at resting potential, the channels have similar properties at more depolarized potentials. Responses to all three agonists were influenced by the concentrations of divalent cations in the perfusion medium. The NMA responses were most sensitive to Mg, increasing in amplitude in the absence of Mg and being depressed by Mg elevation. All responses were sensitive to Ca, with discharges being greatly increased by low Ca and depressed by high Ca. The kainate response was most sensitive to Ca concentration changes. Unlike reports from other preparations the apparent conductance decreases to NMA were not altered by the perfusion of solutions with either no added Mg or no added Ca. The NMA response was very much reduced in either Co (1-2 mM) or Zn (100-200 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

In Vivo Microdialysis Studies on the Effects of Decortication and Excitotoxic Lesions on Kainic Acid-Induced Calcium Fluxes, and Endogenous Amino Acid Release, in the Rat Striatum

The in vivo effects of kainate (1 mM) on fluxes of 45Ca2+, and endogenous amino acids, were examined in the rat striatum using the brain microdialysis technique. Kainate evoked a rapid decrease in dialysate 45Ca2+, and an increase in the concentration of amino acids in dialysates in Ca2+-free dialysates. Taurine was elevated six- to 10-fold, glutamate two- to threefold, and aspartate 1.5- to twofold. There was also a delayed increase in phosphoethanolamine, whereas nonneuroactive amino acids were increased only slightly. The kainic acid-evoked reduction in dialysate 45Ca2+ activity was attenuated in striata lesioned previously with kainate, suggesting the involvement of intrinsic striatal neurons in this response. The increase in taurine concentration induced by kainate was slightly smaller under these conditions. Decortication did not affect the kainate-evoked alterations in either dialysate 45Ca2+ or amino acids. These data suggest that kainate does not release acidic amino acids from their transmitter pools located in corticostriatal terminals.     

Ionic Dependence of Membrane Potential and Glutamate Receptor-Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS-C2-(5)

Membrane potentials of particles present in a subcellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absorbance of a cyanine dye, DiS-C2-5. The membrane potential of the particles seems to be dependent on both Cl- and K+ diffusion potentials, as judged from dependence of the absorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl- was replaced with gluconate. The apparent high Cl- permeability of the membrane preparation was reduced in the presence of picrotoxin, a finding suggesting endogenous activation of receptor-linked Cl- channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4-aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N-methyl-D-aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl- permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmitter receptor-linked membrane potential changes with optical probes of membrane potential.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

去甲肾上腺素引起蟾蜍背根神经节神经细胞膜电位变化的离子机制

本文应用细胞内记录方法,对去甲肾上腺素(NA)引起蟾蜍背根神经节(DRG)神经细胞膜电位去极化或超极化反应时的膜电导及翻转电位值进行了测量,并观察了钾和钙离子通道阻断剂灌流DRG对NA引起膜电位反应的影响。当NA引起去极化反应时,15个细胞的膜电导减小32.6％。少数细胞膜电导开始增加,继而减小(n=4)。NA超极化反应时膜电导增加13.2％(n=8)。NA去极化反应的翻转电位值为-88.5±0.9mV((?)±SE,n=4),NA超极化反应在膜电位处于-89至-92mV时消失。 钾通道阻断剂四乙铵可使NA去极化幅值增加73.7±11.9％((?)±SE,n=7),并使NA超极化幅值减小40.5％(n=4)。细胞内注入氯化铯使苯肾上腺素去极化幅值增加34.5％(n=4)。钙通道阻断剂氯化锰使NA去极化及超极化反应分别减小50.5±9.9％((?)±SE,n=10)和89.5±4.9％((?)±SE,n=7)。结果提示,NA引起DRG神经细胞膜电位的去极化或超极化反应,可能与膜的钾及钙通道活动的改变有关。     

Excitatory amino acid receptor agonists stimulate membrane inositol phospholipid hydrolysis and increase cytoplasmic free Ca2+ in primary cultures of retinal neurons

A variety of neurotransmitters are believed to elicit effects through receptor-stimulated inositol phospholipid metabolism. It appears that most major types of retinal neurons receive a direct glutamatergic input. The aim of the present studies was to characterize excitatory amino acid (EAA) receptor-mediated breakdown of inositol phospholipids and changes in Ca2+ homeostasis in primary avian retinal cell cultures. Cell monolayers, prepared from 8-day-old chick embryo neural retina, were labelled with [3H]inositol for 48 h, and used after 7 days in vitro. Kainic acid stimulated the accumulation of inositol phosphates in a time- and dose-dependent manner (ED50 = 30 microM). The EAA receptor agonists glutamate, N-methyl-D-aspartate (NMDA), ibotenate and quisqualate were all active, with the rank order: glutamate greater than kainate greater than NMDA much greater than ibotenate approximately quisqualate. External Ca2+ was required for these effects. Agonist actions were inhibited by type-specific antagonists, and also Mg2+ in the case of glutamate and NMDA. Glutamate, NMDA and kainate also elevated cytosolic free Ca2+ in individual retinal cells loaded with the Ca2(+)-sensitive dye Fura-2, as assessed by digital fluorescence ratio imaging microscopy. The agonist-induced increases in [Ca2+]i were largely dependent on extracellular Ca2+, independent of membrane depolarization and were blocked by Mg2+ for glutamate and NMDA. These results demonstrate that vertebrate retinal cells possess EAA receptors coupled to intracellular signal transduction pathways.     

Monitoring membrane potentials in Ehrlich ascites tumor cells by means of a fluorescent dye.

1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.     

Activation of AMPA/kainate receptors but not acetylcholine receptors causes Mg2+ influx into Retzius neurones of the leech Hirudo medicinalis

In Retzius neurones of the medicinal leech, Hirudo medicinalis, kainate activates ionotropic glutamate receptors classified as AMPA/kainate receptors. Activation of the AMPA/kainate receptor-coupled cation channels evokes a marked depolarization, intracellular acidification, and increases in the intracellular concentrations of Na+ ([Na+]i) and Ca2+. Qualitatively similar changes are observed upon the application of carbachol, an activator of acetylcholine receptor-coupled cation channels. Using multibarrelled ion-selective microelectrodes it was demonstrated that kainate, but not carbachol, caused additional increases in the intracellular free Mg2+ concentration ([Mg2+]i). Experiments were designed to investigate whether this kainate-induced [Mg2+]i increase was due to a direct Mg2+ influx through the AMPA/kainate receptor-coupled cation channels or a secondary effect due to the depolarization or the ionic changes. It was found that: (a) Similar [Mg2+]i increases were evoked by the application of glutamate or aspartate. (b) All kainate-induced effects were inhibited by the glutamatergic antagonist DNQX. (c) The magnitude of the [Mg2+]i increases depended on the extracellular Mg2+ concentration. (d) A reduction of the extracellular Ca2+ concentration increased kainate-induced [Mg2+]i increases, excluding possible Ca2+ interference at the Mg2+-selective microelectrode or at intracellular buffer sites. (e) Neither depolarizations evoked by the application of 30 mM K+, nor [Na+]i increases induced by the inhibition of the Na+/K+ ATPase caused comparable [Mg2+]i increases. (f) Inhibitors of voltage-dependent Ca2+ channels did not affect the kainate-induced [Mg2+]i increases. Moreover, previous experiments had already shown that intracellular acidification evoked by the application of 20 mM propionate did not cause changes in [Mg2+]i. The results indicate that kainate-induced [Mg2+]i increases in leech Retzius neurones are due to an influx of extracellular Mg2+ through the AMPA/kainate receptor-coupled cation channel. Mg2+ may thus act as an intracellular signal to distinguish between glutamatergic and cholinergic activation of leech Retzius neurones.     

Disopyramide phosphate effects on slow and depressed fast responses

We studied the effects of disopyramide phosphate on explanted neonatal rat ventricle cells exhibiting depressed fast responses or naturally occurring slow response action potentials together with automatic activity. Disopyramide suppressed the spontaneous activity at a concentration of 2.5 micrograms/mL with a half-maximal value of 10 micrograms/mL. Before spontaneous activity was lost, there was an increase in beating rate possibly related to membrane depolarization. In depressed fast and slow response action potentials there was an increase in action potential duration (APD) which was consistently found both at the level of the plateau and at 90% repolarization. Comparison of the APD increase observed after disopyramide treatment and that after exposure to 20 mM tetraethylammonium suggested a block of a potassium conductance as a possible cause underlying the change in APD. The Vmax values of the depressed fast response decreased at constant membrane potential and this was attributed to the local anesthetic effect of the drug. In addition, we report two novel findings: (i) a decrease of Vmax of the slow response action potentials which may be secondary to membrane depolarization, and (ii) an increase in the duration of slow action potentials, possibly caused by inhibition of a potassium conductance.     

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.