Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

Electrical perception of the 'death message' in Chara: characterization of K+ -induced depolarization

When the nodal end of an internodal cell of Chara corallina was subjected to a pulse treatment with artificial cell sap, a depolarization lasted in artificial pond water. This depolarization could also be induced by pulse treatment with KCl solution, indicating that K+ in the artificial cell sap is responsible (K+ -induced depolarization). The depolarization was prolonged in the presence of 2 mM KCl and the prolonged depolarization was terminated by supplementing with either CaCl2, MgCl2 or NaCl. These results supported the hypothesis that K+ released from the killed cell is responsible for generation of the wound-induced membrane depolarization.     

Studies on mechano-perception in the Characeae: transduction of pressure signals into electrical signals

Mechano-perception by Chara cells was studied with an emphasis on the role of the nodal complex in transducing pressure signals into electrical signals. Three types of experimental material were used: (1) tandem internodal cells connected by a single layer of nodal cells; (2) single internodal cells, from which either apical or basal nodes were removed by ligation and cutting; (3) single internodes from which both nodes had been removed. Exposure to a hypertonic solution (sorbitol or sucrose) induced a depolarization at the node in 1 and 2. Depolarization did not occur at the ligated end of the cell in 2, or at all in 3. Addition of K+ increased the magnitude of the response, whilst it was significantly decreased by the divalent cations, Ca2+ and Mg2+. Electrical resistance decreased at the node during the depolarization, showing that a passive diffusion potential was responsible. I suggest that the change in the trans-nodal hydraulic pressure difference mechanically stretches the plasma membrane, and this induces the electrical depolarization.     

Electrical perception of "death message" in Chara: involvement of turgor pressure

Plants show various defense responses upon wounding. Surviving cells must perceive a "death message" from killed cells in order to start the signal processing that results in defense responses. The initial step in perception of the death message by a surviving cell was studied by taking advantage of the filamentous morphology of characean algae. A specimen comprising two adjoining internodal cells was prepared. One cell (the victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were analyzed. Upon cutting the victim cell, at least one of three kinds of response were induced in the receptor cell: (1) slow depolarization lasting more than 10 min, (2) action potentials and (3) small spikes. The first of these response types, slow depolarization, was ubiquitous and is the focus of the present study. Two cell properties were essential for generation of this depolarization. (1) Presence of high cell turgor pressure was necessary. (2) The depolarization was generated only at the nodal end of the receptor cell, not at the flank. I concluded that the death message from the killed cell contains the information that turgor pressure has been lost. The mechanism by which this is translated into the slow depolarization of the receptor cell was discussed.     

High K(+)-induced membrane depolarization attenuates endothelium-dependent pulmonary vasodilation

Impairment of endothelium-dependent pulmonary vasodilation has been implicated in the development of pulmonary hypertension. Pulmonary vascular smooth muscle cells and endothelial cells communicate electrically through gap junctions; thus, membrane depolarization in smooth muscle cells would depolarize endothelial cells. In this study, we examined the effect of prolonged membrane depolarization induced by high K(+) on the endothelium-dependent pulmonary vasodilation. Isometric tension was measured in isolated pulmonary arteries (PA) from Sprague-Dawley rats, and membrane potential was measured in single PA smooth muscle cells. Increase in extracellular K(+) concentration from 4.7 to 25 mM significantly depolarized PA smooth muscle cells. The 25 mM K(+)-mediated depolarization was characterized by an initial transient depolarization (5-15 s) followed by a sustained depolarization that could last for up to 3 h. In endothelium-intact PA rings, ACh (2 microM), levcromakalim (10 microM), and nitroprusside (10 microM) reversibly inhibited the 25 mM K(+)-mediated contraction. Functional removal of endothelium abolished the ACh-mediated relaxation but had no effect on the levcromakalim- or the nitroprusside-mediated pulmonary vasodilation. Prolonged ( approximately 3 h) membrane depolarization by 25 mM K(+) significantly inhibited the ACh-mediated PA relaxation (-55 +/- 4 vs. -29 +/- 2%, P < 0.001), negligibly affected the levcromakalim-mediated pulmonary vasodilation (-92 +/- 4 vs. -95 +/- 5%), and slightly but significantly increased the nitroprusside-mediated PA relaxation (-80 +/- 2 vs. 90 +/- 3%, P < 0. 05). These data indicate that membrane depolarization by prolonged exposure to high K(+) concentration selectively inhibited endothelium-dependent pulmonary vasodilation, suggesting that membrane depolarization plays a role in the impairment of pulmonary endothelial function in pulmonary hypertension.     

Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine

The relationships of the electrical to the mechanical responses of the canine trachealis muscle during stimulation of its cholinergic nerves or exposure to exogenous acetylcholine were recorded in the single or the double sucrose gap. At 27 degrees C, the responses to a train of stimuli consisted of a transient depolarization excitatory junction potential of 10-30 mV followed by fading oscillations and contractions. When stimulus parameters were varied in the single sucrose gap, contractions were more closely associated with the occurrence of and varied in duration with the oscillations rather than with the amplitude of the EJP. Acetylcholine superfused at a concentration of 10(-6) M for 30 s caused a prolonged depolarization of 10-20 mV, but a much larger contraction than could be elicited by nerve stimulation. None of the responses to acetylcholine was significantly affected by the Ca channel antagonists, nifedipine, nitrendipine, or verapamil in Ca channel blocking concentrations. When tissues were exposed to a Ca-free medium, the excitatory junction potentials and oscillations rapidly disappeared, but the electrical and mechanical responses to acetylcholine persisted and only gradually disappeared with repetitive exposures. Furthermore, in a medium with normal Ca2+ in the double sucrose gap, depolarization by 10-15 mV with an applied current caused no contraction, and repolarization to the normal membrane potential during acetylcholine-induced contraction caused no relaxation. Tetraethylammonium ion (20 mM) depolarized the membrane, increased membrane resistance, and enhanced the secondary oscillations and contractions after field stimulation. No other K(+)-channel blocker tested (Ba2+, apamin, 4-aminopyridine, glibenclamide, charybdotoxin) had the effect of prolonging secondary oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of protein synthesis in recovery from refractory period of electrical depolarization induced by osmotic stimulation in <Emphasis Type="Italic">Chara corallina</Emphasis>

Upon addition of sorbitol to the external medium of an internodal cell of Chara corallina, a transient depolarization is induced at its nodal end (Shimmen in Plant Cell Physiol 44:1215–1224, 2003). In the present study, refractory period was found to be very long, 2–4 h. Recovery from refractoriness was completely inhibited by inhibitors of eukaryote-type protein synthesis, cycloheximide or anisomysin, but not by inhibitors of prokaryote-type protein synthesis. This suggested that proteinous factor(s) responsible for generation of the depolarization is lost or inactivated upon depolarization and synthesized during the resting state. Low temperature, which is supposed to inhibit protein synthesis, also inhibited recovery from refractoriness. When unstimulated internodal cells were incubated in the medium containing an inhibitor of eukaryote-type protein synthesis, generation of the depolarization was almost completely inhibited. This result suggested that the factor is slowly turning over even in the absence of osmotic stimulation.     

F-actin does not modulate the initial steps of the protein kinase C activation process in living nerve cells

Actin is a major substrate for protein kinase C (PKC) and PKC is considered a modulator of the actin network. In addition in vitro studies (Biochemistry 39 (2000) 271) have suggested that all PKC isoforms bind to actin during the process of activation of the enzyme. To test the physiological significance of such a coupling we used living PC12 cells and primary cultures of cerebellar granule cells. When PC12 cells were treated with either latrunculin B, which impairs actin polymerization, or phalloidin, which stabilizes actin filaments, we observed a significant reduction of the [Ca2+]i response revealed by Fura-2 fluorescence, while the PKC conformational changes followed by Fim-1 fluorescence were unaffected. The responses induced either by cell depolarization or muscarinic receptor activation were similarly affected by the toxin treatment of PC12 cells. In cerebellar granule cells the [Ca2+]i response induced by KCl depolarization was increased by latrunculin treatment, whereas no effect was observed on the PKC response. Latrunculin had no effect on the NMDA-induced responses in these cells. Finally we also show that the response induced by a long-lasting depolarization, which mimics stimulation leading to neuronal plasticity, was not significantly altered by latrunculin or phalloidin treatment of the cells. These results suggest that the actin network is not involved in the initial steps of the PKC activation process in living nerve cells.     

Effects of PGE2 in guinea pig colonic myenteric ganglia

PGE(2) is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE(2) alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE(2) elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE(2) effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE(2) acts directly and indirectly to depolarize colonic neurons. PGE(2)-evoked depolarization was concentration dependent (approximately 3 microM EC(50)) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE(2) analog PGE(2)-ethanolamide (10 microM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE(2) can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.     

Transduction of pressure signal to electrical signal upon sudden increase in turgor pressure in Chara corallina

By taking advantage of large cell size of Chara corallina, we analyzed the membrane depolarization induced by decreased turgor pressure (Shimmen in J Plant Res 124:639–644, 2011). In the present study, the response to increased turgor pressure was analyzed. When internodes were incubated in media containing 200 mM dimethyl sulfoxide, their intracellular osmolality gradually increased and reached a steady level after about 3 h. Upon removal of dimethyl sulfoxide, turgor pressure quickly increased. In response to the increase in turgor pressure, the internodes generated a transient membrane depolarization at its nodal end. The refractory period was very long and it took about 2 h for full recovery after the depolarizing response. Involvement of protein synthesis in recovery from refractoriness was suggested, based on experiments using inhibitors.     

G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.     

Prolonged membrane potential depolarization in cingulate pyramidal cells after digit amputation in adult rats

The anterior cingulate cortex (ACC) plays an important role in higher brain functions including learning, memory, and persistent pain. Long-term potentiation of excitatory synaptic transmission has been observed in the ACC after digit amputation, which might contribute to plastic changes associated with the phantom pain. Here we report a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo. Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from ~-70 mV to ~-15 mV and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. The depolarization is activity-dependent, since peripheral application of lidocaine significantly reduced it. Furthermore, the depolarization was significantly reduced by a NMDA receptor antagonist MK-801. Our results provide direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain.     

Role of store-dependent influx of Ca2+ and efflux of K+ in apoptosis of CHO cells

Agents mobilising Ca(2+) from the endoplasmic reticulum are known to activate apoptosis. Whatever means are used, the release of Ca(2+) is often followed by a store-dependent entry of Ca(2+). Whether apoptosis is triggered by the depletion of the stores or by the subsequent store-dependent entry of Ca(2+) is still a matter of controversy. Here we studied apoptosis in CHO cells transfected with the rat neurotensin (NT) receptor, in which the store-dependent entry of Ca(2+) is abolished by repressing the transient receptor potential channel 2 (TRPC2) by an antisense oligonucleotide strategy (TRPC2(-) cells) [Cell Calcium 30 (2001) 157]. When stimulated with thapsigargin (TG), apoptosis occurred in both TRPC2(+) and TRPC2(-) cells but 12h earlier in TRPC2(+) cells, suggesting that store-dependent entry of Ca(2+) can accelerate the process. The expression and localisation of caspase-12, an enzyme that has been involved in the apoptosis triggered by a stress on the endoplasmic reticulum, was not different in TRPC2(+) and TRPC2(-) cells. On the contrary, the expression of GADD153 (Growth Arrest and DNA Damage inducible gene 153) triggered by TG treatment depended on external Ca(2+) and occurred earlier in TRPC2(+) than in TRPC2(-) cells. In these cells, we also noted the presence of K(+) channels activated by Ca(2+) (K(Ca) channels). Stimulation of TRPC2(+) cells with TG or with NT triggered a long sustained K(+) current, parallel to [Ca(2+)](i) transients, and resulting in a sustained hyperpolarisation of the cell membrane. K(+) current and hyperpolarisation were transient and not sustained in TRPC2(-) cells. Inhibition of K(Ca) channels with charybdotoxin dramatically reduced the K(+) current and also significantly brought down the level of apoptosis, suggesting that a prolonged efflux of K(+) could be involved in the apoptosis process. We conclude that in CHO cells, store-dependent entry of Ca(2+) can accelerate apoptosis by accelerating the expression of GADD153 and by inducing a prolonged efflux of K(+) out of the cell.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Development of depolarization-induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K(+) concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K(+) (1 min) with a transient increase in [Ca(2+)](i) of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K(+), the amplitude of the [Ca(2+)](i) transients averaging 592 nM. At later stages, all cells responded to 25 mM K(+) with [Ca(2+)](i) transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca(2+)](i) transients. The amplitudes of these [Ca(2+)](i) transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca(2+)](i) transients were greatly reduced in Ca(2+)-free, EGTA-buffered saline, and in the presence of the Ca(2+) channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca(2+) influx through voltage-activated Ca(2+) channels into antennal lobe glial cells. The development of the depolarization-induced Ca(2+) transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna.     

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression.