Transient Membrane Depolarization Research Articles

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore. I. Evidence for two separate Me2+ binding sites with opposing effects on the pore open probability.

This paper reports an investigation on the regulation of the mitochondrial cyclosporin A-sensitive permeability transition pore (MTP). Energized, coupled rat liver mitochondria incubated in sucrose medium in the presence of phosphate maintain a high proton electrochemical gradient (delta microH) and a low permeability to solutes. Addition of a small (10-20 microM) Ca2+ pulse leads to a transient membrane depolarization. After Ca2+ accumulation, a high delta microH is recovered, and mitochondria remain coupled indefinitely. Yet, addition of fully uncoupling concentrations of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) brings about MTP opening within seconds. This finding confirms that MTP opening is the consequence rather than the cause of membrane depolarization, and allowed us to study the operation of the MTP in a synchronized population of mitochondria, since pore opening can be triggered by the addition of uncoupler under a series of experimental conditions. We find that three regulatory sites can be defined: (i) an internal Me2+ binding site: when this site is occupied by Ca2+, the pore "open" probability increases, while other Me2+ ions (Sr2+, Mn2+) have an inhibitory effect; (ii) an external Me2+ binding site: when this site is occupied by Me2+ ions, including Ca2+, the pore open probability decreases; (iii) an independent cyclosporin A binding site: when this site is occupied by cyclosporin A the pore open probability decreases. We show that at variance from the case of cyclosporin A, MTP inhibition by the phospholipase A2 inhibitors nupercaine and trifluoperazine is Ca(2+)-competitive and is presumably related to interference by these drugs with Ca2+ binding to the internal regulatory site.

Depolarizing action of cholecystokinin on rat supraoptic neurones in vitro.

1. Cholecystokinin is co-localized within the oxytocin- and, to a lesser extent, vasopressin-synthesizing magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei. These nuclei are also prominent binding sites for cholecystokinin. In the present study we used intracellular current- and voltage-clamp recordings from fifty-seven supraoptic nucleus cells, maintained in superfused explants of rat hypothalamus, to assess their membrane responses to exogenous cholecystokinin and define the nature of their cholecystokinin receptors. 2. In a majority of the fifty-seven cells tested, bolus infusions into the superfusion media of cholecystokinin fragments (maximum concentrations estimated at 0.3-15 microM) were followed within 1-5 s by a transient and reversible membrane depolarization. Active peptides included sulphated cholecystokinin octapeptide (26-33) (28 of 33 cells responded), non-sulphated cholecystokinin octapeptide (26-33) (21 of 25 cells responded), cholecystokinin tetrapeptide (30-33) (20 of 24 cells responded and caerulein (4 of 4 cells responded). None of five cells responded to cholecystokinin (26-28). Depolarizing responses to cholecystokinin analogues persisted in the presence of tetrodotoxin (0.2-0.4 microM), and in Ca(2+)-free solutions containing MnCl2 (2.5 mM). 3. Under voltage clamp, cholecystokinin fragments evoked an inward current accompanied by an increase in membrane conductance. The amplitude of the inward current varied linearly as a function of membrane voltage, with an extrapolated reversal potential of approximately -15 mV. Reversal potentials were not altered by chloride injection. These features suggest that cholecystokinin activates a non-selective cationic conductance. 4. Active cholecystokinin analogues were approximately equipotent in their depolarizing actions, a feature that supports the activation of cholecystokinin-B type receptors. Moreover bath application of 200 nM L-365,260, an antagonist with a high affinity for cholecystokinin-B receptors, reversibly attenuated the cholecystokinin-induced responses in four of six cells tested. 5. These observations indicate that cholecystokinin can directly influence the excitability of rat supraoptic nucleus neurones and provide evidence for an additional site where this peptide may act within the hypothalamo-neurohypophysial axis.

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings.

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm s-1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a "slow-wave potential". These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.

Ion Channels in Arabidopsis Plasma Membrane : Transport Characteristics and Involvement in Light-Induced Voltage Changes

White light (25 watts per square meter) induced an increase in plasma membrane K(+)-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K(+) on the cytoplasmic side of the patch and 50 millimolar K(+) in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K(+) over Na(+) and Cl(-). One (named PKC1) had a maximum conductance (G(max)) of 44 picosiemens at a membrane voltage (V(m)) of -65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G(max) = 66 picosiemens at V(m) = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K(+) and Na(+) about equally well but not Cl(-). It had G(max) = 109 picosiemens at V(m) = 55 millivolts in (250/50 K) with 10 millimolar Ca(2+) on the cytoplasmic side. Reducing Ca(2+) to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar beta-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.

Transferrin can alter physiological properties of retinal neurons

The role of transferrin as a possible neurotransmitter was examined in cultured chick retinal cells. Brief exposure to transferrin caused a dramatic and transient increase in intracellular calcium levels in approximately 20% of the total population of cultured retinal neurons. The increase in intracellular calcium was observed in cell bodies and neuronal processes. Electrophysiological analysis of a subset of the population, bipolar-like neurons, demonstrated that more than half of these cells responded to the application of transferrin with a transient membrane depolarization. Under voltage clamp conditions, the currents evoked by transferrin were similar to glutamate in that they both displayed non-linear voltage dependence. Furthermore, acute transferrin exposure resulted in a 200% increase in the amount of Na + independent [ 3H]glutamate binding observed in these cultures. These results suggest that transferrin may function as a neurotransmitter or neuromodulator in the developing vertebrate nervous system.

Stimulation of non‐selective cation channels providing Ca2+ influx into platelets by platelet‐activating factor and other aggregation inducers

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.

Spontaneous activity of sodium loaded guinea-pig cardiac myocytes : Contribution of Na+Ca2+exchange

Guinea-pig cardiac myocytes loaded intracellularly with Na+ show spontaneous contractile activity and transient membrane depolarizations or inward currents. We have investigated whether both phenomena can be attributed to release of Ca2+ from overloaded cellular stores causing contractile activation and at the same time generating an inward current due to Ca2+ efflux by the electrogenic Na+/Ca2+ exchange. Variations in membrane potential by voltage clamp or in ionic composition of the superfusion medium changed the amplitude and frequency of spontaneous inward currents in the direction expected from computations of the Na+/Ca2+ exchange. Caffeine in concentrations that activate the Ca2+ release channel of the sarcoplasmic reticulum produced a vigorous contraction accompanied by a large current transient. After exposure to ryanodine both Isp and spontaneous contractions were abolished. Spontaneous inward currents were depressed by inhibitors of the Na+/Ca2+ exchange. The results with caffeine and ryanodine demonstrate that unimpaired sarcoplasmic reticular function is a prerequisite for both the contractile and the electrical event. From the characteristics of Isp it is proposed that this current is due to electrogenic Na+/Ca2+ exchange.

Electrophysiological measurements of volume changes in leech neuropile glial cells

Double-barrelled microelectrodes, sensitive to quaternary ammonium ions, were used for simultaneous measurements of the intracellular free concentrations of choline ([Ch]i) or tetramethylammonium ([TMA]i) as well as membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. Bath application of Ch or TMA (5 mM, 1 min) resulted in a transient membrane depolarization accompanied by a long-lasting (0.5-1 h) intracellular accumulation of these compounds to levels of between 5 and 15 mM. Changes in [Ch]i or [TMA]i were used for the calculation of changes in relative cell volume. Elevation of the extracellular K+ concentration [( K+]e) from 4 to 9, 15, 21, 27.5, or 40 mM elicited a membrane depolarization and a reversible cell swelling by about 7.5, 14, 18.5, 27 and 50%, whereas reduction of [K+]e to 1.5 mM as well as bath application of serotonin (5-HT) produced a membrane hyperpolarization and a concomitant shrinkage by about 6 and 14.3%, respectively. The measured alterations in cell volume were compared with calculated data based on the assumption of an osmotic equilibrium disturbed by potential-dependent changes of the intracellular Cl- concentration. The results indicate, that K(+)- and serotonin-induced changes in the cell volume of the neuropile glial cells are due to passive KCl and water fluxes.

Membrane effects of chloride substitutes in guinea pig gallbladder epithelial cells.

Differences in the responses of guinea pig gallbladder epithelial cells to replacement of luminal Cl- with either isethionate (I), gluconate (G), sulfate (S), or cyclamate (C) were investigated in vitro using intracellular microelectrode techniques. In prostaglandin E1 (PGE1)-treated tissues (10(-6) M, serosal side), where electrodiffusive apical membrane Cl- permeability (PCla) is high, replacement of luminal Cl- caused transient membrane depolarizations of similar magnitudes but different times to peak (C greater than G = S greater than I). The subsequent shifts in membrane voltages were, at steady state, straight correlated with the concomitant increases in apparent ratio of apical to basolateral membrane resistances (Ra/Rb). Increases followed the rank order I greater than G = S greater than C, which was also found to be the case in the peak membrane hyperpolarizations on restoring luminal Cl-. Under control conditions (no PGE1, low PCla), three of the substitutes caused a slow hyperpolarization, C greater than G = S, whereas an I-for-Cl- substitution evoked a transient depolarization and a drop in Ra/Rb. Under both control and PGE1 conditions, a transient depolarization followed luminal I-for-C substitution. Our results are best explained by a stimulatory effect of I (and, less marked, G and S) on PCla. Intrinsic effects of cyclamate are not ruled out; however, among the substitutes examined, it is the most inert.

Neurotensin facilitates release of substance P in the guinea‐pig inferior mesenteric ganglion.

1. Intracellular, electrophysiological techniques were combined with radio-immunological, chromatographic and pharmacological techniques to determine if nerve terminals containing substance P mediated transient depolarizing responses of principal ganglion cells induced by neurotensin. Experiments were performed in vitro on guinea-pig inferior mesenteric ganglia. 2. In 61% of principal ganglion cells tested in normal ganglia, neurotensin caused a transient membrane depolarization. In ganglia which were removed from animals which had been pre-treated with capsaicin, transient responses to neurotensin were virtually abolished. 3. In normal ganglia, neurotensin increased the amplitude and duration of noncholinergic slow EPSPs evoked by electrical stimulation of the lumbar colonic nerve. Such increases were absent in ganglia obtained from animals pre-treated with capsaicin. 4. In guinea-pigs pre-treated with capsaicin, the content of substance P-like material was significantly reduced in inferior mesenteric and coeliac ganglia, dorsal root ganglia and lumbar spinal cord, compared to control animals. The content of substance P-like material in segments of distal colon was slightly reduced. The content of vasoactive intestinal polypeptide-, cholecystokinin- and bombesin-like material in the same tissues from animals pre-treated with capsaicin was not significantly different from control animals. 5. Chromatographic analysis using HPLC (high-performance liquid chromatography) techniques revealed that the material depleted from inferior mesenteric and coeliac ganglia, dorsal root ganglia and lumbar spinal cord by capsaicin pre-treatment co-eluted with synthetic substance P. 6. Electrical stimulation of the lumbar colonic nerve released substance P-like material from isolated inferior mesenteric ganglia as determined by radioimmunoassay of samples of superfusate. Exogenous administration of neurotensin caused a significant increase in the amount of substance P-like material released during nerve stimulation. 7. Transient depolarizing responses evoked by neurotensin were markedly attenuated when ganglion cells were postsynaptically desensitized to exogenously administered substance P. 8. Taken together, these findings suggest that transient depolarizations mediated by an indirect action of neurotensin and facilitation of electrically evoked non-cholinergic slow EPSPs by neurotensin involved presynaptic release of substance P from collateral nerve terminals of primary afferent nerve fibres in the inferior mesenteric ganglion. 9. It was suggested that under normal in vivo conditions, neurotensin or a C-terminal-related peptide contained in central preganglionic nerve endings might function as an excitatory neuromodulator to enhance the release of substance P from primary afferent nerve terminals thereby facilitating non-cholinergic peripheral afferent synaptic input to prevertebral ganglion cells.

Evaluation of Arsenate- and Vanadate-Associated Changes of Electrical Membrane Potential and Phosphate Transport inLemna gibbaG1

Interference of arsenate and vanadate with phosphate uptake in Lemna gibba L. was studied by measuring voltage changes and (32P)phosphate uptake. Arsenate proved to be competitive with the highand low-affinity phosphate uptake system. It induced transient membrane potential changes of up to 120 mV which were similar to those induced by phosphate and indicated a cotransport mechanism with at least 2H+/H2As04. The amplitude of the transient arsenate-induced membrane depolarization was strongly influenced by phosphate starvation. A permanent membrane depolarization to the diffusion potential was achieved within 2 to 6 h in P-starved plants. Thus, arsenate is indeed a strongly competitive physiological analogue of phosphate in higher plants. Vanadate was easily transported into L. gibba as concluded from transient Em changes of up to 110 mV. Vanadate interfered only slightly and non-specifically with the two phosphate transport mechanisms. Like phosphate, vanadate uptake seems to be an H+-cotransport mechanism, both with similar optima at pH 6 0. Unlike phosphate uptake, vanadate-linked membrane depolarization was not affected by high intracellular phosphate concentrations. P-starvation did not enhance the weak long-term effect on Em. Hence, vanadate, in contrast to arsenate, is not regarded as a physiological phosphate analogue. The distinct and rapid vanadate-induced and permanent membrane depolarization of Avena sativa, Triticum aestivum and Glycine max leaves was not seen in Lemna nor in leaves of Gossypium hirsutum and Nicotiana tabacum. Plasmalemma-enriched preparations of L. gibba revealed, however, a high vanadate-sensitive ATPase activity (87%). As a possible explanation for these differences it is suggested that the latter plant species have cytosolic vanadate-detoxifying properties, i.e. they can reduce vanadate to vanadyl ions, in contrast to the former group of plant species.

Electrophysiological characterization of a nicotinic acetylcholine receptor on leech neuropile glial cells

Ion-selective double-barrelled microelectrodes were used to measure the activities of intracellular K+, Na+, Cl-, and H+ (aiK, aiNa, aiCl, pHi) and membrane potential (Em) in neuropile glial cells as well as extracellular K+ activity (aeK) in the neuropile of the leech, Hirudo medicinalis, during bath application of carbachol. As measured with conventional single-barrelled microelectrodes, acetylcholine (ACh), nicotine, carbachol, tetramethylammonium (TMA), and choline elicited concentration-dependent (10(-6)-5 X 10(-3) M) transient membrane depolarizations of up to 60 mV amplitude whereas muscarine (10(-6)-10(-3) M) did not affect Em. alpha-Bungarotoxin (10(-7) M), decamethonium (10(-5) M), d-tubocurarine (5 X 10(-5) M), and strychnine (5 X 10(-5) M) blocked the carbachol depolarization by about 90%. Atropine (5 X 10(-5) M) blocked the response by about 75%, whereas hexamethonium was only effective at millimolar concentrations. Average baseline levels of aeK in the neuropile and of aiK, aiNa, and aiCl in the neuropile glial cells were about 3, 70, 10, and 7 mM, respectively. During the carbachol depolarization aeK and aiNa transiently increased, whereas aiK decreased. In contrast, a rise of aiK and a fall of aiNa were observed during glial depolarizations in solutions with elevated K+ concentration. aiCl increased during both the carbachol- and the K+-induced depolarization. During carbachol, pHi transiently fell by about 0.2 units from its average baseline level of 6.9, whereas an alkalinization of small amplitude was observed in high-K+ solutions. Bath-applied choline, TMA, and decamethonium rapidly accumulated in the neuropile glial cells as intracellularly monitored with double-barrelled microelectrodes filled with Corning K+ exchanger resin, which is highly selective for these agents. The results suggest that leech neuropile glial cells have a nicotinic ACh receptor coupled to a cation channel. It is hypothesized that this channel might also be permeable to choline, TMA, and decamethonium.

Effects of the pyrethroid cypermethrin on l-glutamate-induced changes in the input conductance of the ventrolateral muscles of the larval house fly, Musca domestica

Cypermethrin has been previously demonstrated to block neuromuscular transmission in larval house fly ventrolateral muscles (VLMs). Although the effect is consistent with a presynaptic depolarization as it involves an increase in miniature excitatory postsynaptic current frequency, and decrease in amplitude of evoked responses, the role of a postsynaptic action in the production of these responses has not yet been assessed. Thus, this study was undertaken to observe the effects of both cypermethrin and its solvent acetone upon the postsynaptic aspect of synaptic transmission. VLMs 6a and 7a had similar resting membrane potentials (−57.6 ± 8.0 mV and −59.2 ± 8.3 mV, respectively; n = 20 cells) and input conductances (3.08 ± 0.87 and 2.97 ± 1.00 × 10 −7 siemens, respectively; n = 12 cells). l-Glutamate produced a dose-dependent depolarization and increase in the sarcolemma input conductance. A Hill plot revealed that a minimum of one glutamate molecule was required to bind to each receptor to elicit a unit response. At concentrations of 1 ml liter −1 acetone and 10 n M cypermethrin, no effects were observed on the muscle resting membrane potential, the sarcolemma input conductance, or sensitivity to 10 m M l-glutamate (pretreated with 1 μ M concanavalin A to block glutamate receptor desensitization). However, at higher concentrations both acetone and cypermethrin had postsynaptic effects. Acetone (10 ml liter −1) caused a slight increase in muscle input resistance ( P < 0.05), although higher concentrations produced a decrease in input resistance (EC 50 at 4%) of up to 70% control values. A transient membrane depolarization was observed upon initial application of acetone (> 10 ml liter −1) which was followed by a sustained membrane hyperpolarization. Intense axonal repetitive firing was also noted with acetone concentrations above 10 ml liter −1. Cypermethrin (1 μ M), 30–90 min after application, blocked the l-glutamate induced increases in input conductance and caused substantial oscillations in the sarcolemma resting membrane potential. The initial membrane depolarizations induced by 10 m M l-glutamate in control preparations were still observed in muscles poisoned with 1 μ M cypermethrin. These data demonstrate that the block of neuromuscular transmission at larval house fly nerve terminals by 10 n M cypermethrin does not involve a change in muscle membrane input conductance or a decrease in receptor sensitivity to glutamate, and therefore is likely to be a consequence of its presynaptic action.

Pharmacological characterization of serotonin 5-HT 3 receptor-mediated electrical response in cultured mouse neuroblastoma cells

The aim of this study was to investigate the pharmacological characteristics of the 5-hydroxytryptamine-(5-HT)-induced electrical response in cultured neuroblastoma N1E-115 cells of the mouse. In these cells 5-HT induces a transient membrane depolarization, which is associated with a transient inward current, that has been recorded in voltage clamp experiments on whole cells. The peak amplitude of the inward current depends on the concentration of 5-HT applied. Maximum peak inward current was evoked by 10 μM 5-HT and half maximum effect by 2 μM. Responses to 5-HT were blocked by nanomolar concentrations of selective 5-HT 3-receptor antagonists, whereas the selective agonist 2-methyl-5-HT mimicked the membrane depolarization induced by 5-HT. A number of agonists and antagonists, which are known to act on 5-HT 1-like, 5-HT 2, dopaminergic and adrenergic receptors failed to affect the response to 5-HT in neuroblastoma cells. Observed antagonistic effects of SCH 23390 [(R) − (+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate] and haloperidol are discussed. The inhibitory effect of the 5-HT 3 receptor antagonist, ICS 205–930 [(3α-tropanyl)-1H-indole-3-carboxylic acid ester] has been demonstrated. When cells were exposed to 0.1 nM ICS 205–930 the maximum evoked response was reduced by about 50%, but a surmountable shift of the concentration-response curve of 5-HT was not observed. The kinetics of the 5-HT-induced inward current remained unchanged in the presence of ICS 205–930. Recovery from the block by ICS 205–930 was very slow. The apparent non-competitive nature of this antagonism is discussed. The amplitude of the membrane depolarization evoked by ionophoretic application of 5-HT decreased in the presence of a small concentration of 5-HT. Dopamine (DA) also induced a depolarizing response in N1E-115 cells. Responses to ionophoretically applied 5-HT and dopamine were both potently desensitized by 5-HT. Dopamine induced densensitization only at much greater concentrations. It is concluded that in cultured neuroblastoma N1E-115 cells of the mouse, the 5-HT-induced membrane depolarization and the associated inward current are mediated by a single population of 5-HT 3 receptor-mediated ion channels. These cells are an appropriate in vitro model to study the molecular mechanisms underlying the functioning of 5-HT 3 receptors.

Calcium-activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation

In the isolated parietal cell the following observations were made: gastrin led to an increase in cytoplasmic free Ca 2+ concentration ([Ca 2+] i) even in the absence of medium Ca 2+ and to transient membrane depolarization in both the absence of the medium Ca 2+ and the fura-2-loaded cell. The incorporated Ca 2+ chelator BAPTA inhibited the gastrin-induced membrane depolarization. The magnitude of depolarization caused by gastrin was unchanged on removal of medium Na +. Furosemide but not tetraethylammonium inhibited the gastrin-induced depolarization. The results suggest that the Ca 2+ released from the store(s) induces membrane depolarization, possibly via modulation of a Cl − efflux across the luminal surface during gastrin stimulation.

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH.

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.

Amiloride derivatives that block Na +/Ca 2+ exchange inhibit spontaneous inward currents in Na +-loaded cardiac myocytes

Spontaneous electrical and mechanical activity was observed when single myocytes from guinea-pig hearts were loaded with sodium by direct intracellular application. Transient membrane depolarizations were found to be due to spontaneous inward currents (I sp). Both I sp and spontaneous contractions were abolished by 2′,3′-benzobenzamil or 3′,4′-dichlorobenzamil, two compounds that were previously reported to inhibit Na +/Ca 2+ exchange. These findings suggest that the spontaneous membrane currents in Na +-loaded myocytes could be generated by the Na +/Ca 2+ exchange mechanism.

The dopamine response in mouse neuroblastoma cells is mediated by serotonin 5HT 3 receptors

Serotonin (5HT) and dopamine (DA) induce, in neuroblastoma N1E-115 cells, a transient membrane depolarization associated with an inward current. The half-maximum response is obtained with 2 μM 5HT or 200 μM DA. The maximum response to 5HT is 2–3 times that to DA. The selective 5HT 3 receptor antagonists ICS 205–930 and MDL 72222 at nanomolar concentrations block both the 5HT- and the DA-induced response. High concentrations (10 μM) of 5HT 2 receptor antagonists are without effect. It is concluded that, in N1E-115 cells, 5HT and DA activate a single population of 5HT 3 receptors.

Spontaneous inhibitory postsynaptic potentials in guinea pig neocortex and olfactory cortex neurones

The membrane potential of olfactory cortex and neocortex neurones in vitro was recorded using conventional microelectrode techniques. During recordings with KCl- or CsCl-filled microelectrodes, spontaneous, subthreshold, transient membrane depolarizations were observed. These were abolished by the GABA A-receptor antagonist, bicuculline methiodide, and were prolonged by the barbiturate pentobarbitone. In most cells they were abolished by tetrodotoxin. It is concluded that these spontaneous depolarizations are inhibitory postsynaptic potentials arising from spontaneous activity in inhibitory interneurones.

Intracellular calcium measurements with arsenazo III during cyclic AMP injections into molluscan neurons.

Injections of cyclic adenosine monophosphate into molluscan neurons often produce a transient membrane depolarization. By using the calcium indicator dye arsenazo III, it was found that cyclic nucleotide injections into neurons of Archidoris montereyensis resulted in elevation of internal calcium concentrations. However, this was demonstrated to be a secondary consequence of an induced increase in membrane sodium permeability, and not due to any direct effect of cyclic adenosine monophosphate on cellular calcium influx or internal calcium regulating processes.