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Intracellular ionic concentrations, membrane potential and Na+ and K+ flux were measured in a clonal rat glial cell line, C6. Intracellular concentrations of C6 cells were: (mmole/liter cell water) K+ 145 +/- 4 S.D., Na+ 18 +/- 4 S.D., Cl- 14 +/- 1 S.D. Cells maintained a steady state level of K+ over the duration of the experiments. This was substantiated by the close agreement between absolute values for K+ influx and efflux measured with 42K. When cells were depleted of internal Na+, K+ influx was significantly reduced suggesting that a portion of inward K+ movement is linked to Na+ extrusion. Efflux of Na+, calculated from the half-time of exchange from cells preloaded with 22Na, was higher than passive Na+ influx determined by blocking Na+ extrusion with ouabain. Since cell Na+ concentration remained relatively constant, part of the Na+ efflux may be due to exchange diffusion. The average membrane potential of C6 cells uas -36 mV. The potential showed a 31 mV slope for a 10-fold change in external K+ so that it is determined predominantly by the ratio of external/internal K+. The potential however, consistent with the relatively large passive Na+ influx, was influenced by Na+. Replacing all but 20 mM external Na+ with choline hyperpolarized the membrane by 13 mV. The ratio of PNa/PK of 0.11 suggests that Na+ is outwardly transported to maintain the steady-state concentrations observed. Since these responses are similar to those in non-tumor glia, it suggests that the C6 cell line may provide a useful model for studying ionic regulation in glial cells.
Stimulatory concentrations of glucose induce two patterns of cytosolic Ca2+ concentration ([Ca2+]c) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [Ca2+]c and insulin release from single islets revealed that mixed [Ca2+]c oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single beta-cell within an islet and of [Ca2+]c in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most beta-cells of the islet. Each slow [Ca2+]c increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [Ca2+]c oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA3+/+ beta-cells. We conclude that glucose-induced mixed [Ca2+]c oscillations result from compound bursting in all beta-cells of the islet. Disruption of SERCA3 abolishes mixed [Ca2+]c oscillations and augments beta-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the beta-cell membrane potential during glucose stimulation.
Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about -100 mV relative to the external medium, i.e., inside-negative. ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units. The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about -100 mV. Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N,N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 microM/2 mg protein/ml. These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.
Polypeptide decay has been measured as a function of membrane potential. Mitochondrial translation products were pulse-labeled in vitro with [35S]methionine using isolated rat heart mitochondria in the presence of an energy-generating system. The relative rate of protein degradation was estimated from the specific activity (counts/min/mg of protein) of the labeled translation products following the addition of unlabeled methionine (chase). To modulate membrane potential, inhibitors of oxidative phosphorylation were used singly or in combination; their effect was monitored by following uptake of the nonmetabolizable lipophilic cation triphenylmethylphosphonium. When the potential was dissipated, the rate of polypeptide decay increased and vice versa. These results suggest that the stability of mitochondrial translation products is linked to a process(es) that is dependent upon delta psi; likely candidates include synthesis and/or assembly of mitochondrial gene products.
Apolipoprotein C (apo C) was shown to decrease the Ca2+ capacity and membrane potential of mitochondria isolated from rat liver. The specific ligands of adenine nucleotide carrier, ADP and carboxyatractyloside (CAT), inhibited the effect of apo C on the mitochondrial membrane potential. The effect of ADP and CAT was revealed in the absence of Ca2+. We conclude that in the presence of apo C, adenine nucleosides carrier transforms into a pore, and this causes the decrease in the membrane potential of the mitochondria. ADP and CAT support the primary conformation of the carrier and therefore inhibit the effect of apolipoprotein C.
Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.
The import pathways of the alternative oxidase and the F(A)d subunit of the ATP synthase from soybean were characterised. The F(A)d precursor does not require extramitochondrial ATP for import and this was shown to be a characteristic of the mature protein. The alternative oxidase and F(A)d precursors were shown to differ in their requirement for a membrane potential. The membrane potential was modified using malonate, a competitive inhibitor to complex II. The alternative oxidase could be imported at higher malonate concentrations compared to the F(A)d. This difference could not be ascribed to the number of positive charges in each presequence as would be predicted from similar studies in fungi.
Compound resting membrane potential was recorded by the grease gap technique during normoxic conditions (37 degrees C) in rat optic nerve, a representative CNS myelinated tract. Mean potential was -47 +/- 3 (SD) mV and remained stable for 2-3 h. Input impedance of a single optic nerve axon was calculated to be approximately 5 Gomega. Contribution of the Na+ pump to resting axonal potential is estimated at -7 mV. Ouabain (10 microM to 10 mM) evoked a dose-dependent depolarization that was maximal at >/=1 mM, depolarizing the nerves to approximately 35-40% of control after 60 min. Inhibiting energy metabolism (CN- and iodoacetate) during high-dose ouabain (1-10 mM) exposure caused an additional depolarization, suggesting additional ATP-dependent, ouabain-insensitive ion transport systems. Perfusion with zero-Na+ (choline substituted) caused a transient hyperpolarization, that was greater than with tetrodotoxin (TTX; 1 microM) alone, indicating both TTX-sensitive and -insensitive Na+ influx pathways in resting rat optic nerve axons. Resting probability (P)K:PNa is calculated at 20:1. In contrast to choline-substituted solution, Li+-substituted zero-Na+ perfusate caused a rapid depolarization due to Na+ pump inhibition and the ability of Li+ to permeate the Na+ channel. TTX reduced, but did not prevent, ouabain- or zero-Na+/Li+-induced depolarization. We conclude that the primary Na+ influx path in resting rat optic nerve axons is the TTX-sensitive Na+ channel, with evidence for additional TTX-insensitive routes permeable to Na+ and Li+. In addition, maintenance of membrane potential is critically dependent on continuous Na+ pump activity due to the relatively high exchange of Na+ (via the above mentioned routes) and K+ across the membrane of resting optic axons.
At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.
The effects of microiontophoretic applications of catecholamines and acetylcholine on parotid acinar cell membrane potential and resistance were investigated using intracellular microelectrode recording in superfused segments of mouse parotid or rat submandibular glands. Short pulses of acetylcholine and alpha-adrenergic agonists had similar effects, consisting of a marked decrease in membrane resistance accompanied by an initial depolization or hyperpolarization depending on the level of the resting membrane potential. This initial response was followed by a slow hyperpolarization occurring at a time when the resistance was increasing towards the prestimulation level. The equilibrium potential for the initial potential change caused by excitation of the cholinergic receptors was investigated directly by setting the membrane potential at different levels by injecting direct current and stimulating the same cell repeatedly with equal doses of acetylcholine. The equilibrium potential was found to be about -55 mV. The delayed hyperpolarization could not be reversed by passing hyperpolarizing current, but actually increased in size with higher membrane potentials. The minimum latency of the effect of acetylcholine or alpha-adrenergic agonists was 200-500 msec. Excitation of beta-adrenoceptors caused, after a long latency of several seconds, a small depolarization. Epinephrine induced a combined alpha- and beta-adrenergic response, with the alpha-component predominating. Blocking the alpha-adrenoceptors with phentolamine revealed the beta-adrenergic depolarization, while blocking the beta-adrenoceptors with propranolol caused the components of the alpha-adrenergic response to become more pronounced. All three receptors (alpha- and beta-adrenoceptors and cholinergic receptors) were present in individual acini.
The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, 19F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (alpha = Vin/Vcell) was found to be the same (0.82 +/- 0.06) for all the cell lines in the presence and absence of dexamethasone.
Fluo-3 and di-BA-C4 (3) -loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free [Ca2+]i and the membrane potentials were measured in single thrombin-activated platelets by ACAS-570 (Adherent Cell Analysis and Sorting Cytometer). Fluorescence of fluo-3 and di-BA-C4 (3) was low in rest immobilized platelets without obviously repetitive spiking. When the platelets were activated by thrombin, a rapid rise of cytosolic [Ca2+]i and membrane potential was observed. Subsequently, oscillations in [Ca2+]i to an extent of about 500 fluorescent units were observable, whereas the membrane potential remained at the peak level, i.e., the change of [Ca2+]i in no way of accord with that membrane potential. The above results indicate that the thrombin induced membrane depolarization is not due to influx of Ca2+.
In former work, intraperitoneal injection of 2-mercaptoacetate (MA), an inhibitor of fatty acid oxidation, increased food intake in rats, which was attenuated by hepatic branch vagotomy, and intraportal injection of MA increased the discharge rate in hepatic vagal afferents. In the present study, we investigated, whether intraperitoneal injection or intraportal infusion of MA affects the hepatic membrane potential in rats in vivo. The liver cell membrane potential was measured in anesthetized Sprague-Dawley rats with the microelectrode technique. Intraperitoneal injection of MA at a dose of 800 micromol/kg body wt significantly decreased the hepatocyte membrane potential by 3.8 mV, whereas at a dose of 400 micromol/kg, the depolarization (1.5 mV) of the membrane was not significant. In another strain of Sprague-Dawley rats, however, MA (400 micromol/kg) produced a significant depolarization of the hepatocyte membrane 50 min (2.6 mV) and 2 h (2.9 mV) after intraperitoneal injection. Intraportal infusion of MA (400 micromol/kg) significantly depolarized the membrane 20 and 50 min after infusion by 3.3 and 4.1 mV, respectively. MA at a dose of 800 micromol/kg also depolarized the membrane (4.8 mV after 50 min). These findings in principle are consistent with the "potentiostatic" hypothesis, postulating a link between the hepatic membrane potential, afferent vagal activity, and the control of food intake.
Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.
The individual membrane potentials have been studied in just excised cells of human squamous cell carcinoma (n = 16). The physiological level of the membrane potential amounted to 10.3 mV. The cellular tissue was excised at the region of the rectum. In another experimental group (n = 16), the tumorous tissue was exposed to ionizing radiation by Co-60, and the decreasing membrane potential of individual cells was recorded. The findings are discussed in view of experimental investigation technique for irradiated cells.
1. We previously demonstrated that a balance of Ca2+-activated Cl- current (ICl(Ca)) and K+ current activity sets the resting membrane potential of opossum lower esophageal sphincter (LES) circular smooth muscle at approximately -41 mV, which leads to continuous spike-like action potentials and the generation of basal tone. Ionic mechanisms underlying this basal ICl(Ca) activity and its nitrergic regulation remain unclear. Recent studies suggest that spontaneous Ca2+ release from sarcoplasmic reticulum (SR) and myosin light chain kinase (MLCK) play important roles. The current study investigated this possibility. Conventional intracellular recordings were performed on circular smooth muscle of opossum LES. Nerve responses were evoked by electrical square wave pulses of 0.5 ms duration at 20 Hz. 2. In the presence of nifedipine (1 microm), substance P (1 microm), atropine (3 microm) and guanethidine (3 microm), intracellular recordings demonstrated a resting membrane potential (MP) of -38.1+/-0.7 mV (n=25) with spontaneous membrane potential fluctuations (MPfs) of 1-3 mV. Four pulses of nerve stimulation induced slow inhibitory junction potentials (sIJPs) with an amplitude of 6.1+/-0.3 mV and a half-amplitude duration of 1926+/-147 ms (n=25). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific guanylyl cyclase inhibitor, abolished sIJPs, but had no effects on MPfs. Caffeine, a ryanodine receptor agonist, hyperpolarized MP and abolished sIJPs and MPfs. Ryanodine (20 microm) inhibited the sIJP and induced biphasic effects on MP, an initial small hyperpolarization followed by a large depolarization. sIJPs and MPfs were also inhibited by cyclopiazonic acid, an SR Ca2+ ATPase inhibitor. Specific ICl(Ca) and MLCK inhibitors hyperpolarized the MP and inhibited MPfs and sIJPs. 4. These data suggest that (1). spontaneous release of Ca2+ from the SR activates ICl(Ca), which in turn contributes to resting membrane potential; (2). MLCK is involved in activation of ICl(Ca); (3). inhibition of ICl(Ca) is likely to underlie sIJPs induced by nitrergic innervation.
OBJECTIVE: To investigate the effect of N-acetyl-cysteine (NAC) and depakine (DP) on the changes of membrane potential and peroxidate in rat cortex neurons exposed to ferrous chloride (FeCl(2)). METHODS: Cultured cortex neurons of newly born SD rats were randomly divided into control group (PBS group), model group (FeCl(2) group), NAC pretreatment group (NAC group), DP pretreatment group (DP group) and NAC+DP pretreatment group (NAC+DP group). In the latter three groups, NAC (0.08 mg/ml) and DP (0.1 mg/ml) were added in the cell culture 2 and 3 h before FeCl(2) (1 mmol/L) exposure, respectively. After exposure to FeCl(2), the membrane potential of the neurons was detected with fluorescent dye DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol), and the peroxidate level with 2,7-dichlorofluorescin diacetate (H(2)DCF) by laser confocal scanning microscope (LCSM) and nuclear factor-KappaB (NF-KappaB) level with immunocytochemistry. RESULTS: Compared with FeCl(2) group, the expression of NF-KappaB and peroxidate level in the neurons were decreased significantly in NAC and NAC+DP groups (P<0.01), but not in DP group (P>0.05). FeCl(2) depolarized the membrane potential and increased the expression of NF-KappaB in the neurons. Compared with FeCl(2) group, significant changes in the membrane potential were observed in DP and NAC+DP groups (P<0.01) but not in NAC or PBS group (P>0.05). CONCLUSION: Both NAC and DP can protect the neurons from FeCl(2)-induced damage but through different pathways, and their combined use can significantly alleviate neuronal damages due to FeCl(2) exposure. Antioxidants such as NAC in combination with antiepileptic drugs may produce favorable effect in prevention and treatment of posttraumatic epilepsy.