Regulation of intracellular chloride concentration in rat lactotrope cells and its relation to the membrane resting potential

Rat lactotrope cells in primary culture exhibit physiological properties closely associated with chloride ions (Cl-) homeostasis. In this work, we studied the regulation of intracellular Cl- concentrations ([Cl-]i) and its relation to the membrane resting potential, using a combination of electrophysiology and spectrofluorimetry. Variations in [Cl-]i resulting from the patch clamp technique, pHi, antagonists of Cl(-)-Ca(2+)-dependent channels, an anion exchanger antagonist, and an antagonist of K(+)-Cl- cotransport were considered with respect to their involvement in membrane potential. We show that: (i) The patch-pipette does not always impose its Cl- concentration. (ii) In rat lactotrope cells, membrane resting potential is partially determined by [Cl-]i. (iii) Besides ion channel activity, electroneutral ion transports (cotransports such as K(+)-Cl- and Na(+)-K(+)-2Cl-) participate actively in maintaining a high [Cl-]i. (iv) Finally, Cl- homeostasis is probably linked to cell energetics.     

Membrane Potential Measurements in Cultured Intestinal Villi

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K⁺ concentration ([K]₀], but not significantly on [Na]₀ and [Cl]₀-Deprivation of Ca²⁺ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

Dual-wavelength ratiometric fluorescence measurements of membrane potential

This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.     

Fluorescence emission spectral shift measurements of membrane potential in single cells.

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.     

Quantitative analysis of spontaneous mitochondrial depolarizations

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.     

Depolarization of synaptosomal membranes: a study of mechanism by which rhodamine 6G measures membrane potential

The fluorescence intensity of Rhodamine 6G in synaptosomal suspensions has been measured to monitor the membrane potential changes in pre-synaptic nerve terminals. The fluorescence response of the dye was seen to be a function of potential-dependent partitioning of dye molecules between the synaptosomes and the extracellular medium. Binding of dye molecules to the hydrophobic regions of membranes results in the quenching of fluorescence. Upon depolarization of the synaptosomal membrane, the dye molecules are released from the cells. The effect of changing extracellular ionic composition was also studied. The membrane potential increased linearly with log of [K]0. The resting membrane potential in buffer containing 5 mM K+ was calculated to be -60 mV. Raising the extracellular Ca2+ and Mg2+ from 1.2 mM to 10 mM did not change the membrane potential. Ca2+ ionophore A23187, in the presence of Ca2+ was found to depolarize the membranes.     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

K+ and Cl- uptake by cultured oligodendrocytes

Cultured oligodendrocytes take up K+ triggered by an increase in [K+]o. Simultaneously [Cl-]i increases in the majority of the oligodendrocytes. This KCl uptake, which is not furosemide sensitive, can be explained by the following model. The first event is the entry of Cl- into the cell driven by the discrepancy between the membrane and Cl- equilibrium potential. As a consequence of the movement of negative charge across the membrane, K+ is driven into the cell. The prerequisites of this model, a passive Cl- distribution at resting membrane potential and a Cl- conductance of the membrane were found to exist in most cultured oligodendrocytes. The chloride equilibrium potential (-61 mV, SD +/- 10 mV) was slightly more positive than the membrane potential (-64 +/- 8 mV). Since cell input resistance determined with two independent electrodes increased by 11% (SD +/- 0.07) when [Cl-]o was reduced to 10 mM, part of the membrane conductance appears to be mediated by Cl-. Differences between membrane potential and Cl- equilibrium potential therefore will lead to Cl- fluxes across the membrane. In contrast with oligodendrocytes, [Cl-]i in astrocytes is significantly increased (from 20 to 40 mM) above the equilibrium distribution owing to the activity of an inward directed Cl- pump; this suggests a different mechanism of K+ uptake in these cells.     

Intercellular communications within the rat anterior pituitary XII: immunohistochemical and physiological evidences for the gap junctional coupling of the folliculo-stellate cells in the rat anterior pituitary

Since Farquhar [1957. "Corticotrophs" of the rat adenohypophysis as revealed by electron microscopy. Anat. Rec. 127, 291] was the first to report the presence of agranular folliculo-stellate cells as corticotrophs in the anterior pituitary gland, there were no reports about electro-physiological characteristics of the folliculo-stellate cells because of its no hormonal activity and the chaotic distribution of the parenchyma cells. Male Wistar rats, aged 7 weeks with weighing 250--300 g, were separated into two groups. One group was used for immunohistochemical and light microscopical studies to detect S-100 protein and connexin 43. The other group was used for the electro-physiological study and then for the electron microscopical study to know the fine structural character of folliculo-stellate cells after the electro-physiological experiment. Clusters of S-100 protein cells (agranulated folliculo-stellate cells) and numerous connexin 43 positive sites on S-100 protein cells were clear in the "transitional zone" at which the pituitary tissue made the transition from the pars tuberalis to the proximal part of the anterior lobe. Penetration of electrodes to the cells distributed in the transitional zone showed stable membrane potential ranged between--27 and--67mV with no spontaneous activity. Random penetration of electrode showed that larger populations of cell ( approximately 80%) had membrane potentials with -55.6+/-5.1 mV, and less than 20% of cells had the resting membrane potential with -36.0+/-4.4 mV. There were two types of cell couplings; one major group for the recordings from cells with similar deep resting membrane potentials and the other for the recordings from cells with different resting membrane potentials. The former indicated that two cells were electrically coupled while the latter no electrical couples were observed. Carbenoxolone depolarized the membrane by 12.3+/-5.5 mV and reduced the amplitude of electrotonic potentials, and the response recovered by removal of carbenoxolone by the superfusate. The transitional zones of the pituitary glands examined the electrical coupling were observed by an electron microscopy. Almost cytological profiles were observed as intact. The results clearly indicated that the folliculo-stellate cell system deeply participated in the regulation of the anterior pituitary parallel with the portal vessel system, which was the main regulatory system for pituitary hormone secretion.     

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized. This voltage dependence gave rise to an inwardly rectifying macroscopic current which was half-maximally activated at -65 mV. The current through LCCs was carried by cations because reduction of external [NaCl] shifted the reversal potential of the LCC current towards the predicted Nernst potential for a nonselective cation current. These results suggest that LCCs may contribute to resting membrane potential in the circular muscle of the opossum esophagus.     

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.     

豚鼠耳蜗离体外毛细胞的膜电位和离子电流

利用膜片钳技术对分离的豚鼠耳蜗外毛细胞进行了研究,结果表明：（１）新分离的正常ＯＨＣ呈术状,胞膜光滑,胞核位于底部,静纤毛由顶端表皮板伸出,４小时内形态无明显变化。（２）全细胞电压钳记录结合通道阻断剂实验表明,ＯＨＣ膜电流主要由电压依赖性钾离子流组成。（３）利用全细胞记录方式得到的ＯＨＣ静息电位值为－２６±９ｍＶ．     

豚鼠耳蜗离体外毛细胞的膜电位和离子电流

利用膜片钳技术对分离的豚鼠耳蜗外毛细胞(OHC)进行了研究,结果表明:(1)新分离的正常OHC呈柱状,胞膜先滑,胞核位于底部,静纤毛由顶端表皮板伸出,4小时内形态无明显变化.(2)全细胞电压钳记录结合通道阻断剂实验表明,OHC膜电流主要由电压依赖性钾离子流组成.(3)利用全细胞记录方式得到的OHC静息电位值为-26±9mV((?)±SD,n=10).     

Nonlinear current-voltage relationships in cultured macrophages

Intracellular recordings of cultured mouse thioglycolate-induced peritoneal exudate macrophages reveal that these cells can exhibit two different types of electrophysiological properties characterized by differences in their current-voltage relationships and their resting membrane potentials. The majority of cells had low resting membrane potentials (-20 to -40 mV) and displayed current-voltage relationships that were linear for inward-going current pulses and rectifying for outward-going pulses. Small depolarizing transients, occurring either spontaneously or induced by current pulses, were seen in some cells with low resting membrane potentials. A second smaller group of cells exhibited more hyperpolarized resting membrane potentials (-60 to -90 mV) and S-shaped current-voltage relationships associated with a high- resistance transitional region. Cells with S-shaped current-voltage relationships sometimes exhibited two stable states of membrane potential on either side of the high-resistance transitional region. These data indicate that macrophages exhibit complex electrophysiological properties often associated with excitable cells.     

Cl- transport by gastric mucosa: cellular Cl- activity and membrane permeability

The mechanism of Cl- secretion in the isolated, resting (i.e. cimetidine-treated) gastric mucosa of Necturus has been investigated with radioisotopic and electrophysiological techniques. Measurement of transepithelial 36Cl- fluxes (mucosal to serosal (M leads to S), Jms Cl-; S leads to M, Jsm Cl-) during control conditions show that at open circuit, when the transepithelial potential difference psi ms = 20 mV (S ground), Jms Cl- = Jsm Cl-, i.e. Jnet Cl- = 0, but during short-circuit current conditions Jnet Cl- = I sc = 2 mu equiv cm-2 h. Experiments with low [Cl-] solutions indicate that Cl- exchange diffusion does not contribute significantly to either Jms Cl- or Jsm Cl-. Double-barrelled, Cl- -selective microelectrodes showed that in open circuit, the cellular (C) chemical potential for Cl-, psi c Cl- = 31 mV (apparent [Cl-] = 29 mM), the electrical potential across the M membrane, psi m = -34 mV (mucosa ground) while that across the S membrane, psi s = -52 mV (serosa ground). During short-circuit current conditions, psi m = psi s = -49 mV and [Cl-]c = 30 mM. The permeability of the M membrane to Cl- (Pm Cl-) was calculated both from the tracer experiments and the electrode measurements by using the constant-field equation. Short-term (45 s) uptake of 36Cl- at [Cl]m = 96 mM during short circuit conditions gave Pm Cl- = 2.6 x 10(-5) cm s-1. Measurement of [Cl-]c by means of the electrodes when [Cl-]m was changed from 96 to 2 mM or from 2 to 96 mM gave Pm Cl- = 2.9-5.7 x 10(-5) cm s-1. Our results indicate that during open circuit conditions Cl- is accumulated across the S membrane into gastric cells in an energy-requiring step, but since Jnet Cl- = 0, Cl- must leak back into the S solution at a rate equal to the entry rate. When the tissue is short-circuited, Cl- secretion occurs (Jnet Cl- = Isc) owing to the same energy-requiring accumulation of Cl- by the cells and a passive (apparently electrodiffusive) movement across the mucosal membrane.     

The electrophysiological properties of the resting thyroid follicular cell

Glass microelectrodes have been useful in the study of the electrical properties of the resting thyroid follicular cell membrane. The resting transmembrane potential (RMP) has probably been underestimated in earlier work, possible as a result of leak artefacts, and it is clear that in most species the RMP is certainly greater than -60 mV. The ratio of membrane Na+ permeability to K+ permeability (PNa/PK) is of the order of 0.07 to 0.08, and Cl- is possibly (although not definitely) distributed in a passive fashion across the cell membrane, indicating that the transmembrane K+ gradient is the most important factor in the generation of the RMP. The existence of an electrogenic sodium pump in the follicular cell membrane has been demonstrated: the pump contributes about -2 mV to the RMP under control conditions. Follicular cells are completely electrically coupled, the basic coupled cellular unit probably being equivalent to the individual thyroid follicle, and the specific membrane resistance and specific membrane capacitance have been calculated to be 5 k omega. cm2 and 3.6 microF/cm2 respectively.     

Correlation between membrane potential, cell shape, and motile activity in Amoeba proteus

The various motile activities and cell shapes of Amoeba proteus grown in Chalkley's solution are correlated with definitive electrical membrane potentials. The same correlations were found when definitive motile activities and cell shapes were experimentally induced by changing the pH of the culture medium. The highest values of membrane potential (−70 mV) were measured in monopodial amebae during active locomotion. In resting cells, which prevail in acid or basic media, the membrane potential decreases to −5 mV. In those resting cells, which also stop internal cytoplasmic movement at basic pH, the membrane potential turns positive (+9 mV − +30 mV).     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.     

Calcium-activated and voltage-dependent potassium conductances in clonal pituitary cells

Voltage clamp recordings of GH3/B6 pituitary cells reveal the presence of non linear steady state membrane properties at the level of the resting potential (about ?41 mV). Clamping the cells to potentials more depolarized than ?60 mV is associated with a potential dependent increase in membrane conductance and membrane current variance. Tetra-ethylammonium (TEA), Cobalt (Co²⁺) and methoxy-verapamil (D-600) each attenuate these potential-dependent changes. Spectral analysis of membrane current fluctuations shows that power spectral densities calculated for fluctuations occuring over the ? 70 to ? 40 mV range declin? monotonically as a function of frequency, while spectra derived from fluctuations obtained over the ? 20 mV to 0 mV range decline as the square of frequency and are usually well fitted by a single Lorentzian equation. The half-power frequency of these spectra varies from 45 to 65 Hz. If we assume that the activities of two-state (open-closed) ion channels underlie the electrical behaviour of the membrane at the resting potential and at more depolarized levels, then the results suggests the presence of K⁺ ion channels whose activation depends both on potential and Ca²⁺ ions. These K⁺ ion channels have estimated electrical properties (conductance : 15 ps ; duration : 3 msec) similar to those present in other excitable membranes.