Dielectrospectroscopic monitoring of early embryogenesis in single frog embryos.

Dielectric spectroscopy has been used to monitor the early embryogenesis of frog (Xenopus laevis) eggs. The dielectric spectra of a single egg in suspension over the frequency range 10 Hz to 10 MHz were collected at various stages of its development. The uncleaved egg showed a dielectric dispersion with a narrow distribution of relaxation times. After the first cleavage, the dielectric spectra were mainly composed of two subdispersions. In the cleavage process, up to the morula stage, changes in the spectra were quantitatively simulated by the 'cell-aggregate' model in which the embryo is regarded as a concentrated suspension of shell-spheres that correspond to the blastomeres (i.e. the cells within the embryo). In the stages from the morula to the blastula, the changes in the dielectric spectra were explained as due to a reduction in the size of the blastomere accompanied by an expansion of the blastocoel (i.e. the central cavity in the embryo) using the 'vesicle-inclusion' model that is a cell aggregate covered with a less conducting shell corresponding to the outermost layer of tightly interconnected cells.

Multifrequency method for dielectric monitoring of cold-preserved organs.

To answer a growing need for non-invasive monitoring of biological organs, we have developed an automated system capable of repeated dielectric measurements over the frequency range 10 kHz-100 MHz. Further, we propose a novel method of data analysis that may convert the acquired, individual dispersion curves into a diagram of the time course of specific phenomenological parameters, such as the characteristic frequency. By using this new procedure, unattended, long-term monitoring of temporal changes in the dielectric behaviour of excised liver lobes stored at 4 degrees C was successfully realized. The 'multifrequency' method presented here was definitely superior to the conventional 'fixed-frequency' method in providing reliable results.

A quantitative approach to the dielectric properties of the skin.

The results of measurements using an open-ended coaxial probe of the audio/radiofrequency dielectric properties of human skin in vivo, either dry or moistened with physiological saline, are reported. Permittivity and conductivity dispersion curves were parametrized by using a newly reported dispersion function (Raicu V 1999 Dielectric properties of biological matter: model combining Debye-type and 'universal' responses Phys. Rev. E 60 4677), and the results obtained are discussed in the light of the recent advances in this field. It is suggested that the coaxial probe reports on the properties of the superficial layer, the stratum corneum, when the skin surface is dry, whilst the signal from deeper skin layers becomes dominant after wetting the skin with conductive physiological saline.

Changes in the volume of marginal cells induced by isotonic 'Cl- depletion/restoration': involvement of the Cl- channel and Na+-K+-Cl- cotransporter.

Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl- conductance and Na+-K+-Cl- cotransport in the mechanism of changes in cell volume upon isotonic Cl- depletion/restoration. Changes in cell volume were estimated from video-microscopic images with the aid of an image processor. Marginal cells shrank to approximately 80% of their original volume in 30 s and to 65-70% in 90 s upon total replacement of [Cl]o (approximately 150 mM) by gluconate-, and the original volume of the shrunken cells was restored within 2 min after restoration of Cl-. The order of potency of anions to induce isotonic shrinkage was gluconate > I- > F- > Br-. The cell shrinkage caused by Cl- depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.5 mM), bumetanide (10 microM) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl]o from approximately 150 mM to 7.5 mM was not affected by [K]o in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the 'Cl removal'-induced shrinkage depends on volume-correlated Cl- conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47-62, 1996) and that this pathway(s) is essentially independent of the Na+-K+-Cl- cotransporter, the Na+,K+-ATPase, and the K+-Cl- cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na+, K+ and Cl- in the bath and its substantial inhibition by bumetanide (10 microM) both indicate a major role for Na+-K+-Cl- cotransport. The strong influence on cell volume of solute fluxes working through the Cl- channel and the Na+-K+-Cl- cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.

Intravascularly applied K(+)-channel blockers suppress differently the positive endocochlear potential maintained by vascular perfusion.

We studied the effects of several K+ channel blockers on the positive endocochlear potential (EP) of guinea pigs undergoing perfusion via the anterior inferior cerebellar artery. The EP level was reversibly suppressed by 50-60% in the presence of Ba2+ (2 mM), quinine (2 mM) or verapamil (1 mM) in the perfusate, but not significantly affected by tetraethylammonium (20 mM) or 4-aminopyridine (5 mM). Although the effective site(s) of these blockers at the cell level has not been located yet, these findings indicate an important role for a K+ conductance in the generation of the EP.

Dielectric monitoring of rouleaux formation in human whole blood: a feasibility study.

In search of a method for detecting rouleaux formation in vitro, we studied the dielectric behavior of human blood under both agitated and stationary conditions. Among the parameters examined, relative permittivity ('dielectric constant') at 50-100 kHz was found to be a suitable measure of rouleaux growth, which has been difficult to quantify through conventional optical approaches. The electrical method presented here appears applicable to the kinetic analysis of rouleaux formation in undiluted whole blood.

Maxi-K+ channel in plasma membrane of basal cells dissociated from the stria vascularis of gerbils.

The plasma membrane of isolated strial basal cells has been probed for conductive pathways by the patch-clamp single-channel recording technique. Maxi-K+ channels were identified in 28 excised patches (i.e., 29%) out of 95, and these active patches each contained an average of 2.4 channel activities. In the cell-attached mode, activity of the maxi-K+ channel was also observed. Properties of the maxi-K+ channel thus revealed include: (1) linear I-V relations with 150 mM K+ on both sides of the membrane, (2) a unit conductance of 246.2 +/- 4.0 pS (n = 14). (3) Ca2- sensitivity, (4) activation by membrane depolarization. (5) a complete block by Ba2- (2 mM) from either side of the membrane. (6) a flickering block by quinine (0.1 mM) or verapamil (0.1 mM) from either side of the membrane, and (7) a complete block by tetraethylammonium (1 mM) from the outside only. The maxi-K+ channel may play a role in the generation of endocochlear potentials.

A novel, volume-correlated Cl- conductance in marginal cells dissociated from the stria vascularis of gerbils.

Using the whole-cell patch-clamp technique, we examined Cl-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl-selective conductance of approximately 18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mM Cl-. Under a quasi-symmetrical Cl- condition, the "instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN- > Br- approximately = Cl- > F- > NO3- approximately = I- > gluconate-, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl- currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl- concentration, anion substitution and altered holding potentials. The Cl- conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl- current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mM) and diphenylamine-2-carboxylic acid (DPC, 1.0 mM), but 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.5 mM) was without effect. The properties of the present whole-cell Cl- current resembled those of the single Cl- channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89-100, 1995), suggesting that the volume-correlated Cl- conductance could be ascribed predominantly to the basolateral membrane. This Cl- conductance may function not only in cell volume regulation but also for the transport of Cl- and the setting of membrane potential in marginal cells under physiological conditions.

Ion channels in basolateral membrane of marginal cells dissociated from gerbil stria vascularis.

The basolateral membrane of isolated strial marginal cells has been probed for conductive pathways by the patch-clamp technique. Two types of voltage-insensitive channels were identified in both cell-attached and excised patches. Of these, frequently (69% of excised patches) observed was a Ca(2+)-activated nonselective cation channel having a unit conductance of 24.9 +/- 0.5 pS (N = 16). Other characteristics of this type in excised patches include: 1) linear I-V relations with 150 mM K+ (pipette)/150 mM Na+ (bath), 2) a permeability sequence of NH4+ > Na+ = K+ = Rb+ > Li+, 3) a flickering block by quinine or quinidine (both 1 mM), and 4) a dose dependent block of its activity by ADP or ATP (IC50,ATP/IC50,ADP = 20-35), both from the cytosolic side. Channels with similar characteristics were found in the apical membrane of the same cell; however, the basolateral channels were 2-4 times more densely distributed than the apical counterparts. Also frequently (57%) detected was a Cl- channel of 80.0 +/- 0.5 pS (N = 6), whose activity was Ca2+ independent. Additionally, this Cl- channel had: 1) linear I-V relations with symmetric Cl-, 2) a permeability sequence of Cl- > Br- > I- > or = NO3- > or = gluconate-, and 3) a complete and reversible block by 1 mM diphenylamine-2-carboxylate. In contrast to the apical Cl- channels, the basolateral ones had a much higher density (57% vs. < 1%) as well as a higher unit conductance (80 pS vs. 50 pS) than the apical counterpart. The relative abundance of these two types as the major conductive pathways for Na+, K+, and Cl- in the basolateral region must be taken into account when addressing the role of strial marginal cells in generating the positive endocochlear potential. The Cl- channel may facilitate Cl- distribution across the basolateral membrane.

Cl- and nonselective cation channels in the basolateral membrane of strial marginal cells in the cochlea.

The mechanisms underlying the marginal cells' ion transport and endocochlear potential (EP) generation are still controversial. Conductive pathways in the basolateral membrane of isolated strial marginal cells of the gerbil have been investigated by the patch-clamp technique. Two types of ion channel, 80 pS Cl- and 25 pS Ca(2+)-activated nonselective cation, were identified frequently. The abundance of these two channels as the conductive pathways for Na+, K+, and Cl- in the basolateral membrane must be taken into account when addressing the role of strial marginal cells in generating EP.

[Dielectric measurements on the rabbit cornea using a surface electrode].

Using a concentric surface electrode, we measured in situ electrical impedance in the 10 kHz-100 MHz range for the anterior portion of eyeballs isolated from white rabbits. Replacement of fluid in the anterior chamber with an equal volume of air led to decreases in conductivity, resulting in a marked alteration of impedance behavior as expressed in a loss tangent plot. Eyeballs either with corneal erosin induced through mechanical abrasion or with endothelial injury caused by a 70% ethanol replacement in the anterior chamber were subjected to impedance measurements. Dielectric analysis based on the two-term Cole-Cole equation revealed that the observed overall dielectric dispersion could be divided into two subdispersions. Of these, the first dispersion, occurring at low frequencies was associated with the endothelial layer and the second, higher-frequency dispersion was due to the epithelial layer of the cornea.

Dielectric behavior of the frog lens in the 100 Hz to 500 MHz range. Simulation with an allocated ellipsoidal-shells model.

In an attempt to correlate the passive electrical properties of the lens tissue with its structure, we measured ac admittances for isolated frog lenses, lens nuclei, and homogenate of cortical fiber cells, over the frequency range 10(2)-5.10(8) Hz. The whole lenses molded into discoid shape show a characteristic "two-step" dielectric dispersion with a huge permittivity increment of the order of 10(5) at 1 kHz. Of the two subdispersions disclosed, dispersion 1 has a permittivity increment (delta epsilon) of 2.10(5) with a characteristic frequency (fc) of 2 kHz, and dispersion 2 has a delta epsilon of 400 with an fc of 2 MHz. In terms of loss tangent, these dispersions are more clearly located as two separate peaks. Data are analyzed using an allocated ellipsoidal-shells model which has been developed by taking into account fiber orientation inside the lens tissue. Dispersion 1 is assigned to the equatorial cortex, where fiber cells run parallel to the applied electric field, and dispersion 2 to the nucleus with a complex fiber arrangement and also to the polar cortex, in which the fiber alignment is predominantly perpendicular. In addition, the model analysis reveals that, in the frog lens, the nucleus occupies approximately 30% in volume and that relative permittivity and conductivity for the cell interior are, respectively, 45 and 3 mS/cm for the cortical cells, and 28 and 0.3 mS/cm for the nuclear cells.

Fluorescent probe studies of sodium/sugar cotransport and membrane potential changes in isolated chicken enterocytes.

The fluoroprobe technique using a potential-sensitive dye, diS-C3(5), was applied to isolated enterocytes in order to correlate membrane potential changes with Na+/sugar cotransport. Cells were prepared from the chicken small intestine by enzymatic (Dispase) treatment combined with mechanical agitation. In the presence of Na+, addition of D-glucose (2 mM) to the suspension of cells doped with the dye (1.8 microM) gave rise to Na+-dependent increases in fluorescence indicative of depolarization of the cell membrane potential. The pH optimum for this response was ca. 7.3. Similar but smaller fluorescence increases were also evoked by D-galactose, alpha-methyl-D-glucoside, and 3-O-methyl-D-glucose, all known to be actively accumulated by enterocytes in a Na+-dependent fashion. These monosaccharide-evoked fluorescence changes were suppressed by lowering the extracellular Na+ concentration or by phlorizin. Monosaccharides with no reported Na+ requirement induced no such fluorescence responses. A disaccharide, maltose, elicited a smaller fluorescence increase which also was Na+ dependent and phlorizin inhibitable; this was interpreted as due to a possible supply of D-glucose by the membrane-bound disaccharidase systems. No change was observed with lactose, sucrose, or trehalose, however. The glucose-evoked fluorescence changes correlatable with depolarization were also confirmed using another carbocyanine, diO-C5(3). These results demonstrate that fluorescence from diS-C3(5) or diO-C5(3) can be a good measure for cotransport-associated membrane potential changes in isolated intestinal epithelial cells.

Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. I. Dielectric behavior of cell suspensions in 0.01-500 MHz and its simulation with a single-shell model.

Frequency dependence of relative permittivity (dielectric constant) and conductivity, or the 'dielectric dispersion', of cultured cells (RBL-1 line) in suspension was measured using a fast impedance analyzer system capable of scanning 92 frequency points over a 10 kHz-500 MHz range within 80 s. Examination of the resulting dispersion curves of an improved reliability revealed that the dispersions consisted of at least two separate components. The low-frequency component (dispersion 1) had a permittivity increment (delta epsilon) of 10(3)-10(4) and a characteristic frequency (fc) at several hundred kHz; for the high-frequency component (dispersion 2), delta epsilon was smaller by a factor of 10(2) and fc = 10-30 MHz. Increments delta epsilon for both components increased with the volume fraction of cell suspension, while fc did not change appreciably as long as the conductivity of suspending medium was fixed. By fitting a model for shelled spheres (the 'single-shell' model) to the data of dispersion 1, the dielectric capacity of the plasma membrane phase (Cm) was estimated to be approx. 1.4 microF/cm2 for the cells in an isotonic medium. However, simulation by this particular shell model failed to reproduce the entire dispersion profile leaving a sizable discrepancy between theory and experiment especially at frequencies above 1 MHz where dispersion 2 took place. This discrepancy could not be filled up even by taking into consideration either the effect of cell size distribution actually determined or that of possible heterogeneity in the intracellular conductivity. The present data strongly indicate the need for a more penetrating model that effectively accounts for the behavior of dispersion 2.

Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. II. Effects of osmotic perturbation.

The effects of osmotic perturbation on the dielectric behavior of cultured rat basophilic leukemia (RBL-1) cells were examined. Cells exposed to osmolalities (pi) of 145-650 mosmolal showed dielectric dispersions of the following characteristics: Permittivity increment delta epsilon(= epsilon l - epsilon h where epsilon l and epsilon h refer to the low- and high-frequency limit values) for a fixed volume concentration increased with pi; gross permittivity behavior was apparently of a typical Cole-Cole type; however, frequency dependence of conductivity was undulant and could be simulated by a superposition of two separate Cole-Cole type dispersions; separation of these subdispersions along the frequency axis was an increasing function of pi, and so was conductivity increment in the high-frequency region. As examined by light microscopy, the cells were spherical in spite of imposed anisotonic stresses and behaved as osmometers at 200-410 mosmolal. When normalized by dividing by number (not volume) concentration, delta epsilon remained relatively constant irrespective of pi. Apparent membrane capacities (Cm), analyzed by applying a single-shell model, increased systematically from a hypotonic value of approx. 1 microF/cm2 up to 5 microF/cm2 at 650 mosmolal. This increase was interpreted as due to increased cellular 'surface/volume' ratios that were confirmed by scanning electron microscopy. Cole-Cole's beta parameter, which culminated around 0.9 for isotonic cells and declined to approx. 0.8 for anisotonic cells, did not parallel the broadening of cell volume distribution but appeared to reflect changes in the intracellular conductivity caused by the anisotonic challenge. The results indicate that the dispersion method can probe changes in surface morphology as well as subcellular organelles' constitution of living cells.

Dielectric analysis of mitochondria isolated from rat liver. I. Swollen mitoplasts as simulated by a single-shell model.

A re-evaluation of the dielectric studies on isolated mitochondria (Pauly, H., Packer, L. and Schwan, H.P. (1960) J. Biophys. Biochem. Cytol. 7, 589-601, and ibid. 7, 603-612) is presented. The suspensions of 'mitoplasts' prepared from rat liver mitochondria by a hyposmotic (10 mM KCl) treatment showed a dielectric dispersion with its characteristic frequency lying in the 1-100 MHz range. In the analysis of data special emphasis was put on the choice of the theoretical models to employ after scrutiny of their applicability to the suspensions tested. As such we adopted the theory of Hanai et al. (Hanai, T., Asami, K., and Koizumi, N. (1979) Bull. Inst. Chem. Res., Kyoto Univ. 57, 297-305) that was advanced to include concentrated suspensions of shelled spheres. Curve fittings based on that theory resulted in a better agreement with experiment than the fittings based on a conventional theory for dilute suspensions. Major findings from our analyses on the swollen mitoplasts are that: (i) the limiting membrane of the mitoplasts has a specific electrical capacity of 1 microF/cm2, (ii) the ratio of permittivity (or dielectric constant) for the mitoplast interior and permittivity for the external medium is 0.6-0.7, and (iii) the conductivity ratio between the interior phase and the medium is approx. 0.6. Reasons for discrepancy between the results of Pauly et al. and ours are discussed.

Dielectric analysis of mitochondria isolated from rat liver. II. Intact mitochondria as simulated by a double-shell model.

The dielectric dispersion of isolated intact mitochondria in suspension has been measured between 10 kHz and 500 MHz. In isotonic KCl media at 4 degrees C, the mitochondria maintained their characteristic 'double membrane' structure as examined by electron microscopy, and the observed dispersion curves were successfully simulated in terms of a superposition of two sub-dispersions having different characteristic frequencies and different permittivity magnitudes. Taking these observations into account we analyzed the dispersion data on the basis of a 'double-shell' model in which two concentric shells are meant to represent the mitochondrial outer and inner membranes. The analyses by a computerized curve-fitting method revealed that: (i) electric capacities for the outer and the inner membrane are 1.7 and 0.5 microF/cm2, respectively, (ii) relative permittivity for the inner compartment (or the equivalent homogeneous matrical space) = 50-60, (iii) outer compartment-to-external conductivity ratio = 0.4-0.6, and (iv) inner compartment-to-external conductivity ratio = 0.14. The implications of these parameter values are discussed with due attention paid to the limitations inherent in our 'double-shell' model approach.

Adrenal chromaffin cells form functional cholinergic synapses in culture.

Adrenomedullary cells and autonomic ganglion cells originate from the neural crest. Both cell types synthesize, store and release catecholamines; however, their structural and functional properties are distinctly different. Aloe and Levi-Montalcini have shown in vivo that when the adrenal medulla is exposed to exogenous nerve growth factor (NGF) most cells differentiate into neuronal cells substantially similar to sympathetic neurones. Experiments in vitro have also shown that neonatal as well as adult adrenal chromaffin cells and their neoplastic correlate (PC12 cells) undergo neurone-like morphologic differentiation in response to NGF. From these morphological and biochemical studies alone, however, it remains uncertain whether the functional neuronal transformation is also accompanied. We report here that the adrenal chromaffin cells in culture can differentiate into neuronal cells having functional synapses which were found to be cholinergic in nature. Furthermore, the type of synaptic vesicles in the newly formed synapses was apparently dependent upon K+ levels in the culture medium.

Contribution of an electrogenic sodium pump to the membrane potential in the intestinal epithelial cell.

The transmural potential difference of isolated rat duodenum and the membrane potential of its epithelial cell were examined during changes of the ionic medium at both warm (31--35 degrees C) and cold temperatures (2--5 degrees C). In the range of low external K+ concentrations, cooling produced an immediate and reversible depolarization of the membrane potential (around -10 mV) greater than that estimated from a simple thermodynamic temperature effect (RT/F). Such a temperature sensitive component of the membrane potential at 31 degrees C was completely abolished by anoxia and serosal ouabain. Readmission of K+ to the bathing fluid of duodenum previously deprived of K+ and loaded by Na+ caused a remarkable transient hyperpolarization of the epithelial membrane, which was abolished by serosal ouabain. Decreases in the value of transmural potential difference were also produced by cooling, anoxia and serosal ouabain, but these were so small that concurrent changes in the membrane potential could be regarded as chiefly reflecting changes in the effective emf of the cell. Thus, an electrogenic Na+-K+ exchange pump located on the serosal membrane of the duodenal epithelial cells was shown to contribute significantly to the membrane potential under normal conditions (up to around -7 mV). The coupling ratio of Na+-efflux and K+-influx associated with this pump was estimated by the Mullins-Noda equation (1963) to be approximately 4:3 under the physiological steady-state conditions.