Electrical properties of hydrated collagen. I. Dielectric properties

The effect of water on the low-frequency (10²-10⁵ H_z) complex permittivitv of native, sold-state collagen has been investigated experimentally. Measurements at ambient temperature show that dry collagen exhibits essentially no frequency or temperature dependence. As water is absorbed, both dielectric constant and loss factor increase simultaneously and rise sharply upward at a hydration level which may be associated with the completion of the primary absorption layer as determined from independent water absorption studies. The behaviour is qualitatively identical to that observed for other proteins and related materials. Temperature-dependent measurements made under vacuum conditions in the range ?196°C to +100°C are characteristic of the dielectric properties of the water in the sample. Dehydration produced by successive temperature recycling to the maximum temperature effectively eliminates any temperature or frequency dependence. A maximum in the temperature-dependent curves is found at about +40°C and is explained as the superposition of two processes: (1) the transition of water molecules from bound to free states, and (2) the difffusion of water molecules out of the system. The dielectric constant of dry collagen, after desorption at ambient temperature, is about 4.5. Desorption at elevated temperatures reduced the room temperature value to about 2.3 and the liquid nitrogen temperature value to a number indistinguishable from the optical value of n² = 2.16.     

Electrical properties of hydrated collagen. II. Semiconductor properties

The dc conductivity of hydrated bovine Achilles' tendon collagen has been determined as a function of hydration over a limited temperature range. At ambient temperature the conductivity changes from 10^?15 (Ω cm)^?1 in the dry state to about 10^?8 (Ω cm)^?1 at ～24% water content by weight. For all temperatures the conductivity increases exponentially with hydration obeying σ(h) = A exp (βh), where h is a measure of the hydration, A is independent of temperature, and the parameter β ～ T^?1. It is shown that the data may be described by an impurity-type mechanism in which the effective activation energy for the process is dependent on temperature and hydration. Conduction is assumed to be electronic with the impurity (water) acting as a donor. In the solid state the effect of water on the conductivity is reversible indicating the absence of chemical alteration of the hydrated collagen.     

Electrical properties of sheep Purkinje strands. Electrical and chemical potentials in the clefts

The impedence of sheep Purkinje strands, measured to 3-5 kHz, is interpreted with circuit models based on morphology. The strand is described as a one-dimensional electrical cable. Clefts between myocytes of the strand allow radial current to flow in parallel with current across the outer membrane. A lumped model of the clefts, in which all the cleft membrane is in series with 100 omega-cm2, fits only below 20 Hz. Two distributed models, pie and disk, fit at all frequencies with somewhat different (31%) luminal resistivities, but with similar membrane parameters. Series resistance representing the endothelial sheath is small. Simulations of voltage clamp experiments include measured linear parameters and nonlinear membrane channels, as well as radial variation of cleft concentration, membrane flux, voltage, and current. Cleft potential is drastically nonuniform when sodium current flows. Cleft potential is reasonably uniform when calcium and potassium currents flow, but the calcium and potassium concentrations change markedly, enough to turn off the calcium current, even if the calcium channel did not inactivate. We conclude that physiological current flows produce significant nonuniformities in electrochemical potentials in the clefts of this cardiac preparation.     

Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties.

We have developed a calcium diffusion model for a spherical neuron which incorporates calcium influx and extrusion through the plasma membrane as well as three calcium buffer systems with different capacities, mobilities, and kinetics. The model allows us to calculate the concentration of any of the species involved at all locations in the cell and can be used to account for experimental data obtained with high-speed Ca imaging techniques. The influence of several factors on the Ca2+ transients is studied. The relationship between peak [Ca2+]i and calcium load is shown to be nonlinear and to depend on buffer characteristics. The time course of the Ca2+ signals is also shown to be dependent on buffer properties. In particular, buffer mobility strongly determines the size and time course of Ca2+ signals in the cell interior. The model predicts that the presence of exogenous buffer, such as fura-2, modifies the Ca2+ transients to a variable extent depending on its proportion relative to the natural, intrinsic buffers. The conclusions about natural calcium buffer properties that can be derived from Ca imaging experiments are discussed.     

Electrical properties ofValonia ventricosa

Summary The cytoplasmic electrical potential and membrane resistance of mature cells ofValonia ventricosa have been measured by inserting a microelectrode concentric with another electrode into the vacuole of the cell. The cytoplasmic region was investigated by advancing the microelectrode into the cell wall from the vacuolar side.The results revealed a unique region where the vacuolar electric potential and membrane resistance changed in a simultaneous single step to values close to zero. The measured potential always remained positive immediately after the step.At no time was a highly negative potential region encountered. Further penetration of the microelectrode revealed a low resistance negative potential region of –12.6±1.1 mV associated with the cell wall. Experiments were also carried out on aplanospores ofV. ventricosa to compare mature and immature cells. The chemical composition of the vacuolar and protoplasmic phases of mature cells was determined. The results agreed with previous results except that the Cl^– ion content of the protoplasm was significantly higher at 381±20 mmoles/liter (H₂O). It was concluded that mature cells ofValonia are significantly different from immature cells in that no highly negative potential cytoplasmic region was found in mature cells.It was considered that the measured step change in electric potential and membrane resistance occurred at the plasmalemma and that the tonoplast was a region of very low resistance. The implications of these findings in terms of models of ion transport intoValonia are discussed.     

Electrical properties of developing rat heart. Effects of dexamethasone

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.     

Dielectric properties of spherical macroion suspensions. I. Study on monodisperse polystyrene latex

Dielectric properties of polystyrene latex suspended in aqueous media are investigated with special attention to the effect of volume fraction of the latex and salt concentration. The experimental data show the existence of two dispersions, one in the low-frequency range from 10(3) to 10(5) Hz. and the other in the high-frequcncy range from 10(5) to 10(7) Hz. In the salt-free system. both dispersions are of the Debye type and their relaxation limes arc insensitive to the volume fraction. Addition of H2SO4 to the suspension enlarges the magnitude of the low-frequency dispersion and reduces that of the high-frequency dispersion. whereas it does not affect the relaxation times. In the mixture of two species of counterions, e.g. H+ and Na(+), the low-frequency dispersion deviates from the Debye type. while the high-frequency dispersion docs not. From these facts, the high- and low-frequency dispersions are thought to be due to the radial and tangential components of the displacement current at the surface of the latex. The latter process is consistent with the Schwarz theory of the dielectric dispersion of colloidal suspensions.     

Electrical properties and structure of the frog arachnoid membrane.

We have used an in vitro preparation of the frog arachnoid membrane to study the role of this membrane in the maintenance of the "blood-cerebrospinal fluid (csf) barrier". Electron microscopy showed that the membrane was made up of 10-15 layers of flat epithelial cells joined together by numerous cell junctions. The electrical resistance of the preparation was about 2000 ohms cm2. The steady-state transmural potential difference (pd) ranged up to 45 mV, csf positive, and this eliminated by either the addition of ouabain to the csf, or by replacing the NaC1 with TEA C1. The pd across the membrane increased when bicarbonate was added to the external bathing solutions. We conclude that this pd is due to the active transport of sodium from the subural fluid to the csf. In some preparations the transmural pd was reversed, i.e., csf negative, and this was also abolished by the addition of ouabain to the csf, or by replacing chloride with isethionate. We conclude that this pd is related to active chloride transport. These, and other experiments, lead us to the conclusion that the arachnois membrane is involved in the production of the cerebrospinal fluid and the maintenance of the blood-cerebrospinal fluid barrier.     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Electrical properties of vertebrate oocyte membranes

The electrical properties of vertebrate oocyte and egg cell membranes are reviewed. Ion channels of these oocytes generate transcellular currents and action potentials as well as responses to neurotransmitters. Electrical properties change during meiotic maturation and fertilization. Available information about the electrical properties of sperm is also discussed.     

An improved syncytia infectivity assay for the bovine leukemia virus.

Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 X 10(6) viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay.     

Electrical properties of an excitable epithelium

The exumbrellar epithelium of the hydromedusa, Euphysa japonica, is composed of a single layer of broad (70 micrometers), thin (1--2 micrometers) cells which are joined by gap junctions and simple appositions. Although the epithelium lacks nerves, it is excitable; electrically stimulating the epithelium initiates a propagated action potential. The average resting potential of the epithelial cells is -46 mV. The action potential, recorded with an intracellular electrode, is an all-or-nothing, positive, overshooting spike. The epithelial cells are electrically coupled. The passive electrical properties of the epithelium were determined from the decrement in membrane hyperpolarization with distance from an intracellular, positive current source. The two-dimensional space constant of the epithelium is 1.3 mm, the internal longitudinal resistivity of the cytoplasm and intercellular junctions is 196 omega cm, and the resistivity of both apical and basal cell membranes is greater than 23 k omega cm2. Although the membrane resistivity is high, the transverse resistivity of the epithelium is quite low (7.5 omega cm2), indicating that the epithelium is leaky with a large, transverse, paracellular shunt.     

Electrical properties of Neurospora crassa. Respiration and the intracellular potential

The internal potential of Neurospora appears to have two components, one (a) which is reduced by anoxia or abolished by respiratory inhibitors such as azide and 2,4-dinitrophenol, and (b) a fraction that remains in the presence of respiratory inhibitors and is sensitive to the external potassium concentration. Under standard conditions 1 mM azide or dinitrophenol diminishes internal potentials from near -200 mv to about -30 mv within 1 minute and at a maximal rate of 20 mv/second. The internal potential usually recovers within 10 minutes after the inhibitor has been removed. The effect of carbon monoxide on the internal potential is similar to that of azide or dinitrophenol, but can be reversed by visible light, specifically of the wavelengths (430 mµ and 590 mµ) known to decompose cytochrome-CO complexes in yeast. Respiration and internal potentials vary proportionally with azide concentration, but dinitrophenol at low (3 x 10^-6 M) concentrations enhances oxygen consumption without affecting the internal potential. In the presence of 0.1 mM calcium, the fraction of the internal potential which persists during respiratory inhibition increases (becomes more negative) about 30 mv for each tenfold decrease of external potassium over the range 10 to 0.1 mM. The surface resistivity of Neurospora, normally about 5000 ohm.cm², is unchanged by respiratory inhibitors during the period of rapid potential shift.     

Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia.

The fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the paramyxovirus simian virus 5 (SV5) were expressed individually or coexpressed in CV-1 cells by using SV40-based vectors and recombinant vaccinia viruses. The extent of detectable fusion in a syncytium formation assay was found to be affected by the expression system used. In addition, when HN was coexpressed with F, it was found that the expression vector system influenced the contribution of HN in forming syncytia. The abilities of the SV5, human parainfluenza virus type 3, and Newcastle disease virus F glycoproteins to cause fusion, when expressed alone or coexpressed with HN, were directly compared by using the SV40-based vector system in CV-1 cells. The F proteins exhibited various degrees of fusion activity independent of HN expression, but the formation of syncytia could be enhanced to different extents by the coexpression of the homotypic HN protein.     

Acidic pH induces fusion of cells infected with baculovirus to form syncytia.

The enveloped baculovirus insect cell system has been used extensively for expression of recombinant proteins, including viral fusion proteins. We tested wild-type baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance changes were observed after incubating infected Spodoptera frugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse of 10 s is needed to trigger syncytia formation. Identical results were obtained with recombinant baculovirus. This new system is convenient for studying pH activated cell-cell fusion. However, using this enveloped virus to study the mechanism of recombinant fusion proteins requires caution.