Rafi Korenstein

Capacitative pulsed electric stimulation of bone cells. Induction of cyclic-AMP changes and DNA synthesis.

Pulsed electric stimulation, coupled capacitively to bone cells isolated from rat embryo calvaria, caused changes in the intracellular level of cyclic AMP and enhanced DNA synthesis. The capacitive method of electrical stimulation was characterized in terms of displacement currents (0.7-4.0 A) and voltages (10-54 V/cm) prevailing in the stimulation chamber. Changes, both in cyclic AMP and in incorporation of [3H]thymidine into DNA, were correlated with the strength of the applied electric field. Unlike the mechanical stimulation of bone cells, the electrical stimulus was not mediated by de novo synthesis of prostaglandins. The findings suggest that cyclic-AMP changes, induced by the capacitive electrical stimulation of bone cells, trigger DNA synthesis.

Kinetic isotope effects in the photochemical cycle of bacteriorhodopsin.

Kinetics were determined for the four transients K590, L540, M410, O660 of the photochemical cycle of bacteriorhodopsin (BR570) both in 1H2O and in 2H2O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25‡–32‡ for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L540, and is present in the subsequent formation and decay of M410 and O660. Thus in the light-driven proton pump of BR570, proton ejection from the Schiff base correlates with decay of L504 and reprotonation occurs with the decay of both M410 and O660 back to BR570.

Bacteriorhodopsin: Biphasic kinetics of phototransients and of light‐induced proton transfer by sub‐bacterial Halobacterium halobium particles and by reconstituted liposomes

It is well established that the bacteriorhodopsin of Halobacterium halobium acts as a proton-pump in intact bacteria [l-3] as well as in vesicles [4,.5] and reconstituted liposomes [6-91. Isolated purple membrane fragments release protons upon illur&nation [lo] . Reversible de-protonation of the Schiff-base linkage between retinal and lysine [ 1 l] occurs on forming the 412 nm-intermediate of the photochemical reaction cycle [ 12171. Proton pumping by bacteriorhodopsin is probably a direct consequence of protonation and deprotonation during the cycle. In this communication it is shown that lightinduced proton release and proton uptake by subbacterial particles or reconstituted proteoliposomes (which have a reversed membrane orientation [ 161) show biphasic kinetics. Furthermore, the decay of the 412 nm-intermediate of bacteriorhodopsin also shows biphasic kinetics. This similarity in the kinetics of the macroscopic and microscopic processes rnr/, however, be coincidental. It is suggested that the biphasic proton movement results from a combination of two processes: a faster process of association or dissociation, and a slower process reflecting transport across the membrane.

Rotation of a single swollen thylakoid vesicle in a rotating electric field. Electrical properties of the photosynthetic membrane and their modification by ionophores, lipophilic ions and pH

Abstract Rotation of single swollen thylakoid vesicles (‘blebs’) was induced by means of a rotating electric field of strength 104 V · cm −1 , inducing a membrane voltage of 72 mV peak. Within the range of medium conductives described (40–300 μ S · cm −1 ), measurement of the field frequency (2–100 kHz) giving maximum rotation rate is equivalent to measuring the electrical time constant of the bleb membrane. Hence the membrane capacity (specific capacitance) was determined, and the value found at pH 8.1 (0.93 ± 0.07 μ F · cm −2 ) is in agreement with values deduced from measurements using other techniques. However, the capacity was also found to decreased with pH: a minimum value of 0.77 ± 0.01 μ F · cm −2 was measured at pH 4.4. The present study was extended to measurements of the effects of the lipid-soluble anion of dipicrylamine on the membrane capacity. At pH 7.2 and dipicrylamine concentration of 1.0 μM, a minimum estimate of the apparent membrane capacity was found to be 2.0 ± 0.2 μ F · cm −2 , with 2.6 ± 0.2 μ F · cm −2 being observed at 5.0 μM concentration. In addition, it was found possible to measure the membrane resistivity (specific resistance) in the presence of either gramicidin (1.0 to 10 nM) or valinomycin (1.0 to 10 μM). In the case of gramicidin, it was possible to derive a maximum estimate of the mean channel conductance, and this agrees very well with the values for individual, single channels that may be deduced from artificial bilayer work. Unless the gramicidin channels in blebs are in fact substantially more conductive than in artificial bilayers, this indicates that a high percentage of the added gramicidin forms channels which are open for most of the time. In the case of valinomycin, a much greater amount had to be added to produce the same reduction of membrane resistivity as seen with a given concentration of gramicidin. However, calculations indicate that the majority of this effect is due to the difference in partioning behaviour of the two ionophores.

Energetics and chronology of phototransients in the light response of the purple membrane of Halobacterium halobium

Arrhenius parameters for formation and decay of phototransients in suspensions of purple membrane fragments in H2O and 2H2O have been determined in the temperature range 0-60 degree C. Kinetic isotope effects are found which show that proton transfer steps are involved in both formation and decay of the two longest-lived transients absorbing at 410 nm and 660 nm, respectively. The results also suggest that these transients do not occupy a single pathway in the spontaneous deexcitation of bacteriorhodopsin within the purple membrane. Purple membrane undergoes a phase transition at 25-30 degrees C in both H2O and 2H2O.

Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific

Pulsed electric stimulation, coupled capacitively to different cell cultures of skeletal origin, caused immediate changes in the cellular levels of cyclic AMP and a later enhanced DNA synthesis. Changes both in cyclic AMP level and DNA synthesis were correlated with the strength of the applied electric field. Cultures of calvaria bone cells which contain mainly two cell types, parathyroid hormone responsive cells (osteoblast-like) and prostaglandin E2 responsive cells (fibroblast-like), respond to both low (13 V/cm) and to high (54 V/cm) electric field strength, with no response at intermediate (24 V/cm) field strength. Rat epiphyseal cartilage responded like bone cells both to low and high field intensities, while rat condylar cartilage responded only to the intermediate field strength. Moreover, subcultures of calvaria bone cells, which lost their osteoblastic phenotype expression during subculturing, were responsive only to low field strength. On the other hand, osteoblast-enriched cultures, derived from calvaria bone grown in low calcium, were responsive only to the high field strength. These findings suggest that the response to various electric field intensities is cell-specific and might be used as an additional parameter to characterize cell types. Our study points to the possibility that when exposing a whole organ to an electrical stimulation it is possible to affect specifically only one cell population out of the many cell types existing in the organ.

Electroselection in the photosynthetic membrane: polarized luminescence induced by an external electric field

Delayed luminescence emission (a general property of photosynthetic systems) is closely related to primary electron flow and energetic events in photosynthesis [ 1,2]. In particular, electrical phenomena at the membrane level have a marked influence on the emission; thus, for example, an artificially induced diffusion electric potential (positive inside) [3] or a change in the dielectric constant of the membrane [2] have been shown to stimulate delayed luminescence. An efficient and kinetically unique method to induce transmembrane potentials in a suspension of membrane-bound vesicles is the application of an external macroscopic electric field [4]. In this way, delayed luminescence can be significantly enhanced (by l-3 orders of magnitude) [5,6], especially in hypotonically extensively swollen particles originating from the chloroplasts (blebs [7]). This phenomenon, termed electrophotoluminescence (EPL) [6] has interesting kinetic features, not yet understood. The role of the electric field is explained [6,8] generally in terms of the primary photosynthetic charge separation in photosystem II, its vectorial nature in the membrane [9] and an additional term in the activation energy for its back reaction introduced by the transmembrane field [8]. The resulting charge recombination produces the chlorophyll excited singlet state, giving rise to luminescence. In order to obtain additional information on the mechanism of EPL production and its relation to membrane topology, one can make use of the directional nature of the external electric field as a triggering agent. Since the electric field induced within the membrane by the external field has a strong angular dependence on the external field direction [6], one

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. II. Electric field-induced electrical breakdown of the photosynthetic membrane and its recovery

Preilluminated suspensions of swollen thylakoid vesicles (‘blebs’) were exposed to uni- and bipolar pairs of identical electric field pulses of variable duration, intensity and spacing. The resulting field-stimulated luminescence (electrophotoluminescence) was used as an intrinsic, voltage-sensitive optical probe to monitor electrical phenomena at the membrane level. The application of a pair of voltage pulses of opposite polarity made it possible to produce electric changes in the membrane by the first pulse and to analyse these effects by a second pulse of opposite polarity. It was found that the relative amplitudes of the two electrophoto-luminescence signals depended on the intensity of the applied electric field and on the time interval (t∗) between the two pulses. When t∗ varied from 0.4 to 12 ms, the second stimulated luminescence signal was at first much smaller than the first one and then increased exponentially until the two signals were equal for t∗ ≥ 3 ms. We analysed these differences between the two field-stimulated luminescence signals as a measure of the electrical breakdown of the membrane, induced during the first pulse. In this way a distinction between irreversible and reversible breakdown could be made with an estimation of the recovery kinetics of the reversible breakdown, which was found to be complete within 3 ms. Irreversible breakdown of the membrane was found to increase with lengthening the exposure time from 0.1 to 1.3 ms especially when applying high electric field of at least 2000 V/cm.

External electric field effects on photosynthetic membrane vesicles. Kinetic characterization of two electrophotoluminescence phases in hypotonically swollen chloroplasts

Abstract Strong externally applied electrical field pulses are known to stimulate delayed luminescence from preilluminated blebs (hypotonically swollen vesicles originating from thylakoid membranes of broken chloroplasts) by up to 3 orders of magnitude. This phenomenon is known as electrophotoluminescence. Previous analysis showed the kinetics of the electrophotoluminescence to be biphasic, displaying a rapid (R) phase which decays towards a slower one (S) (Ellenson, J.L. and Sauer, K. (1976) Photochem. Photobiol. 23, 113–123). We demonstrate that these two components represent different processes. At low pH, a good kinetic separation is obtained between the two phases, which become distinct, with the S phase manifesting also an initial rise period. Under these conditions, it is possible to estimate separately the approximate rise times of the two phases. It is shown that the R and S components have a different dependence on the pH and on the time between the actinic flash and onset of the field. The field dependence is also different, with the S phase requiring a lower threshold field than R. From these observations, it is concluded that the R and S luminescence components are formed by different precursors. The difference in behaviour of the two phases during formation of the bleb indicates that the precursors of the R and S phases belong to different parts of the bleb. We suggest that R precursors are located in the wall of the swollen thylakoid and S precursors in the membrane formations which are attached to this wall.

Dielectric spectroscopy of energy coupling membranes: Chloroplast thylakoids

Abstract (1) The dielectric properties (complex permittivity) of suspensions of chloroplast thylakoids have been determined in the range 10 Hz to 13 MHz. In common with other charged membrane vesicles, thylakoids exhibit two broad dielectric dispersions corresponding to the classical α- and β-dispersions. (2) Heat treatment of thylakoids, to produce blebs of a greater average radius, increases, as expected, the magnitude of the β-dispersion, but not that of the α-dispersion. This result suggests that the α-dispersion is not caused solely by the unrestricted tangential relaxation of the ions of the diffuse double layer. (3) The magnitude of the α-dispersion is strongly pH-dependent, and is negligible at the pI ( ca . pH 4.3–4.4) of thylakoids. The α-dispersion is much less sensitive to pH. Measurements of the magnitude of the a dispersion may therefore be used to obtained the pI of charged membrane vesicles. (4) Both α- and β-dispersions are significantly decreased by treatment of the thylakoids with the cross-linking reagent glutaraldehyde, suggesting that the (lateral) motions (in particular) of charged membrane components contribute to the dielectric properties in this frequency range. The relaxation times observed, however, are not consistent with the view that such motions are restricted by hydrodynamic forces alone. The breadth of the dispersion remains very large, however, suggesting also a variable restriction on the genuinely tangential motions of double ions.