Electrical effects in bone

This paper presents a review of the work carried out on the electromechanical properties of bone over the the past three decades. Research in this field has established the piezoelectric nature of bone and identified collagen as the generating source in dry bone. Some of the characteristics of the strain generated potential (SGP) signal from dry and hydrated bone were found to be unaccountable in terms of a classical piezoelectric theory. Modifications of the theory were suggested and in the case of fully hydrated bone, a new mechanism (streaming potential) has emerged. The paper also reports on recent developments in the field and presents results from microstructural (osteonic) studies and from fluid-filled bone. The review indicates the need for actual in vivo work because most of the reported data were obtained, in the last decade, from in vitro work and were considered valid in vivo. Modelling of the mechanism which produces the SGP has been considered to explain the characteristics of these potentials. A representative model recently developed by the present authors and co-workers is reported. This model relates the generated potential to reorientation of spontaneous dipoles and differentiates between the generated and recorded signal, thus identifying effects from the measuring circuitry. The clinical aspects of electricity of bone in assisting fracture healing and the different techniques employed are mentioned briefly. Emphasis on new techniques of piezoelectric implants and their future development is also reported.     

Light-triggered action potentials in the liverwort Conocephalum conicum

The response to light of a liverwort, Conocephalum conicum L., measured as a change in the resting potential, consists of two stages. The first stage is a slight depolarization dependent on light intensity. This plays the role of a generator potential (GP) which induces the second stage - an action potential of the all-or-none character. Action potentials induced by light and by electrical stimuli have the same properties, i.e. identical time course, propagation velocity, and refractory periods. A summation occurs of sub threshold light stimuli and of light and electrical stimuli. The presence of 5⋅10-⁻⁶ M DCMU cancelled the light response and blocked - by inhibition of the electron transport chain - the mechanism leading to GP generation. However, this effect did not produce any change in the response to electrical stimuli.     

A correlation between mechanical and electrical properties of the synthetic hydrogel chosen as an experimental model of cytoskeleton

The correlation between the electrochemical (Donnan) potential and volume swelling was studied for synthetic polyelectrolyte hydrogels considered as models of cytoskeleton gel-forming biopolymers. Hydrogels involving polyacrylic and polymethacrylic acids with varying network density were synthesized by a radical polymerization in aqueous solution. Electrical charge was introduced into the gel network by partial neutralization of monomer acids with several alkali and alkali earth (hydr)oxides. The electrochemical (Donnan) potential of synthetic gels was determined using conventional microelectrode tools for cell potential determination. It was demonstrated that the negative electrical potential of many anionic gels with various charges and network densities decreased with the decrease of equilibrium swelling, i.e., with the decrease in water content in the gel. It was shown that a drastic phase transition in the gel structure from a swollen to a compressed state induced by K⁺/Ca²⁺ exchange is accompanied by an analogous decrease in the absolute Donnan potential of the gels. A kinetic study demonstrated that the gel volume changed ahead of its electrical potential. This suggests that the volume phase transition in gel is the main cause of the electrical response. A similarity between the swelling/compression transition in synthetic gels and the volume changes in the cytoskeleton in the vicinity of the cell membrane was demonstrated. Based on the universal analogy between the properties of synthetic and natural polymer gels, a possible involvement of swelling of the gel-like cytoskeleton structures in electrical regulation in the cell was postulated.     

Ion-dependence of Z-line and M-line response to calcium in striated muscle fibres in rigor.

The calcium-dependent contraction of vertebrate skeletal muscle is thought to be primarily controlled through the interaction of the thick and thin filaments. Through measurement of the Donnan potential, we have shown that an electrical switching mechanism (sensitive to both anions and cations) is present in both A- and I-bands [1]. Here we show that this mechanism is not confined to the contractile apparatus and report for the first time the presence of M-line potentials. The Z-line responds to Ca2+ ions in a similar manner to the A-band under the same solution conditions (phosphate-chloride and imidazole buffers), even though it has no reported Ca2+ binding sites. Z-line potentials were not observed in tris-acetate buffer. The M-line has a markedly different response to any of the other subsarcomeric regions, however, and can only be detected in the phosphate-chloride buffer. Preliminary observations of the M-line potential in creatine kinase-deficient mouse muscle (phosphate-chloride buffer) reveal significant differences in the calcium-induced transitions between two of the genotypes and demonstrate definitively that it is the M-line potential that is being recorded. From these results, it seems likely that the charge response of the Z-line and M-line is being mediated by titin in an anion-dependent manner. Our evidence comes from several observations. First, the similarity between the response of the Z-line potentials to the A-band potentials, where titin is the only link between these structures and second, the differential observation of M-line and Z-line potentials in a range of buffers containing different anion(s). Both Z-line and M-line potentials were seen in phosphate-chloride buffer, but only the Z-line potentials could be detected in chloride-only (imidazole) buffer and neither was observed in the acetate buffer. The latter observations can be attributed to two sources. The first is the effect of acetate buffer on the conformation of myosin [2]; the second is the absence of binding of the M-line protein, myomesin, to titin in the absence of phosphate ions [3].     

Low frequency changes in skin surface potentials by skin compression: experimental results and theories

Human living skin generates an increase in the skin potential when compressed. This was measured on eight subjects with a matrix of nine Ag/AgCl electrodes. The potential increased with the pressure until it reached a maximum. When the pressure was increased stepwise, the response showed an overshoot at each step. Human cadaver skin did not show these potential increments. Neither did pads of collagen, paper tissue soaked in a KCl solution, nor layers of cultured keratinocytes. Three theories are described that may explain the origin of the measured skin potentials. The first is based on the piezoelectric characteristics of proteins in the skin. The second theory assumes that the skin is a charged membrane which generates a streaming potential when deformed. A third theory is proposed in which deformation of absorbed charged protein layers on structures in the skin change the alignment of Donnan potentials in the surrounding tissue.     

23Na-nuclear magnetic resonance study of ionophore-mediated cation exchange between two populations of liposomes.

A model system to observe and investigate the transfer of Na+ ions between different internal compartments in suspension of liposomes was developed, and the exchange was followed by nuclear magnetic resonance spectroscopy. The experiments were performed under conditions of a Donnan equilibrium. Quantitative analysis of this three-site transmembrane exchange system allowed us to distinguish between direct and indirect exchange between liposomes. It also disclosed a "confining" effect on the exchange between the two populations of liposomes. This confining effect may have been due to an electrostatic field in the presence of a membrane potential. Donnan potentials and ionic compositions at equilibrium for the three-compartment system were calculated numerically. The model system may be used to explore further the effects of membrane potentials, surface potentials, and ionic mobilities on ion transport in biological (model) systems in general.     

The Origin of Bioelectrical Potentials in Plant and Animal Cells

The electrical potential difference across a plant or animalcell membrane can be caused by at least three different mechanisms,acting alone or in concert. First, a Donnan equilibrium canaccount for a sizable membrane potential without the participationof any active transport process. In a Donnan equilibrium themembrane potential is generated by the diffusion of permeatingions down their concentration gradients. The asymmetric distributionof permeating ions is caused by the presence of charged, nondiffusibleions, e.g., proteins inside the cell. The second mechanism isan electrically neutral ion pump, e.g., the coupled sodium-potassiumpump found in many types of cells. An electrically neutral pumpcan generate a large membrane potential if the membrane hasa high passive permeability to one of the actively transportedions, usually potassium. The third mechanism is an electrogenicion pump, which makes a substantial contribution to the membranepotential in several types of plant and animal cells. An electrogenicpump directly causes a net movement of charge across the cellmembrane. The membrane voltage generated by the pump then causesa passive flow of diffusible ions which partially short circuitsthe potential difference generated by the pump.     

Theoretical evaluation of a possible nature of the outer membrane potential of mitochondria

A possibility of generation of the outer membrane potential in mitochondria has been suggested earlier in the literature, but the potential has not been directly measured yet. Even its nature, metabolic impact and a possible range of magnitudes are not clear, and require further theoretical and experimental analysis. Here, using simple mathematical model, we evaluated a possible contribution of the Donnan and metabolically derived potentials to the outer membrane potential, concluding that the superposition of both is most probable; exclusively Donnan origin of the potential is doubtful because unrealistically high concentrations of charged macromolecules are needed for maintaining its relatively high levels. Regardless of the mechanism(s) of generation, the maximal possible potential seems to be less than 30 mV because significant osmotic gradients, created at higher values, increase the probability of the outer membrane rupture. New experimental approaches for direct or indirect determination of true value of the outer membrane potential are suggested here to avoid a possible interference of the surface electrical potential of the inner membrane, which may change as a result of the extrusion of matrix protons under energization of mitochondria.     

Synaptic repression at crayfish neuromuscular junctions. II. Evidence for calcium involvement

The inability of synaptic junctions to generate normalsized postsynaptic potentials under normal physiological conditions was studied at crayfish neuromuscular synapses. Synaptic repression in the superficial flexor muscle system of the crayfish was induced by surgery: the nerve was cut in the middle of the target field, and the lateral muscle fibers were removed. After this surgery, the remaining medial synapses were unable to generate normal-sized junction potentials (jp) over the medial muscle population. In an attempt to study the mechanism underlying this response, we varied the extracellular calcium concentration of the Ringers solution bathing the preparation, in both repressed and control animals, while monitoring the size of the same junction potential. The junction potential generated by the spontaneous activity of the nerve increased in size with increasing calcium concentrations in control animals, but failed to do so in repressed animals, that is, changes in external calcium concentrations did not affect repressed synapses. However, in the presence of the calcium ionophore A23187, control and repressed synapses both show an increase in the junction potential sizes they generate. Our data suggest that calcium is involved in the mechanisms that underlie synaptic repression in this crustacean neuromuscular system. © 1993 John Wiley & Sons, Inc.     

Induction of action potentials in fish red muscle by denervation

The effects of denervation on the electrical membrane properties of fish red muscle were investigated. Forty to fifty hours after denervation, miniature endplate potentials disappeared abruptly and field stimulation of the nerve within the muscle failed to evoke endplate potentials, indicating that transmission failure occurred at this time. The membrane resistance of the red muscle fibre increased after denervation. Normally innervated fish red muscles do not generate action potentials in response to either nerve or direct muscle stimulation. However, approximately 3 weeks after nerve sectioning, action potentials could be induced in the muscles. The action potential was sodium-dependent, and was sensitive to tetrodotoxin. Actinomycin D injected in the early phase after operation suppressed the induction of the action potential. These results indicate that RNA synthesis is preliminary to the induction of the action potential mechanism, and that this mechanism is under neural control.     

Transient and cyclic responses of strain-generated potential in rabbit patellar tendon are frequency and pH dependent

The goal of this study was to expand understanding of strain-generated potential (SGP) in ligamentous or tendinous tissues. Most SGP studies in the past have focused on cartilage or bone. Herein, rabbit patellar tendon (PT) was used as a model. Each patellar tendon had two Ag/AgCl electrodes inserted at axial positions of 1/4 and 1/2 from patellar to tibial insertions. Each specimen was electrically isolated, gripped in a servohydraulic test system, and then subjected to a short session of uniaxial haversine tension (2.5 percent maximum strain) at a frequency of 0.5, 1.0, 2.0, or 5.0 Hz. A cyclic (sinusoidal) electrical potential superimposed upon a larger transient (exponentially asymptotic) potential was consistently observed. Upon termination of loading, the cyclic SGP ended, and the shifted baseline of the SGP exponentially decayed and asymptotically returned to a residual potential which over all specimens was not different than the original potential. The transient and cyclic SGPs were frequency dependent (P < 0.001, P = 0.06, respectively). To our knowledge, this transient portion of the SGP, although theoretically predicted by Suh (1996, Biorheology, 33, pp. 289-304) and Chen (1996, Ph.D. thesis, University of Wisconsin-Madison) has not been observed in other experiments using different protocols. Additional PTs were dehydrated and the rehydrated in solution at different pH levels. The magnitude of SGPs increased in basic solution (pH 9.5) but diminished in pH 4.7 buffer. This pH dependency suggests that electrokinetics is the dominant mechanism for the transient and cyclic responses of the SGPs, although this study does not provide direct evidence.     

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.     

Donnan potential measurements in extended hexagonal polyelectrolyte gels such as muscle.

In this paper we reconsider the theoretical and practical aspects of using KCl-filled microelectrodes in extended polyelectrolyte gels such as muscle to measure Donnan potentials, and then calculate protein fixed-charge concentrations. An analytical calculation of the electrical potential function between muscle filaments shows that whether the microelectrode averages the ionic concentration or the local potentials the results are indistinguishable in the practical regime. After consideration of this and other possible sources of error, we conclude that the charge-concentrations measurements that have appeared in the literature are legitimate.     

Influence of propagating electrical signals on delayed luminescence in pelargonium leaves: Experimental analysis

Local damaging stimuli delivered to the Pelargonium leafstalk induce propagating electrical signals (variation potentials) that alter the parameters of delayed luminescence in the leaf blade. The response includes two phases with apparently different mechanisms.     

Note on the Donnan equilibrium

The solution for the spatial distribution of ions in a Donnan equilibrium has been given by J. H. Bartlett and R. A. Kromhout (1952). The present note gives an explicit solution for the case in which the length of the region containing the membrane is large; in biological situations this requires only that the length considered should be greater than a few hundred Ångstrom units. The Donnan equilibrium may be considered to be a special case of a situation in which forces other than electrical act upon the ions; in particular, it represents the case in which only one ion is acted upon and the energy difference on the two sides of the membrane is infinite. An expression is given for the difference in energy of theith in terms of the electrical potential and of the ion concentrations. As an illustration, the results are applied to nerve membrane potentials.     

Immunopotentiating effects of the adjuvants SGP and Quil A. I. Antibody responses to T-dependent and T-independent antigens

The present study was undertaken to compare the effects of two adjuvants, SGP (a starch-acrylamide polymer) and Quil A (purified saponin), with that of aluminum hydroxide (Al(OH)3) on murine primary antibody responses to T-independent (TI) and T-dependent (TD) antigens. All three adjuvants augmented the responses to the TD antigens, dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), and sheep erythrocytes (SRBC). SGP was the most potent adjuvant and increased the primary IgG response to DNP-KLH as much as 90-fold. Quil A and Al(OH)3 had comparable effects on the primary response to DNP-KLH, but Quil A was less effective than Al(OH)3 for augmenting the primary response to SRBC. Quil A and SGP both augmented the primary IgM and IgG responses to trinitrophenyl-lipopolysaccharide (TNP-LPS), TNP-Brucella (TI-1 antigens), and TNP-Ficoll (TI-2 antigens). Al(OH)3, like most commonly used adjuvants, had little or no effect on responses to TI antigens. The kinetics of the response to TNP-Ficoll was altered by SGP, since peak responses were maintained for at least 7 days, while the response to TNP-Ficoll alone peaked on Day 4 and had declined considerably by Day 7. Both SGP and Quil A could augment responses to both optimal and suboptimal doses of antigen. The adjuvant activity of SGP was diminished, but still effective, when smaller amounts of SGP were used with the immunizing antigen, and all three adjuvants were able to augment primary responses when given in separate injections from the antigen. These results demonstrate that SGP is a very effective adjuvant, and show that both Quil A and SGP have a unique ability to increase antibody responses to TI antigens, suggesting that their effects may be mediated at least partially through B cells.     

Plants as Environmental Biosensors

Plants are continuously exposed to a wide variety of perturbations including variation of temperature and/or light, mechanical forces, gravity, air and soil pollution, drought, deficiency or surplus of nutrients, attacks by insects and pathogens, etc., and hence, it is essential for all plants to have survival sensory mechanisms against such perturbations. Consequently, plants generate various types of intracellular and intercellular electrical signals mostly in the form of action and variation potentials in response to these environmental changes. However, over a long period, only certain plants with rapid and highly noticeable responses for environmental stresses have received much attention from plant scientists. Of particular interest to our recent studies on ultra fast action potential measurements in green plants, we discuss in this review the evidence supporting the foundation for utilizing green plants as fast biosensors for molecular recognition of the direction of light, monitoring the environment, and detecting the insect attacks as well as the effects of pesticides, defoliants, uncouplers, and heavy metal pollutants.Key Words: plant signaling, plant electrophysiology, action potential, biosensor     

Biological and artificial ion exchangers: Electrical measurements with glass microelectrodes

Summary Biological (stratum corneum) and artificial (cation-exchange resin beads, Bio-Rad AG 50W-X2) ion exchangers were impaled by glass microelectrodes filled with KCl solution. The electrical potential difference recorded in these structures in reference to the external bathing medium was shown to be dependent on the KCl concentration of both the external and the microelectrode filling solutions. The potentials were interpreted on the grounds of the fixed charge theory of membrane potentials as a consequence of two phase boundary potentials (Donnan potentials), one at the matrix-external solution interface and the other at the matrix-microelectrode solution interface. The contribution of a diffusion component for the recorded potential was considered.     

Glutamatergic elements in an excitability and circumnutation mechanism

In plants, an electrical potential and circumnutation disturbances are a part of a response to environmental and internal stimuli. Precise relations between electrical potential changes and circumnutation mechanisms are unclear. We have found recently that glutamate (Glu) injection into Helianthus annuus stem induced a series of action potentials (APs) and a transient decrease in circumnutation activity. A theoretical explanation for this finding is discussed here taking into considerations data about the ion mechanism of AP and circumnutation as well as about the metabolic and signaling pathways of glutamate and their possible interactions.Key words: action potential, circumnutation, elongation, glutamate, Helianthus annuus, plant movement