Direct electric field effects and sequential processes in biosystems

455

Bioelectrochemistry and Bioenergetics, 25 (1991) 455-442 A section of J. Electroannl. Chem., and constituting Vol. 320 (1991) Elsevier Sequoia S.A., Lausanne

Short communication

Direct electric field effects and sequential processes in biosystems * Dean Astumian Chemical Process Metrology Division, National Institute of Stana&& Gaithersburg. MD (USA)

and Technology,

Hermann Berg Central Institute of Microbiology and Experimental

Therapy, Academy of Sciences, Jena (Germany)

(Received 20 February 1990; in revised form 22 August 1990)

INTRODUCTION

Especially since the 5th Symposium of the Bioelectrochemical Society held at Weimar in 1979, a lot of new electric field effects on membrane bilayers, cells, tissues and animals have been found by several methods, e.g. inductive or capacitative coupling applications of dc or ac field pulses. For these effects and methods several synonyms and definitions are used in the current literature [l-3]: electroporation pore formation in lipid bilayers electrolesion formation of lesions in a cell wall permeabilization electropercolation uptake of substances from solution into a cell electroincorporation electrotransconformation changes of conformation of membrane proteins release of cell ingredients after electroporation or elecelectrorelease trolesion electroadsorption affinity changes of polyelectrolytes etc. to the cell envelope by modulation of the surface potential electroinsertion fixation of biologically effective chain polymers in pores of membranes in such a way that active groups can react outside of the membrane electrotransfection incorporation of genetic material (DNA, RNA, phages, etc.) into a cell

* Presented at the International Symposium: “ Electromagnetic Field Effects on Molecules and Biological Cells - Biotechnological Applications”, l-7 April 1990, at the University of Bielefeld, Germany. 0302-4598/91/$03.50

0 1991 - Elsevier Sequoia S.A.

456

insertion of new genes into the genome of a cell after electrotransfection fusion of contacted cells (without cell walls) electrofusion vesicle conformation during an electrofusion process electrovesiculation enhancement of channels and changes of genetic and electrostimulation metabolic processes by pulsating fields (electroinduction) cellular spinning by rotating fields electrorotation Moreover, other terms are used today, e.g.: electroinjection instead of electrotransfection; electroporative gene transfer instead of electrotransformation, reversible breakdown for electroporation etc. From this list, it can be seen that either high voltage single or weak alternating electric pulses can influence the behaviour of cells in many ways and consequently many biophysical mechanisms have been proposed [3-51. Our aim in this short communication is to distinguish between the usual nomenclature of the methods mentioned on the one hand and relations between their main subsequent partial processes on the other, based on the general principle of electroconformational coupling (ECC), i.e. the influence of the transmembrane voltage A+,,, on the conformational state of membrane structures and of bound proteins, especially enzymes. electrotransformation

RESULTS

AND

DISCUSSION

The transmembrane voltage ranges between - 5 and - 60 mV for mainly mitotic cells (tumour cells, fibroblasts), and for “non-proliferating” cells (muscle and glia cells, neurons) up to -100 mV. Variations of A&, caused by changes outside or inside the cell (local alterations of pH, ionic strength; adsorption on membranes, lipid and protein translocations; cell-cell interactions etc.) influence cellular functions [4]: membrane transport ions, metabolites, drugs; membrane structure lipid composition, endocytosis-exocytosis, aggregation, fusion; mitosis, growth, shape, fertilisation, movement, etc. cell behaviour These correlations are due to the electrolyte, polyelectrolyte and dipolar structure of the cell’s components. A&, = 100 mV translates into a high field strength of 2 X lo5 V/cm, because the thickness of the phospholipid bilayer is between 5 and 10 nm. An electric field pulse of 10 X lo3 V/cm is suitable for increasing A+,, to 1000 mV, leading to electroporation of membranes or electrolesion of cell walls and - of course - to disturbances of functions (mentioned above) and to additional effects of high significance (Scheme 1). Moreover, pulsating weak fields in the mV range are also applied, leading to some other changes in cellular behaviour, too (Scheme 1 and Table 1). In Scheme 1, which depicts many consecutive reactions, three time regions can be distinguished:

457

Region h/2

ELECTROPOLARIZATION

I

G w

Disturbances

Enhancement

of charge distributions

of TRANSMEMBRANE

and dipoles

VOLTAGE

(A&,,)

-----------f------------------------------------------------------------------------------------------.-----Region II h/2:

ELECTROCONFORMATIONAL

PS

COUPLING

I ELECTROPERMEABILIZATION

ELECTROADSORPTION

Cell walls

Cell membranes

[Proteins, Effecters, (second messengers)]

[Glycoproteins]

Production

‘t/s ’ ns

[Metabolites]

I

I

I ELECTROSTIMULATION

Region III

I

[Lipids(domains)]

ELECTROLESION

ELECTROPORATION

Activation

Fusion

Incorporation

(Replication,

(Vesicu-

(Transfection,

e

Release

-+

Leakage

j

Gene Expression Cell death Cell proliferation Scheme 1. Main targets and processes (included in some processes are: membrane transitions, channels, hybridization, etc.). The regions of the half-life times (t,,2) are estimated.

widening

of protein

458 TABLE

1

Electrostimulation

of biological

processes

via effecters

Syntheses: DNA synthesis (replication) in rat embryonic bone cells determined by 3H-thyrnidine incorporation [24] m-RNA synthesis (transcription) in X-chromosomes of insects, determined by ‘H-uridine incorporation

P51 protein synthesis (translation) in rat skin cells, determined proteins similar to TNF and IFN in leukocytes (401 ATP synthesis in E. cob [27] Enzyme activation Omithindecarboxylase in brain cells: increase of production Tyrosinase in melanoma cells: increase of melanine [29]

by incorporation

of polyamine

of labeled

amino

acids [26],

[28]

Membrane transport Human erythrocyte: increase of release of Na+ [30] Chicken bone cells: increase of Ca*+ uptake [31] Proliferation Chicken ganglia: enhanced neurite outgrowth Human lymphocytes: enhanced division [33]

[32]

Morphological changes Amphibia: limb regeneration [34] Erythrocytes, amphibia: changes of shape [35]

(I) A region of very fast electromagnetic field effects on ions, predominantly at cell surfaces, causing polarization and changes of the double layer capacitor with concomitant changes in A&,,. (II) A region of fast sequential electric effects at the envelope and inside cells with targets such as proteins, effecters and lipid domains and glycoproteins. (III) A region of slow afterfield effects on morphology as well as on metabolism, gene expression, and proliferation. The processes in region I are well known in the electrochemistry of polarized electrodes and there is a strong analogy to membranes. The phenomena of region II exhibit new origins and for their mechanisms first models were presented recently [3]. One of them takes into account changes in membrane voltage and protein behaviour, which will be discussed briefly. Basic principles of electroconformational

coupling (ECC [SIOJ)

The basic working hypothesis is that the most likely site for an interaction between an applied field and metabolism is at the cell membrane. We can focus on two possible targets for this interaction, namely lipid structures, such as domains with pores, and membrane proteins. The electroconformational coupling model considers the effect of an electric field on the equilibrium between various states, either of the protein or of the lipid.

459

Imagine a simple transition A *KB

(1)

where A and B could represent, e.g., a conducting and a non-conducting pore, or two conformations of a membrane protein, etc.; then K is the thermodynamic equilibrium constant between them. The fundamental relative governing the field dependence of such an equilibrium is the Van ‘t Hoff equation (a In K/aE),,==

AM/RT

(2)

where E is the effective field acting on the reaction and AM is the difference in molar polarization between A and B. The probability for an ECC mechanism is higher under the condition: AMAE>

RT(AK/K),,=

(3)

according to the integrated eqn. (2). AM of a protein changes if its motion is restricted in the membrane and E is related to the transmembrane voltage A+,,, by the Maxwell relation A$,,=lSrEcos8

.

(4)

where r is the radius of the dielectric sphere. 0 is the angle formed by a line normal to the membrane surface with electric. That means that at the two loci facing the electrodes A+,, is roughly 1.5r times the applied field E. This may well explain those results in which very small input electric fields are able to produce very significant effects in many biological systems. So a stimulus of E = 20 V/cm could induce a change of the surface potential A$ of about 12 mV and the effect of this on the activity of e.g. (Na,K)-ATPase is also frequency and wave-form dependent

[ill.

Besides the well-known electroporation (according directions in region II are the following (Scheme 1).

to E. Neumann [3]) further

Electrolesion Treatment of microbiological and plant cells with high-energy pulses causes destruction and opening of envelopes (cell wall and protoplast membrane) leading either to cell death [12] and release of ingredients [13] or repair [14]. The time course of repair can be gauged from the penetration of dye or an antibiotic substance through the lesions at different times after the pulse. This process needs more time - several minutes - than the closing of membrane pores after electroporation at 25°C. Electroinsertion For medical purposes: fixation of antibodies (receptors) in membranes of blood cells attracting viruses (e.g. HIV [15]).

460

Electrostimulation Predominantly weak periodic fields - inducing currents in the mV range [16] are suitable for influencing metabolic processes, gene expression [17,18], enzyme activity [16,19] and membrane transport [20,21] mostly via effecters (second messengers as Ca’+, c-AMP, ATP, inositoltriphosphate, etc.) and consequently proliferation [22,23], too. Some important examples are listed in Table 1. It can be seen that basic processes of living beings may be altered, if the so-called “electric window” for the response of each cell type or organism is detected. One can conclude from these results that in future all biosciences will use this new tool, last but not least for biotechnological production, too. Region III of Scheme 1 shows essential afterfield effects. They take place according to the same or slightly modified mechanism as are known in cell biology and genetics. Therefore the important processes of this region III will be mentioned only briefly. Cell fusion [3,36] A slow process after electroporation of animal cells or microbiological and plant protoplasts leading to a new spherical “daughter cell” with mixed protoplasm (plasmogamy). Later on in some cases fusion of nuclei (karyogamy) may be possible, leading to a more or less stable new organism. Transformation [3,36] Slow processes after electroporation and transfection leading to the insertion of genetic material (DNA, plasmid DNA, etc.) into the genome of the cell, establishing a new stable cell line. Vesiculation [3] Formation of little “liposomes” from an excess of membrane material during cell fusion in the melting region in between the parent cells. Hybridization [3,36] In the broadest sense, any cross-mating of two genetically different individuals. Especially, the hybridization between spleen cell and tumour cell called a hybridoma is used for the production of monoclonal antibodies. Gene expression The phenotypic manifestation of genes by the process of gene activation of nuclear and extra-nuclear genomes: besides replication of DNA, the transcription into complementary RNA sequences e.g. messenger RNA; subsequent translation into polypeptide chains by the messenger RNA; further metabolic pathways. Leakage, release Outflow of cell ingredients.

461 DISCUSSION

Nowadays, electric field effects are of increasing interest not only for genetics, biotechnology, and medicine but also for ecology, because industrialization needs high-power electric plants and devices generating field strengths of more than 100 V/cm in their environment. The basic research of the past ten years proves its immense consequences for modem biology, of which only a few examples are shown in Table 1. Nevertheless it is evident that the most important syntheses in living beings, namely of ATP, DNA, RNA, and protein, can be influenced - mainly activated either by weak inductive or galvanic stimulations or by strong single pulses [37,39]. Transformation of electrical energy into chemical energy, (e.g. by conformational changes (ECC) influencing activation energies of reactions, e.g.: disturbance of the transcription process, causing changes in the sequence of m-RNA (mutations of the code); stopping syntheses of high molar mass proteins [18]; changes in ion movement [38]. Further possibilities of electric field effects are under study [ll], especially using weak fields. In this vein, a parametric resonance model was presented by V. Lednev [41] at this Bielefeld Symposium, which was verified by the magnetic field induced inhibition of the Ca*+-calmodulin dependent phosphorylation of myosin. Nowadays such weak alternating fields are of considerable interest in the biosciences. REFERENCES 1 H. Berg, Stud. Biophys., 119 (1987) 17. 2 E. Neumann, Ferroelectrics, 86 (1988) 325, 3 E. Neumann, A. Sowers and C. Jordan (Eds.) Electroporation and Electrofusion in Cell Biology, Plenum Press, New York, 1989. 4 R. Glaser in R. Glaser and D. Gingel (Eds.), Biophysics of the Cell Surface, Springer Verlag, Berlin, 1990, p. 175. 5 T. Tsong and D. Astumian, Bioelectrochem. Bioenerg., 15 (1986) 457. 6 T. Tsong and D. Astumian, Prog. Biophys. Mol. Biol., 50 (1987) 1. 7 D. Astumian, P. Chock, T. Tsong and H. Westerhoff, Phys. Rev. A, 39 (1989) 6416. 8 D. Astumian and B. Robertson, J. Chem. Phys., 19 (1989) 489. 9 B. Robertson and D. Astumian, Biophys. J., in press. 10 J. Weaver and D. Astumian, Science, 247 (1990) 459. 11 T. Tsong, Bioelectrochem. Bioenerg., 24 (1990) 271. 12 A. Sale and W. Hamilton, B&him. Biophys. Acta, 148 (1967) 781. 13 E. Bauer, I. Hones, H.-E. Jacob and H. Berg, Stud. Biophys., 130 (1989) 189. 14 H.-E. Jacob, W. Fijrster and H. Berg, Z. Allg. Mikrobiol., 21 (1980) 225. 15 Y. Mouneimne, P. Tosi, Y. Gazittand and C. Nicolau, B&hem. Biophys. Res. Commun., 159 (1989) 34. 16 H. Berg, Stud. Biopbys., 130 (1989) 219. 17 A. Pilla, P. Sechaud and B. McLeod, J. Biol. Phys., 11 (1983) 51. 18 M. Blank and R. Goodman, Bioelectrochem. Bioenerg., 19 (1988) 569. 19 E. Serpesu and T. Tsong, J. Biol. Chem., 259 (1984) 7155. 20 J. Bond, Bioelectrochem. Bioenerg., 12 (1984) 177.

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