Ionic Nonequilibrium Phenomena in Tissue ... - ACS Publications

16 Ionic Nonequilibrium Phenomena in Tissue Interactions with Electromagnetic Fields

Downloaded by UNIV OF MISSOURI COLUMBIA on August 5, 2013 | http://pubs.acs.org Publication Date: August 4, 1981 | doi: 10.1021/bk-1981-0157.ch016

W. R. ADEY

Jerry L. Pettis Memorial Veterans Hospital, Loma Linda, CA 92357 and Departments of Physiology and Surgery, Loma Linda University School of Medicine, Loma Linda, CA 92350 With few exceptions, plant and animal cells maintain internal concentrations of cations that differ sharply from levels in the surrounding fluid. Typically, this partitioning involves the selective intracellular accumulation of K + and the extrusion of Na + . The lipid bilayer or plasma membrane forms the essential skeleton of the cell against free ionic movement in either direction. Transmembrane movement of ions is said to occur through pores or channels which are highly hydrophobic and behave selectively towards different ionic species. Removal of Na+ from the interior of the living cell involves active transport by "pumps" that require metabolic energy. To a first approximation, these ionic relationships in the "resting" condition of the cell have been effectively modeled on the basis of ionic equilibrium states, as expressed in the Goldman and Nernst equations (1). These equations offer an adequate basis for the development of the negative membrane potential of 70 to 90 mV. Excitation as a process characterizing nerve and muscle cells is associated with a transient reduction or abolition of this membrane potent i a l , and in some cases with a temporary "overshoot" or reversal of its polarity. Just as for the membrane potential, these major but transient perturbations in the production of action potentials have been adequately modeled in dynamics of ionic equilibria by Hodgkin and Huxley (2). The Hodgkin-Huxley model elegantly describes major ionic phenomena in excitation of the squid giant axon. Its subsequent application to motor nerve cells of the mammalian spinal cord (3) was based on some of the first microelectrode records from within nerve cells, which disclosed with striking clarity the hyperpolarizing and depolarizing shifts in membrane potential associated with activation of inhibitory and excitatory synaptic nerve fiber endings on the surface of the cell. Interest and excitement occasioned by these findings were universal, but scarcely justified the giant intellectual leap in the inference that the neurobiologist held within his grasp "the neurophysiolgical basis of mind." In the interim, it has become clear that we should This chapter not subject to U.S. copyright. Published 1981 American Chemical Society

In Biological Effects of Nonionizing Radiation; Illinger, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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approach an understanding of s t r u c t u r a l and f u n c t i o n a l s u b s t r a t e s of b r a i n as the organ of mind i n more s u b t l e ways, p l a c i n g renewed emphasis on neglected or discarded aspects of i t s t i s s u e o r g a n i z a t i o n . Even so, we may remain b a f f l e d and confused by apparent c a u s a l i t y that we would a t t r i b u t e to b r a i n maps and models of p a r t i c u l a r regions or systems, s i n c e f o r example, any one b r a i n region may be depicted i n terms of molecular b i o l o g y of i t s synaptic t r a n s m i t t e r substances, or of the immunological i d e n t i t y of i t s membrane surface g l y c o p r o t e i n s , or of the extent of dend r i t i c branching of i t s neurons, or of the " l o c a l c i r c u i t r y " of i t s i n t r i n s i c neuronal networks, or by the major a f f e r e n t and e f f e r e n t paths connecting i t w i t h other c e r e b r a l n u c l e i . We have come to recognize the importance of h i e r a r c h i c a l o r g a n i z a t i o n i n these superimposable schemata that d e p i c t a p a r t i c u l a r b r a i n region. Nevertheless, i t i s not c l e a r that any of these models has considered the p o s s i b i l i t y that one c e l l may i n f l u e n c e another i n b r a i n t i s s u e through modulation of t h e i r shared e l e c t r o c h e m i c a l environment; whether t h i s be by a l t e r e d c a t i o n i c concentrations i n the e x t r a c e l l u l a r medium, or by changing the b i n d i n g of o p i o i d s and other peptides at receptor s i t e s on c e l l surface glycopro t e i n s , or by p o s s i b l e s u s c e p t i b i l i t y of an i n d i v i d u a l c e r e b r a l neuron to the weak o s c i l l a t i n g e l e c t r o c h e m i c a l f i e l d that s u r rounds i t as the electroencephalogram (EEG). This o s c i l l a t i n g p e r i c e l l u l a r f i e l d appears t o a r i s e p r i n c i p a l l y by "leakage" from the multitude of f i n e d e n d r i t i c branches that spread from the small c e l l body of t y p i c a l c e r e b r a l neurons. Though weaker by about s i x orders of magnitude than the e l e c t r i c gradient of 10 V/cm across the l i p i d b i l a y e r of the c e l l membrane, evidence from a wealth of experiments that have mimicked these o s c i l l a t i o n s i n b r a i n , bone and p a n c r e a t i c t i s s u e u n e q u i v o c a l l y confirm t h e i r c a p a c i t y to modify i n t e r r e l a t e d c e l l surface receptor mechanisms that i n c l u d e i o n b i n d i n g c h a r a c t e r i s t i c s , hormonal b i n d i n g a b i l i t y and immunological c a p a c i t i e s . This growing body of evidence from q u i t e d i s p a r a t e t i s s u e s suggests that c e l l - t o - c e l l communication through weak e l e c t r o c h e m i c a l p e r t u r b a t i o n s i n the p e r i c e l l u l a r environment may be a q u i t e general b i o l o g i c a l property. The e f f e c t i v e n e s s of such weak s t i m u l i i n t r i g g e r i n g a cascade of i n t r a c e l l u l a r processes a t f a r higher energy l e v e l s i m p l i e s a s e r i e s of a m p l i f i c a t i o n mechanisms before or during the t r a n s membrane coupling of the i n i t i a l s t i m u l u s . At l e a s t a p a r t i a l answer to t h i s a m p l i f i c a t i o n and extension of the weak t r i g g e r i n g event at the membrane surface may l i e i n cooperative processes w i t h i n and around those g l y c o p r o t e i n molecules that form a s p e c i f i c receptor s i t e . I n seeking an understanding of aspects of these elemental processes of i n t e r c e l l u l a r communication, there i s a modest prospect that they may u l t i m a t e l y be equated w i t h the processes by which storage of experience and i t s r e c a l l occur i n b r a i n t i s s u e . 5



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Tissue Interactions with EM Fields

Imposition of weak electromagnetic f i e l d s o s c i l l a t i n g or modulated a t low frequencies i s a v a l u a b l e t o o l i n s t u d i e s o f t r a n s d u c t i v e c o u p l i n g of p e r i c e l l u l a r e l e c t r o c h e m i c a l s t i m u l i to c e l l u l a r mechanisms. Observed s e n s i t i v i t i e s i n v o l v e responses to s t i m u l i f a r weaker than those i n i t i a t i n g massive i o n i c p e r t u r b a t i o n s c h a r a c t e r i z i n g e x c i t a t i o n according to the Hodgkin-Huxley model. Some of these responses a l s o show s h a r p l y lowered thresholds i n c e r t a i n narrow ranges of frequency and amplitude. J o i n t l y , these data i n d i c a t e that these s e n s i t i v i t i e s are based on n o n e q u i l i b r i u m r e a c t i o n s . F u r t h e r s u p p o r t i n g evidence comes from s t u d i e s of membrane phase t r a n s i t i o n s that show sharp thermal dependence. Nonlinear

T r a n s i t i o n s i n C e l l Membrane S t a t e s

As discussed below, new knowledge about c e l l membrane u l t r a s t r u c t u r e emphasizes the importance of a l a y e r of stranded g l y c o p r o t e i n m a t e r i a l e x t e r n a l to the l i p i d b i l a y e r i n a wide range of f u n c t i o n s . These f u n c t i o n s range from modulation of transmembrane ion f l u x e s through membrane i o n channels to formation of r e c e p t o r s i t e s f o r antibody molecules. These c a p a b i l i t i e s depend i n great measure on the p o l y a n i o n i c t e r m i n a l s t r u c t u r e of these stranded p r o t r u s i o n s . C l e a r l y , the two examples c i t e d present extremes i n the c o m p l e x i t i e s of membrane o r g a n i z a t i o n . N e v e r t h e l e s s , both types of process e x h i b i t s i m i l a r sharp t r a n s i t i o n s as a f u n c t i o n of temperature that are c o n s i s t e n t w i t h cooperative processes. The accumulation of potassium i n smooth muscle shows a c r i t i c a l thermal t r a n s i t i o n that can be d e s c r i b e d i n terms of a coop e r a t i v e accumulation process ( 4 ) . Steady-state l e v e l s of potassium and sodium of taeniae c o l i of guinea-pig are c r i t i c a l l y a f f e c t e d by v a r y i n g temperature i n the narrow range 12° to 17°C. E l e c t r o l y t e d i s t r i b u t i o n w i t h i n these muscle c e l l s undergoes abrupt readjustment around a c r i t i c a l temperature. For both c a t i o n s , the c r i t i c a l temperature, T , i s 13.8°C i n the presence of 5 mM e x t e r n a l potassium. T decreases to 10.0°C w i t h an e x t e r n a l potassium c o n c e n t r a t i o n of 10 mM (Figure 1 ) . These f i n d i n g s are i n good agreement w i t h the f o l l o w i n g p r e d i c t i v e model of a cooperative mechanism. Using a cooperative a d s o r p t i o n isotherm developed by L i n g (5) to d e r i v e a c o o p e r a t i v e a b s o r p t i o n isotherm that e x p l i c i t l y r e l a t e s i n t r a c e l l u l a r potassium content to e x t e r n a l potassium c o n c e n t r a t i o n , R e i s i n and G u l a t i (4·) have expressed the coopera t i v e isotherm f o r the i n t r a c e l l u l a r potassium as a f u n c t i o n of K x / N a . This isotherm K j i s defined c

c

e

ex

a(

K

a

d

=

F T /

2 {[« - D

2

+

(D

where F represents the amount of f i x e d negative charge s i t e s on the membrane s u r f a c e ( i n μΜ/g) which can i n t e r a c t c o o p e r a t i v e l y T



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Figure 1. (A) Effect of temperature on the steady-state potassium (O) and sodium (Φ) contents of taenia coli smooth muscle fibers. The high sodium content above 17°C is mainly attributable to the large extracellular space in smooth muscles. (B) Effect of K .r on T for the thermal transition, developed from theoretical curves derived from Equations 1 and 3 (see text). For these curves, the potassium levels in the sorbitol space (2.15 μmol/g at 5mM K , and 4.3 \xmol/g at WmM K ) were added to the theoretically calculated values. (4) r

r

r r


cx

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K

K

Na

w

n

e

r

e

K

i

s t

n

e

w i t h c a t i o n s ; ξ i s d e f i n e d as §a->K4 ex/ ex» §a-»K i n t r i n s i c e q u i l i b r i u m constant ( s e l e c t i v i t y r a t i o of potassium over sodium); K and N a a r e the e x t e r n a l potassium and sodium c o n c e n t r a t i o n s ; and η i s a f u n c t i o n of the energy of nearest neighbor i n t e r a c t i o n s , and gives a measure of cooperation among s i t e s . When the potassium c o n c e n t r a t i o n approaches H F-j., Eq. 1 can be rearranged e x

e x

In/

K

a

d

4 = η In Κ χ + η 1 η FaT- ad / Na,ex 6

Κ

4

( 2 )


K

I f F and η are not s i g n i f i c a n t l y a f f e c t e d by temperature, Eq. 2 can be d i f f e r e n t i a t e d w i t h respect t o the r e c i p r o c a l of absolute temperature 81n[K /F - K ] = η T

ad

T

a d

3(1/T)

9(1/T) (3)

ΔΗ°° Na->K where -RT.lnK4

K

= F°° = ΔΗ°° - T A S

0 0

(4)

and AF°°, ΔΗ°° and S°° a r e changes i n the i n t r i n s i c f r e e energy, i n t r i n s i c enthalpy, and i n t r i n s i c entropy, r e s p e c t i v e l y , f o r the exchange of potassium by sodium on a d s o r p t i v e s i t e s . The r e l a t i o n s h i p i n Eq. 3 emphasizes that f o r an a d s o r p t i v e process there must be a c r i t i c a l thermal t r a n s i t i o n . I n non-cooperative mechanisms (when η = 1 ) , thermal t r a n s i t i o n s a l s o occur, but f o r a f i x e d value of ΔΗ°° the cooperative t r a n s i t i o n i s steeper by a f a c t o r of n. Reisen and G u l a t i conclude that t h i s model c o r r e c t l y p r e d i c t s the behavior of smooth muscle around the t r a n s i t i o n temperature w i t h respect to c e l l e l e c t r o l y t e l e v e l s as a f u n c t i o n of temperature, and i n p r e d i c t i n g a lower t r a n s i t i o n temperature when K i s r a i s e d . For sodium-rich f r o g muscles, the reaccumu l a t i o n of potassium to a steady s t a t e c o n d i t i o n i s r e l a t e d t o K by a s i g m o i d a l c o o p e r a t i v e curve f o r experiments performed a t 0°, 10O and 25°C (6)_. Accumulation of potassium w i t h i n the c e l l by a c o o p e r a t i v e process i s a transmembrane event. However, there i s a l s o good evidence that movement of potassium ions through membrane channels i s modulated by the b i n d i n g of c a l c i u m ions a t channel s i t e s ~ the " p l u g i n the bath" model ( 7 ) . Membrane s u r f a c e g l y c o p r o t e i n s w i t h p o l y a n i o n i c t e r m i n a l strands o f f e r a broad and powerful sub s t r a t e f o r these c a t i o n i c i n t e r a c t i o n s . Competition between hydrogen and c a l c i u m ions a t these s i t e s has been modeled as the i n i t i a l t r a n s d u c t i v e step i n e x c i t a t i o n ( 8 ) . These same s u r f a c e g l y c o p r o t e i n s have been modeled as recep t o r s f o r antibody molecules and i n the b i n d i n g of l e c t i n mole c u l e s , such as concanavalin A. On the lymphocyte membrane, e x

e x



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cross l i n k i n g between s i t e s i n these processes recodes the sur face i n length and i n area by "patching" and "capping." Patching and capping are i n h i b i t e d by p r i o r treatment of the c e l l surface w i t h l e c t i n molecules, a process described as anchorage modulat i o n (9, H)> 11). Patching and capping occur a t a more complex l e v e l of s t r u c t u r a l o r g a n i z a t i o n than i n simple c a t i o n b i n d i n g to a p o l y a n i o n i c s u b s t r a t e . By comparison, the dimensions of Yshaped antibody molecules o r of t e t r a v a l e n t concanavalin A molecules, and the s i z e of r e s u l t i n g patches and caps, a l l bespeak processes on a g a l a c t i c s c a l e . Y e t these, too, are calcium dependent and e x h i b i t sharp thermal t r a n s i t i o n s i n the range 20-25°C, c o n s i s t e n t w i t h cooperative processes. They are dependent on metabolic energy s u p p l i e d from w i t h i n the c e l l , and t h i s requirement f o r n o n e q u i l i b r i u m c o n d i t i o n s a l s o supports a cooperative model. Tissue S e n s i t i v i t i e s and Membrane Models of Transductive to Weak EM F i e l d s

Coupling

For the squid axon, the transmembrane current d e n s i t y t o pro duce a nerve impulse has a t h r e s h o l d around 1.0 mA/cm . Many nerve c e l l s , i n c l u d i n g the motor nerve c e l l s of the mammalian s p i n a l cord, r e q u i r e a s i m i l a r current d e n s i t y through the membrane of the c e l l body to induce f i r i n g . The neuronal membrane o f f e r s a r e s i s t a n c e of 2000-5000 Ω/cm . By c o n t r a s t , t i s s u e f l u i d surrounding nerve c e l l s i n b r a i n and other nervous g a n g l i a has a much lower s p e c i f i c r e s i s t a n c e i n the range 5-25 Ω.αιΓ . Thus, i n the context of e i t h e r i n t r i n s i c t i s s u e f i e l d s generated by the leakage of b i o e l e c t r i c a c t i v i t y from c e l l s t o the f l u i d that surrounds them, o r i n e f f e c t s a t t r i b u t a b l e to t i s s u e components o f environmental EM f i e l d s , transmembrane paths do not form a p r e f e r r e d pathway f o r t i s s u e current f l o w , and only a s m a l l f r a c t i o n of the t o t a l t i s s u e current a c t u a l l y penetrates c e l l membranes. Based on d i f f e r e n t i a l conductance c h a r a c t e r i s t i c s c i t e d above, these transmembrane components would be about three orders o f magnitude l e s s than those i n the e x t r a c e l l u l a r space. Thus, i f we assume a transmembrane current d e n s i t y of 1.0 mA/cm a t t h r e s h o l d f o r nervous e x c i t a t i o n , a current d e n s i t y of 1.0 A/cm i n e x t r a c e l l u l a r f l u i d would be necessary t o achieve t h i s transmembrane l e v e l . We may e x t r a p o l a t e from these b a s i c c o n s i d e r a t i o n s i n two ways. We may evaluate the e x t r a c e l l u l a r e l e c t r i c gradients a s s o c i a t e d w i t h i n t r i n s i c or imposed t i s s u e f i e l d s against the magnitude of the g r a d i e n t s i n the membrane p o t e n t i a l , both i n r e s t i n g c o n d i t i o n s and i n a s s o c i a t i o n w i t h m o d i f i c a t i o n of the membrane p o t e n t i a l during s y n a p t i c e x c i t a t i o n . I n t h i s way, we may appraise the p r o b a b i l i t y of d i r e c t e f f e c t s of e x t r a c e l l u l a r t i s s u e f i e l d s i n e x c i t a t i o n of nerve c e l l s . A second approach w i l l consider the observed b i o l o g i c a l s e n s i t i v i t i e s to these f i e l d s . This w i l l lead to the crux of our current dilemma. A 2

2

1

2

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r a p i d l y widening s e r i e s of observations i n c e l l s of bone (12, 13, 14) and blood (15), as w e l l as i n nervous t i s s u e (16, 17, 18) have confirmed s e n s i t i v i t i e s to e l e c t r o c h e m i c a l gradients i n f l u i d around these c e l l s as much as s i x orders of magnitude l e s s than the e l e c t r i c a l gradient across the c e l l membrane (the membrane p o t e n t i a l ) . What are the mechanisms by which these weak o s c i l l a t i n g e l e c t r o c h e m i c a l gradients are coupled to the vast t i d e s of c a t i o n s known to move across the c e l l membrane i n the ensuing stages of e x c i t a t o r y d e p o l a r i z a t i o n and i n h i b i t o r y h y p e r p o l a r i z a t i o n ? H i t h e r t o , the l a t t e r processes have h e l d p r i d e of place i n a l l models of e x c i t a t i o n , d e s p i t e the very l a r g e t r i g g e r i n g energies necessary f o r t h e i r i n i t i a t i o n . The c l e a r evidence f o r e f f e c t i v e n e s s of s t i m u l i much weaker than those producing c e l l membrane d e p o l a r i z a t i o n through massive d i r e c t p e r t u r b a t i o n of i o n i c e q u i l i b r i a r a i s e s a second q u e s t i o n . What i s the s i g n i f i c a n c e of these newly d i s c l o s e d s e n s i t i v i t i e s f o r i n t e r n a l communication i n normal and abnormal t i s s u e ? How are they u t i l i z e d i n c e l l - t o - c e l l communication i n d e f i n i n g aspects of homeostasis, or i n modulating s e n s i t i v i t y to the myriad e x t r i n s i c e l e c t r o c h e m i c a l and neurohumoral s t i m u l i that c o n s t a n t l y pervade a t i s s u e ? Recent s t u d i e s provide p a r t i a l answers t o some of these q u e s t i o n s , and a l s o p o i n t the way to needed f u t u r e research. B i o e l e c t r i c O r g a n i z a t i o n of Nervous T i s s u e . The membrane p o t e n t i a l of 70 mV i s developed across the l i p i d b i l a y e r of the c e l l membrane. This l a y e r i s approximately 40 8 t h i c k , so that the transmembrane e l e c t r i c gradient i s of the order of 1 0 V/cm. This e x t r a o r d i n a r y d i e l e c t r i c s t r e n g t h i s not e a s i l y r e p l i c a t e d i n a r t i f i c i a l m a t e r i a l s . I t i s noteworthy that the r e s t i n g membrane p o t e n t i a l maintains t h i s d i e l e c t r i c b i l a y e r w i t h i n a f a c t o r of two of e l e c t r i c a l breakdown (19). Release of n e u r a l t r a n s m i t t e r substances from s y n a p t i c terminals on the nerve c e l l surface t r a n s i e n t l y s h i f t s the membrane p o t e n t i a l a t the s i t e of r e l e a s e by a few m i l l i v o l t s . I n terms of an a l t e r e d transmembrane g r a d i e n t , t h i s s h i f t i s of the order of 1.0 kV/cm. C e r e b r a l cortex has an i n t r i n s i c o s c i l l a t i n g f i e l d i n the e x t r a c e l l u l a r f l u i d , the E E C I t appears to o r i g i n a t e p r i n c i p a l l y i n the enormously branched dendrite s that c h a r a c t e r i z e c e r e b r a l neurons. Measured over c e l l u l a r dimensions, the EEG has a t y p i c a l gradient of 50 mV/cm (20, 21), and a frequency spectrum from 1 to 100 Hz, w i t h most energy i n the band from 1 to 20 Hz. Records from i n t r a c e l l u l a r m i c r o e l e c t r o d e s i n many but not a l l c e r e b r a l neurons d i s p l a y l a r g e slow o s c i l l a t i o n s up to 15 mV i n amplitude that resemble the EEG from the same c o r t i c a l r e g i o n i n s p e c t r a l analyses. However, w i t h an amplitude of 20-50 uV, the EEG i s l e s s than 1.0 percent of the amplitude of the i n t r a c e l l u l a r neuronal waves from which i t i s d e r i v e d , due to i t s a t t e n u a t i o n i n the neuronal membrane. 5



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U n t i l r e c e n t l y , e x t r a c e l l u l a r g r a d i e n t s as weak as the EEG i n c e r e b r a l t i s s u e have not been considered p h y s i o l o g i c a l l y s i g n i f i cant i n i n f o r m a t i o n t r a n s f e r , nor i n the modulation of t i s s u e f u n c t i o n a l s t a t e s . As discussed below, there i s now good evidence that impressed gradients that mimic the EEG, e i t h e r simply as low frequency f i e l d s or as RF o r microwave f i e l d s amplitude modulated at low frequencies i n the EEG range, can e l i c i t a l t e r e d e f f l u x of calcium ions and of t r a n s m i t t e r substances (22-26). Moreover, these weak imposed e x t r a c e l l u l a r f i e l d s are capable of e i t h e r modifying or e n t r a i n i n g the EEG a t the r a t e of low frequency f i e l d components (27, 2 8 ) , implying that these f i e l d s w i t h gradients of 50 mV/cm are capable of i n f l u e n c i n g the much l a r g e r membrane p o t e n t i a l of 1 0 V/cm. C l e a r l y , t h i s r e q u i r e s a form of t r a n s d u c t i v e coupling w i t h c o n s i d e r a b l e " a m p l i f i c a t i o n " of the weak t r i g g e r i n g o s c i l l a t i o n . P o s s i b l e models of these i n t e r a c t i o n s w i l l be d i s c u s s e d . 5

B i o e l e c t r i c S e n s i t i v i t i e s of Organisms to Environmental EM F i e l d s . As background to current research on b i o p h y s i c a l mechanisms t h a t might u n d e r l i e these s e n s i t i v i t i e s a t the molecu l a r l e v e l , we may enumerate some of the i n t e r a c t i o n s reported i n whole organisms ( f o r reviews, see Adey, 249, 30) . B e h a v i o r a l e f f e c t s have been reported w i t h t i s s u e components of environmental extremely low frequency (ELF) f i e l d s between 10~ and 1 0 ~ V/cm i n the spectrum below 10 Hz. They i n c l u d e n a v i g a t i o n and prey d e t e c t i o n by sharks and rays (31, 32), b i r d n a v i g a t i o n (33, 3 4 ) , a l t e r e d d a i l y b i o l o g i c a l rhythms i n man and b i r d s (35, 3 6 ) , and s u b j e c t i v e time estimates i n monkeys (37, 38). Rats exposed to 60 Hz 1 kV/m f i e l d s show reduced n o c t u r n a l motor a c t i v i t y i n the f i r s t 2 or 3 days of exposure and increased a l e r t i n g behavior during and f o l l o w i n g 30 days of exposure to these f i e l d s (39). Rays could be t r a i n e d t o seek a food reward i n a c i r c u l a r tank i n which the earth's magnetic f i e l d became an e s s e n t i a l cue (40). C a l c u l a t e d t i s s u e e l e c t r i c g r a d i e n t s based on the r a t e of the animal's movement through the n a t u r a l magnetic f i e l d were 0.5 yV/cm. These responses faded out w i t h magnetic f i e l d s weaker than the n a t u r a l l e v e l , and were not observed w i t h f i e l d s two orders of magnitude g r e a t e r , suggesting an i n t e n s i t y "window." These low frequency p e r t u r b a t i o n s are sensed by t u b u l a r receptors opening on the s k i n of the head. T h e i r extremely high w a l l r e s i s t a n c e (6 ΜΩ/cm ) c o n t r i b u t e s to a low-pass frequency c h a r a c t e r i s t i c w i t h an e f f e c t i v e upper frequency l i m i t of about 10 Hz, thus e s t a b l i s h i n g a frequency "window." No such s p e c i a l i z e d e l e c t r o r e c e p t o r s are known t o e x i s t i n the mammalian p e r i p h e r a l o r c e n t r a l nervous systems. However, i n t r a c e l l u l a r f e r r o magnetic bodies have been described i n b a c t e r i a that o r i e n t to the earth's magnetic f i e l d (41), i n the t h o r a c i c banding of bees (42), and i n the s k u l l or muscles of pigeons (see 4k3). The p o s s i b l e r o l e of these i n c l u s i o n s i n t i s s u e s of bees and pigeons i n o r i e n t i n g to environmental magnetic f i e l d s i s not y e t known. 7

8

2


16.

ADEY

279


These b e h a v i o r a l responses t o ELF f i e l d s suggest "windowed" s e n s i t i v i t i e s to nervous t i s s u e gradients around 1 0 " V/cm, w i t h an e f f e c t i v e upper frequency l i m i t between 10 and 100 Hz. This t i s s u e component i s produced by f i e l d s i n a i r of 10-100 V/m. I t i s approximately s i x orders of magnitude weaker than the EEG gradient of 10-100 mV/cm over c e l l u l a r dimensions. I n d u c t i o n of EEG l e v e l gradients by environmental low frequency e l e c t r i c f i e l d s i s not p r a c t i c a l , by reason of weak c o u p l i n g of such a f i e l d i n a i r t o the aqueous conducting medium of t i s s u e (29). I t would r e q u i r e an environmental f i e l d of 500 kV/m. However, EEG l e v e l f i e l d s are e a s i l y induced i n t i s s u e s by weak r a d i o and microwave f i e l d s a t frequencies of 100-1000 MHz w i t h i n c i d e n t energies around 1.0 mW/cm , and e l e c t r i c gradients i n a i r of 60100 V/m. When s i n u s o i d a l l y modulated a t ELF f r e q u e n c i e s , p a r t i c u l a r l y below 20 Hz, they a l t e r c a t i o n b i n d i n g i n c e r e b r a l t i s s u e . These are a l s o "windowed" r e a c t i o n s , as discussed below, and r e l a t e to the low frequency modulation of the imposed f i e l d . Only a few b e h a v i o r a l s t u d i e s have so f a r been reported w i t h these modulated RF f i e l d s . Chicks exposed t o 450 MHz, 1 o r 5 mW/cm f i e l d s s i n u s o i d a l l y modulated a t 3 o r 16 Hz d u r i n g performance of a f i x e d - t i m e 30 sec schedule of water r e i n f o r c e ment showed trends towards longer l a t e n c y of response (44) . I n Soviet s t u d i e s , r a b b i t s and r a t s c h r o n i c a l l y exposed f o r 120 days to extremely low l e v e l s of 50 o r 2500 MHz continuous f i e l d s o r 10 GHz f i e l d s pulsed a t 1000 o r 20 Hz showed s t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s i n conditioned reflexes with f i e l d i n t e n s i t i e s between 1.9 and 2.0 pW/cm . A 2450 MHz, 1 mW/cm microwave f i e l d pulsed at 1000/sec had a s y n e r g i c e f f e c t on the a c t i o n o f Valium ( c h l o r d i a z e p o x i d e ) on f i x e d - i n t e r v a l behavior i n r a t s (46).


7

2

2

2

2

C e l l Membrane Models of Transductive Coupling of Weak EM F i e l d s . The arrangement of stranded g l y c o p r o t e i n molecules outs i d e the l i p i d b i l a y e r (plasma membrane) of the c e l l membrane was noted above. T h e i r o r g a n i z a t i o n and f u n c t i o n a l r o l e have been addressed i n a s e r i e s of membrane models which have developed the concepts of "the greater membrane" (47) and the " f l u i d mosaic" model (48). The f l u i d mosaic model embraces most of the recent data on membrane molecular b i o l o g y . The p h o s o p h l i p i d b i l a y e r of the plasma membrane behaves as a f l u i d whose v i s c o s i t y i s modified by the presence of intramembranous p a r t i c l e s (IMPs) w i t h i n i t (49). The IMPs range i n molecular weight from about 300 f o r the p r o s t a g l a n d i n s and r e l a t e d "molecular s w i t c h i n g " molecules to about 50,000 f o r B-microglobulins a s s o c i a t e d w i t h antibody receptor s i t e s ( 9 ) . Many of these molecules have both e x t e r n a l and i n t e r n a l t e r m i n i . The i n t e r n a l p r o t r u s i o n s may connect w i t h microtubules and thus w i t h the nucleus o f the c e l l . Many of the e x t e r n a l p r o t r u s i o n s are g l y c o p r o t e i n s w i t h t e r m i n a l amino-sugars ( s i a l i c acids) that have numerous fixed-charge a n i o n i c b i n d i n g s i t e s . The c e l l surface thus becomes a p o l y a n i o n i c sheath (50), as described above, w i t h a strong a f f i n i t y


280

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2 +

EFFECTS

OF

RADIATION

+

f o r c a t i o n s , and w i t h C a and H more s t r o n g l y bound than other monovalent c a t i o n s (51). They a l s o p a r t i c i p a t e i n receptor s i t e s f o r n e u r o t r a n s m i t t e r , hormonal and antibody molecules. For muscle end-plate, t h i s s u r f a c e p o t e n t i a l i s t y p i c a l l y about -50 mV. When the surface p o t e n t i a l i s modified by i n c r e a s i n g c o n c e n t r a t i o n of d i v a l e n t c a t i o n s C a or M g , f o r example, the e f f e c t i v e n e s s of tubocurarine as an antagonist f o r a c e t y l c h o l i n e i s reduced (50). Increased d i v a l e n t c a t i o n c o n c e n t r a t i o n i n the surrounding f l u i d decreases the n e g a t i v i t y of the s u r f a c e potent i a l and thereby lowers the c o n c e n t r a t i o n of c a t i o n s at the membrane-solution i n t e r f a c e , suggesting that e f f e c t s of s u r f a c e p o t e n t i a l on c a t i o n i c concentrations at a c t i v e s i t e s may be important i n drug i n t e r a c t i o n s . The S i n g e r - N i c o l s o n f l u i d - m o s a i c model i s a l s o c o n s i s t e n t w i t h two major sequences of events i n the t r a n s d u c t i v e c o u p l i n g of a wide range of e l e c t r o c h e m i c a l s t i m u l i at the membrane s u r face. One i n v o l v e s Ca-dependent conformational changes i n these intramembranous p a r t i c l e s by which a s i g n a l i s t r a n s m i t t e d to the i n t e r i o r of the c e l l ( 9 ) , as f o r example, i n the a c t i v a t i o n of membrane-bound enzymes, such as adenyl c y c l a s e , f o l l o w i n g b i n d i n g of a hormone molecule or an antibody to a membrane surface recept o r s i t e . The second concerns e l e c t r o c h e m i c a l i n t e r a c t i o n s o c c u r r i n g i n the length and area of the membrane s u r f a c e , and probably i n v o l v i n g c r o s s - l i n k i n g between a d j o i n i n g molecular s t r a n d s , as f i r s t suggested by Katchalsky (51) from i n v i t r o s t u d i e s of Ca i o n b i n d i n g to p o l y v i n y l c h l o r i d e . C l e a r l y , l o n g range i n t e r a c t i o n s between charge s i t e s along the membrane s u r face suggest a mechanisms by which weak o s c i l l a t i n g e l e c t r o c h e m i c a l gradients may be sensed and " a m p l i f i e d " as steps i n a coope r a t i v e i n t e r a c t i o n i n v o l v i n g b i n d i n g and r e l e a s e of Ca i o n s , as discussed below. 2 +


NONIONIZING

2+

Evidence f o r Nonequilibrium Aspects of F i e l d I n t e r a c t i o n s i n B r a i n T i s s u e , using *+ Ca E f f l u x as an I n d i c a t o r 5

2+

2

E f f l u x of 4Ca "*" from i n t a c t , awake cat c e r e b r a l cortex responds i n a h i g h l y n o n l i n e a r f a s h i o n to changing e x t r a c e l l u l a r Ca c o n c e n t r a t i o n (53). Increased c o n c e n t r a t i o n of C a r e s u l t e d i n increased e f f l u x of both C a and H-GABA, but the e f f e c t of a 1 mM increment i n C a c o n c e n t r a t i o n was only s l i g h t l y l e s s than that of a 20 mM increment. These i n i t i a l observations suggested membrane r e a c t i o n s t r i g g e r i n g the r e l e a s e or turnover of calcium. The observed k i n e t i c s are c o n s i s t e n t w i t h displacement of C a bound to p o l y a n i o n i c s i t e s at the surface of the membrane (54). 2 +

2 +

4 5

2 +

3

2 +

4 5

Ca Ca4

2 +

+

4 5

Ca —

2 +

M

n_

•+ Ca,45

Ca-M ( n - 2 ) - _ 4 5 2 + + Ca

Ca-M C a

(n-2)-

_ nM


16.

ADEY

281


where M represents a membrane a n i o n i c s p e c i e s . Thus, the e f f l u x of Ca + from the membrane i s p r o p o r t i o n a l to a h i g h power of the c o n c e n t r a t i o n of bound C a . This h i g h l y n o n l i n e a r p a t t e r n of autogenic C a e f f l u x l e d to a s e r i e s of s t u d i e s of EM f i e l d i n t e r a c t i o n s w i t h c e r e b r a l t i s s u e , i n a search f o r t h e i r o r i g i n s i n cooperative mechanisms (55). We proceeded on the premise that i f long-range i n t e r a c t i o n s were i n volved a t fixed-charge s i t e s on membrane surface macromolecules, major changes i n C a e f f l u x might occur w i t h very weak o s c i l l a t i n g e l e c t r i c f i e l d s around the c e l l s , and that the magnitude of these responses would be comparable w i t h those induced a t f a r higher l e v e l s of f i e l d i n t e n s i t y . I n t u r n , t h i s l e d to a search f o r p o s s i b l e "windows" i n the range of f i e l d amplitudes i n d u c i n g a response. A second premise concerning p o s s i b l e long-range i n t e r a c t i o n s was that they might occur i n narrow bands of f r e quencies as "windows" i n the range from 1 t o 100 Hz. This concept arose from p o s s i b l e f i e l d i n t e r a c t i o n s w i t h the dominant frequencies of i n t r i n s i c EEG rhythms i n b r a i n t i s s u e , and from measurements of membrane e l e c t r i c a l t i s s u e constants i n none x c i t a b l e c e l l s , i n c l u d i n g bone and connective t i s s u e . Based on these c r i t e r i a , i t was hoped to a r r i v e a t the l e v e l s of coopérâtivity to be expected i n these membrane i o n b i n d i n g mechanisms. R e s u l t s of these s t u d i e s have been e x t e n s i v e l y reviewed elsewhere (29, 3 0 ) . Weak, ELF f i e l d s at frequencies from 1 to 75 Hz and 5 t o 100 V/m i n a i r were t e s t e d f o r t h e i r e f f e c t s on C a + e f f l u x from f r e s h l y i s o l a t e d c h i c k and c a t c e r e b r a l t i s s u e (23). Tissue gradients were estimated to be i n the range of 0.1 yV/cm. F i e l d exposures r e s u l t e d i n a general trend toward a r e d u c t i o n i n the r e l e a s e of preincubated C a . Maximum decreases occurred a t 6 and 16 Hz and were i n the range from 12 to 15 percent. Threshold f i e l d s were around 10 and 56 V/m i n a i r f o r c h i c k and c a t t i s s u e s , r e s p e c t i v e l y , but s e n s i t i v i t y decreased at 100 V/m. Thus, calcium b i n d i n g i n c h i c k and c a t c e r e b r a l t i s s u e e x h i b i t e d frequency and amplitude windows i n responses t o these extremely weak f i e l d s (Figure 2B). I n t r i n s i c o s c i l l a t i o n s of the EEG are about s i x orders of magnitude l a r g e r than those j u s t d e s c r i b e d , t y p i c a l l y i n the range 10"" to 10"" V/cm i n f l u i d surrounding a neuron. E l e c t r i c a l s t i m u l a t i o n of awake c a t c e r e b r a l c o r t e x w i t h 200/sec t r a i n s of pulses (1.0 msec duration) a t t h i s i n t e n s i t y increased c o r t i c a l e f f l u x of ** Ca by about 13 percent (26) , and simultaneous e f f l u x of the i n h i b i t o r y n e u r o t r a n s m i t t e r GABA rose by about 12 percent. When f r e s h l y i s o l a t e d c h i c k c e r e b r a l t i s s u e was exposed to s i n u s o i d a l l y modulated 147 MHz f i e l d s that a l s o produced EEG l e v e l s of e l e c t r i c gradient i n the t i s s u e , there were s t r i k i n g changes i n 4Ca *" e f f l u x as the s i n u s o i d a l modulation frequency covered the spectrum from 0.5 to 35 Hz (22) . No e f f e c t s were seen from the unmodulated c a r r i e r wave, but the 4 C a * e f f l u x f o l l o w e d a smooth " t u n i n g curve," w i t h an i n c r e a s e 2

2 +

2 +


4 5

2 +

4 5

i + 5

1

2

2 +

2

5

2+

2-

2


BIOLOGICAL

282

EFFECTS

O F NONIONIZING R A D I A T I O N

147 MHz EM FIELDS AMPLITUDE MODULATED BY ELF SINE WAVES 1.2 ρ Tissue Gradient IO" V/cm Τ 1

̶I

C

Ε£Β

U

3

6 6


I

9

16 16

20

ÎL

25 35 32

Τ E L F SINE WAVE ELECTRIC FIELDS Tissue Gradient IO" V/cm 7

45

2+

Figure 2. Comparison of (A) Ca from freshly isolated chick cerebral hemi spheres exposed to a weak radiofrequencyfield(147 MHz, 0.8 mW/cm ), ampli tude-modulated at low frequencies, and (B) C + efflux changes from exposure to far weaker electricfields(56 V/m) in the same frequency spectrum from 1 to 32 Hz. The peak magnitude of the efflux change is similar for the twofields,but oppo site in direction. For the radiofrequencyfield(A), the unmodulated carrier wave U had no effect when compared with controls C. Field gradients differ by about six orders of magnitude between (A) and (B) (22, 23). 2

45

2

INTENSITY OF RADIATION, mW/cm*

Radio Science

Figure 3. Effects of changing power density of a 147-MHz radiofrequency field, sinusoidally amplitude*modulated at 16 Hz, on Ca + efflux from freshly isolated chick cerebral hemispheres. Ordinate shows mean difference (with S.E.) between exposed (\ ) and control (V ) samples (25) · 45

T

2

c


16.

ADEY


283

of about 15 percent i n the range from 6 to 20 Hz, and a maximum e f f e c t at 16 Hz (Figure 2A). A s i m i l a r but sharper e f f l u x curve, a l s o peaking i n the range 9-16 Hz, was found i n independent s t u d i e s (25). Blackman e_t al. a l s o reported an amplitude "window" for t h i s response at i n c i d e n t f i e l d energies around 1.0 mW/cm , confirmed independently by Bawin, Sheppard and Adey (56) f o r 450 MHz f i e l d s s i n u s o i d a l l y modulated at 16 Hz (Figure 3). The windowed character of these responses i n frequency and amplitude domains u n e q u i v o c a l l y e s t a b l i s h e s a s e r i e s of h i g h l y n o n l i n e a r mechanisms i n the c o n t r o l of c e r e b r a l calcium i o n b i n d i n g . I t a l s o p o i n t s to long-range i n t e r a c t i o n s r e q u i r i n g ordered molecular s t a t e s that p e r s i s t f o r appreciable time over considerable atomic distances (30, 57, 58). I t i s a l s o c l e a r that the l e v e l s of c o o p e r a t i v i t y i n these i n t e r a c t i o n s must be h i g h , s i n c e f o r the ELF f i e l d e f f e c t s c i t e d here, the e f f e c t i v e s t i m u l i may appear to be below the l e v e l of Boltzmann thermal noise (kT). Though t h i s i s probably not the case (see d i s c u s s i o n on models below), both the b i o l o g i c a l thresholds c i t e d above and these chemical thresholds f o r ELF e l e c t r i c and magnetic f i e l d s are mutually c o n s i s t e n t w i t h s e n s i t i v i t i e s w i t h i n an order o f magnitude of thermal noise l e v e l s (23, 59). The search f o r a p h y s i c a l b a s i s f o r t h i s c o o p e r a t i v i t y may prove d i f f i c u l t . As a prelude to l a t e r d i s c u s s i o n of p o s s i b l e models, a d d i t i o n a l evidence may be c i t e d on modulation of Ca + b i n d i n g and r e l e a s e i n c e r e b r a l t i s s u e by other i o n i c species (17). Increased Ca e f f l u x from c h i c k c e r e b r a l hemispheres induced by a 450 MHz 0.75 mW/cm f i e l d s i n u s o i d a l l y modulated a t 16 Hz was i n s e n s i t i v e to v a r i a t i o n s i n Ca c o n c e n t r a t i o n between 0 and 4.16 mM i n the bathing s o l u t i o n . I t was enhanced by r a i s e d H* concentrations (0.108 mM HC1), and i n h i b i t e d i n the absence o f normal l e v e l s of HCO3 i o n s . L a ions added to the NaHC03-free medium r e s t o r e d e l e c t r i c a l responsiveness, but the 450 MHz f i e l d then reduced Ca e f f l u x by about 15 percent, compared to the t y p i c a l f i e l d - i n d u c e d increase i n e f f l u x of about the same s i z e w i t h normal NaHC0 l e v e l s (2.4 mM). These observations suggest two c o n c l u s i o n s . F i r s t , s i n c e lanthanum blocks transmembrane movement of C a i n both d i r e c t i o n s , p e r s i s t i n g e f f l u x changes to f i e l d s t i m u l a t i o n a f t e r L a p e r f u s i o n are i n t e r p r e t e d as i n d i c a t i n g a s i t e at c e l l membrane surfaces f o r EM f i e l d i n t e r actions with C a b i n d i n g s i t e s . Second, the i n f l u e n c e of H concentration suggests that weak, low-frequency e x t r a c e l l u l a r e l e c t r i c gradients may be transduced i n a s p e c i f i c c l a s s o f e x t r a c e l l u l a r negative b i n d i n g s i t e s normally occupied by C a and s u s c e p t i b l e to competitive H* b i n d i n g . We may f u r t h e r hypothesize t h a t , by the windowed character of these i n t e r a c t i o n s , the r o l e of the hydrogen i o n may be as a proton i n t u n n e l i n g i n t e r a c t i o n s (59). These cooperative i n t e r a c t i o n s i n i s o l a t e d c e r e b r a l t i s s u e have suggested two f u r t h e r l i n e s of research.


2

2

i + 5

2 +

2

3 +

l + 5

2 +

3

2 +

3 +

2 +

+

2 +


284

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Do s i m i l a r i n t e r a c t i o n s occur i n l i v i n g cortex? Can they be detected i n u l t r a s t r u c t u r a l elements of the c e n t r a l nervous system, such as synaptosomes? The 4Ca "*" e f f l u x was s t u d i e d i n i n t a c t awake cat c o r t e x , using a c o r t i c a l cup technique (18). Tissue f i e l d s measured w i t h a m i n i a t u r i z e d , implantable e l e c t r i c f i e l d probe were of the order of 500 mV/cm w i t h an i n c i d e n t f i e l d of 3.0 mW/cm , amplitude modulated by a 16 Hz sinewave. I r r a d i a t i o n began 60 min a f t e r completion of 90 min i n c u b a t i o n of the cortex w i t h 4Ca " . R e s u l t i n g e f f l u x curves were d i s rupted by waves of increased C a e f f l u x . These waves showed periods of 10 to 30 min. For a n a l y s i s , the e f f l u x curves were t r e a t e d by s t r a i g h t - l i n e f i t s to the l o g a r i t h m i c a l l y transformed data, w i t h two epochs f o r each curve. One curve covered the pref i e l d - exposure p e r i o d , the other from 20 min a f t e r f i e l d onset to the end of the experiment. D i f f e r e n c e s a s s o c i a t e d w i t h the wavelike d i s r u p t i o n s between pre-exposure/exposure epochs and control/exposure curves were s i g n i f i c a n t at an 0.96 confidence l e v e l , using o n e - t a i l e d b i n o m i a l p r o b a b i l i t y s t a t i s t i c s . I n p a r a l l e l s t u d i e s , synaptosomes from male r a t c e r e b r a l c o r t e x w i t h a t y p i c a l diameter of 0.5 ym were incubated at 22°C f o r 10 min i n a p h y s i o l o g i c a l s o l u t i o n c o n t a i n i n g C a (60, 61). They were then a p p l i e d to M i l l i p o r e f i l t e r s and perfused at 2.0 ml/min f o r 30 min. I n experiments i n which exposure t o 450 MHz f i e l d s (as used i n experiments w i t h i n t a c t cats) began when the synaptosomes were a p p l i e d to the M i l l i p o r e f i l t e r , and onset of p e r f u s i o n was delayed u n t i l 5-10 min l a t e r , continuous exposure to a f i e l d of 0.5 mW/cm increased the r a t e constant of the 4 C a * e f f l u x curve (p < 0.01). Conversely, i n e x p e r i ments at 34°C, where the synaptosomes were continuously perfused from the time of t h e i r a p p l i c a t i o n to the f i l t e r , increased Ca e f f l u x occurred only when f i e l d exposure s t a r t e d 10 min a f t e r onset of p e r f u s i o n . Moreover, slow o s c i l l a t o r y p e r t u r b a tions i n C a e f f l u x w i t h periods of 5-10 min followed terminat i o n of a 10 min f i e l d exposure, resembling those i n i n t a c t c o r t e x . We may conclude from these s t u d i e s that responses seen p r e v i o u s l y i n f r e s h l y i s o l a t e d but n o n - r e s p i r i n g cortex a l s o occur i n the i n t a c t b r a i n , and that they are a property of t i s s u e elements s u b s t a n t i a l l y s m a l l e r than a whole c e l l . Indeed, for a synaptosome w i t h a diameter of 0.5 ym or l e s s , t i s s u e f i e l d l e v e l s measured i n the cat's b r a i n i n these s t u d i e s would not exceed a gradient of 25 yV across i t s f u l l diameter, f a r s m a l l e r than i t s membrane p o t e n t i a l of the order of 50 mV. From the viewpoint of a cooperative system, t h i s cooperative macromolecul a r o r g a n i z a t i o n at membrane C a b i n d i n g s i t e s may be presumed f u n c t i o n a l l y e f f e c t i v e over dimensions as s m a l l as these, s i n c e even weaker f i e l d s r e l e a s e both 4 C a and s t o r e d t r a n s m i t t e r substances, such as GABA, apparently from s y n a p t i c s t o r e s (26). Despite t h i s progress i n d e f i n i n g some i n t e r a c t i o n s w i t h weak EM f i e l d s , p a r t i c u l a r l y i n s p e c i a l i z e d preparations o f i s o l a t e d c e l l s and t i s s u e s , we must a l s o take account of recent 2

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d i s c l o s u r e of i n t e r a c t i o n s between n a t u r a l o p i o i d p e p t i d e s , the enkephalins and endorphins, and the b i n d i n g of C a to c e l l membrane s u r f a c e s . I n the i n t a c t b r a i n , they exert powerful modulating i n f l u e n c e s on s t a t e s of e x c i t a b i l i t y and a s s o c i a t e d behavior. T h e i r s i t e s of a c t i o n are now known to extend to t i s s u e s o u t s i d e the c e n t r a l nervous system, and to i n c l u d e the g a s t r o i n t e s t i n a l t r a c t . I n synaptosome f r a c t i o n s , 3-endorphin has a r e g u l a t o r y r o l e on C a movement, i n h i b i t i n g i n f l u x and enhancing e f f l u x (62). I n t h i n s l i c e s of b r a i n hippocampal t i s s u e , e x c i t a t o r y e f f e c t s of 200 nM s o l u t i o n s of ( D - A l a , M e t ) enkephalin are s t r o n g l y modulated by h i g h or low C a concen t r a t i o n s (63). Since amounts of these substances i n b r a i n t i s s u e s e n s i t i v e l y r e f l e c t s t r e s s f u l experiences, we may hypo t h e s i z e that s e n s i t i v i t y of the i n t a c t subject to weak n o n i o n i z ing EM r a d i a t i o n , as a r e s u l t of m o d i f i e d C a b i n d i n g at c e l l membrane s u r f a c e s i t e s , may be i n f l u e n c e d by concomitant enkepha l i n l e v e l s . These may vary from day t o day, and thus become an important determinant of s u s c e p t i b i l i t y t o weak f i e l d s (64) . I n a broader context, we may hypothesize that development of r e a l i s t i c s a f e t y standards f o r human exposure, based on needed know ledge of s u b t l e but presumably important neuroendocrine and neurohumoral e f f e c t s induced by these weak f i e l d s , w i l l r e q u i r e c l o s e a t t e n t i o n to p o s s i b l e v a r i a t i o n s i n thresholds a t t r i b u t a b l e to these n a t u r a l o p i o i d substances, occasioned by t h e i r d i u r n a l and even seasonal v a r i a t i o n s . 2 +

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We have already considered the f l u i d i t y of c e l l membranes, and the long-range m o b i l i t y of enclosed p r o t e i n s and l i p i d s i n the plane of the membrane. I m p o s i t i o n of weak EM f i e l d s o f f e r s a t o o l f o r study of both r e c e p t o r m o b i l i t y and of c o u p l i n g mechanisms between humoral agents and receptor s i t e s . Embryonic muscle f i b e r s are i n i t i a l l y s e n s i t i v e to a c e t y l c h o l i n e (ACh) over the e n t i r e membrane s u r f a c e , but w i t h the onset of synaptogenesis, ACh receptors are e s s e n t i a l l y confined to the n e u r o t r a n s m i t t e r f u n c t i o n . When a steady e l e c t r i c f i e l d of 30 mV i s a p p l i e d across a s i n g l e s p h e r i c a l embryonic muscle c e l l of Xenopus, ACh receptors aggregate toward the cathodal pole of the c e l l w i t h i n 1 hour. The movement i s e l e c t r o p h o r e t i c i n nature and r e s u l t s i n formation of s t a b l e , m e t a b o l i c a l l y inde pendent receptor aggregates (65). I n c o n t r a s t , concanavalin A r e c e p t o r s , which a l s o accumulate toward the cathodal p o l e , com p l e t e l y resume t h e i r general d i s t r i b u t i o n w i t h i n 10 min of ending of f i e l d exposure. At 22°C, the average e l e c t r o p h o r e t i c m o b i l i t y of the e l e c t r o p h o r e t i c a l l y mobile p o p u l a t i o n of the concanavalin A receptors i s about 1.9 χ 10~ ym/sec/V/cm, w h i l e t h e i r average d i f f u s i o n c o e f f i c i e n t i s 5.1 χ 10~" cm /sec (66). Apart from displacement of receptors by the f i e l d , i s there evidence of a m o d i f i c a t i o n of c o u p l i n g of s i g n a l s , u s u a l l y 3

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a s s o c i a t e d w i t h b i n d i n g of humoral and hormonal molecules at membrane receptor s i t e s , to the chain of i n t r a c e l l u l a r metabolic sequences? When c u l t u r e s of a c l o n a l l i n e of o s t e o b l a s t c e l l s were exposed to pulsed magnetic f i e l d s f a r weaker than those i n the myosphere experiments (72 Hz pulse t r a i n , pulse d u r a t i o n 325 ysec w i t h 5 msec reverse p o l a r i t y , peak i n t e n s i t y 35 gauss, induced current d e n s i t y i n c u l t u r e 1.0 μΑ/cm , induced e l e c t r i c gradient 3 mV/cm), there was a 90-99 percent i n h i b i t i o n of the adenyl c y c l a s e response to p a r a t h y r o i d hormone (PTH). At the same time, P T H - s p e c i f i c decreases i n c o l l a g e n s y n t h e s i s and a l k a l i n e phosphatase r e l e a s e d i d not occur. F l u o r i d e - a c t i v a t e d adenyl c y c l a s e formation was unchanged, suggesting that the magnetic f i e l d blocked PTH a c t i o n e i t h e r by m o d i f i c a t i o n of the PTH receptor on the membrane, or by a l t e r e d c o u p l i n g between the receptor and the enzyme adenyl c y c l a s e i n s i d e the membrane (12). Bone c e l l c u l t u r e s from f e t a l r a t c a l v a r i a responded to s i m i l a r magnetic f i e l d s w i t h s i g n i f i c a n t l y increased DNA s y n t h e s i s , but the response depended on pulse waveform and c u l t u r e techniques (14). Cultured chick t i b i a e showed s i m i l a r s e n s i t i v i t i e s i n formation of adenyl c y c l a s e , but d i f f e r e n t i a l responses were noted between s h a f t and epiphyseal regions i n the amount of c o l l a g e n formed (13).


2

Models of Nonequilibrium Phenomena i n B i o e f f e c t s of Weak EM F i e l d s We have considered b i o e f f e c t s from two q u i t e d i f f e r e n t l e v e l s of induced e l e c t r i c f i e l d s ; those o c c u r r i n g w i t h t i s s u e g r a d i e n t s around 10" V/cm i n e x t r a c e l l u l a r f l u i d and those at s u b s t a n t i a l l y higher l e v e l s from 10~ to 10"" V/cm. In the f i r s t c l a s s , there can be no doubt that the coupling process or processes must be h i g h l y c o o p e r a t i v e , as discussed below. For b i o e f f e c t s w i t h f i e l d s i n the higher range, i t i s necessary to evaluate the p o s s i b i l i t y that these responses occurred i n s t e a d by a thermal energy t r a n s f e r mechanism. This problem has been formulated t h e o r e t i c a l l y by c a l c u l a t i n g the minimum e l e c t r i c f i e l d s t r e n g t h r e q u i r e d to produce the alteration i n C a b i n d i n g observed i n c h i c k c e r e b r a l t i s s u e exposed to 147 and 450 MHz f i e l d s (22, 57), i f the f i e l d i n t e r a c t i o n occurred by a thermal energy t r a n s f e r mechanism (67). This approach i n v o l v e s two stages: (1) a change i n the f r a c t i o n of C a i o n b i n d i n g s i t e s at the c e l l surface i s r e l a t e d to an a l t e r a t i o n i n the temperature and the f r e e energy of b i n d i n g ; (2) the temperature and f r e e energy changes are r e l a t e d to absorbed energy from the a p p l i e d EM f i e l d (dependent on the square of the e l e c t r i c f i e l d amplitude). Tenforde concludes that the value of the absorbed energy p r e d i c t e d t h e o r e t i c a l l y to achieve the observed r e l e a s e of bound C a from c e l l membrane surfaces i s at l e a s t 100 times greater than the f i e l d s a c t u a l l y present i n the exposed b r a i n t i s s u e . Therefore, the a l t e r e d Ca b i n d i n g "very l i k e l y proceeds by a mechanism other than 7

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thermal energy t r a n s f e r . " Tenforde f u r t h e r p o i n t s out that modulation "windows" f o r these e f f e c t s suggest a resonant o r cooperative energy t r a n s f e r mechanism. However, the path t o an acceptable cooperative model i s fraught w i t h d i f f i c u l t y . By comparison w i t h t i s s u e e l e c t r i c gradients induced by environmental EM f i e l d s , the r e q u i s i t e g r a d i e n t s f o r some known cooperative macromolecular t r a n s i t i o n s are very l a r g e . The h e l i x - c o i l conformational change i n poly(Y-benzyl L-glutamate) can be induced as a cooperative t r a n s i t i o n by a gradient of 260 kV/cm (68), based on nearest neighbor i n t e r a c t i o n s . L o n g - l a s t i n g conformation changes occur i n poly(A).2poly(U) and i n ribosomal RNA w i t h pulsed e l e c t r i c f i e l d s of 20 kV/cm and w i t h a decay time of 10 sec (69, 70). They are viewed as a model f o r nerve e x c i t a b i l i t y . I t i s c l e a r t h a t these phenomena and r e l a t e d models i n v o l v e energy l e v e l s orders of magnitude greater than those a s s o c i a t e d w i t h weak EM f i e l d s that e l i c i t such a wide range of proven b i o e f f e c t s . We may hypothesize that one o r more t r a n s d u c t i v e steps a r e necessary before the p e r i c e l l u l a r e l e c t r i c f i e l d becomes an e f f e c t i v e transmembrane s i g n a l . Models of these processes f a l l i n t o two c a t e g o r i e s , one c o n s i d e r i n g p r o p e r t i e s of p o l y a n i o n i c membrane surface macromolecules, and the other the behavior of the p h o s p h o l i p i d b i l a y e r . They w i l l be summari z e d ( f o r more extensive review, see 30). Models of T i s s u e - F i e l d I n t e r a c t i o n s a t Membrane Surface Macromolecules. There are no known mechanisms to e x p l a i n b i o e f f e c t s at extremely low frequencies (ELF) on the b a s i s of d i r e c t i n t e r a c t i o n s w i t h component d i p o l e s of molecular systems o s c i l l a t i n g a t these low frequencies. Grodsky (71) has hypothes i z e d that e x c i t a b l e membranes are e n e r g e t i c a l l y e q u i v a l e n t to sheets of g i a n t d i p o l e s bathed i n c o n t r o l l e d e x t e r n a l f i e l d s . Grodsky s f o r m u l a t i o n envisaged the outer l a y e r of p h o s p h o l i p i d p o l a r heads as a two-dimensional c r y s t a l mosaic of m u l t i p o l a r charge s i t e s ( p - s i t e s ) , s p r i n k l e d w i t h i s l a n d s of g l y c o p r o t e i n s w i t h c a t i o n i c b i n d i n g s i t e s ( c - s i t e s ) . The p - s i t e s are taken to be occupied by i d e a l d i p o l e s . Grodsky hypothesized t h a t , w i t h the a d d i t i o n of an e x t e r n a l e l e c t r i c f i e l d to the system, when the frequency of an allowed o s c i l l a t i o n mode reaches zero, the system would become a macroscopic quantum a m p l i f i c a t i o n d e v i c e , and would e x h i b i t long-range order phase changes that generate i n t o the zero-frequency mode (Einstein-Bose condensation). The model has merit i n seeking a b a s i s i n membrane u l t r a s t r u c t u r e , but u n f o r t u n a t e l y recent r e a p p r a i s a l of the p o s s i b i l i t i e s of an Einstein-Bose phase t r a n s i t i o n as the b a s i s f o r the low frequency s e n s i t i v i t i e s i n d i c a t e s that i t would not occur w i t h t h i s formulat i o n ( I . T. Grodsky and H. F r o h l i c h , personal communications). Kaczmarek (72, 73) has considered the p o s s i b l e occurrence of resonant phenomena i n chemical r e a c t i o n s analyzed as l i n e a r systems. For appropriate values of the k i n e t i c constants and 1



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the extent of c o o p e r a t i v i t y , h i s model d i s p l a y s m u l t i p l e steady s t a t e s , l i m i t c y c l e behavior, or both. When the system i s i n the l i m i t c y c l e mode, i t s dynamics were very s e n s i t i v e to the frequen cy of any p e r t u r b a t i o n . Kaczmarek p o i n t s out that i f calcium b i n d i n g i n n e u r a l membranes can e x h i b i t l i m i t - c y c l e behavior due to r e a c t i o n steps being maintained f a r from chemical e q u i l i b r i u m , weak e x t e r n a l p e r t u r b a t i o n s could e a s i l y d i s r u p t the e l e c t r o c h e m i c a l balance. I n essence, the s t a t u s of molecular populations would then determine low frequency m a n i f e s t a t i o n s of f i e l d i n t e r actions i n tissue. I n seeking some form of a m p l i f i c a t i o n of i n i t i a l t r a n s d u c t i v e steps at an e x t r a c e l l u l a r l o c a t i o n , we may presume that i t i n volves systems capable of i n t e g r a t i n g the weak f i e l d over some d i s t a n c e , and would thus occur i n the length and area of the membrane s u r f a c e , r a t h e r than i n a transmembrane a x i s ( 7 4 ) . This problem has been addressed by E i n o l f and Carstensen (75) i n a study of the behavior of micron-sized r e s i n p a r t i c l e s con s i d e r e d as porous p a r t i c l e s w i t h uniformly d i s t r i b u t e d f i x e d charge s i t e s . Porous charged p a r t i c l e s are c h a r a c t e r i z e d by a low frequency d i e l e c t r i c r e l a x a t i o n l e a d i n g to l a r g e s t a t i c d i e l e c t r i c constants. This r e s u l t s i n p o l a r i z a t i o n of the i o n i c atmosphere at the surface of the p a r t i c l e i n the presence of an e x t e r n a l e l e c t r i c f i e l d . This produces an a d d i t i o n a l "apparent" d i e l e c t r i c constant of the p a r t i c l e , exceeding the d i e l e c t r i c constant by s e v e r a l orders of magnitude at low f r e q u e n c i e s . I t i s p r o p o r t i o n a l t o the diameter of the p a r t i c l e and the square root of the fixed-charge c o n c e n t r a t i o n i n the porous m a t e r i a l , w i t h values as high as 1 0 a t frequencies below 1 kHz f o r microns i z e d r e s i n p a r t i c l e s . S i m i l a r p r o p e r t i e s may be expected at the surface of t u b u l a r s t r u c t u r e s w i t h diameters i n the micron range, i n c l u d i n g d e n d r i t e s w i t h p o l y a n i o n i c g l y c o p r o t e i n surface l a y e r s . We have considered the E i n o l f and Carstensen model as a b a s i s f o r those b i o e f f e c t s i n which i t appears that thermal noise a t normal t i s s u e temperature i s s u b s t a n t i a l l y l a r g e r than the t i s s u e components of imposed e l e c t r i c f i e l d s (23, 30). The Boltzmann equation may be w r i t t e n i n terms that model the c e l l s u r f a c e i n the r e g i o n of the counterion l a y e r as a low-pass f i l t e r : 6

e

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= 4 kT BR

where the t r a n s f e r f u n c t i o n f o r the root mean square noise v o l t age e i s a f u n c t i o n of the frequency bandwidth Ji and the s p e c i f i c r e s i s t a n c e of the noise pathway R. With a s p e c i f i c r e s i s t a n c e for b r a i n t i s s u e of the order of 300 Ω.οπΓ and an e f f e c t i v e frequency bandwidth from 0 t o 100 Hz, the e q u i v a l e n t noise v o l t age gradient would be of the order of 10~ V/cm. This i s i n c l o s e agreement w i t h observed s e n s i t i v i t i e s i n marine v e r t e b r a t e s , b i r d s and mammals f o r c e r t a i n low frequency f i e l d s , and these thresholds are c o n s i s t e n t w i t h a thermal " f l o o r " as the l i m i t i n g factor. 1

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We have shown that the E i n o l f and Carstensen model of d i e l e c t r i c d i s p e r s i o n at the surface of s m a l l charged spheres a l s o a p p l i e d to l o n g , t h i n c e l l processes which t y p i f y the dendrites of neurons (76) . The degree of p o l a r i z a t i o n induced a t the surface depends on the o r i e n t a t i o n of the i n c i d e n t f i e l d w i t h respect to the length of the d e n d r i t e or axon, r a d i u s of the c y l i n d r i c a l s u r f a c e , i o n m o b i l i t y , and temperature. We may speculate that the e x t r a o r d i n a r y degree of p o l a r i z a t i o n produced by counterions at the c e l l s u r f a c e may b r i n g about a dynamic d i s t r i b u t i o n of weakly bound ions of reduced momenta. From the Heisenberg equation r e l a t i n g p o s i t i o n a l and momentum u n c e r t a i n t i e s , i t i s seen that ions of s u f f i c i e n t l y low momentum would e x h i b i t quantum behavior u s u a l l y not a s s o c i a t e d w i t h such massive bodies at b i o l o g i c a l temperatures. For example, the momentum of a thermal proton at 300°K i s o r d i n a r i l y 4.5 χ 1 0 kg-m/sec. The r e s u l t i n g p o s i t i o n a l u n c e r t a i n t y , Δχ, i s about 1 0 " m, so s m a l l that c l a s s i c a l notions are e n t i r e l y adequate f o r dynamic con s i d e r a t i o n s . However, an i o 4 w i t h attenuated momentum ρε4 has a p o s i t i o n a l u n c e r t a i n t y of ε4Δχ. I f we take a value f o r ε i n the r e g i o n surrounding the i o n of the same order of magnitude as for the macroscopic ε measured w i t h a c e l l suspension of 10** t o 1 0 ) , we see that the i o n would have a p o s i t i o n a l u n c e r t a i n t y of about 1 0 ~ to 10"~ m. Tunneling would then be p o s s i b l e from one p o t e n t i a l w e l l t o another, because the i o n i c wave f u n c t i o n would extend through the r e g i o n of a t y p i c a l p o t e n t i a l b a r r i e r and even over a p p r e c i a b l e regions along the s u r f a c e . I f momenta are s u f f i c i e n t l y reduced, s m a l l e r ions — p a r t i c u l a r l y protons — would have s i g n i f i c a n t p r o b a b i l i t i e s of t u n n e l i n g from one p o t e n t i a l minimum to another. I t i s suggested that proton t u n n e l i n g may occur between charge s i t e s that i n t e r a c t across the boundary between coherent and incoherent charge zones on c e l l membrane surface g l y c o p r o t e i n s (57, 74, 76, 77). Such a process would be most e f f e c t i v e i n the a x i s of the f i e l d e l e c t r i c g r a d i e n t , along t h i s surface biopolymer sheet. Development of long-range order among s u r f a c e macromolecules i n t e r a c t i n g v i a low-momentum ions may induce s i g n i f i c a n t changes over the e n t i r e polarized region. 2lt

1 1

6

8

9

Models of T i s s u e - F i e l d I n t e r a c t i o n s I n v o l v i n g the C e l l Membrane L i p i d B i l a y e r . Although concepts of t r a n s d u c t i v e coupling of weak e l e c t r o c h e m i c a l s t i m u l i by membrane glycopro t e i n s are w e l l supported by a v a i l a b l e evidence, very l i t t l e i s known of ways i n which events i n the g l y c o p r o t e i n l a y e r are coupled to the u n d e r l y i n g l i p i d b i l a y e r i n the ensuing steps of e x c i t a t i o n . Indeed, there remains the p o s s i b i l i t y that the e x t e r n a l f i e l d may i n t e r a c t d i e r e c t l y w i t h the l i p i d b i l a y e r , d e s p i t e the enormous d i f f e r e n c e i n t h e i r r e s p e c t i v e e l e c t r i c g r a d i e n t s . Barnes and Hu (78) have estimated s h i f t s i n i o n c o n c e n t r a t i o n and magnitude of current flow r e s u l t i n g from membrane r e c t i f i c a t i o n of r a d i o and microwave f i e l d s , using the


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n o n l i n e a r Boltzmann equation. I n t h i s system, where charged p a r t i c l e concentrations vary i n space, i f there i s no net curr e n t , the f i e l d - d r i v e n d r i f t current must balance the d i f f u s i o n c u r r e n t , so that a p o t e n t i a l b a r r i e r i s formed to maintain the c o n c e n t r a t i o n d i f f e r e n c e . At e q u i l i b r i u m , even s m a l l values of an a l t e r n a t i n g f i e l d superimposed on t h i s steady p o t e n t i a l w i l l s h i f t e q u i l i b r i u m concentrations. For example, an i n c i d e n t f i e l d of 10 mW/cm produces a gradient of 4.4 V/cm across a membrane enclosed i n an aqueous d i e l e c t r i c medium. This corresponds to an e l e c t r i c gradient of 9 yV across a 200 8 membrane, w i t h a concent r a t i o n s h i f t of 1 p a r t i n 1 0 and a flow of 400 ions/sec f o r a c e l l w i t h a surface area of 10" cm . The b i o l o g i c a l s i g n i f i c a n c e of such s m a l l s h i f t s i s unknown. Rather than assuming that changes i n i o n i c p e r m e a b i l i t i e s through channels i n the l i p i d b i l a y e r i n the course of the a c t i o n p o t e n t i a l account f o r l a r g e t r a n s i e n t membrane conductances, Vaccaro and Green (79) suggest that the sodium p e r m e a b i l i t y hypothesis be replaced by one a t t r i b u t i n g the a c t i o n p o t e n t i a l to n o n l i n e a r plasma o s c i l l a t i o n s and v o l t a g e clamp records to s t r o n g l y damped o s c i l l a t o r y c u r r e n t s . I n t h i s model, the membrane phase i s t r e a t e d as a s e m i - e l e c t r o l y t e w i t h connected regions i n which the i o n i c concentrations are not very d i f f e r e n t from those of the adjacent e l e c t r o l y t e s . M o b i l i t i e s of d i f f e r e n t i o n i c species i n the membrane are i d e n t i f i e d w i t h those measured i n the steady s t a t e , and are not assumed to have l a r g e v a r i a t i o n s during e x c i t a t i o n . T h e i r s o l u t i o n to the plasma model y i e l d s periods i n the m i l l i s e c o n d range, c o n s i s t e n t w i t h the time course of the a c t i o n p o t e n t i a l and v o l t a g e clamp records. P o l y v a l e n t c a t i o n s , such as C a , have a more pronounced damping e f f e c t on the e l e c t r o l y t e systems than monovalent c a t i o n s . Plasma o s c i l l a t i o n s would a l l o w t r a n s f e r of ions a t a much f a s t e r r a t e than could be achieved by o r d i n a r y conductance, an e s s e n t i a l f e a t u r e of the a c t i o n p o t e n t i a l . I n t h i s i n i t i a l f o r m u l a t i o n , the i n v e s t i g a t o r s have not considered the p o s s i b i l i t y of entrainment of plasma o s c i l l a t i o n s by weak imposed f i e l d s , although t h i s would appear a t e s t a b l e aspect of t h e i r model. 2


6

6

2

2 +

Summary There i s good evidence that i n i t i a l events i n c o u p l i n g of weak o s c i l l a t i n g e l e c t r o c h e m i c a l f i e l d s to c e l l membranes occur on membrane surface polyanions c h a r a c t e r i z e d by t r a n s i e n t coherent s t a t e s of nearest neighbor fixed-charge s i t e s . These coherent s t a t e s may e x i s t f o r considerable d i s t a n c e s along the membrane s u r f a c e . The f i r s t steps i n t r a n s d u c t i v e coupling would thus occur i n the length of the membrane. Binding and r e l e a s e of calcium ions to these membrane surface s i t e s c o r r e l a t e s c l o s e l y w i t h exposure to c e r t a i n weak electromagnetic f i e l d s . Sensitivity to these f i e l d s i n narrow frequency and amplitude "windows" supports a s e r i e s of models based on h i g h l y cooperative i n t e r a c t i o n s


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at c e l l membrane surface g l y c o p r o t e i n s . Evidence from calciumhydrogen i o n i n t e r a c t i o n s i n the presence of these f i e l d s r a i s e s the p o s s i b i l i t y of proton t u n n e l i n g , perhaps a t the margins between coherent and incoherent fixed-charge zones. Recent b i o p h y s i c a l models of c e l l membrane o r g a n i z a t i o n are reviewed, w i t h c o n s i d e r a t i o n of i o n i c behavior as a plasma i n the l i p i d b i l a y e r . There i s good experimental evidence from a v a r i e t y of t i s s u e s that a form of c e l l - t o - c e l l communication can occur through weak e l e c t r o c h e m i c a l s t i m u l i that are sensed by h i g h l y cooperative processes a t c e l l membrane s u r f a c e s . I n v a r y i n g degrees, t h i s may be a general property of t i s s u e o r g a n i z a t i o n . Acknowledgments Studies from our l a b o r a t o r y described here were supported by the O f f i c e of Naval Research, the N a t i o n a l I n s t i t u t e of E n v i r o n mental Health Sciences, the Department of Energy, the Bureau of R a d i o l o g i c a l H e a l t h , and the Southern C a l i f o r n i a Edison Company. I t i s a pleasure to acknowledge t h e i r p a t i e n t help and guidance throughout the d i f f i c u l t i n i t i a l phases of t h i s research.

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