Electromagnetic modulation of biological processes

119

Bioelectrochemistry and Bioenergetics, 10 (1983) 119- 131 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

W-ELECTROMAGNETIC

MODULATION

OF BIOLOGICAL

CDEMICAL, PHYSICAL AND BIOLOGICAL CORRELATIONS Ca-UPTAKE BY EMBRYONAL CHICK TIBIA IN VITRO

PROCESSES IN THE

GIUSEPPE COLACICCO * and ARTHUR A. PILLA * BioelectrochemistryL.&oratory, Columbia Uniwrsity, 650 West 168th Street, New York, NY 10032 (U.S.A.) (Manuscript received October 8th 1982)

SUMMARY Ca-uptake by embryonal chick tibia in short-term culture is herewith used as an empirical model to probe the coupling of electromagnetic signals with biological processes. Tibiae from 8- to lo-day-old chick embryos were incubated 60 to 120 minutes in media of given composition at 37.5 f0.5“C in the presence of 45Ca, either inside or outside a very low frequency pulsating electromagnetic field. Ca-uptake by the chick tibia rudiment was best determined by 45Ca counting in aliquots of the culture medium. The field produced an increase in Ca-uptake only in the presence of sufficient quantities of bicarbonate, which was also the most effective of the ions to bring about Ca-uptake outside the field (bicarbonate > phosphate > chloride > acetate). Pharmacologic agents, as ethanol, Mg*+, and NaF, stimulated the positive effect of the field on Ca-uptake, but only in the presence of bicarbonate. The magnitude of the effect was also dependent on the characteristics of the induced electromagnetic signal, whereby the dose-response curves had peaks at certain signal amplitudes. No molecular mechanisms have been as yet identified for the observed electromagnetic effects. However, because of the passivity of non-living systems, it is clearly the Ca-uptake by living tissue that is being modified by the pulsating electromagnetic field. Probable candidates in the electromagnetic coupling that results in an increased Ca-uptake may have to be found in the membrane processes of active ion transport and related phenomena.

INTRODUCTION

Use of pulsating current with designed variations in signal parameters has been proposed as an experimental approach to test the electrochemical information transfer hypothesis [l]. The non-faradaic response by the living tissue to the pulsating signal of given amplitude (a few mv), frequency, and certain other time constants [ 1,2], resulted into increased Ca-uptake by chick embryo tibia in vitro [2], facilitated bone growth and repair in oiuo [3], accelerated DNA synthesis by cartilage cells in culture [4], and a faster Ca ‘+ efflux from brain tissue in vitro [5]. In a preliminary report [6], we pointed out that pulsating electromagnetic fields with certain signal characteristics stimulated Ca-uptake by embryonal chick tibia rudil Present address: Bioelectrochemistry Laboratory, Mount Sinai School of Medicine, Room 1702 Anne&erg Building, New York, NY 10029, U.S.A.

0302-4598/83/$03.00

0 1983 Elsevier Sequoia S.A.

120

ments in short-term culture; but this was possible only in the presence of sufficient quantities of sodium bicarbonate. The bicarbonate effect provides a clue toward identifying the molecular mechanisms at the basis of the role of Ca2’ in bone growth and other biological processses. This and other clues must be related to ongoing speculations on the central or focal position that Ca” occupies in the information transfer connecting the extracellular microenvironment, the membrane with its complex messenger systems of ATPases and nucleotidyl cyclases, cahnodulin and microtubules, and finally the nucleus and the genetic apparatus [7-91. Inasmuch as pulsating electromagnetic waves influenced the synthesis and uncoiling of nuclear DNA [4,10-121, a search for the mechanisms of such electromagnetic actions poses the question as to if and how Ca2+, or whichever species, picks up the signal and sends the message to the subsequent biological steps over a chain of interconnected electrochemical and metabolic events. Another obvious question is if Ca2’ primes all of so many [4,7,8] and other effects, or if the field influences also systems that have no dependence on Ca2+. Far from answering all those questions, the present communication provides some novel information about the effect of pulsating electromagnetic fields on the Ca-uptake by embryonal chick tibia rudiments under two major experimental conditions. In one, the Ca-uptake was studied as a function of the chemical composition of the medium; in the other, the effect of the field on the Ca-uptake was examined as a function of the amplitude of the electromagnetic signal. EXPERIMENTAL

PROCEDURES

Chemicals Water was doubly distilled, the second time from Pyrex glass. Salts and other chemicals were reagent grade. Hepes (N-2-dihydroethyl piperazineN-Zethane sodium sulfonate) was obtained from Sigma Chemical Company, St. Louis, MO, U.S.A. Radioactive “‘CaCl, was purchased from New England Nuclear, Cambridge, MA, U.S.A. Solutions for bathing and washing in the isolation of tibiae consisted of either a Hepes buffer: 20 mM Hepes, 120 mM NaCl, 4.8 mM KCI, 11 mM glucose, 1.0 mM NaH,PO,, 1.2 mM CaCl,, 1.2 mM MgSO,, or a glucose-rich phosphate buffer (basal Y) containing 120 mM glucose, 90 mM NaCl, 6.0 mM KCI, 1.0 mM NaH,PO,, 1.2 mM CaCl,, 1.2 mM MgSO,. The pH of these bicarbonate-free media was 7.30. Culture media Two types of media were mostly used. One, referred to as Y medium, was obtained by adding small measured volumes of fresh solutions of 0.1 M NaH,PO, and 1 M NaHCO, to basal Y until final concentrations of 5 mM phosphate and 10 mM bicarbonate were obtained. The initial pH of this medium was 6.70, and it was adjusted to 7.00 or any other desired value. The other culture medium was the

121

commercially available BGJ, which was obtained from GIBCO, Grand Island, NY U.S.A. Two simpler media, A and B, are described under the pertinent experiments. Inductors The electromagnetic

devices were provided by Electra-Biology, Inc., Fairfield, NJ, U.S.A. Each one consisted of a power supply that delivered pulsating current into two approximately square, 10 cm X 10 cm, 7 cm apart, Helmholtz air gap coils. The device produced a signal of desired characteristics, such as amplitude, frequency or repetition rate, and certain selected time constants; the coils and signal specifications have been described in detail elsewhere [ 1,2,11]. The following general characteristics may be of interest. The signals mostly used had a main peak amplitude of 15 mV, a duration of 200 ps for the main and 20 ps for the opposite polarity. The pulses were in bursts or trains of 5 ms duration. The repetition rate or frequency of the pulse train was between 2 and 1000 Hz, mostly around 15 Hz, all of which are in the very low frequency range. The energy content of this pulse is very small since the extracellular field is less than 1 mV/cm, the rate of change of the magnetic field is 0.1 G/ps, and the current density is 10 to 50 PA/cm’. Tibiae

Fresh fertile White Leghorn eggs were kept for 8 to 10 days in Marsh Roll-X incubators (Garden Grove, CA, U.S.A.) at 38.5 f 0.5OC and 85% humidity. At the desired time, the tibiae were isolated and placed in bathing medium for 30 to 60 minutes at room temperature (25 f IOC). Then each tibia rudiment was transferred to a round-bottomed cylindrical Falcon culture tube (clear polystyrene, 12 mm X 75 mm) and submerged with 600 mm3 (~1) culture medium containing about 0.4 PCi 45CaC1,. The cultures were then incubated for 60 to 120 minutes at 37.5 + 0.5”C, either outside the field (controls) or inside the field (inside coil). A plastic holder capable of up to 16 culture tubes was placed in the field with the sample tibia rudiment at mid-distance between the two coils. Analyses

Immediately after incubation, 50 n-& aliquots were pipetted from the culture medium and submitted to counting of the 45Ca that remained after uptake by the tibia rudiment; a Packard scintillation spectrometer was used. The % Ca-uptake was calculated as 100 x (initial - final)/initial, where initial and final stand for 45Ca c.p.m. of blank and test (or before and after culture) respectively. This indirect approach provided the most accurate method for determination of Ca-uptake by the tibia rudiment. Such a conclusion was reached in separate experiments, in which a total balance of 45Ca radioactivity was made; the activity from the Ca-uptake was distributed in three portions: (a) 80 to 40% in the chick tibia rudiment itself, (b) 20 to 60% in the biologial material that was either shedded or detached from the organ and had adhered to the wall of the tube, and (c) not more than IS in biological material that was suspended in the medium.

122

Because of the large and variable quantities of material adhering to the tube’s wall, counting of “Ca in the tibia rudiment provided an erroneous and erratic measure of the Ca-uptake. Similarly, because of the negligible quantity of radioactivity in (c), the 45Ca count remaining in the supernatant was an accurate expression of the total calcium that had been taken up by the tibia’s material and distributed among the rudiment and debris on the tube’s wall. RESULTS

The number of tibiae, age of embryos, and % deviation are indicated under each experiment. In the reported data, the average deviation was less than f 10%; in the cases in which the deviation was greater than that, the data were not utilized. Provided that sufficient bicarbonate was present, a field effect was obtained with a variety of culture media. Effects of bicarbonate, phosphate, and other ions In order to study the influence of certain ions without interference by others, we prepared media that were simpler than Y and BGJ. Medium A consisted of 300 mM glucose, 1.0 mM phosphate, and 1.2 mM CaCI,; medium B contained 150 mM NaCl (instead of glucose), and the same 1.0 mM phosphate, and 1.2 mM CaCl,. The relevant data are summarized in Table 1. In the absence of bicarbonate (Columns I, II, III, IV, V), Ca-uptake was relatively small and the field effect was nil; under these conditions, the order of effectiveness of anions in producing Ca-uptake was acetate < chloride < phosphate. Interestingly, acetate, which is known to be a good calcium transporter in mitochondria [ 131, abolished the effect of 1.2 mM phosphate of medium A (Columns I, III). The

TABLE 1 Effects of bicarbonate (BC) and phosphate (ph) on % Ca-uptake by embryonal chick tibia rudiments after 60 minute culture in media of various compositions. Medium A: 300 mM glucose, 1.0 mM phosphate, 1.2 mM CaCl,; Medium B: 150 mM NaCl, 1.0 mM phosphate, 1.2 mM CaCl,. Embryos’ age: 8 days 6 hours. 6 Samples; average deviation < f 10%. Initial pH 7.20. Medium

A

B

A +NaAc 10 mM

A

A

+ph 1.6 mM

+ph 8mM

A +BC 8mM

A +ph8mM +BC8mM

V

VI

VII

Column No.

I

II

III

IV

Initial pH

7.40

7.40

7.00

6.70

Outside field Inside field

4.7 4.5

2.5 2.4

1.2 1.1

7.5 7.8

12.1 12.2

23.5 28.9

32.8 48.4

Field effect, A

None

None

None

None

None

5.4

15.6

5.95

7.40

7.40

123

positive effect of increasing concentrations of phosphate on the Ca-uptake is apparent in Columns I, IV, V. Although all the media in Table 1 contained phosphate, separate experiments [14] showed that, outside the field, bicarbonate alone was markedly more effective than phosphate alone in producing Ca-uptake. It is also apparent that, outside and inside the field as well, acetate inhibited the Ca-uptake that was typical of phosphate. When 8 mM bicarbonate was added to medium A, the Ca-uptake (Column VI) rose appreciably over that of medium A (Column I) and that of a phosphate-enriched A (Column V), and a significant effect of the electromagnetic field ensued. Numberless experiments proved that bicarbonate was indispensable for a field effect. However, the field effect nearly trebled (Column VII) when 8 mit4 phosphate was added to the 8 mM bicarbonate. This proves two points. First, in the absence of bicarbonate, there was no field effect no matter how large the quantity of phosphate (Column V). Secondly, although by itself it had no field effect, phosphate promoted markedly the effect of the bicarbonate on the electromagnetic stimulation of the Ca-uptake, or the effect of the field on the function of bicarbonate in the Ca-uptake. The unique role of bicarbonate in the electromagnetic stimulation of Ca-uptake is intriguing. Other stimulants of Ca-uptake Previous studies have shown that Mg2+, ethanol, and NaF stimulate accretion of bone calcium and mineralization in vitro as well as in uivo [ 15-181. Under different experimental conditions, we found that all three agents (NaF > ethanol > Mg) stimulated Ca-uptake by embryonal chick tibia rudiments outside the field, and, provided that bicarbonate was present, the electromagnetic field stimulated further the effect of those chemical agents on the Ca-upake. Magnesium In line with a prior finding [16], magnesium salts (sulfate and chloride indifferently) produced an appreciable increase of Ca-uptake by the tibia rudiments in Y as

TABLE 2 Effect of additional MgSO, on the I Ca-uptake by embryonal chick tibia rudiments after 60 minute culture in BGJ. Embryos’ age: 8 days 20 hours. 6 samples; average deviation -z *5%. Initial pH 7.50.

1MgSQlmM

0

0.8

2.0

4.0

Outside field Inside field

8.3 11.3

8.9 12.9

12.1 16.7

14.5 18.0

Field effect, A A /mM MgSO,

-

3.0

4.0 1.25

4.6 0.80

3.5 0.13

124

well as in BGJ media. Significant was also the stimulation of Ca-uptake inside the electromagnetic field under the same conditions. Some results are presented in Table 2, with 3 different concentrations of MgSO, added to BGJ. The stimulatory effect of Mg2+ on Ca-uptake, outside the field as well as inside the field, increased with greater Mg2+ concentrations up to 2 mM MgSO, (in excess of 0.8 mM already present in BGJ) but declined with higher Mg2+ concentrations. Clearly, the rise in Ca-uptake in going from 0.8 to 2.0 mM MgSO, was greater than between 2 mM and 4 mM MgSO,. At the lower concentrations, Mg2+ stimulated Ca-uptake least in the control, thus making greater the field effect per unit concentration of stimulant (A/mM Mg”). This observation has some important pharmacologic implications. Since the field seems to accelerate what nature would do more slowly, application of the electromagnetic device could render invaluable services by stimulating the effect of very small doses of a chemical agent, which in greater doses would be toxic. Ethanol The stimulatory effect of modest concentrations of ethanol (1.1% to 3.4%) on Ca-uptake by the tibia rudiments (Table 3) are consistent with the bone mineralization effects (of ethanol) that were obtained under different conditions with live chicks and embryonal chick tibiae in longer term culture [ 171. The example in Table 3 describes the events with 1.7% ethanol in BGJ. The effect of ethanol on the Ca-uptake in the field (A = 8.6) was appreciably greater than that outside the field (A = 6.0), and much greater than the effect of the field in the absence of ethanol (A = 2.2). In other words, once the chemical stimulating Ca-uptake was available, the pulsating electromagnetic field accelerated the action of the stimulant (provided, however, that bicarbonate was present). Sodium fluoride Although for our good reasons we used non-physiological concentrations of NaF, our results on the Ca-uptake by chick embryo tibia rudiments are consistent with the findings of other studies [18,19], which used non-toxic concentrations of NaF (< 100

TABLE 3 Effect of 1.7% (282 mM) ethanol on % Ca-uptake by embryonal chick tibia rudiments after 2 hour culture in BGJ. The small volume ethanol was added immediately before culture. Embryos’ age: 8 days 16 hours, 4 samples; average deviation < &-5%. Initial pH 7.50. Notice effect of ethanol outside field (A = 6.0) and inside field (A = 8.6). Ethanol 1.7% Outside field Inside field Field effect, A

17.3 19.5

23.3 28.1

2.2

4.8

125 TABLE 4 Effect of NaF (1 mM) on 46Ca-uptake by embryonal chick tibia rudiments after 2 hours culture in BGJ, initial pH 7.50. The small volume of aqueous NaF was added immediately before culture. Embryos’ age: 8 days 15 hours. 6 samples; average deviation < rt5%. Notice effect of NaF outside field (A = 1.9) and inside field (A = 6.5). 1 mM NaF Outside field Inside field Field effect, A

17.3 19.3

19.2 25.8

2.0

6.6

PM), longer culture times (3 days instead of 2 hours), and different other experimental conditions. The data with 1 mM NaF in BGJ are presented in Table 4. This relatively small, though still unphysiological, concentration of NaF caused a modest stimulation of Ca-uptake (A = 1.9 outside field), which was the same as that inside the field in the absence of NaF (A = 2.0); however, the electromagnetic stimulation in the presence of NaF (A = 6.6) was more than 3 times as large as that in the absence of NaF. This confirms once more the pharmacologic potential of the technology, particularly when certain chemical agents must be used in the very small concentrations at which they are not toxic but also not effective. Undoubtedly, the potentialities and the mechanics of the approach will be better engineered when we will understand the molecular correlates of the electromagnetic stimulation. Observations about pH in culture medium

No systematic investigation was carried out regarding the pH dependence of Ca-uptake and coil effect. However, from inspection of several thousands of experiments with chick tibia rudiments, two phenomena stood out: the influence of the initial pH, and the correlation between final pH and Ca-uptake. The value of the initial pH, between 6.00 and 8.00, did not really seem to matter as far as Ca-uptake and field effect were concerned. Most of the experiments were carried out with initial pH values between 6.70 and 7.50. Within such wide pH limits, as hard as we tried, a correlation between initial pH and Ca-uptake seemed elusive. A dependence of the field effect on initial pH was observed however under different experimental conditions, in which a stable pH of the culture medium was maintained by CO* supply [2]. In contrast, the value of the pH at the end of the culture had some correlation with the Ca-uptake and the field effect. Because of the bicarbonate content, after 60 minute incubation at 37’C, the pH of the culture medium Y rose from an initial value of 7.00 to’ a final value of 8.60. This pH increase is due primarily to the alkalinity that results upon loss of CO2 gas to the atmosphere. Such a CO2 loss and pH change occur in the presence as well as in the absence of tibia.

126

. Because of the complexity of the biological system, it is quite impossible to isolate the various contributions to the CO2 losses and utilization of bicarbonate and to the pH changes. However, empirically, when a pronounced field effect was obtained, the final pH of the medium tended to be up to 0.1 pH unit higher than in the control (outside the field); similarly, a chemical agent that stimulated Ca-uptake produced also a greater pH increase. This phenomenon was unmistakeable in experiments in which NaF (1 mM to 10 mM) was used to stimulate Ca-uptake. In general, a greater pH increase accompanied a greater Ca-uptake because of either electromagnetic stimulation or chemical stimulation, or both. In the last case, the pH effects were cumulative. An example is summarized in Table 5, where Ca-uptake data are correlated with the final pH values and with the effects of 10 mM NaF. The results may also serve to show that such a large concentration of NaF has some deleterious effects. Although NaF (10 mA4) still stimulated Ca-uptake, the effects per unit NaF concentration were much smaller than when 1 mM NaF was used (see Table 4); unlike .then, the total field ‘effect with 10 mM NaF was smaller than that without NaF. At 50 mM concentration, NaF was destructive of both Ca-uptake and field effect. These comments should not detract from the significance of the correlation between Ca-uptake and final pH, for that correlation existed also with smaller NaF concentrations. It remains to be established whether said correlations of final pH and Ca-uptake are accidental, due to coincidental or even causal increase in CO2 escape and bicarbonate utilization, or they have some mechanistic dependence on the electromagnetic or chemical stimulation of Ca-uptake in chick tibia. Needless to say, further extrication of this pH effect from the biochemical complexities of the biological system may require a model other than the chick tibia under the present experimental conditions.

TABLE 5 Correlations between final pH of culture medium and Ca-uptake by embryonal chick tibia rudiments after 2 hours culture in BGJ and in BGJ containing 10 mM NaF. Initial pH 7.50. Small volumes of aqueous 500 mM NaF were added to the system immediately before culture. Age of embryos: 9 days 1 hour. 4 samples; average deviation < k58. 10 mA4 NaF Outside field Inside field

23.5 30.7

32.1 38.7

Field effect, A

7.2

6.6

Final pH outside Final pH inside

8.60 8.70

8.70 8.80

127

Effect of amplitude of electromagnetic signal

Just as the stimulating action of the field on the Ca-uptake was influenced by the concentration of certain chemical agents (above), for a given medium composition the Ca-uptake depended also on the amplitude of the electromagnetic signal. At constant repetition rate, 15 cycles, with all the media examined (Y, BGJ, and modified Fitton-Jackson), the Ca-uptake peaked at amplitudes between 10 mV and 20 mV of the electromagnetically induced current. The data in Figs. 1 and 2 are expressed in % field effect, which is 100 x A/control. The same medium, Y, was used in the two cases; the only difference was in initial pH, 7.20 in Fig. 1 and 7.00 in Fig. 2. The marked difference in % yield between the two experiments should not be attributed to any small or large difference in initial pH; it could instead be due to intrinsic biological differences between the two batches of eggs used in the two experiments. As noted elsewhere [14], in this sort of work, the trends were always reproducible; however, quantitative disparities in % Ca-uptake and % field effect were not surprising. They are to be expected [20]. The optimal amplitude of 10 mV to 20 mV was verified also in the electromagnetic stimulation of the effect of 1.6 mM NaF on the Ca-uptake (Fig. 2). The largest difference between control (lower curve) and the NaF curve occurred also in that amplitude range. Interestingly, the 15 mV-15 cycle/s coil was found to be most effective in clinical osteogenesis (unpublished data; and Ref. 3). DISCUSSION

The foregoing experiments considered a limited number of factors-chemical and physical-that influenced the Ca-uptake by embryonal chick tibia rudiments in 2520-x 15-2 F 10 - 8 5-s 0

z!

Peak induced voltage asC+O(mV)

d -5-s fiI -10-g -15-2o-25-

Fig. 1. Effect of amplitude of electromagnetic signal on Ca-uptake by embryonal chick tibia rudiments after 60 minutes culture in Y medium, initial pH 7.20. On the ordinate is the %Ca-uptake in field relative to control (% C&uptake in field relative to control (% Ca-uptake in field- % Ca-uptake outside field)/% Ca-uptake outside field. Embryos’ age: 8 days 7 hours. 4 samples; average deviation < f 10%.

128

Fig. 2. Effect of amplitude of electromagnetic signal on Ca-uptake by embryonal chick tibia rudiments after 60 minutes culture in Y medium, initial pH 7.00. On the ordinate is the X field effect relative to control as in Fig. 1. Embryos’ age: 9 days 1 hour. 3 samples; average deviation c f 10%. Lower curve, stimulated control; upper curve, with 1.6 mM NaF

short-term culture. We have established: (a) The order of effectiveness of certain anions was bicarbonate > phosphate > chloride > acetate; (b) the electromagnetic signal stimulated Ca-uptake only in the presence of adequate concentrations of bicarbonate; (c) pharmacologic agents such as NaF > ethanol > Mg*+ stimulated the effect of the field (or vice versa, the field stimulated the effect of the chemical agent) only in the presence of bicarbonate; (d) at a given frequency or repetition rate, stimulation of the Ca-uptake by the pulsating signal depended on the latter’s amplitude, whose effect peaked between 10 and 20 mV. A general objective of our work is to define the molecular mechanisms that couple the signal or first electrochemical messenger with Ca” or some other species that either controls or triggers the reactions leading to Ca-uptake. In view of the fact that the molecular mechanisms for the known biological roles of Ca**, phosphate, and bicarbonate are only speculative [4,7,8,21,22], we shall not add new speculations, but we shall use the available ones to shed some light onto the empirical leads that our work has provided. Basically, our data are consistent with the knowledge that phosphate and bicarbonate are good calcium transporters [21,22] and are quite effective in bone mineralization [23-251. The single portentous novelty of the present work is the fact that the electromagnetic field stimulated Ca-uptake only when a good dose of bicarbonate was present. Although this unique role of bicarbonate is intriguing and still far from understood, bicarbonate is the only known empirical link between elecromagnetic stimulation and Ca-uptake by embryonal chick tibia. Although any attempt at describing a

129

molecular mechanism for this bioelectrochemical coupling would be futile at this stage, the story transpiring from the foregoing and other experiments [12,14] is straightforward and consistent. It should now be also emphasized that the field acted on the Ca-uptake only with the living tibia rudiment and not at all with a dead (glutaraldehyde fixed) tibia, nor with artificial ion-sorbing systems, all of which had a good to huge capacity for Ca-uptake [12]. It is, therefore, possible that the target of the electromagnetic signal is in the plasma membrane and the active ion transport, which is a unique feature of live biological membranes. Since such a transport is coupled with the hydrolysis of ATP and with the utilization of mineral phosphate [21], the data are suggestive of (a) an involvement of the membrane’s ion pumps in bicarbonate, phosphate, and Ca” transport and metabolism [21], and (b) a coupling of these systems with the electromagnetic stimulation of Ca-uptake. This working model is consistent with two hypotheses or speculations. One is the electrochemical information transfer [ 11.The other is the belief that bicarbonate and phosphate play a role in the transport of H+ and Ca” across biological membranes [21]. In that case, the electromagnetic signal could couple with the bicarbonate-phosphate-Ca2+ triangle and with the H+ gradient, both of which are at the basis of transmembrane energetics and whose consequences reach far into the cytoplasm and the nucleus [4,7,8]. This view is supported by the observation that the combination of bicarbonate and phosphate potentiated markedly the stimulation of the Ca-uptake by the field, and is consistent with the (first) hypothesis of the electrochemical information transfer [ 1,261. CONCLUSIONS, SPECULATIONS, AND PERSPECTIVES

Future investigations may profit from a simple concept that has developed from the foregoing experiments. Namely, what nature does, the electromagnetic field helps to do better. Everything seems to suggest that the field stimulates naturally on-going processes and thus potentiates the action of metabolic and pharmacologic agents; the latter naturally stimulate Ca-uptake by themselves but not as far as the field may help them to do. The evidence was amply provided by the experiments using ethanol, Mg2+, and sodium fluoride (Tables 2-4). Among the several questions that this work has generated, a fundamental one is whether the stimulation of the biological processes effected by the field is electrical or magnetic. According to the electrochemical information transfer hypothesis [26], stimulation and response are electrical, non-faradaic; thereby, loops of electromagnetically induced current couple with transmembrane ionic currents. Interestingly, the frequencies adopted in this and other work [2,5,12], of the order of 1 to 100 Hz, are extremely low as compared to those, 1 kHz to 2 MHz, used to produce spinning of cells in elecromagnetic fields, where the role of the plasma membrane was also invoked for the coupling [27]. This may imply that the two widely different fields may be coupling with totally different or only quantitatively different processes, albeit at the membrane level in both cases.

130

Since bicarbonate was indispensable for electromagnetic stimulation that leads to increased Ca-uptake, it is conceivable that the loop of induced current couples with bicarbonate ion transport or some other ion transport that is connected with or dependent on bicarbonate. The mechanism would therefore be one that couples an induced current of certain amplitude with the kinetics of some bicarbonate related process, whose parameters are consistent with the characteristics of the wave signal. Evidence for this and other electrical models, and possibly for strictly magnetic or catalytic models, awaits further investigation. Meanwhile, a recurrent phenomenon in all these studies [2,5,28] is the relatively small window or sharp peak in the effective amplitude and frequency of the signal, as well as in the concentration of the given chemical agent. This picture may facilitate identification of the coupling mechanisms and recognition of the classes of processes involved. The purely magnetic effects, if any, remain to be identified. Finally, irrespective of whether the coupling is electrical or magnetic, irrespective of whether the electrical coupling is with the bicarbonate, Ca*‘, or other ionic current, a thought-provoking consideration is the enormous disparity between the small energy put in by the electrical signal and the large yield in Ca-uptake effect. The quantity of induced current used is by several orders of magnitude smaller than that needed to account energetically for the calcium that is taken up under that stimulation. Another instructive consideration may be the energetics of the extremely low frequency signal. The main frequency contents of the current calculated by Fourier or Laplace analyses [ 11,291 are also very small, namely below 100 Hz; such values are fairly comparable with the signal-train frequency of 15 Hz. Irrespective of whichever of these frequency values are used in the calculation of the energy from the wave equation E = hf, with f= 100 Hz, the energy of the signal is 0.42 erg mole- ‘. Such an astoundingly small energy value can hardly be attached to any process that we know of from chemical bond physics. As long as the exact coupling is not identified, one could speculate that the signal (with a wavelength of 3 000 km) may perturb some metastable equilibrium thus increasing the chances for activation transfer and speeding up the reaction rates. Interestingly, the same kind of signal was effective in (a) inhibiting an adenyl cyclase which had been stimulated by parathyroid hormone in bone cells in vitro [28]; (b) stimulating Na+K+ATPase and DNA synthesis that had been inhibited by exogenous ATP in Raji cells in uifro [30], and the ATPase-dependent Na+ flux in human erythrocytes [31]; (c) promoting Ca-uptake probably via stimulation of a bicarbonate-dependent Ca*+-ATPase in chick tibia rudiments [14]. It appears as if the signal finds its way in systems that are under perturbation by other agents such as activators and inhibitors, whereas it may have little effect in normal undisturbed systems.

131 REFERENCES 1 A.A. Pilla, Adv. Chem. Ser., 188 (1980) 339. 2 J. Assailly, J.D. Monet, Y. Goureau, P. Christel and A.A. Pilla, Bioelectrochem. Bioenerg., 8 (1981) 515. 3 C.A.L. Bassett, A.A. Pilla and R.J. Pawluk, J. Clin. Orthop., 124 (1977) 117. 4 G.A. Rodan, L.A. Bourret, and L.A. Norton, Science, 199 (1978) 690. 5 S.M. Bawin, W.R. Adey and I.M. Sabbot, Proc. Natl. Acad. Sci. (USA), 75 (1978) 6314. 6 G. Colacicco and A.A. Pilla, Abstracts 5th International Symposium on Bioelectrochemistry, Weimar, F.R.G., September 1979. 7 J.G. Kaplan, Annu. Rev. Physiol., 40 (1978) 19. 8 R.H. Kretsinger, Adv. Cyclic Nucleotide Res., 11 (1979) 1. 9 M. Tada, F. Ohmori, N. Kinoshita and H. Abe, Adv. Cyclic Nucleotide Res., 9 (1978) 355. 10 A. Chiabrera, M. Hinsenkamp, A.A. Pilla, J.P. Ryaby, D. Porta, F. Beltrame, M. Grattarole and C. Nicolini, J. Histochem. Cytochem., 27 (1979) 375. 11 A.A. Pilla in Bioelectrochemistry, H. Keyzer and F. Gutmann (Editors), Plenum Press, New York, 1980, pp. 353-396. 12 G. Colacicco and A.A. Pilla, J. Electrochem. Sot., 130 (1983) 12lC. 13 A.L. Lehninger, Proc. Natl. Acad. Sci. (USA), 71 (1974) 1520. 14 G. Cola&co and A.A. Pilla, Z. Naturforschung, 38C (1983), in press. 15 S.P. Nielsen, Calcif. Tissue Res., 11 (1973) 78. 16 W.K. Ramp, J.R. Thomas and P.D. Nifong, Calcif. Tissue Res., 24 (1977) 93. 17 W.K. Ramp, W.C. Murdock, W.A. Gonnerman and T.-C. Peng, Calcif. Tissue Res., 17 (1975) 195. 18 E.P. Hicks and W.K. Ramp, Calcif. Tissue Res., 17 (1975) 205. 19 G.R. Spencer, A.L. Cohen and G.E. Garner, Calcif. Tissue Res., 15 (1974) 111. 20 V. Hamburger and H.L. Hamilton, J. Morphol., 88 (1951) 49. 21 A. Lehninger, Biochemistry, Worth, New York, 1975. 22 F.L. Bygrave, Biol. Rev., 53 (1978) 43. 23 W.K. Ramp and W.F. Newman, Calcif. Tissue Res., 11 (1973) 171. 24 W.H. Harris, R.P. Heaney, L.A. Davis, E.H. Weinberg, R.D. Coutts and A.L. Schiller, Calcif. Tissue Res., 22 (1976) 85. 25 M.B. Orsatti, L.L. Fucci, J.L. Valenti and R.C. Puche, Calcif. Tissue Res., 21 (1976) 195. 26 A.A. Pilla, Ann. N.Y. Acad. Sci., 238 (1974) 149. 27 Z. Zimmerman. J. Vienken and G. Pilwat, Z. Naturforschung, Teil C, 36 (1981) 173. 28 R.A. Luben, C.D. Cain, M.C.-Y. Chen, D.M. Rosen and W.R. Adey, Proc. Natl. Acad. Sci. (USA), 79 (1982) 4180. 29 A.A. Pilla, J. Electiochem. Sot., 118 (1971) 1295. 30 G. Cola&co and A.A. Pilla, Z. Naturforschung, 38C (1983), in press. 31 K. Gary, A.A. Pilla and C. Mayaud, J. Electrochem. Sot., 130 (1983) 12OC.